U.S. patent application number 08/954771 was filed with the patent office on 2003-03-20 for vertebrate embryonic pattern-inducing proteins and uses related thereto.
Invention is credited to INGham, PHILIP w, MCMAHON, ANDREW P., TABIN, CLIFFORD J.
Application Number | 20030054437 08/954771 |
Document ID | / |
Family ID | 46279360 |
Filed Date | 2003-03-20 |
United States Patent
Application |
20030054437 |
Kind Code |
A1 |
INGham, PHILIP w ; et
al. |
March 20, 2003 |
VERTEBRATE EMBRYONIC PATTERN-INDUCING PROTEINS AND USES RELATED
THERETO
Abstract
The present invention concerns the discovery that proteins
encoded by a family of vertebrate genes, termed here
hedgehog-related genes, comprise morphogenic signals produced by
embryonic patterning centers, and are involved in the formation of
ordered spatial arrangements of differentiated tissues in
vertebrates. The present invention makes available compositions and
methods that can be utilized, for example to generate and/or
maintain an array of different vertebrate tissue both in vitro and
in vivo.
Inventors: |
INGham, PHILIP w; (OXFORD,
GB) ; MCMAHON, ANDREW P.; (LEXINGTON, MA) ;
TABIN, CLIFFORD J; (CAMBRIDGE, MA) |
Correspondence
Address: |
ROPES & GRAY
ONE INTERNATIONAL PLACE
BOSTON
MA
02110-2624
US
|
Family ID: |
46279360 |
Appl. No.: |
08/954771 |
Filed: |
October 20, 1997 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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08954771 |
Oct 20, 1997 |
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08462386 |
Jun 5, 1995 |
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08462386 |
Jun 5, 1995 |
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08435093 |
May 4, 1995 |
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08435093 |
May 4, 1995 |
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08356060 |
Dec 14, 1994 |
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5844079 |
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08356060 |
Dec 14, 1994 |
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08176427 |
Dec 30, 1993 |
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5789543 |
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Current U.S.
Class: |
435/69.1 ;
435/226; 435/320.1; 435/368; 536/23.2 |
Current CPC
Class: |
C12N 15/8509 20130101;
A01K 2227/40 20130101; A01K 2267/03 20130101; A01K 2227/30
20130101; A61K 38/00 20130101; C07K 14/46 20130101; A01K 67/0339
20130101; C07K 2319/00 20130101; A01K 2207/15 20130101; A01K
67/0275 20130101; A01K 67/0271 20130101; C12N 2800/30 20130101;
A01K 67/033 20130101; A01K 2227/50 20130101; A01K 2267/0318
20130101; A01K 2227/105 20130101; A01K 2217/00 20130101; A01K
2267/0368 20130101; C07K 14/4702 20130101; A01K 2217/05 20130101;
C07K 14/475 20130101 |
Class at
Publication: |
435/69.1 ;
435/320.1; 435/226; 435/368; 536/23.2 |
International
Class: |
C12N 009/64; C07H
021/04; C12N 005/08; C12P 021/02 |
Goverment Interests
[0002] Work described herein was supported by funding from the
National Institutes of Health. The United States Government has
certain rights in the invention.
Claims
What is claimed is:
1. A method for modulating growth, differentiation, or survival of
a cell comprising contacting said cell with an effective amount of
a hedgehog polypeptide.
2. A method for modulating one or more of growth, differentiation,
or survival of a mammalian cell responsive to hedgehog induction,
comprising treating the cell with an effective amount of a hedgehog
polypeptide thereby altering, relative to the cell in the absence
of hedgehog treatment, at least one of (i) rate of growth, (ii)
differentiation, or (iii) survival of the cell.
3. The method of claim 2, which polypeptide mimics the effects of a
naturally-occurring hedgehog protein on said cell.
4. The method of claim 2, which polypeptide antagonizes the effects
of a naturally-occurring hedgehog protein on said cell.
5. The method of claim 2, which polypeptide comprises an amino acid
sequence identical or homologous to an amino acid sequence
designated in one of SEQ ID No:8, SEQ ID No:9, SEQ ID No:10, SEQ ID
No:11, SEQ ID No:12, SEQ ID No:13 or SEQ ID No:14.
6. The method of claim 5, which polypeptide is a bioactive fragment
of a hedgehog polypeptide.
7. The method of claim 2, which polypeptide comprises an amino acid
sequence identical or homologous to an amino acid sequence
designated in SEQ ID No:34.
8. The method of claim 2, wherein the cell is a testicular cell,
and the polypeptide modulates spermatogenesis.
9. The method of claim 2, wherein the cell is an osteogenic cell,
and the polypeptide modulates osteogenesis.
10. The method of claim 2, wherein the cell is a chondrogenic cell,
and the polypeptide modulates chondrogenesis.
11. The method of claim 2, wherein the polypeptide modulates the
differentiation of neuronal cells.
12. The method of claim 11, which neuronal cells are selected from
the group consisting of motor neurons, cholinergic neurons,
dopanergic neurons, serotenergic neurons, and peptidergic
neurons.
13. The method of claim 11, wherein the polypeptide promotes
survival of the neuronal cells.
14. A method for modulating, in an animal, cell growth, cell
differentiation or cell survival, comprising administering a
therapeutically effective amount of a hedgehog polypeptide to
alter, relative the absence of hedgehog treatment, at least one of
(i) rate of growth, (ii) differentiation, or (iii) survival of one
or more cell-types in the animal.
15. The method of claim 14, which polypeptide mimics the effects of
a naturally-occurring hedgehog protein on cells in the animal.
16. The method of claim 14, which polypeptide antagonizes the
effects of a naturally-occurring hedgehog protein on cells in the
animal
17. The method of claim 14, which polypeptide comprises an amino
acid sequence identical or homologous to amino acid sequence
designated in one of SEQ ID No:8, SEQ ID No:9, SEQ ID No:10, SEQ ID
No:11, SEQ ID No:12, SEQ ID No:13, SEQ ID No: 14, SEQ ID No. 34,
SEQ ID No. 40, SEQ ID No. 41, or homologs thereof.
18. The method of claim 17, which polypeptide is a bioactive
fragment of a hedgehog polypeptide.
19. The method of claim 14, which method modulates spermatogenesis
in the animal.
20. The method of claim 14, which method modulates osteogenesis in
the animal.
21. The method of claim 14, which method modulates chondrogenesis
in the animal.
22. The method of claim 14, which method modulates differentiation
of neuronal cells in the animal.
23. A method for inducing a cell to differentiate to a neuronal
cell phenotype, comprising contacting said cell with a hedgehog
polypeptide.
24. The method of claim 23, which polypeptide comprises an amino
acid sequence identical or homologous to amino acid sequence
designated in one of SEQ ID No:8, SEQ ID No:9, SEQ ID No:10, SEQ ID
No:11, SEQ ID No:12, SEQ ID No:13, SEQ ID No:14, SEQ ID No. 34, SEQ
ID No. 40, SEQ ID No. 41, or homologs thereof.
25. The method of claim 24, which polypeptide is a bioactive
fragment of a hedgehog polypeptide.
26. The method of claim 23, wherein said neuronal cell phenotype is
selected from the group consisting of motor neurons, cholinergic
neurons, dopanergic neurons, serotenergic neurons, and peptidergic
neurons.
27. A method of modulating skeletogenesis comprising contacting a
target tissue with an effective amount of a hedgehog polypeptide so
as to cause one or both of chrondrogenesis and oseteogenesis in the
target tissue.
28. The method of claim 27, wherein said target tissue is selected
from the group consisting of bone, connective tissue and a
combination thereof.
29. A method for treating a degenerative disorder of the nervous
system characterized by neuronal cell death, comprising
administering to a patient a therapeutically effective amount of a
pharmaceutical preparation of a hedgehog polypeptide thereby
causing, relative to the absence of hedgehog treatment, prolonged
survival of neural cells in said patient.
30. The method of claim 29, wherein said hedgehog polypeptide
comprises an amino acid sequence identical or homologous to a
polypeptide selected from the group consisting of SEQ ID No:8, SEQ
ID No:9, SEQ ID No:10, SEQ ID No:11, SEQ ID No:12, SEQ ID No:13,
and SEQ ID No:14, or is a bioactive fragment thereof.
31. The method of claim 29, wherein said hedgehog polypeptide
comprises an amino acid designated in SEQ ID No. 41.
32. The method of claim 29, wherein said hedgehog polypeptide
comprises an amino acid identical or homologous to SEQ ID No. 34,
or a bioactive fragment thereof.
33. The method of claim 29, wherein said therapeutically effective
amount of hedgehog polypeptide inhibits the de-differentiation of
neural cells of said patient.
34. The method of claim 33, wherein said neural cell is a glial
cell.
35. The method of claim 33, wherein said neural cell is a nerve
cell.
36. The method of claim 29, wherein said degenerative disorder is a
neuromuscular disorder.
37. The method of claim 29, wherein said degenerative disorder is a
autonomic disorder.
38. The method of claim 29, wherein said degenerative disorder is a
central nervous system disorder.
39. The method of claim 29, wherein said degenerative disorder is
selected from a group consisting of Alzheimer's disease,
Parkinson's disease, amyotrophic lateral sclerosis, Pick's disease,
Huntington's disease, multiple sclerosis, neuronal damage resulting
from anoxia-ischemia, neuronal damage resulting from trauma, and
neuronal degeneration associated with a natural aging process.
40. The method of claim 29, further comprising administering to
said patient a therapeutically effective amount of a growth factor
having neurotrophic activity.
41. The method of claim 40, wherein said growth factor is selected
from a group consisting of a nerve growth factor, cilliary
neurotrophic growth factor, schwanoma-derived growth factor, glial
growth factor, striatal-derived neuronotrophic factor,
platelet-derived growth factor.
Description
RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S. Ser. No.
08/435,093, filed May 4, 1995, which is a continuation-in-part of
U.S. Ser. No. 08/356,060, filed Dec. 14, 1994, which is a
continuation-in-part of U.S. Ser. No. 08/227,371 filed Dec. 30,
1993 and entitled "Vertebrate Embryonic Pattern-Inducing Proteins
and Uses Related Thereto", the teachings of which are incorporated
herein by reference.
BACKGROUND OF THE INVENTION
[0003] Pattern formation is the activity by which embryonic cells
form ordered spatial arrangements of differentiated tissues. The
physical complexity of higher organisms arises during embryogenesis
through the interplay of cell-intrinsic lineage and cell-extrinsic
signaling. Inductive interactions are essential to embryonic
patterning in vertebrate development from the earliest
establishment of the body plan, to the patterning of the organ
systems, to the generation of diverse cell types during tissue
differentiation (Davidson, E., (1990) Development 108: 365-389;
Gurdon, J. B., (1992) Cell 68: 185-199; Jessell, T. M. et al.,
(1992) Cell 68: 257-270). The effects of developmental cell
interactions are varied. Typically, responding cells are diverted
from one route of cell differentiation to another by inducing cells
that differ from both the uninduced and induced states of the
responding cells (inductions). Sometimes cells induce their
neighbors to differentiate like themselves (homoiogenetic
induction); in other cases a cell inhibits its neighbors from
differentiating like itself. Cell interactions in early development
may be sequential, such that an initial induction between two cell
types leads to a progressive amplification of diversity. Moreover,
inductive interactions occur not only in embryos, but in adult
cells as well, and can act to establish and maintain morphogenetic
patterns as well as induce differentiation (J. B. Gurdon (1992)
Cell 68:185-199).
[0004] The origin of the nervous system in all vertebrates can be
traced to the end of gastrulation. At this time, the ectoderm in
the dorsal side of the embryo changes its fate from epidermal to
neural. The newly formed neuroectoderm thickens to form a flattened
structure called the neural plate which is characterized, in some
vertebrates, by a central groove (neural groove) and thickened
lateral edges (neural folds). At its early stages of
differentiation, the neural plate already exhibits signs of
regional differentiation along its anterior posterior (A-P) and
mediolateral axis (M-L). The neural folds eventually fuse at the
dorsal midline to form the neural tube which will differentiate
into brain at its anterior end and spinal cord at its posterior
end. Closure of the neural tube creates dorsal/ventral differences
by virtue of previous mediolateral differentiation. Thus, at the
end of neurulation, the neural tube has a clear anterior-posterior
(A-P), dorsal ventral (D-V) and mediolateral (M-L) polarities (see,
for example, Principles in Neural Science (3rd), eds. Kandel,
Schwartz and Jessell, Elsevier Science Publishing Company: NY,
1991; and Developmental Biology (3rd), ed. S. F. Gilbert, Sinauer
Associates: Sunderland, Mass., 1991). Inductive interactions that
define the fate of cells within the neural tube establish the
initial pattern of the embryonic vertebrate nervous system. In the
spinal cord, the identify of cell types is controlled, in part, by
signals from two midline cell groups, the notochord and floor
plate, that induce neural plate cells to differentiate into floor
plate, motor neurons, and other ventral neuronal types (van
Straaten et al. (1988) Anat. Embryol. 177:317-324; Placzek et al.
(1993) Development 117:205-218; Yamada et al. (1991) Cell
64:035-647; and Hatta et al. (1991) Nature 350:339-341). In
addition, signals from the floor plate are responsible for the
orientation and direction of commissural neuron outgrowth (Placzek,
M. et al., (1990) Development 110: 19-30). Besides patterning the
neural tube, the notochord and floorplate are also responsible for
producing signals which control the patterning of the somites by
inhibiting differentiation of dorsal somite derivatives in the
ventral regions (Brand-Saberi, B. et al., (1993) Anat. Embryol.
188: 239-245; Porquie, O. et al., (1993) Proc. Natl. Acad. Sci. USA
90: 5242-5246).
[0005] Another important signaling center exists in the posterior
mesenchyme of developing limb buds, called the Zone of Polarizing
Activity, or "ZPA". When tissue from the posterior region of the
limb bud is grafted to the anterior border of a second limb bud,
the resultant limb will develop with additional digits in a
mirror-image sequence along the anteroposterior axis (Saunders and
Gasseling, (1968) Epithelial-Mesenchymal Interaction, pp. 78-97).
This finding has led to the model that the ZPA is responsible for
normal anteroposterior patterning in the limb. The ZPA has been
hypothesized to function by releasing a signal, termed a
"morphogen", which forms a gradient across the early embryonic bud.
According to this model, the fate of cells at different distances
from the ZPA is determined by the local concentration of the
morphogen, with specific thresholds of the morphogen inducing
successive structures (Wolpert, (1969) Theor. Biol. 25:1-47). This
is supported by the finding that the extent of digit duplication is
proportional to the number of implanted ZPA cells (Tickle, (1981)
Nature 254:199-202).
[0006] A candidate for the putative ZPA morphogen was identified by
the discovery that a source of retinoic acid can result in the same
type of mirror-image digit duplications when placed in the anterior
of a limb bud (Tickle et al., (1982) Nature 296:564-565;
Summerbell, (1983) J. Embryol 78:269-289). The response to
exogenous retinoic acid is concentration dependent as the morphogen
model demands (Tickle et al., (1985) Dev. Biol. 109:82-95).
Moreover, a differential distribution of retinoic acid exists
across the limb bud, with a higher concentration in the ZPA region
(Thaller and Eichele, (1987) Nature 327:625-628).
[0007] Recent evidence, however, has indicated that retinoic acid
is unlikely to be the endogenous factor responsible for ZPA
activity (reviewed in Brockes, (1991) Nature 350:15; Tabin, (1991)
Cell 66:199-217). It is now believed that rather than directly
mimicking an endogenous signal, retinoic acid implants act by
inducing an ectopic ZPA. The anterior limb tissue just distal to a
retinoic acid implant and directly under the ectoderm has been
demonstrated to acquire ZPA activity by serially transplanting that
tissue to another limb bud (Summerbell and Harvey, (1983) Limb
Development and Regeneration pp. 109-118; Wanek et al., (1991)
Nature 350:81-83). Conversely, the tissue next to a ZPA graft does
not gain ZPA activity (Smith, (1979) J. Embryol 52:105-113).
Exogenous retinoic acid would thus appear to act upstream of the
ZPA in limb patterning.
[0008] The immediate downstream targets of ZPA action are not
known. However, one important set of genes which are ectopically
activated during ZPA-induced pattern duplications are the 5' genes
of the Hoxd cluster. These genes are normally expressed in a nested
pattern emanating from the posterior margin of the limb bud (Dolle
et al., (1989) Nature 342:767-772; Izpisua-Belmonte et al., (1991)
Nature 350:585-589). This nested pattern of Hox gene expression has
been directly demonstrated to determine the identity of the
structures produced along the anteroposterior axis of the limb
(Morgan et al., (1993) Nature 358:236-239). As this would predict,
ZPA grafts which produce mirror-image duplication of structures at
an anatomical level first lead to the ectopic activation of the
Hoxd genes in a mirror-image duplication at the molecular level.
(Nohno et al., (1991) Cell 64:1197-1205; Izpisua-Belmonte et al.,
(1991) Nature 350:585-589). The molecular signals which regulate
the expression of these important genes are currently not
understood.
SUMMARY OF THE INVENTION
[0009] The present invention relates to the discovery of a novel
family of genes, and gene products, expressed in vertebrate
organisms, which genes referred to hereinafter as the "hedgehog"
gene family, the products of which are referred to as hedgehog
proteins. The products of the hedgehog gene have apparent broad
involvement in the formation and maintenance of ordered spatial
arrangements of differentiated tissues in vertebrates, both adult
and embryonic, and can be used to generate and/or maintain an array
of different vertebrate tissue both in vitro and in vivo.
[0010] In general, the invention features hedgehog polypeptides,
preferably substantially pure preparations of one or more of the
subject hedgehog polypeptides. The invention also provides
recombinantly produced hedgehog polypeptides. In preferred
embodiments the polypeptide has a biological activity including: an
ability to modulate proliferation, survival and/or differentiation
of mesodermally-derived tissue, such as tissue derived from dorsal
mesoderm; the ability to modulate proliferation, survival and/or
differentiation of ectodermally-derived tissue, such as tissue
derived from the neural tube, neural crest, or head mesenchyme; the
ability to modulate proliferation, survival and/or differentiation
of endodermally-derived tissue, such as tissue derived from the
primitive gut. Moreover, in preferred embodiments, the subject
hedgehog proteins have the ability to induce expression of
secondary signaling molecules, such as members of the Transforming
Growth Factor .beta. family, as well as members of the fibroblast
growth factor (FGF) family.
[0011] In a preferred embodiment, the polypeptide is identical with
or homologous to a Sonic hedgehog (Shh) polypeptide, such as a
mammalian Shh represented by SEQ ID Nos:13 or 11, an avian Shh
represented by SEQ ID No: 8, or a fish Shh represented by SEQ ID
No: 12. For instance, the Shh polypeptide preferably has an amino
acid sequence at least 60% homologous to a polypeptide represented
by any of SEQ ID Nos: 8, 11, 12 or 13, though polypeptides with
higher sequence homologies of, for example, 80%, 90% or 95% are
also contemplated. Exemplary Shh proteins are represented by SEQ ID
No. 40. The Shh polypeptide can comprise a full length protein,
such as represented in the sequence listings, or it can comprise a
fragment of, for instance, at least 5, 10, 20, 50, 100, 150 or 200
amino acids in length. Preferred hedgehog polypeptides include Shh
sequences corresponding approximately to the natural proteolytic
fragments of the hedgehog proteins, such as from about Cys-24
through about the region that contains the proteolytic processing
site, e.g., Ala-194 to Gly-203, or from about Cys-198 through
Ala-475 of the human Shh protein, or analogous fragments
thereto.
[0012] In another preferred embodiment, the polypeptide is
identical with or homologous to an Indian hedgehog (Ihh)
polypeptide, such as a human Ihh represented by SEQ ID No:14, or a
mouse Ihh represented by SEQ ID No: 10. For instance, the Ihh
polypeptide preferably has an amino acid sequence at least 60%
homologous to a polypeptide represented by either of SEQ ID Nos: 10
or 14, though Ihh polypeptides with higher sequence homologies of,
for example, 80%, 90% or 95% are also contemplated. The polypeptide
can comprise the full length protein represented by in part by
these sequences, or it can comprise a fragment of, for instance, at
least 5, 10, 20, 50, 100, 150 or 200 amino acids in length.
Preferred Ihh polypeptides comprise an N-terminal fragment from
Cys-28 through the region that contains the proteolytic processing
site, e.g., Ala-198 to Gly-207, or a C-terminal fragment from about
Cys-203 through Ser-411 of the mouse Ihh represented by SEQ ID
No:10, or analogous fragments thereto.
[0013] In still a further preferred embodiment, the polypeptide is
identical with or homologous to a Desert hedgehog (Dhh)
polypeptide, such as a mouse Dhh represented by SEQ ID No: 9. For
instance, the Dhh polypeptide preferably has an amino acid sequence
at least 60% homologous to a polypeptide represented by SEQ ID No:
9, though Dhh polypeptides with higher sequence homologies of, for
example, 80%, 90% or 95% are also contemplated. The polypeptide can
comprise the full length protein represented by this sequence, or
it can comprise a fragment of, for instance, at least 5, 10, 20,
50, 100, 150 or 200 amino acids in length. Preferred Dhh
polypeptides comprise Dhh sequences corresponding to the N-terminal
portion of the protein from about Cys-23 through about the region
that contains the proteolytic processing site, e.g., Val-124 to
Asn-203 or C-terminal fragment from about Cys-199 through Gly-396
of SEQ ID No:9, or analogous fragments thereto.
[0014] In another preferred embodiment, the invention features a
purified or recombinant polypeptide fragment of a hedgehog protein,
which polypeptide has the ability to modulate, e.g., mimic or
antagonize, a the activity of a wild-type hedgehog protein.
Preferably, the polypeptide fragment comprises a sequence identical
or homologous to an amino acid sequence designated in one of SEQ ID
No:8, SEQ ID No:9, SEQ ID No:10, SEQ ID No:11, SEQ ID No:12, SEQ ID
No:13, or SEQ ID No:14. More preferably, the polypeptide fragment
comprises an amino acid sequence designated in SEQ ID No: 40, e.g.,
includes the fragment of Cys-1 to Gly-174.
[0015] In yet another preferred embodiment, the invention features
a purified or recombinant polypeptide, which polypeptide has a
molecular weight of approximately 19 kDa and has the ability to
modulate, e.g., mimic or antagonize, a the activity of a wild-type
hedgehog protein. Preferably, the polypeptide comprises an amino
acid sequence identical or homologous to an sequence designated in
one of SEQ ID No:8, SEQ ID No:9, SEQ ID No:10, SEQ ID No:11, SEQ ID
No:12, SEQ ID No:13, or SEQ ID No:14. More preferably, the
polypeptide comprises an amino acid sequence designated in SEQ ID
No:40.
[0016] In still another preferred embodiment, the invention
features a purified or recombinant hedgehog polypeptide comprising
an amino acid sequence represented by the formula A-B wherein, A
represents all or the portion of the amino acid sequence designated
by residues 1-168 of SEQ ID No:40; and B represents at least one
amino acid residue of the amino acid sequence designated by
residues 169-221 of SEQ ID No:40; wherein A and B together
represent a contiguous polypeptide sequence represented by SEQ ID
No:40, and the polypeptide modulates, e.g., mimics or antagonizes,
the biological activity of a hedgehog protein. Preferably, B can
represent at least 5, 10 or 20 amino acid residues of the amino
acid sequence designated by residues 169-221 of SEQ ID No:40.
[0017] In another embodiment, the invention features a purified or
recombinant polypeptide comprising an amino acid sequence
represented by the formula A-B, wherein A represents all or the
portion of the amino acid sequence designated by residues 24-193 of
SEQ ID No:13; and B represents at least one amino acid residue of
the amino acid sequence designated by residues 194-250 of SEQ ID
No:13; wherein A and B together represent a contiguous polypeptide
sequence designated in SEQ ID No:13, and the polypeptide modulates,
e.g., mimics or antagonizes, the biological activity of a hedgehog
protein.
[0018] In yet another preferred embodiment, the invention features
a purified or recombinant polypeptide comprising an amino acid
sequence represented by the formula A-B, wherein A represents all
or the portion of the amino acid sequence designated by residues
25-193, or analogous residues thereof, of a vertebrate hedgehog
polypeptide identical or homologous to SEQ ID No:11; and B
represents at least one amino acid residue of the amino acid
sequence designated by residues 194-250, or analogous residues
thereof, of a vertebrate hedgehog polypeptide identical or
homologous to SEQ ID No:11; wherein A and B together represent a
contiguous polypeptide sequence designated in SEQ ID No:11, and the
polypeptide modulates, e.g., agonizes or antagonizes, the
biological activity of a hedgehog protein.
[0019] In another embodiment, the invention features a purified or
recombinant polypeptide comprising an amino acid sequence
represented by the formula A-B, wherein A represents all or the
portion of the amino acid sequence designated by residues 23-193 of
SEQ ID No:9; and B represents at least one amino acid residue of
the amino acid sequence designated by residues 194-250 of SEQ ID
No:9; wherein A and B together represent a contiguous polypeptide
sequence designated in SEQ ID No:9, and the polypeptide modulates,
e.g., agonizes or antagonizes, the biological activity of a
hedgehog protein.
[0020] In yet another embodiment, the invention features a purified
or recombinant polypeptide comprising an amino acid sequence
represented by the formula A-B, wherein A represents all or the
portion of the amino acid sequence designated by residues 28-197 of
SEQ ID No:10; and B represents at least one amino acid residue of
the amino acid sequence designated by residues 198-250 of SEQ ID
No:10; wherein A and B together represent a contiguous polypeptide
sequence designated in SEQ ID No:10, and the polypeptide modulates,
e.g., agonizes or antagonizes, the biological activity of a
hedgehog protein.
[0021] In yet a further preferred embodiment, the invention
features a purified or recombinant polypeptide comprising an amino
acid sequence represented by the formula A-B, wherein A represents
all or the portion of the amino acid sequence designated by
residues 1-98, or analogous residues thereof, of a vertebrate
hedgehog polypeptide identical or homologous to SEQ ID No:14; and B
represents at least one amino acid residue of the amino acid
sequence designated by residues 99-150, or analogous residues
thereof, of a vertebrate hedgehog polypeptide identical or
homologous to SEQ ID No:14; wherein A and B together represent a
contiguous polypeptide sequence designated in SEQ ID No:14, and the
polypeptide modulates, e.g., agonizes or antagonizes, the
biological activity of a hedgehog protein.
[0022] In another preferred embodiment, the invention features a
nucleic acid encoding a polypeptide fragment of a hedgehog protein,
e.g. a fragment described above. Preferably, the polypeptide
fragment comprises an amino acid sequence identical or homologous
with a sequence designated in one of SEQ ID No:8, SEQ ID No:9, SEQ
ID No:10, SEQ ID No:11, SEQ ID No:12, SEQ ID No:13, or SEQ ID
No:14. More preferably, the polypeptide fragment comprises an amino
acid sequence designated in SEQ ID No:40.
[0023] In yet another preferred embodiment, the invention features
a nucleic acid encoding a polypeptide, which polypeptide has a
molecular weight of approximately 19 kDa and has the ability to
modulate, e.g., either mimic or antagonize, atleast a portion of
the activity of a wild-type hedgehog protein. Preferably, the
polypeptide comprises an amino acid sequence identical or
homologous with a sequence designated in one of SEQ ID No:8, SEQ ID
No:9, SEQ ID No:10, SEQ ID No:11, SEQ ID No:12, SEQ ID No:13, or
SEQ ID No:14. More preferably, the polypeptide comprises an amino
acid sequence designated in the general formula SEQ ID No:40.
[0024] In another preferred embodiment, the invention feature a
nucleic acid which encodes a polypeptide that modulates, e.g.,
mimics or antagonizes, the biological activity of a hedgehog
protein, which nucleic acid comprises all or a portion of the
nucleotide sequence of the coding region of a gene identical or
homologous to the nucleotide sequence designated by one of SEQ ID
No:1, SEQ ID No:2, SEQ ID No:3, SEQ ID No:4, SEQ ID No:5, SEQ ID
No:6 or SEQ ID No:7. Preferably, the nucleic acid comprises a
hedgehog-encoding portion that hybridizes under stringent
conditions to a coding portion of one or more of the nucleic acids
designated by SEQ ID No:1-7.
[0025] Moreover, as described below, the hedgehog polypeptide can
be either an agonist (e.g. mimics), or alternatively, an antagonist
of a biological activity of a naturally occurring form of the
protein, e.g., the polypeptide is able to modulate differentiation
and/or growth and/or survival of a cell responsive to authentic
hedgehog proteins. Homologs of the subject hedgehog proteins
include versions of the protein which are resistant to proteolytic
cleavage, as for example, due to mutations which alter potential
cleavage sequences or which inactivate an enzymatic activity
associated with the protein. Other forms are secreted and
isolatable from a cell with no further proteolytic cleavage
required beyond cleavage of a signal sequence, e.g., truncated
forms of the protein, such as corresponding to the natural
proteolytic fragments described below.
[0026] The hedgehog polypeptides of the present invention can be
glycosylated, or conversely, by choice of the expression system or
by modification of the protein sequence to preclude glycosylation,
reduced carbohydrate analogs can also be provided. Glycosylated
forms include derivatization with glycosaminoglycan chains.
Likewise, hedgehog polypeptides can be generated which lack an
endogenous signal sequence (though this is typically cleaved off
even if present in the pro-form of the protein).
[0027] The subject proteins can also be provided as chimeric
molecules, such as in the form of fusion proteins. For instance,
the hedgehog protein can be provided as a recombinant fusion
protein which includes a second polypeptide portion, e.g., a second
polypeptide having an amino acid sequence unrelated (heterologous)
to the hedgehog polypeptide, e.g. the second polypeptide portion is
glutathione-S-transferase, e.g. the second polypeptide portion is
an enzymatic activity such as alkaline phosphatase, e.g. the second
polypeptide portion is an epitope tag.
[0028] Yet another aspect of the present invention concerns an
immunogen comprising a hedgehog polypeptide in an immunogenic
preparation, the immunogen being capable of eliciting an immune
response specific for a hedgehog polypeptide; e.g. a humoral
response, e.g. an antibody response; e.g. a cellular response. In
preferred embodiments, the immunogen comprising an antigenic
determinant, e.g. a unique determinant, from a protein represented
by one of SEQ ID Nos. 8-14.
[0029] A still further aspect of the present invention features
antibodies and antibody preparations specifically reactive with an
epitope of the hedgehog immunogen.
[0030] In another preferred embodiment, the invention features a
nucleic acid encoding a polypeptide fragment of a hedgehog protein,
e.g. a fragment described above. Preferably, the polypeptide
fragment comprises an amino acid sequence identical or homologous
with a sequence designated in one of SEQ ID No:8, SEQ ID No:9, SEQ
ID No:10, SEQ ID No:11, SEQ ID No:12, SEQ ID No:13, or SEQ ID
No:14. More preferably, the polypeptide fragment comprises an amino
acid sequence designated in SEQ ID No:40.
[0031] In yet another preferred embodiment, the invention features
a nucleic acid encoding a polypeptide, which polypeptide has a
molecular weight of approximately 19 kDa and has the ability to
modulate, e.g., either mimic or antagonize, atleast a portion of
the activity of a wild-type hedgehog protein. Preferably, the
polypeptide comprises an amino acid sequence identical or
homologous with a sequence designated in one of SEQ ID No:8, SEQ ID
No:9, SEQ ID No:10, SEQ ID No:11, SEQ ID No:12, SEQ ID No:13, or
SEQ ID No:14. More preferably, the polypeptide comprises an amino
acid sequence designated in the general formula SEQ ID No:40.
[0032] In another preferred embodiment, the invention feature a
nucleic acid which encodes a polypeptide that modulates, e.g.,
mimics or antagonizes, the biological activity of a hedgehog
protein, which nucleic acid comprises all or a portion of the
nucleotide sequence of the coding region of a gene identical or
homologous to the nucleotide sequence designated by one of SEQ ID
No:1, SEQ ID No:2, SEQ ID No:3, SEQ ID No:4, SEQ ID No:5, SEQ ID
No:6 or SEQ ID No:7. Preferably, the nucleic acid comprises a
hedgehog-encoding portion that hybridizes under stringent
conditions to a coding portion of one or more of the nucleic acids
designated by SEQ ID No:1-7.
[0033] Another aspect of the present invention provides a
substantially isolated nucleic acid having a nucleotide sequence
which encodes a hedgehog polypeptide. In preferred embodiments, the
encoded polypeptide specifically mimics or antagonizes inductive
events mediated by wild-type hedgehog proteins. The coding sequence
of the nucleic acid can comprise a sequence which is identical to a
coding sequence represented in one of SEQ ID Nos: 1-7, or it can
merely be homologous to one or more of those sequences. For
instance, the hedgehog encoding sequence preferably has a sequence
at least 60% homologous to a nucleotide sequence in one or more of
SEQ ID Nos: 1-7, though higher sequence homologies of, for example,
80%, 90% or 95% are also contemplated. The polypeptide encoded by
the nucleic acid can comprise an amino acid sequence represented in
one of SEQ ID Nos: 8-14 such as one of those full length proteins,
or it can comprise a fragment of that nucleic acid, which fragment
may, for instance, encode a fragment which is, for example, at
least 5, 10, 20, 50 or 100 or 200 amino acids in length. The
polypeptide encoded by the nucleic acid can be either an agonist
(e.g. mimics), or alternatively, an antagonist of a biological
activity of a naturally occurring form of a hedgehog protein.
[0034] Furthermore, in certain preferred embodiments, the subject
hedgehog nucleic acid will include a transcriptional regulatory
sequence, e.g. at least one of a transcriptional promoter or
transcriptional enhancer sequence, which regulatory sequence is
operably linked to the hedgehog gene sequence. Such regulatory
sequences can be used in to render the hedgehog gene sequence
suitable for use as an expression vector.
[0035] In yet a further preferred embodiment, the nucleic acid
hybridizes under stringent conditions to a nucleic acid probe
corresponding to at least 12 consecutive nucleotides of either
sense or antisense sequence of one or more of SEQ ID Nos:1-7;
though preferably to at least 20 consecutive nucleotides; and more
preferably to at least 40, 50 or 75 consecutive nucleotides of
either sense or antisense sequence of one or moreof SEQ ID
Nos:1-7.
[0036] The invention also features transgenic non-human animals,
e.g. mice, rats, rabbits, chickens, frogs or pigs, having a
transgene, e.g., animals which include (and preferably express) a
heterologous form of a hedgehog gene described herein, or which
misexpress an endogenous hedgehog gene, e.g., an animal in which
expression of one or more of the subject hedgehog proteins is
disrupted. Such a transgenic animal can serve as an animal model
for studying cellular and tissue disorders comprising mutated or
mis-expressed hedgehog alleles or for use in drug screening.
[0037] The invention also provides a probe/primer comprising a
substantially purified oligonucleotide, wherein the oligonucleotide
comprises a region of nucleotide sequence which hybridizes under
stringent conditions to at least 10 consecutive nucleotides of
sense or antisense sequence of SEQ ID No:1, or naturally occurring
mutants thereof. Nucleic acid probes which are specific for each of
the classes of vertebrate hedgehog proteins are contemplated by the
present invention, e.g. probes which can discern between nucleic
acid encoding an Shh versus an Ihh versus a Dhh versus an Mhh. In
preferred embodiments, the probe/primer further includes a label
group attached thereto and able to be detected. The label group can
be selected, e.g., from a group consisting of radioisotopes,
fluorescent compounds, enzymes, and enzyme co-factors. Probes of
the invention can be used as a part of a diagnostic test kit for
identifying dysfunctions associated with mis-expression of a
hedgehog protein, such as for detecting in a sample of cells
isolated from a patient, a level of a nucleic acid encoding a
subject hedgehog protein; e.g. measuring a hedgehog mRNA level in a
cell, or determining whether a genomic hedgehog gene has been
mutated or deleted. These so called "probes/primers" of the
invention can also be used as a part of "antisense" therapy which
refers to administration or in situ generation of oligonucleotide
probes or their derivatives which specifically hybridize (e.g.
bind) under cellular conditions, with the cellular mRNA and/or
genomic DNA encoding one or more of the subject hedgehog proteins
so as to inhibit expression of that protein, e.g. by inhibiting
transcription and/or translation. Preferably, the oligonucleotide
is at least 10 nucleotides in length, though primers of 20, 30, 50,
100, or 150 nucleotides in length are also contemplated.
[0038] In yet another aspect, the invention provides an assay for
screening test compounds for inhibitors, or alternatively,
potentiators, of an interaction between a hedgehog protein and a
hedgehog receptor. An exemplary method includes the steps of (i)
combining a hedgehog receptor, either soluble or membrane bound
(including whole cells), a hedgehog polypeptide, and a test
compound, e.g., under conditions wherein, but for the test
compound, the hedgehog protein and the hedgehog receptor are able
to interact; and (ii) detecting the formation of a complex which
includes the hedgehog protein and the receptor either by directly
quantitating the complex or by measuring inductive effects of the
hedgehog protein. A statistically significant change, such as a
decrease, in the formation of the complex in the presence of a test
compound (relative to what is seen in the absence of the test
compound) is indicative of a modulation, e.g., inhibition, of the
interaction between the hedgehog protein and the receptor.
[0039] Yet another aspect of the present invention concerns a
method for modulating one or more of growth, differentiation, or
survival of a mammalian cell responsive to hedgehog induction. In
general, whether carries out in vivo, in vitro, or in situ, the
method comprises treating the cell with an effective amount of a
hedgehog polypeptide so as to alter, relative to the cell in the
absence of hedgehog treatment, at least one of (i) rate of growth,
(ii) differentiation, or (iii) survival of the cell. Accordingly,
the method can be carried out with polypeptides mimics the effects
of a naturally-occurring hedgehog protein on the cell, as well as
with polypeptides which antagonize the effects of a
naturally-occurring hedgehog protein on said cell. In preferred
embodiments, the hedgehog polypeptide provided in the subject
method are derived from verterbrate sources, e.g., are vertebrate
hedgehog polypeptides. For instance, preferred polypeptides
includes an amino acid sequence identical or homologous to an amino
acid sequence (e.g., including bioactive fragments) designated in
one of SEQ ID No:8, SEQ ID No:9, SEQ ID No:10, SEQ ID No:11, SEQ ID
No:12, SEQ ID No:13 or SEQ ID No:14. Furthermore, the present
invention contemplates the use of invertebrate hedgehog
polypeptides, such as the Dros-HH polypeptide designated by SEQ ID
No:34, or bioactive fragments thereof equivalent to the subject
vertebrate fragments.
[0040] In one embodiment, the subject method includes the treatment
of testicular cells, so as modulate spermatogenesis. In another
embodiment, the subject method is used to modulate osteogenesis,
comprising the treatment of osteogenic cells with a hedgehog
polypeptide. Liekwise, where the treated cell is a chondrogenic
cell, the present method is used to modulate chondrogenesis. In
still another embodiment, hedgehog polypeptides can be used to
modulate the differentiation of neural cells, e.g., the method can
be used to cause differentiation of a neuronal cell, to maintain a
neuronal cell in a differentiated state, and/or to enhance the
survival of a neuronal cell, e.g., to prevent apoptosis or other
forms of cell death For instance, the present method can be used to
affect the differentiation of such neuronal cells as motor neurons,
cholinergic neurons, dopanergic neurons, serotenergic neurons, and
peptidergic neurons.
[0041] The present method is applicable, for example, to cell
culture technique, such as in the culturing of neural and other
cells whose survival or differentiative state is dependent on
hedgehog function. Moreover, hedgehog agonists and antagonists can
be used for therapeutic intervention, such as to enhance survival
and maintenance of neurons and other neural cells in both the
central nervous system and the peripheral nervous system, as well
as to influence other vertebrate organogenic pathways, such as
other ectodermal patterning, as well as certain mesodermal and
endodermal differentiation processes. In an exemplary embodiment,
the method is practiced for modulating, in an animal, cell growth,
cell differentiation or cell survival, and comprises administering
a therapeutically effective amount of a hedgehog polypeptide to
alter, relative the absence of hedgehog treatment, at least one of
(i) rate of growth, (ii) differentiation, or (iii) survival of one
or more cell-types in the animal.
[0042] Another aspect of the present invention provides a method of
determining if a subject, e.g. a human patient, is at risk for a
disorder characterized by unwanted cell proliferation or aberrant
control of differentiation. The method includes detecting, in a
tissue of the subject, the presence or absence of a genetic lesion
characterized by at least one of (i) a mutation of a gene encoding
a hedgehog protein, e.g. represented in SEQ ID No: 2, or a homolog
thereof; or (ii) the mis-expression of a hedgehog gene. In
preferred embodiments, detecting the genetic lesion includes
ascertaining the existence of at least one of: a deletion of one or
more nucleotides from a hedgehog gene; an addition of one or more
nucleotides to the gene, a substitution of one or more nucleotides
of the gene, a gross chromosomal rearrangement of the gene; an
alteration in the level of a messenger RNA transcript of the gene;
the presence of a non-wild type splicing pattern of a messenger RNA
transcript of the gene; or a non-wild type level of the
protein.
[0043] For example, detecting the genetic lesion can include (i)
providing a probe/primer including an oligonucleotide containing a
region of nucleotide sequence which hybridizes to a sense or
antisense sequence of a hedgehog gene, e.g. a nucleic acid
represented in one of SEQ ID Nos: 1-7, or naturally occurring
mutants thereof, or 5' or 3' flanking sequences naturally
associated with the hedgehog gene; (ii) exposing the probe/primer
to nucleic acid of the tissue; and (iii) detecting, by
hybridization of the probe/primer to the nucleic acid, the presence
or absence of the genetic lesion; e.g. wherein detecting the lesion
comprises utilizing the probe/primer to determine the nucleotide
sequence of the hedgehog gene and, optionally, of the flanking
nucleic acid sequences. For instance, the probe/primer can be
employed in a polymerase chain reaction (PCR) or in a ligation
chain reaction (LCR). In alternate embodiments, the level of a
hedgehog protein is detected in an immunoassay using an antibody
which is specifically imrnmunoreactive with the hedgehog
protein.
[0044] The practice of the present invention will employ, unless
otherwise indicated, conventional techniques of cell biology, cell
culture, molecular biology, transgenic biology, microbiology,
recombinant DNA, and immunology, which are within the skill of the
art. Such techniques are explained fully in the literature. See,
for example, Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by
Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory
Press: 1989); DNA Cloning, Volumes I and II (D. N. Glover ed.,
1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et
al. U.S. Pat. No.: 4,683,195; Nucleic Acid Hybridization (B. D.
Hames & S. J. Higgins eds. 1984); Transcription And Translation
(B. D. Hames & S. J. Higgins eds. 1984); Culture Of Animal
Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells
And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To
Molecular Cloning (1984); the treatise, Methods In Enzymology
(Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian
Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor
Laboratory); Methods In Enzymology, Vols. 154 and 155 (Wu et al.
eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer
and Walker, eds., Academic Press, London, 1987); Handbook Of
Experimental Immunology, Volumes I-IV (D. M. Weir and C. C.
Blackwell, eds., 1986); Manipulating the Mouse Embryo, (Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).
[0045] Other features and advantages of the invention will be
apparent from the following detailed description, and from the
claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0046] FIG. 1 represents the amino acid sequences of two chick hh
clones, chicken hedgehog-A (pCHA; SEQ ID No:35) and chicken
hedgehog-B (pCHB; SEQ ID No:36). These clones were obtained using
degenerate primers corresponding to the underlined amino acid
residues of the Drosophila sequence (corresponding to residues
161-232 of SEQ ID No:34) also shown in FIG. 1, followed by nested
PCR using chicken genomic DNA.
[0047] FIG. 2 is an alignment comparing the amino acid sequences of
chick Shh (SEQ ID No:8) with its Drosophila homolog (SEQ ID No:34).
Shh residues 1-26 correspond to the proposed signal peptide.
Identical residues are enclosed by boxes and gaps in order to
highlight similarity The nucleotide sequence of Shh has been
submitted to Genbank.
[0048] FIG. 3 is a hydropathy plot for the predicted chick Shh
protein, generated by the methods of Kyte and Doolittle (1982). The
values of hydrophobicity are plotted against the amino acid
positions. Negative values predict a hydrophobic domain of the
protein.
[0049] FIG. 4 is an alignment comparing the amino acid sequences of
various hh proteins. The white region on the amino terminus of
chicken Shh corresponds to the putative signal peptide. The black
box refers to a highly conserved region from aa residues 26-207 of
SEQ ID No:8). The arrows point to exon boundaries in the Drosophila
gene (Lee et al. (1992) Cell 71: 33-50). In each case, the proteins
are compared to chicken Shh (SEQ ID No:8) and the percent amino
acid identity is indicated in each region's box.
[0050] FIG. 5A is a "pileup" alignment of predicted amino acid
sequences which compares Drosophila hh (D-hh; SEQ ID No:34), mouse
hh (M-Dhh; SEQ ID No:9; M-Ihh; SEQ ID No:10; M-Shh; SEQ ID No:11),
chicken hh (C-Shh; SEQ ID No:8), and zebrafish hh (Z-Shh; SEQ ID
No:12). The predicted hydrophobic transmembrane/signal sequences
are indicated in italics and the predicted signal sequence
processing site is arrowed. The positions of introns interrupting
the Drosophila hh and M-Dhh open reading frames are indicated by
arrowheads. All amino acids shared among the six predicted hh
proteins are indicated in bold. FIG. 5B is a sequence alignment of
the N-terminal portion of vertebrate hedgehog proteins, and the
predicted degenerate sequence "CON" (SEQ ID No: 41).
[0051] FIG. 6 is an inter- and cross-species comparison of amino
acid identities among the predicted processed hh proteins shown in
FIG. 5A. All values are percentages. Figures in parentheses
represent similarities allowing for conservative amino acid
substitutions.
[0052] FIG. 7 is a representation of the DNA constructs used in
transgenic studies to study ectopic expression of chick Shh in
mouse embryos. Constructs were generated for ectopic expression of
cDNA clones in the Wnt-l expression domain and tested in transgenic
mice embryos using a lac-Z reporter (pWEXP-lacZ (used as a
control)) and a chick Shh reporter (pWEXP-CShh). The pWEXP-CShh
construct contained two tandem head to tail copies of a chick Shh
cDNA. The results of WEXP2-CShh transgenic studies are shown in
Table 1.
[0053] FIG. 8 is a model for anterioposterior limb patterning and
the Zone of Polarizing Activity (ZPA), based on Saunders and
Gasseling (1968). The left portion of the diagram schematizes a
stage 20 limb bud. The somites are illustrated as blocks along the
left margin of the limb bud; right portion of the same panel
illustrates the mature wing. The hatched region on the posterior
limb is the ZPA. Normally, the developed wing contains three digits
II, III, and IV. The figure further shows the result of
transplanting a ZPA from one limb bud to the anterior margin of
another. The mature limb now contains six digits IV, III, II, II,
III, and IV in a mirror-image duplication of the normal pattern.
The large arrows in both panels represent the signal produced by
the ZPA which acts to specify digit identity.
[0054] FIGS. 9A and 9B illustrate the comparison of zebrafish Shh
(Z-Shh) and Drosophila hh (hh) amino acid sequences. FIG. 9A is an
alignment of zebrafish Shh and Drosophila hh amino acid sequences.
Identical amino acids are linked by vertical bars. Dots indicate
gaps introduced for optimal alignment. Putative
transmembrane/signal peptide sequences are underlined (Kyte and
Doolittle (1982) J Mol Biol 157:133-148). The position of exon
boundaries in the Drosophila gene are indicated by arrowheads. The
region of highest similarity between Z-Shh and hh overlaps exon 2.
FIG. 9B is a schematic comparison of Z-Shh and drosophila hh. Black
boxes indicate the position of the putative transmembrane/signal
peptide sequences. relative to the amino-terminus. Sequence
homologies were scored by taking into account the alignment of
chemically similar amino acids and percentage of homology in the
boxed regions is indicated.
[0055] FIG. 10 is an alignment of partial predicted amino acid
sequences from three different zebrafish hh homologs. One of these
sequences corresponds to Shh, while the other two define additional
hh homologs in zebrafish, named hh(a) and hh(b). Amino acid
identities among the three partial homologs are indicated by
vertical bars.
[0056] FIG. 11 is a schematic representations of chick and mouse
Shh proteins. The putative signal peptides and Asn-linked
glycosylation sites are shown. The numbers refer to amino acid
positions.
[0057] FIG. 12 is a schematic representation of myc-tagged Shh
constructs. The positions of the c-myc epitope tags are shown, as
is the predicted position of the proteolytic cleavage site. The
shaded area following the signal peptide of the carboxy terminal
tagged construct represents the region included in the
Glutathione-S-transferase fusion protein used to generate antisera
in rabbits.
[0058] FIG. 13 is a schematic diagram of Shh processing.
Illustrated are cleavage of the signal peptide (black box),
glycosylation at the predicted Asn residue (N), and the secondary
proteolytic cleavage. The question marks indicate that the precise
site of proteolytic cleavage has not been determined. The different
symbols representing the carbohydrate moiety indicated maturation
of this structure in the Golgi apparatus. The dashed arrow leading
from the signal peptide cleaved protein indicates that secretion of
this species may be an artifact of the incomplete proteolytic
processing of Shh seen in Xenopus oocytes and cos cells.
[0059] FIG. 14 is a schematic diagram of a model for the
coordinated growth and patterning of the limb. Sonic is proposed to
signal directly to the mesoderm to induce expression of the Hoxd
and Bmp-2 genes. The induction of these mesodermal genes requires
competence signals from the overlying AER. One such signal is
apparently Fgf-4. Expression of Fgf-4 in the AER can be induced by
Sonic providing an indirect signaling pathway from Sonic to the
mesoderm. FGFs also maintain expression of Sonic in the ZPA,
thereby completing a positive feedback loop which controls the
relative positions of the signaling centers. While Fgf-4 provides
competence signals to the mesoderm, it also promotes mesodermal
proliferation. Thus patterning of the mesoderm is dependent on the
same signals which promote its proliferation. This mechanism
inextricably integrates limb patterning with outgrowth.
[0060] FIG. 15 is a schematic diagram of patterning of the
Drosophila and vertebrate gut. Regulatory interactions responsible
for patterning of Drosophila midgut (A) are compared to a model for
patterning of the vertebrate hindgut (B) based on expression data.
Morphologic regional distinctions are indicated to the left (A and
B), genes expressed in the visceral mesoderm are in the center
panel, those in the gut lumenal endoderm are on the right. HOM/Hox
gene expression domains are boxed. Regionally expressing secreted
gene products are indicated by lines. Arrows indicate activating
interactions, barred lines, inhibiting interactions. Regulatory
interactions in Drosophila gut (A) have been established by genetic
studies except for the relationship between dpp and hedgehog, which
is hypothesized based on their interactions in the Drosophila
imaginal discs, hedgehog appears to be a signal from the endoderm
to the mesoderm, and that dpp is expressed in the mesoderm.
[0061] FIG. 16 is a schematic diagram of chromosomal locations of
Ihh, Shh and Dhh in the mouse genome. The loci were mapped by
interspecific backcross analysis. The segregation patterns of the
loci and flanking genes in backcross animals that were typed for
all loci are shown above the chromosome maps. For individual pairs
of loci more animals were typed. Each column represents the
chromosome identified in the backcross progeny that was inherited
from the (C57BL/6J.times.M. spretus) F1 parent. The shaded boxes
represent the presence of a C57BL/6J allele and white boxes
represent the presence of a M. spretus allele. The number of the
offsprings inheriting each type of chromosome is listed at the
bottom of each column. Partial chromosome linkage maps showing
location of Ihh, Shh and Dhh in relation too linked genes is shown.
The number of recombinant N.sub.2 animals is presented over total
number of N.sub.2 animals typed to the left of the chromosome maps
between each pair of loci. The recombinant frequencies, expressed
as genetic distance in centimorgans (.+-.one standard error) are
also shown. When no recombination between loci was detected, the
upper 95% confidence limit of the recombination distance is
indicated in parentheses. Gene order was determined by minimizing
the number of recombinant events required to explain the allele
distribution patterns. The position of loci in human chromosomes
can be obtained from GDB (Genome Data Base), a computerized
database of human linkage information maintained by the William H.
Welch Medical Library of the John Hopkins University (Baltimore,
Md.).
DETAILED DESCRIPTION OF THE INVENTION
[0062] Of particular importance in the development and maintenance
of tissue in vertebrate animals is a type of extracellular
communication called induction, which occurs between neighboring
cell layers and tissues (Saxen et al. (1989) Int J Dev Biol
33:21-48; and Gurdon et al. (1987) Development 99:285-306). In
inductive interactions, chemical signals secreted by one cell
population influence the developmental fate of a second cell
population. Typically, cells responding to the inductive signals
are diverted from one cell fate to another, neither of which is the
same as the fate of the signaling cells.
[0063] Inductive signals are key regulatory proteins that function
in vertebrate pattern formation, and are present in important
signaling centers known to operatex embryonically, for example, to
define the organization of the vertebrate embryo. For example,
these signaling structures include the notochord, a transient
structure which initiates the formation of the nervous system and
helps to define the different types of neurons within it. The
notochord also regulates mesodermal patterning along the body axis.
Another distinct group of cells having apparent signaling activity
is the floorplate of the neural tube (the precursor of the spinal
cord and brain) which also signals the differentiation of different
nerve cell types. It is also generally believed that the region of
mesoderm at the bottom of the buds which form the limbs (called the
Zone of Polarizing Activity or ZPA) operates as a signaling center
by secreting a morphogen which ultimately produces the correct
patterning of the developing limbs.
[0064] The present invention concerns the discovery that
polypeptides encoded by a family of vertebrate genes, termed here
hedgehog genes, comprise the signals produced by these embryonic
patterning centers. As described herein, each of the disclosed
vertebrate hedgehog (hh) homologs exhibits spatially and temporally
restricted expression domains indicative of important roles in
embryonic patterning. For instance, the results provided below
indicate that vertebrate hh genes are expressed in the posterior
limb bud, Hensen's node, the early notochord, the floor plate of
the neural tube, the fore- and hindgut and their derivatives. These
are all important signaling centers known to be required for proper
patterning of surrounding embryonic tissues.
[0065] The hedgehog family of vertebrate inter-cellular signaling
molecules provided by the present invention consists of at least
four members. Three of these members, herein referred to as Desert
hedgehog (Dhh), Sonic hedgehog (Shh) and Indian hedgehog (Ihh),
apparently exist in all vertebrates, including fish, birds, and
mammals. A fourth member, herein referred to as Moonrat hedgehog
(Mhh), appears specific to fish. According to the appended sequence
listing, (see also Table 1) a chicken Shh polypeptide is encoded by
SEQ ID No:1; a mouse Dhh polypeptide is encoded by SEQ ID No:2; a
mouse Ihh polypeptide is encoded by SEQ ID No:3; a mouse Shh
polypeptide is encoded by SEQ ID No:4 a zebrafish Shh polypeptide
is encoded by SEQ ID No:5; a human Shh polypeptide is encoded by
SEQ ID No:6; and a human Ihh polypeptide is encoded by SEQ ID
No:7.
1TABLE 1 Guide to hedgehog sequences in Sequence Listing Nucleotide
Amino Acid Chicken Shh SEQ ID No. 1 SEQ ID No. 8 Mouse Dhh SEQ ID
No. 2 SEQ ID No. 9 Mouse Ihh SEQ ID No. 3 SEQ ID No. 10 Mouse Shh
SEQ ID No. 4 SEQ ID No. 11 Zebrafish Shh SEQ ID No. 5 SEQ ID No. 12
Human Shh SEQ ID No. 6 SEQ ID No. 13 Human Ihh SEQ ID No. 7 SEQ ID
No. 14
[0066] Certain of the vertebrate hedgehog (hh) proteins of the
present invention are defined by SEQ ID Nos:8-14 and can be cloned
from vertebrate. organisms including fish, avian and mammalian
sources. These proteins are distinct from the drosophila hedgehog
protein which, for clarity, will be referred to hereinafter as
"Dros-HH". In addition to the sequence variation between the
various hh homologs, the vertebrate hedgehog proteins are
apparently present naturally in a number of different forms,
including a pro-form, a full-length mature form, and several
processed fragments thereof. The pro-form includes an N-terminal
signal peptide for directed secretion of the extracellular domain,
while the full-length mature form lacks this signal sequence.
Further processing of the mature form apparently occurs in some
instances to yield biologically active fragments of the protein.
For instance, sonic hedgehog undergoes additional proteolytic
processing to yield two peptides of approximately 19 kDa and 27
kDa, both of which are secreted. In addition to proteolytic
fragmentation, the vertebrate hedgehog proteins can also be
modified post-translationally, such as by glycosylation, though
bacterially produced (e.g. unglycosylated) forms of the proteins
apparently still maintain some of the activity of the native
protein.
[0067] As described in the following examples, the cDNA clones
provided by the present invention were first obtained by screening
a mouse genomic library with a partial Drosophila hh cDNA clone (7
kb). Positive plaques were identified and one mouse clone was
selected. This clone was then used as a probe to obtain a genomic
clone containing the full coding sequence of the Mouse Dhh gene. As
described in the attached Examples, Northern blots and in situ
hybridization demonstrated that Mouse Dhh is expressed in the
testes, and potentially the ovaries, and is also associated with
sensory neurons of the head and trunk. Interestingly, no expression
was detected on the nerve cell bodies themselves (only the axons),
indicating that Dhh is likely produced by the Shwann cells.
[0068] In order to obtain cDNA clones encoding chicken hh genes,
degenerate oligonucleotides were designed corresponding to the
amino and carboxy ends of Drosophila hh exon 2. As described in the
Examples below, these oligonucleotides were used to isolate PCR
fragments from chicken genomic DNA. These fragments were then
cloned and sequenced. Ten clones yielded two different hh homologs,
chicken Dhh and chicken Shh. The chicken Shh clone was then used to
screen a stage 21/22 limb bud cDNA library which yielded a full
length Shh clone.
[0069] In order to identify other vertebrate hedgehog homologs, the
chicken clones (Dhh and Shh) were used to probe a genomic southern
blot containing chicken DNA. As described below, genomic DNA was
cut with various enzymes which do not cleave within the probe
sequences. The DNA was run on a gel and transferred to a nylon
filter. Probes were derived by ligating each 220 bp clone into a
concatomer and then labeling with a random primer kit. The blots
were hybridized and washed at low stringency. In each case, three
hybridizing bands were observed following autoradiography, one of
which was significantly more intense (a different band with each
probe), indicating that there are at least three vertebrate hh
genes. Additional cDNA and genomic screens carried out have yielded
clones of three hh homologs from chickens and mice (Shh, Dhh and
Ihh), and four hh homologs from zebrafish (Shh, Dhh, Ihh and Mhh).
Weaker hybridization signals suggested that the gene family may be
even larger. Moreover, a number of weakly hybridizing genomic
clones have been isolated. Subsequently, the same probes derived
from chicken hedgehog homologs have been utilized to screen a human
genomic library. PCR fragments derived from the human genomic
library were then sequenced, and PCR probes derived from the human
sequences were used to screen human fetal cDNA libraries.
Full-length cDNA encoding human sonic hedgehog protein (Shh) and
partial cDNA encoding human Indian hedgehog protein (Ihh) were
isolated from the fetal library, and represent a source of
recombinant human hedgehog proteins.
[0070] To order to determine the expression patterns of the various
vertebrate hh homologs, in situ hybridizations were performed in
developing embryos of chicken, mice and fish. As described in the
Examples below, the resulting expression patterns of each hh
homolog were similar across each species and revealed that hh genes
are expressed in a number of important embryonic signaling centers.
For example, Shh is expressed in Hensen's node, the notochord, the
ventral floorplate of the developing neural tube, and the ZPA at
the base of the limb buds; Ihh is expressed in the embryonic
yolksac and hindgut, and appear also to be involved in
chondrogenesis; Dhh is expressed in the testes; and Mhh (only in
zebrafish) is expressed in the notochord and in certain cranial
nerves.
[0071] Furthermore, experimental evidence indicates that certain
hedgehog proteins initiate expression of secondary signaling
molecules, including Bmp-2 (a TGF-.beta. relative) in the mesoderm
and Fgf-4 in the ectoderm. The mesoderm requires
ectodermally-derived competence factor(s), which include Fgf-4, to
activate target gene expression in response to hedgehog signaling.
The expression of, for example, Sonic and Fgf-4 is coordinately
regulated by a positive feedback loop operating between the
posterior mesoderm and the overlying AER, which is the ridge of
pseudostratified epithelium extending antero-posteriorly along the
distal margin of the bud. These data provide a basis for
understanding the integration of growth and patterning in the
developing limb which can have important implications in the
treatment of bone disorders described in greater detail herein.
[0072] To determine the role hedgehog proteins plays in inductive
interactions between the endoderm and mesoderm, which are critical
to gut morphogenesis, in situ hybridizations and recombinant
retroviral injections were performed in developing chick embryos.
The ventral mesoderm is induced to undergo gut-specific
differentiation by the adjacent endoderm. As described in Examples
below, at the earliest stages of chick gut formation Shh is
expressed by the endoderm, and BMP-4 (a TGF-.beta. relative) is
expressed in the adjacent visceral mesoderm. Ectopic expression of
Sonic is sufficient to induce expression of BMP-4 in visceral
mesoderm, suggesting that Sonic serves as an inductive signal from
the endoderm to the mesoderm. Subsequent organ-specific endodermal
differentiation depends on regional inductive signal from the
visceral mesoderm. Hox genes are expressed in the undifferentiated
chick hind gut mesoderm with boundaries corresponding to
morphologic borders, suggesting a role in regulating gut
morphogenesis.
[0073] Bioactive fragments of hedgehog polypeptides of the present
invention have been generated and are described in great detail in
U.S. Ser. No. 08/435,093, filed May 4 1995, herein incorporated by
reference.
[0074] Accordingly, certain aspects of the present invention relate
to nucleic acids encoding vertebrate hedgehog proteins, the
hedgehog proteins themselves, antibodies immunoreactive with hh
proteins, and preparations of such compositions. Moreover, the
present invention provides diagnostic and therapeutic assays and
reagents for detecting and treating disorders involving, for
example, aberrant expression of vertebrate hedgehog homologs. In
addition, drug discovery assays are provided for identifying agents
which can modulate the binding of vertebrate hedgehog homologues to
hedgehog-binding moieties (such as hedgehog receptors, ligands, or
other extracellular matrix components). Such agents can be useful
therapeutically to alter the growth and/or differentiation of a
cell. Other aspects of the invention are described below or will be
apparent to those skilled in the art in light of the present
disclosure.
[0075] For convenience, certain terms employed in the
specification, examples, and appended claims are collected
here.
[0076] As used herein, the term "nucleic acid" refers to
polynucleotides such as deoxyribonucleic acid (DNA), and, where
appropriate, ribonucleic acid (RNA). The term should also be
understood to include, as equivalents, analogs of either RNA or DNA
made from nucleotide analogs, and, as applicable to the embodiment
being described, single (sense or antisense) and double-stranded
polynucleotides.
[0077] As used herein, the term "gene" or "recombinant gene" refers
to a nucleic acid comprising an open reading frame encoding one of
the vertebrate hh polypeptides of the present invention, including
both exon and (optionally) intron sequences. A "recombinant gene"
refers to nucleic acid encoding a vertebrate hh polypeptide and
comprising vertebrate hh-encoding exon sequences, though it may
optionally include intron sequences which are either derived from a
chromosomal vertebrate hh gene or from an unrelated chromosomal
gene. Exemplary recombinant genes encoding the subject vertebrate
hh polypeptide are represented by SEQ ID No:1, SEQ ID No:2, SEQ ID
No:3, SEQ ID No:4, SEQ ID No:5, SEQ ID No:6 or SEQ ID No:7. The
term "intron" refers to a DNA sequence present in a given
vertebrate hh gene which is not translated into protein and is
generally found between exons.
[0078] As used herein, the term "transfection" means the
introduction of a nucleic acid, e.g., an expression vector, into a
recipient cell by nucleic acid-mediated gene transfer.
"Transformation", as used herein, refers to a process in which a
cell's genotype is changed as a result of the cellular uptake of
exogenous DNA or RNA, and, for example, the transformed cell
expresses a recombinant form of a vertebrate hh polypeptide or,
where anti-sense expression occurs from the transferred gene, the
expression of a naturally-occurning form of the vertebrate hh
protein is disrupted.
[0079] As used herein the term "bioactive fragment of a hedgehog
protein" refers to a fragment of a hedgehog polypeptide, wherein
the encoded polypeptide specifically agonizes or antagonizes
inductive events mediated by wild-type hedgehog proteins. The
hedgehog biactive fragment preferably is, for example, at least 5,
10, 20, 50, 100, 150 or 200 amino acids in length.
[0080] An "effective amount" of a hedgehog polypeptide, or a
bioactive fragment thereof, with respect to the subject method of
treatment, refers to an amount of agonist or antagonist in a
preparation which, when applied as part of a desired dosage
regimen, provides modulation of growth, differentiation or survival
of cells, e.g., modulation of spermatogenesis, skeletogenesis,
e.g., osteogenesis, chondrogenesis, or limb patterning, or neuronal
differentiation.
[0081] As used herein, "phenotype" refers to the entire physical,
biochemical, and physiological makeup of a cell, e.g., having any
one trait or any group of traits.
[0082] The terms "induction" or "induce", as relating to the
biological activity of a hedgehog protein, refers generally to the
process or act of causing to occur a specific effect on the
phenotype of cell. Such effect can be in the form of causing a
change in the phenotype, e.g., differentiation to another cell
phenotype, or can be in the form of maintaining the cell in a
particular cell, e.g., preventing dedifferentation or promoting
survival of a cell.
[0083] As used herein the term "animal" refers to mammals,
preferably mammals such as live stock or humans. Likewise, a
"patient" or "subject" to be treated by the subject method can mean
either a human or non-human animal.
[0084] As used herein, the term "vector" refers to a nucleic acid
molecule capable of transporting another nucleic acid to which it
has been linked. One type of preferred vector is an episome, i.e.,
a nucleic acid capable of extra-chromosomal replication. Preferred
vectors are those capable of autonomous replication and/expression
of nucleic acids to which they are linked. Vectors capable of
directing the expression of genes to which they are operatively
linked are referred to herein as "expression vectors". In general,
expression vectors of utility in recombinant DNA techniques are
often in the form of "plasmids" which refer generally to circular
double stranded DNA loops which, in their vector form are not bound
to the chromosome. In the present specification, "plasmid" and
"vector" are used interchangeably as the plasmid is the most
commonly used form of vector. However, the invention is intended to
include such other forms of expression vectors which serve
equivalent functions and which become known in the art subsequently
hereto.
[0085] "Transcriptional regulatory sequence" is a generic term used
throughout the specification to refer to DNA sequences, such as
initiation signals, enhancers, and promoters, which induce or
control transcription of protein coding sequences with which they
are operably linked. In preferred embodiments, transcription of one
of the recombinant vertebrate hedgehog genes is under the control
of a promoter sequence (or other transcriptional regulatory
sequence) which controls the expression of the recombinant gene in
a cell-type in which expression is intended. It will also be
understood that the recombinant gene can be under the control of
transcriptional regulatory sequences which are the same or which
are different from those sequences which control transcription of
the naturally-occurring forms of hedgehog proteins.
[0086] As used herein, the term "tissue-specific promoter" means a
DNA sequence that serves as a promoter, i.e., regulates expression
of a selected DNA sequence operably linked to the promoter, and
which effects expression of the selected DNA sequence in specific
cells of a tissue, such as cells of neural origin, e.g. neuronal
cells. The term also covers so-called "leaky" promoters, which
regulate expression of a selected DNA primarily in one tissue, but
cause expression in other tissues as well.
[0087] As used herein, the term "target tissue" refers to
connective tissue, cartilage, bone tissue or limb tissue, which is
either present in an animal, e.g., a mammal, e.g., a human or is
present in in vitro culture, e.g, a cell culture.
[0088] As used herein, a "transgenic animal" is any animal,
preferably a non-human mammal, bird or an amphibian, in which one
or more of the cells of the animal contain heterologous nucleic
acid introduced by way of human intervention, such as by transgenic
techniques well known in the art. The nucleic acid is introduced
into the cell, directly or indirectly by introduction into a
precursor of the cell, by way of deliberate genetic manipulation,
such as by microinjection or by infection with a recombinant virus.
The term genetic manipulation does not include classical
cross-breeding, or in vitro fertilization, but rather is directed
to the introduction of a recombinant DNA molecule. This molecule
may be integrated within a chromosome, or it may be
extrachromosomally replicating DNA. In the typical transgenic
animals described herein, the transgene causes cells to express a
recombinant form of one of the vertebrate hh proteins, e.g. either
agonistic or antagonistic forms. However, transgenic animals in
which the recombinant vertebrate hh gene is silent are also
contemplated, as for example, the FLP or CRE recombinase dependent
constructs described below. The "non-human animals" of the
invention include vertebrates such as rodents, non-human primates,
sheep, dog, cow, chickens, amphibians, reptiles, etc. Preferred
non-human animals are selected from the rodent family including rat
and mouse, most preferably mouse, though transgenic amphibians,
such as members of the Xenopus genus, and transgenic chickens can
also provide important tools for understanding and identifying
agents which can affect, for example, embryogenesis and tissue
formation. The term "chimeric animal" is used herein to refer to
animals in which the recombinant gene is found, or in which the
recombinant is expressed in some but not all cells of the animal.
The term "tissue-specific chimeric animal" indicates that one of
the recombinant vertebrate hh genes is present and/or expressed in
some tissues but not others.
[0089] As used herein, the term "transgene" means a nucleic acid
sequence (encoding, e.g., one of the vertebrate hh polypeptides),
which is partly or entirely heterologous, i.e., foreign, to the
transgenic animal or cell into which it is introduced, or, is
homologous to an endogenous gene of the transgenic animal or cell
into which it is introduced, but which is designed to be inserted,
or is inserted, into the animal's genome in such a way as to alter
the genome of the cell into which it is inserted (e.g., it is
inserted at a location which differs from that of the natural gene
or its insertion results in a knockout). A transgene can include
one or more transcriptional regulatory sequences and any other
nucleic acid, such as introns, that may be necessary for optimal
expression of a selected nucleic acid.
[0090] As is well known, genes for a particular polypeptide may
exist in single or multiple copies within the genome of an
individual. Such duplicate genes may be identical or may have
certain modifications, including nucleotide substitutions,
additions or deletions, which all still code for polypeptides
having substantially the same activity. The term "DNA sequence
encoding a vertebrate hh polypeptide" may thus refer to one or more
genes within a particular individual. Moreover, certain differences
in nucleotide sequences may exist between individual organisms,
which are called alleles. Such allelic differences may or may not
result in differences in amino acid sequence of the encoded
polypeptide yet still encode a protein with the same biological
activity.
[0091] "Homology" refers to sequence similarity between two
peptides or between two nucleic acid molecules. Homology can be
determined by comparing a position in each sequence which may be
aligned for purposes of comparison. When a position in the compared
sequence is occupied by the same base or amino acid, then the
molecules are homologous at that position. A degree of homology
between sequences is a function of the number of matching or
homologous positions shared by the sequences. An "unrelated" or
"non-homologous" sequence shares less than 40 percent identity,
though preferably less than 25 percent identity, with one of the
vertebrate hh sequences of the present invention.
[0092] "Cells," "host cells" or "recombinant host cells" are terms
used interchangeably herein. It is understood that such terms refer
not only to the particular subject cell but to the progeny or
potential progeny of such a cell. Because certain modifications may
occur in succeeding generations due to either mutation or
environmental influences, such progeny may not, in fact, be
identical to the parent cell, but are still included within the
scope of the term as used herein.
[0093] A "chimeric protein" or "fusion protein" is a fusion of a
first amino acid sequence encoding one of the subject vertebrate hh
polypeptides with a second amino acid sequence defining a domain
foreign to and not substantially homologous with any domain of one
of the vertebrate hh proteins. A chimeric protein may present a
foreign domain which is found (albeit in a different protein) in an
organism which also expresses the first protein, or it may be an
"interspecies", "intergenic", etc. fusion of protein structures
expressed by different kinds of organisms. In general, a fusion
protein can be represented by the general formula X-hh-Y, wherein
hh represents a portion of the protein which is derived from one of
the vertebrate hh proteins, and X and Y are independently absent or
represent amino acid sequences which are not related to one of the
vertebrate hh sequences in an organism, including naturally
occurring mutants.
[0094] As used herein, the terms "transforming growth factor-beta"
and "TGF-.beta." denote a family of structurally related paracrine
polypeptides found ubiquitously in vertebrates, and prototypic of a
large family of metazoan growth, differentiation, and morphogenesis
factors (see, for review, Massaque et al. (1990) Ann Rev Cell Biol
6:597-641; and Spom et al. (1992) J Cell Biol 119:1017-1021).
Included in this family are the "bone morphogenetic proteins" or
"BMPs", which refers to proteins isolated from bone, and fragments
thereof and synthetic peptides which are capable of inducing bone
deposition alone or when combined with appropriate cofactors.
Preparation of BMPs, such as BMP-1, -2, -3, and -4, is described
in, for example, PCT publication WO 88/00205. Wozney (1989) Growth
Fact Res 1:267-280 describes additional BMP proteins closely
related to BMP-2, and which have been designated BMP-5, -6, and -7.
PCT publications WO89/09787 and WO89/09788 describe a protein
called "OP-1," now known to be BMP-7. Other BMPs are known in the
art.
[0095] The term "isolated" as also used herein with respect to
nucleic acids, such as DNA or RNA, refers to molecules separated
from other DNAs, or RNAs, respectively, that are present in the
natural source of the macromolecule. For example, an isolated
nucleic acid encoding one of the subject vertebrate hh polypeptides
preferably includes no more than 10 kilobases (kb) of nucleic acid
sequence which naturally immediately flanks the vertebrate hh gene
in genomic DNA, more preferably no more than 5 kb of such naturally
occurring flanking sequences, and most preferably less than 1.5 kb
of such naturally occurring flanking sequence. The term isolated as
used herein also refers to a nucleic acid or peptide that is
substantially free of cellular material, viral material, or culture
medium when produced by recombinant DNA techniques, or chemical
precursors or other chemicals when chemically synthesized.
Moreover, an "isolated nucleic acid" is meant to include nucleic
acid fragments which are not naturally occurring as fragments and
would not be found in the natural state.
[0096] As used herein the term "approximately 19 kDa" with respect
to N-terminal bioactive fragments of a hedgehog protein, refers to
a polypeptide which can range in size from 16 kDa to 22 kDa, more
preferably 18-20 kDa. In a preferred embodiment, "approximately 19
kDa" refers to a mature form of the peptide after the cleavage of
the signal sequence and proteolysis to release an N-terminal
portion of the mature protein. For instance, in the case of the
Sonic hedgehog polypeptide, a fragment of approximately 19 kDa is
generated when the mature polypeptide is cleaved at a proteolytic
processing site which is located in the region between Ala-169 and
Gly-178 of SEQ ID No:40, e.g., a fragment from Cys-1 to Gly-174 of
SEQ ID No:40.
[0097] Likewise, the term "approximately 27 kDa" with respect to
C-terminal fragments of a hedgehog protein, refers to a polypeptide
which can range in size from 24 kDa to 30 kDa, more preferably
26-29 kDa. In a preferred embodiment, "approximately 27 kDa" refers
to a mature form of the C-terminal polypeptide after proteolysis to
release an N-terminal portion of the mature protein.
[0098] As described below, one aspect of the invention pertains to
isolated nucleic acids comprising the nucleotide sequences encoding
vertebrate hh homologues, and/or equivalents of such nucleic acids.
The term nucleic acid as used herein is intended to include
fragments as equivalents. The term equivalent is understood to
include nucleotide sequences encoding functionally equivalent
hedgehog polypeptides or functionally equivalent peptides having an
activity of a vertebrate hedgehog protein such as described herein.
Equivalent nucleotide sequences will include sequences that differ
by one or more nucleotide substitutions, additions or deletions,
such as allelic variants; and will, therefore, include sequences
that differ from the nucleotide sequence of the vertebrate hedgehog
cDNAs shown in SEQ ID Nos:1-7 due to the degeneracy of the genetic
code. Equivalents will also include nucleotide sequences that
hybridize under stringent conditions (i.e., equivalent to about
20-27.degree. C. below the melting temperature (T.sub.m) of the DNA
duplex formed in about 1M salt) to the nucleotide sequences
represented in one or more of SEQ ID Nos:1-7. In one embodiment,
equivalents will further include nucleic acid sequences derived
from and evolutionarily related to, a nucleotide sequences shown in
any of SEQ ID Nos: 1-7.
[0099] Moreover, it will be generally appreciated that, under
certain circumstances, it may be advantageous to provide homologs
of one of the subject hedgehog polypeptides which function in a
limited capacity as one of either a hedgehog agonist (mimetic) or a
hedgehog antagonist, in order to promote or inhibit only a subset
of the biological activities of the naturally-occurring form of the
protein. Thus, specific biological effects can be elicited by
treatment with a homolog of limited function, and with fewer side
effects relative to treatment with agonists or antagonists which
are directed to all of the biological activities of naturally
occurring forms of hedgehog proteins.
[0100] Homologs of one of the subject hedgehog proteins can be
generated by mutagenesis, such as by discrete point mutation(s), or
by truncation. For instance, mutation can give rise to homologs
which retain substantially the same, or merely a subset, of the
biological activity of the hh polypeptide from which it was
derived. Alternatively, antagonistic forms of the protein can be
generated which are able to inhibit the function of the naturally
occurring form of the protein, such as by competitively binding to
an hh receptor.
[0101] In general, polypeptides referred to herein as having an
activity (e.g., are "bioactive") of a vertebrate hh protein are
defined as polypeptides which include an amino acid sequence
corresponding (e.g., identical or homologous) to all or a portion
of the amino acid sequences of a vertebrate hh proteins shown in
any of SEQ ID No:8, SEQ ID No:9, SEQ ID No:10, SEQ ID No:11, SEQ ID
No:12, SEQ ID No:13 or SEQ ID No:14 and which mimic or antagonize
all or a portion of the biological/biochemical activities of a
naturally occuring hedgehog protein. Examples of such biological
activity include the ability to induce (or otherwise modulate)
formation and differentiation of the head, limbs, lungs, central
nervous system (CNS), digestive tract or other gut components, or
mesodermal patterning of developing vertebrate embryos. As set out
in U.S. Ser. Nos. 08/356,060 and 08/176,427, the vertebrate
hedgehog proteins, especially Shh, can constitute a general
ventralizing activity. For instance, the subject polypeptides can
be characterized by an ability to induce and/or maintain
differentiation of neurons, e.g., motorneurons, cholinergic
neurons, dopanergic neurons, serotenergic neurons, peptidergic
neurons and the like. In preferred embodiments, the biological
activity can comprise an ability to regulate neurogenesis, such as
a motor neuron inducing activity, a neuronal differentiation
inducing activity, or a neuronal survival promoting activity.
Hedgehog proteins of the present invention can also have biological
activities which include an ability to regulate organogensis, such
as through the ability to influence limb patterning, by, for
example, skeletogenic activity. The biological activity associated
with the hedgehog proteins of the present invention can also
include the ability to induce stem cell or germ cell
differentiation, including the ability to induce differentiation of
chondrocytes or an involvement in spermatogenesis.
[0102] Hedgehog proteins of the present invention can also be
characterized in terms of biological activities which include: an
ability to modulate proliferation, survival and/or differentiation
of mesodermally-derived tissue, such as tissue derived from dorsal
mesoderm; the ability to modulate proliferation, survival and/or
differentiation of ectodermally-derived tissue, such as tissue
derived from the neural tube, neural crest, or head mesenchyme; the
ability to modulate proliferation, survival and/or differentiation
of endodermally-derived tissue, such as tissue derived from the
primitive gut. Moreover, as described in the Examples below, the
subject hedgehog proteins have the ability to induce expression of
secondary signaling molecules, such as members of the Transforming
Growth Factor .beta. (TGF.beta.) family, including bone morphogenic
proteins, e.g. BMP-2 and BMP-4, as well as members of the
fibroblast growth factor (FGF) family, such as Fgf-4. Other
biological activities of the subject hedgehog proteins are
described herein or will be reasonably apparent to those skilled in
the art. According to the present invention, a polypeptide has
biological activity if it is a specific agonist or antagonist of a
naturally-occurring form of a vertebrate hedgehog protein.
[0103] Preferred nucleic acids encode a vertebrate hedgehog
polypeptide comprising an amino acid sequence at least 60%
homologous, more preferably 70% homologous and most preferably 80%
homologous with an amino acid sequence selected from the group
consisting of SEQ ID Nos:8-14. Nucleic acids which encode
polypeptides at least about 90%, more preferably at least about
95%, and most preferably at least about 98-99% homology with an
amino acid sequence represented in one of SEQ ID Nos:8-14 are also
within the scope of the invention. In one embodiment, the nucleic
acid is a cDNA encoding a peptide having at least one activity of
the subject vertebrate hh polypeptide. Preferably, the nucleic acid
includes all or a portion of the nucleotide sequence corresponding
to the coding region of SEQ ID Nos:1-7.
[0104] Preferred nucleic acids encode a bioactive fragment of a
vertebrate hedgehog polypeptide comprising an amino acid sequence
at least 60% homologous, more preferably 70% homologous and most
preferably 80% homologous with an amino acid sequence selected from
the group consisting of SEQ ID Nos:8-14. Nucleic acids which encode
polypeptides at least about 90%, more preferably at least about
95%, and most preferably at least about 98-99% homology, or
identical, with an amino acid sequence represented in one of SEQ ID
Nos:8-14 are also within the scope of the invention.
[0105] With respect to bioctive fragments of sonic clones, a
preferred nucleic acid encodes a polypeptide including a hedgehog
portion having molecular weight of approximately 19 kDa and which
polyptide can modulate, e.g., mimic or antagonize, a hedgehog
biological activity. Preferably, the polypeptide encoded by the
nucleic acid comprises an amino acid sequence identical or
homologous to an amino acid sequence designated in one of SEQ ID
No:8, SEQ ID No:9, SEQ ID No:10, SEQ ID No:11, SEQ ID No:12, SEQ ID
No:13, or SEQ ID No:14. More preferably, the polypeptide comprises
an amino acid sequence designated in SEQ ID No:40.
[0106] A preferred nucleic acid encodes a hedgehog polypeptide
comprising an amino acid sequence represented by the formula A-B
wherein, A represents all or the portion of the amino acid sequence
designated by residues 1-168 of SEQ ID No:40; and B represents at
least one amino acid residue of the amino acid sequence designated
by residues 169-221 of SEQ ID No:40; wherein A and B together
represent a contiguous polypeptide sequence designated by SEQ ID
No:40. Preferably, B can represent at least five, ten or twenty
amino acid residues of the amino acid sequence designated by
residues 169-221 of SEQ ID No:40.
[0107] To further illustrate, another preferred nucleic acid
encodes a polypeptide comprising an amino acid sequence represented
by the formula A-B, wherein A represents all or the portion of the
amino acid sequence designated by residues 24-193 of SEQ ID No:13;
and B represents at least one amino acid residue of the amino acid
sequence designated by residues 194-250 of SEQ ID No:13; wherein A
and B together represent a contiguous polypeptide sequence
designated in SEQ ID No:13, and the polypeptide modulates, e.g.,
agonizes or antagonizes, the biological activity of a hedgehog
protein.
[0108] Yet another preferred nucleic acid encodes a polypeptide
comprising an amino acid sequence represented by the formula A-B,
wherein A represents all or the portion, e.g., 25, 50, 75 or 100
residues, of the amino acid sequence designated by residues 25-193,
or analogous residues thereof, of a vertebrate hedgehog polypeptide
identical or homologous to SEQ ID No:11; and B represents at least
one amino acid residue of the amino acid sequence designated by
residues 194-250, or analogous residues thereof, of a vertebrate
hedgehog polypeptide identical or homologous to SEQ ID No:11;
wherein A and B together represent a contiguous polypeptide
sequence designated in SEQ ID No:11.
[0109] Another preferred nucleic acid encodes a polypeptide
comprising an amino acid sequence represented by the formula A-B,
wherein A represents all or the portion, e.g., 25, 50, 75 or 100
residues, of the amino acid sequence designated by residues 23-193
of SEQ ID No:9; and B represents at least one amino acid residue of
the amino acid sequence designated by residues 194-250 of SEQ ID
No:9; wherein A and B together represent a contiguous polypeptide
sequence designated in SEQ ID No:9, and the polypeptide modulates,
e.g., agonizes or antagonizes, the biological activity of a
hedgehog protein.
[0110] Another preferred nucleic acid encodes a polypeptide
comprising an amino acid sequence represented by the formula A-B,
wherein A represents all or the portion, e.g., 25, 50, 75 or 100
residues, of the amino acid sequence designated by residues 28-197
of SEQ ID No:10; and B represents at least one amino acid residue
of the amino acid sequence designated by residues 198-250 of SEQ ID
No:10; wherein A and B together represent a contiguous polypeptide
sequence designated in SEQ ID No:10, and the polypeptide modulates,
e.g., agonizes or antagonizes, the biological activity of a
hedgehog protein.
[0111] Yet another preferred nucleic acid encodes a polypeptide
comprising an amino acid sequence represented by the formula A-B,
wherein A represents all or the portion, e.g., 25, 50 or 75
residues, of the amino acid sequence designated by residues 1-98,
or analogous residues thereof, of a vertebrate hedgehog polypeptide
identical or homologous to SEQ ID No:14; and B represents at least
one amino acid residue of the amino acid sequence designated by
residues 99-150, or analogous residues thereof, of a vertebrate
hedgehog polypeptide identical or homologous to SEQ ID No:14;
wherein A and B together represent a contiguous polypeptide
sequence designated in SEQ ID No:14.
[0112] Another aspect of the invention provides a nucleic acid
which hybridizes under high or low stringency conditions to a
nucleic acid represented by one of SEQ ID Nos:1-7. Appropriate
stringency conditions which promote DNA hybridization, for example,
6.0.times.sodium chloride/sodium citrate (SSC) at about 45.degree.
C., followed by a wash of 2.0.times.SSC at 50.degree. C., are known
to those skilled in the art or can be found in Current Protocols in
Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.
For example, the salt concentration in the wash step can be
selected from a low stringency of about 2.0.times.SSC at 50.degree.
C. to a high stringency of about 0.2.times.SSC at 50.degree. C. In
addition, the temperature in the wash step can be increased from
low stringency conditions at room temperature, about 22.degree. C.,
to high stringency conditions at about 65.degree. C.
[0113] Nucleic acids, having a sequence that differs from the
nucleotide sequences shown in one of SEQ ID No:1, SEQ ID No:2, SEQ
ID No:3, SEQ ID No:4, SEQ ID No:5, SEQ ID No:6 or SEQ ID No:7 due
to degeneracy in the genetic code are also within the scope of the
invention. Such nucleic acids encode functionally equivalent
peptides (i.e., a peptide having a biological activity of a
vertebrate hh polypeptide) but differ in sequence from the sequence
shown in the sequence listing due to degeneracy in the genetic
code. For example, a number of amino acids are designated by more
than one triplet. Codons that specify the same amino acid, or
synonyms (for example, CAU and CAC each encode histidine) may
result in "silent" mutations which do not affect the amino acid
sequence of a vertebrate hh polypeptide. However, it is expected
that DNA sequence polymorphisms that do lead to changes in the
amino acid sequences of the subject hh polypeptides will exist
among vertebrates. One skilled in the art will appreciate that
these variations in one or more nucleotides (up to about 3-5% of
the nucleotides) of the nucleic acids encoding polypeptides having
an activity of a vertebrate hh polypeptide may exist among
individuals of a given species due to natural allelic
variation.
[0114] As used herein, a hedgehog gene fragment refers to a nucleic
acid having fewer nucleotides than the nucleotide sequence encoding
the entire mature form of a vertebrate hh protein yet which
(preferably) encodes a polypeptide which retains some biological
activity of the full length protein.
[0115] As indicated by the examples set out below, hedgehog
protein-encoding nucleic acids can be obtained from mRNA present in
any of a number of eukaryotic cells. It should also be possible to
obtain nucleic acids encoding vertebrate hh polypeptides of the
present invention from genomic DNA obtained from both adults and
embryos. For example, a gene encoding a hh protein can be cloned
from either a cDNA or a genomic library in accordance with
protocols described herein, as well as those generally known to
persons skilled in the art. A cDNA encoding a hedgehog protein can
be obtained by isolating total mRNA from a cell, e.g. a mammalian
cell, e.g. a human cell, including embryonic cells. Double stranded
cDNAs can then be prepared from the total mRNA, and subsequently
inserted into a suitable plasmid or bacteriophage vector using any
one of a number of known techniques. The gene encoding a vertebrate
hh protein can also be cloned using established polymerase chain
reaction techniques in accordance with the nucleotide sequence
information provided by the invention. The nucleic acid of the
invention can be DNA or RNA. A preferred nucleic acid is a cDNA
represented by a sequence selected from the group consisting of SEQ
ID Nos:1-7.
[0116] Another aspect of the invention relates to the use of the
isolated nucleic acid in "antisense" therapy. As used herein,
"antisense" therapy refers to administration or in situ generation
of oligonucleotide probes or their derivatives which specifically
hybridizes (e.g. binds) under cellular conditions, with the
cellular mRNA and/or genomic DNA encoding one or more of the
subject hedgehog proteins so as to inhibit expression of that
protein, e.g. by inhibiting transcription and/or translation. The
binding may be by conventional base pair complementarity, or, for
example, in the case of binding to DNA duplexes, through specific
interactions in the major groove of the double helix. In general,
"antisense" therapy refers to the range of techniques generally
employed in the art, and includes any therapy which relies on
specific binding to oligonucleotide sequences.
[0117] An antisense construct of the present invention can be
delivered, for example, as an expression plasmid which, when
transcribed in the cell, produces RNA which is complementary to at
least a unique portion of the cellular mRNA which encodes a
vertebrate hh protein. Alternatively, the antisense construct is an
oligonucleotide probe which is generated ex vivo and which, when
introduced into the cell causes inhibition of expression by
hybridizing with the mRNA and/or genomic sequences of a vertebrate
hh gene. Such oligonucleotide probes are preferably modified
oligonucleotide which are resistant to endogenous nucleases, e.g.
exonucleases and/or endonucleases, and is therefore stable in vivo.
Exemplary nucleic acid molecules for use as antisense
oligonucleotides are phosphoramidate, phosphothioate and
methylphosphonate analogs of DNA (see also U.S. Pat. Nos.
5,176,996; 5,264,564; and 5,256,775). Additionally, general
approaches to constructing oligomers useful in antisense therapy
have been reviewed, for example, by Van der Krol et al. (1988)
Biotechniques 6:958-976; and Stein et al. (1988) Cancer Res
48:2659-2668.
[0118] Accordingly, the modified oligomers of the invention are
useful in therapeutic, diagnostic, and research contexts. In
therapeutic applications, the oligomers are utilized in a manner
appropriate for antisense therapy in general. For such therapy, the
oligomers of the invention can be formulated for a variety of loads
of administration, including systemic and topical or localized
administration. Techniques and formulations generally may be found
in Remmington's Pharmaceutical Sciences, Meade Publishing Co.,
Easton, Pa. For systemic administration, injection is preferred,
including intramuscular, intravenous, intraperitoneal, and
subcutaneous for injection, the oligomers of the invention can be
formulated in liquid solutions, preferably in physiologically
compatible buffers such as Hank's solution or Ringer's solution. In
addition, the oligomers may be formulated in solid form and
redissolved or suspended immediately prior to use. Lyophilized
forms are also included.
[0119] Systemic administration can also be by transmucosal or
transdermal means, or the compounds can be administered orally. For
transmucosal or transdermal administration, penetrants appropriate
to the barrier to be permeated are used in the formulation. Such
penetrants are generally known in the art, and include, for
example, for transmucosal administration bile salts and fusidic
acid derivatives. In addition, detergents may be used to facilitate
permeation. Transmucosal administration may be through nasal sprays
or using suppositories. For oral administration, the oligomers are
formulated into conventional oral administration forms such as
capsules, tablets, and tonics. For topical administration, the
oligomers of the invention are formulated into ointments, salves,
gels, or creams as generally known in the art.
[0120] In addition to use in therapy, the oligomers of the
invention may be used as diagnostic reagents to detect the presence
or absence of the target DNA or RNA sequences to which they
specifically bind. Such diagnostic tests are described in further
detail below.
[0121] Likewise, the antisense constructs of the present invention,
by antagonizing the normal biological activity of one of the
hedgehog proteins, can be used in the manipulation of tissue, e.g.
tissue differentiation, both in vivo and in ex vivo tissue
cultures.
[0122] Also, the anti-sense techniques (e.g. microinjection of
antisense molecules, or transfection with plasmids whose
transcripts are anti-sense with regard to an hh mRNA or gene
sequence) can be used to investigate role of hh in developmental
events, as well as the normal cellular function of hh in adult
tissue. Such techniques can be utilized in cell culture, but can
also be used in the creation of transgenic animals.
[0123] This invention also provides expression vectors containing a
nucleic acid encoding a vertebrate hh polypeptide, operably linked
to at least one transcriptional regulatory sequence. Operably
linked is intended to mean that the nucleotide sequence is linked
to a regulatory sequence in a manner which allows expression of the
nucleotide sequence. Regulatory sequences are art-recognized and
are selected to direct expression of the subject vertebrate hh
proteins. Accordingly, the term transcriptional regulatory sequence
includes promoters, enhancers and other expression control
elements. Such regulatory sequences are described in Goeddel; Gene
Expression Technology: Methods in Enzymology 185, Academic Press,
San Diego, Calif. (1990). For instance, any of a wide variety of
expression control sequences, sequences that control the expression
of a DNA sequence when operatively linked to it, may be used in
these vectors to express DNA sequences encoding vertebrate hh
polypeptides of this invention. Such useful expression control
sequences, include, for example, a viral LTR, such as the LTR of
the Moloney murine leukemia virus, the early and late promoters of
SV40, adenovirus or cytomegalovirus immediate early promoter, the
lac system, the trp system, the TAC or TRC system, T7 promoter
whose expression is directed by T7 RNA polymerase, the major
operator and promoter regions of phage .lambda., the control
regions for fd coat protein, the promoter for 3-phosphoglycerate
kinase or other glycolytic enzymes, the promoters of acid
phosphatase, e.g., Pho5, the promoters of the yeast .alpha.-mating
factors, the polyhedron promoter of the baculovirus system and
other sequences known to control the expression of genes of
prokaryotic or eukaryotic cells or their viruses, and various
combinations thereof. It should be understood that the design of
the expression vector may depend on such factors as the choice of
the host cell to be transformed and/or the type of protein desired
to be expressed. Moreover, the vector's copy number, the ability to
control that copy number and the expression of any other proteins
encoded by the vector, such as antibiotic markers, should also be
considered. In one embodiment, the expression vector includes a
recombinant gene encoding a peptide having an agonistic activity of
a subject hedgehog polypeptide, or alternatively, encoding a
peptide which is an antagonistic form of the hh protein. Such
expression vectors can be used to transfect cells and thereby
produce polypeptides, including fusion proteins, encoded by nucleic
acids as described herein.
[0124] Moreover, the gene constructs of the present invention can
also be used as a part of a gene therapy protocol to deliver
nucleic acids encoding either an agonistic or antagonistic form of
one of the subject vertebrate hedgehog proteins. Thus, another
aspect of the invention features expression vectors for in vivo or
in vitro transfection and expression of a vertebrate hh polypeptide
in particular cell types so as to reconstitute the function of, or
alternatively, abrogate the function of hedgehog-induced signaling
in a tissue in which the naturally-occurring form of the protein is
misexpressed; or to deliver a form of the protein which alters
differentiation of tissue, or which inhibits neoplastic
transformation.
[0125] Expression constructs of the subject vertebrate hh
polypeptide, and mutants thereof, may be administered in any
biologically effective carrier, e.g. any formulation or composition
capable of effectively delivering the recombinant gene to cells in
vivo. Approaches include insertion of the subject gene in viral
vectors including recombinant retroviruses, adenovirus,
adeno-associated virus, and herpes simplex virus-1, or recombinant
bacterial or eukaryotic plasmids. Viral vectors transfect cells
directly; plasmid DNA can be delivered with the help of, for
example, cationic liposomes (lipofectin) or derivatized (e.g.
antibody conjugated), polylysine conjugates, gramacidin S,
artificial viral envelopes or other such intracellular carriers, as
well as direct injection of the gene construct or CaPO.sub.4
precipitation carried out in vivo. It will be appreciated that
because transduction of appropriate target cells represents the
critical first step in gene therapy, choice of the particular gene
delivery system will depend on such factors as the phenotype of the
intended target and the route of administration, e.g. locally or
systemically. Furthermore, it will be recognized that the
particular gene construct provided for in vivo transduction of
hedgehog expression are also useful for in vitro transduction of
cells, such as for use in the ex vivo tissue culture systems
described below.
[0126] A preferred approach for in vivo introduction of nucleic
acid into a cell is by use of a viral vector containing nucleic
acid, e.g. a cDNA, encoding the particular form of the hedgehog
polypeptide desired. Infection of cells with a viral vector has the
advantage that a large proportion of the targeted cells can receive
the nucleic acid. Additionally, molecules encoded within the viral
vector, e.g., by a cDNA contained in the viral vector, are
expressed efficiently in cells which have taken up viral vector
nucleic acid.
[0127] Retrovirus vectors and adeno-associated virus vectors are
generally understood to be the recombinant gene delivery system of
choice for the transfer of exogenous genes in vivo, particularly
into humans. These vectors provide efficient delivery of genes into
cells, and the transferred nucleic acids are stably integrated into
the chromosomal DNA of the host. A major prerequisite for the use
of retroviruses is to ensure the safety of their use, particularly
with regard to the possibility of the spread of wild-type virus in
the cell population. The development of specialized cell lines
(termed "packaging cells") which produce only replication-defective
retroviruses has increased the utility of retroviruses for gene
therapy, and defective retroviruses are well characterized for use
in gene transfer for gene therapy purposes (for a review see
Miller, A. D. (1990) Blood 76:271). Thus, recombinant retrovirus
can be constructed in which part of the retroviral coding sequence
(gag, pol, env) has been replaced by nucleic acid encoding one of
the subject proteins rendering the retrovirus replication
defective. The replication defective retrovirus is then packaged
into virions which can be used to infect a target cell through the
use of a helper virus by standard techniques. Protocols for
producing recombinant retroviruses and for infecting cells in vitro
or in vivo with such viruses can be found in Current Protocols in
Molecular Biology, Ausubel, F. M. et al. (eds.) Greene Publishing
Associates, (1989), Sections 9.10-9.14 and other standard
laboratory manuals. Examples of suitable retroviruses include pLJ,
pZIP, pWE and pEM which are well known to those skilled in the art.
Examples of suitable packaging virus lines for preparing both
ecotropic and amphotropic retroviral systems include .psi.Crip,
.psi.Cre, .psi.2 and .psi.Am. Retroviruses have been used to
introduce a variety of genes into many different cell types,
including neuronal cells, in vitro and/or in vivo (see for example
Eglitis, et al. (1985) Science 230:1395-1398; Danos and Mulligan
(1988) Proc. Natl. Acad. Sci. USA 85:6460-6464; Wilson et al.
(1988) Proc. Natl. Acad. Sci. USA 85:3014-3018; Armentano et al.
(1990) Proc. Natl. Acad. Sci. USA 87:6141-6145; Huber et al. (1991)
Proc. Natl. Acad. Sci. USA 88:8039-8043; Ferry et al. (1991) Proc.
Natl. Acad. Sci. USA 88:8377-8381; Chowdhury et al. (1991) Science
254:1802-1805; van Beusechem et al. (1992) Proc. Natl. Acad. Sci.
USA 89:7640-7644; Kay et al. (1992) Human Gene Therapy 3:641-647;
Dai et al. (1992) Proc. Natl. Acad. Sci USA 89:10892-10895; Hwu et
al. (1993) J. Immunol. 150:4104-4115; U.S. Pat. Nos. 4,868,116,
4,980,286; PCT Application WO 89/07136; PCT Application WO
89/02468; PCT Application WO 89/05345; and PCT Application WO
92/07573).
[0128] Furthermore, it has been shown that it is possible to limit
the infection spectrum of retroviruses and consequently of
retroviral-based vectors, by modifying the viral packaging proteins
on the surface of the viral particle (see, for example PCT
publications WO93/25234 and WO94/06920). For instance, strategies
for the modification of the infection spectrum of retroviral
vectors include: coupling antibodies specific for cell surface
antigens to the viral env protein (Roux et al. (1989) PNAS
86:9079-9083; Julan et al. (1992) J. Gen Virol 73:3251-3255; and
Goud et al. (1983) Virology 163:251-254); or coupling cell surface
receptor ligands to the viral env proteins (Neda et al. (1991) J
Biol Chem 266:14143-14146). Coupling can be in the form of the
chemical cross-linking with a protein or other variety (e.g.
lactose to convert the env protein to an asialoglycoprotein), as
well as by generating fusion proteins (e.g. single-chain
antibody/env fusion proteins). This technique, while useful to
limit or otherwise direct the infection to certain tissue types,
can also be used to convert an ecotropic vector in to an
amphotropic vector.
[0129] Moreover, use of retroviral gene delivery can be further
enhanced by the use of tissue- or cell-specific transcriptional
regulatory sequences which control expression of the hh gene of the
retroviral vector.
[0130] Another viral gene delivery system useful in the present
invention utilizes adenovirus-derived vectors. The genome of an
adenovirus can be manipulated such that it encodes and expresses a
gene product of interest but is inactivated in terms of its ability
to replicate in a normal lytic viral life cycle. See for example
Berkner et al. (1988) BioTechniques 6:616; Rosenfeld et al. (1991)
Science 252:431-434; and Rosenfeld et al. (1992) Cell 68:143-155.
Suitable adenoviral vectors derived from the adenovirus strain Ad
type 5 dl324 or other strains of adenovirus (e.g., Ad2, Ad3, Ad7
etc.) are well known to those skilled in the art. Recombinant
adenoviruses can be advantageous in certain circumstances in that
they can be used to infect a wide variety of cell types, including
airway epithelium (Rosenfeld et al. (1992) cited supra),
endothelial cells (Lemarchand et al. (1992) Proc. Natl. Acad. Sci.
USA 89:6482-6486), hepatocytes (Herz and Gerard (1993) Proc. Natl.
Acad. Sci. USA 90:2812-2816) and muscle cells (Quantin et al.
(1992) Proc. Natl. Acad. Sci. USA 89:2581-2584). Furthermore, the
virus particle is relatively stable and amenable to purification
and concentration, and as above, can be modified so as to affect
the spectrum of infectivity. Additionally, introduced adenoviral
DNA (and foreign DNA contained therein) is not integrated into the
genome of a host cell but remains episomal, thereby avoiding
potential problems that can occur as a result of insertional
mutagenesis in situations where introduced DNA becomes integrated
into the host genome (e.g., retroviral DNA). Moreover, the carrying
capacity of the adenoviral genome for foreign DNA is large (up to 8
kilobases) relative to other gene delivery vectors (Berkner et al.
cited supra; Haj-Ahmand and Graham (1986) J. Virol. 57:267). Most
replication-defective adenoviral vectors currently in use and
therefore favored by the present invention are deleted for all or
parts of the viral E1 and E3 genes but retain as much as 80% of the
adenoviral genetic material (see, e.g., Jones et al. (1979) Cell
16:683; Berkner et al., supra; and Graham et al. in Methods in
Molecular Biology, E. J. Murray, Ed. (Humana, Clifton, N.J., 1991)
vol. 7. pp. 109-127). Expression of the inserted hedgehog gene can
be under control of, for example, the E1A promoter, the major late
promoter (MLP) and associated leader sequences, the E3 promoter, or
exogenously added promoter sequences.
[0131] Yet another viral vector system useful for delivery of one
of the subject vertebrate hh genes is the adeno-associated virus
(AAV). Adeno-associated virus is a naturally occurring defective
virus that requires another virus, such as an adenovirus or a
herpes virus, as a helper virus for efficient replication and a
productive life cycle. (For a review see Muzyczka et al. Curr.
Topics in Micro. and Immunol. (1992) 158:97-129). It is also one of
the few viruses that may integrate its DNA into non-dividing cells,
and exhibits a high frequency of stable integration (see for
example Flotte et al. (1992) Am. J. Respir. Cell. Mol. Biol.
7:349-356; Samulski et al. (1989) J. Virol. 63:3822-3828; and
McLaughlin et al. (1989) J. Virol. 62:1963-1973). Vectors
containing as little as 300 base pairs of AAV can be packaged and
can integrate. Space for exogenous DNA is limited to about 4.5 kb.
An AAV vector such as that described in Tratschin et al. (1985)
Mol. Cell. Biol. 5:3251-3260 can be used to introduce DNA into
cells. A variety of nucleic acids have been introduced into
different cell types using AAV vectors (see for example Hermonat et
al. (1984) Proc. Natl. Acad. Sci. USA 81:6466-6470; Tratschin et
al. (1985) Mol. Cell. Biol. 4:2072-2081; Wondisford et al. (1988)
Mol. Endocrinol. 2:32-39; Tratschin et al. (1984) J. Virol.
51:611-619; and Flotte et al. (1993) J. Biol. Chem.
268:3781-3790).
[0132] In addition to viral transfer methods, such as those
illustrated above, non-viral methods can also be employed to cause
expression of a subject hedgehog polypeptide in the tissue of an
animal. Most nonviral methods of gene transfer rely on normal
mechanisms used by mammalian cells for the uptake and intracellular
transport of macromolecules. In preferred embodiments, non-viral
gene delivery systems of the present invention rely on endocytic
pathways for the uptake of the subject hh polypeptide gene by the
targeted cell. Exemplary gene delivery systems of this type include
liposomal derived systems, poly-lysine conjugates, and artificial
viral envelopes.
[0133] In clinical settings, the gene delivery systems for the
therapeutic hedgehog gene can be introduced into a patient by any
of a number of methods, each of which is familiar in the art. For
instance, a pharmaceutical preparation of the gene delivery system
can be introduced systemically, e.g. by intravenous injection, and
specific transduction of the protein in the target cells occurs
predominantly from specificity of transfection provided by the gene
delivery vehicle, cell-type or tissue-type expression due to the
transcriptional regulatory sequences controlling expression of the
receptor gene, or a combination thereof. In other embodiments,
initial delivery of the recombinant gene is more limited with
introduction into the animal being quite localized. For example,
the gene delivery vehicle can be introduced by catheter (see U.S.
Pat. No. 5,328,470) or by stereotactic injection (e.g. Chen et al.
(1994) PNAS 91: 3054-3057). A vertebrate hh gene, such as any one
of the clones represented in the group consisting of SEQ ID NO:1-7,
can be delivered in a gene therapy construct by electroporation
using techniques described, for example, by Dev et al. ((1994)
Cancer Treat Rev 20:105-115).
[0134] The pharmaceutical preparation of the gene therapy construct
can consist essentially of the gene delivery system in an
acceptable diluent, or can comprise a slow release matrix in which
the gene delivery vehicle is imbedded. Alternatively, where the
complete gene delivery system can be produced intact from
recombinant cells, e.g. retroviral vectors, the pharmaceutical
preparation can comprise one or more cells which produce the gene
delivery system.
[0135] Another aspect of the present invention concerns recombinant
forms of the hedgehog proteins. Recombinant polypeptides preferred
by the present invention, in addition to native hedgehog proteins,
are at least 60% homologous, more preferably 70% homologous and
most preferably 80% homologous with an amino acid sequence
represented by any of SEQ ID Nos:8-14. Polypeptides which possess
an activity of a hedgehog protein (i.e. either agonistic or
antagonistic), and which are at least 90%, more preferably at least
95%, and most preferably at least about 98-99% homologous with a
sequence selected from the group consisting of SEQ ID Nos:8-14 are
also within the scope of the invention.
[0136] The term "recombinant protein" refers to a polypeptide of
the present invention which is produced by recombinant DNA
techniques, wherein generally, DNA encoding a vertebrate hh
polypeptide is inserted into a suitable expression vector which is
in turn used to transform a host cell to produce the heterologous
protein. Moreover, the phrase "derived from", with respect to a
recombinant hedgehog gene, is meant to include within the meaning
of "recombinant protein" those proteins having an amino acid
sequence of a native hedgehog protein, or an amino acid sequence
similar thereto which is generated by mutations including
substitutions and deletions (including truncation) of a naturally
occurring form of the protein.
[0137] The present invention further pertains to recombinant forms
of one of the subject hedgehog polypeptides which are encoded by
genes derived from a vertebrate organism, particularly a mammal
(e.g. a human), and which have amino acid sequences evolutionarily
related to the hedgehog proteins represented in SEQ ID Nos:8-14.
Such recombinant hh polypeptides preferably are capable of
functioning in one of either role of an agonist or antagonist of at
least one biological activity of a wild-type ("authentic") hedgehog
protein of the appended sequence listing. The term "evolutionarily
related to", with respect to amino acid sequences of vertebrate
hedgehog proteins, refers to both polypeptides having amino acid
sequences which have arisen naturally, and also to mutational
variants of vertebrate hh polypeptides which are derived, for
example, by combinatorial mutagenesis. Such evolutionarily derived
hedgehog proteins polypeptides preferred by the present invention
are at least 60% homologous, more preferably 70% homologous and
most preferably 80% homologous with the amino acid sequence
selected from the group consisting of SEQ ID Nos:8-14. Polypeptides
having at least about 90%, more preferably at least about 95%, and
most preferably at least about 98-99% homology with a sequence
selected from the group consisting of SEQ ID Nos:8-14 are also
within the scope of the invention.
[0138] The present invention further pertains to methods of
producing the subject hedgehog polypeptides. For example, a host
cell transfected with a nucleic acid vector directing expression of
a nucleotide sequence encoding the subject polypeptides can be
cultured under appropriate conditions to allow expression of the
peptide to occur. The polypeptide hedgehog may be secreted and
isolated from a mixture of cells and medium containing the
recombinant vertebrate hh polypeptide. Alternatively, the peptide
may be retained cytoplasmically by removing the signal peptide
sequence from the recombinant hh gene and the cells harvested,
lysed and the protein isolated. A cell culture includes host cells,
media and other byproducts. Suitable media for cell culture are
well known in the art. The recombinant hh polypeptide can be
isolated from cell culture medium, host cells, or both using
techniques known in the art for purifying proteins including
ion-exchange chromatography, gel filtration chromatography,
ultrafiltration, electrophoresis, and immunoaffinity purification
with antibodies specific for such peptide. In a preferred
embodiment, the recombinant hh polypeptide is a fusion protein
containing a domain which facilitates its purification, such as an
hh/GST fusion protein.
[0139] This invention also pertains to a host cell transfected to
express a recombinant form of the subject hedgehog polypeptides.
The host cell may be any prokaryotic or eukaryotic cell. Thus, a
nucleotide sequence derived from the cloning of vertebrate hedgehog
proteins, encoding all or a selected portion of the full-length
protein, can be used to produce a recombinant form of a vertebrate
hh polypeptide via microbial or eukaryotic cellular processes.
Ligating the polynucleotide sequence into a gene construct, such as
an expression vector, and transforming or transfecting into hosts,
either eukaryotic (yeast, avian, insect or mammalian) or
prokaryotic (bacterial cells), are standard procedures used in
producing other well-known proteins, e.g. insulin, interferons,
human growth hormone, IL-1, IL-2, and the like. Similar procedures,
or modifications thereof, can be employed to prepare recombinant
hedgehog polypeptides by microbial means or tissue-culture
technology in accord with the subject invention.
[0140] The recombinant hedgehog genes can be produced by ligating
nucleic acid encoding an hh protein, or a portion thereof, into a
vector suitable for expression in either prokaryotic cells,
eukaryotic cells, or both. Expression vectors for production of
recombinant forms of the subject hh polypeptides include plasmids
and other vectors. For instance, suitable vectors for the
expression of a hedgehog polypeptide include plasmids of the types:
pBR322-derived plasmids, pEMBL-derived plasmids, pEX-derived
plasmids, pBTac-derived plasmids and pUC-derived plasmids for
expression in prokaryotic cells, such as E. coli.
[0141] A number of vectors exist for the expression of recombinant
proteins in yeast. For instance, YEP24, YIP5, YEP51, YEP52, pYES2,
and YRP17 are cloning and expression vehicles usefull in the
introduction of genetic constructs into S. cerevisiae (see, for
example, Broach et al. (1983) in Experimental Manipulation of Gene
Expression, ed. M. Inouye Academic Press, p. 83, incorporated by
reference herein). These vectors can replicate in E. coli due the
presence of the pBR322 ori, and in S. cerevisiae due to the
replication determinant of the yeast 2 micron plasmid. In addition,
drug resistance markers such as ampicillin can be used. In an
illustrative embodiment, an hh polypeptide is produced
recombinantly utilizing an expression vector generated by
sub-cloning the coding sequence of one of the hedgehog genes
represented in SEQ ID Nos:1-7.
[0142] The preferred mammalian expression vectors contain both
prokaryotic sequences, to facilitate the propagation of the vector
in bacteria, and one or more eukaryotic transcription units that
are expressed in eukaryotic cells. The pcDNAI/amp, pcDNAI/neo,
pRc/CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7,
pko-neo and pHyg derived vectors are examples of mammalian
expression vectors suitable for transfection of eukaryotic cells.
Some of these vectors are modified with sequences from bacterial
plasmids, such as pBR322, to facilitate replication and drug
resistance selection in both prokaryotic and eukaryotic cells.
Alternatively, derivatives of viruses such as the bovine
papillomavirus (BPV-1), or Epstein-Barr virus (pHEBo, pREP-derived
and p205) can be used for transient expression of proteins in
eukaryotic cells. The various methods employed in the preparation
of the plasmids and transformation of host organisms are well known
in the art. For other suitable expression systems for both
prokaryotic and eukaryotic cells, as well as general recombinant
procedures, see Molecular Cloning A Laboratory Manual, 2nd Ed., ed.
by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory
Press: 1989) Chapters 16 and 17.
[0143] In some instances, it may be desirable to express the
recombinant hedgehog polypeptide by the use of a baculovirus
expression system. Examples of such baculovirus expression systems
include pVL-derived vectors (such as pVL1392, pVL1393 and pVL941),
pAcUW-derived vectors (such as pAcUW1), and pBlueBac-derived
vectors (such as the .beta.-gal containing pBlueBac III).
[0144] When it is desirable to express only a portion of an hh
protein, such as a form lacking a portion of the N-terminus, i.e. a
truncation mutant which lacks the signal peptide, it may be
necessary to add a start codon (ATG) to the oligonucleotide
fragment containing the desired sequence to be expressed. It is
well known in the art that a methionine at the N-terminal position
can be enzymatically cleaved by the use of the enzyme methionine
aminopeptidase (MAP). MAP has been cloned from E. coli (Ben-Bassat
et al. (1987) J. Bacteriol. 169:751-757) and Salmonella typhimurium
and its in vitro activity has been demonstrated on recombinant
proteins (Miller et al. (1987) PNAS 84:2718-1722). Therefore,
removal of an N-terminal methionine, if desired, can be achieved
either in vivo by expressing hedgehog-derived polypeptides in a
host which produces MAP (e.g., E. coli or CM89 or S. cerevisiae),
or in vitro by use of purified MAP (e.g., procedure of Miller et
al., supra).
[0145] Alternatively, the coding sequences for the polypeptide can
be incorporated as a part of a fusion gene including a nucleotide
sequence encoding a different polypeptide. This type of expression
system can be useful under conditions where it is desirable to
produce an immunogenic fragment of a hedgehog protein. For example,
the VP6 capsid protein of rotavirus can be used as an immunologic
carrier protein for portions of the hh polypeptide, either in the
monomeric form or in the form of a viral particle. The nucleic acid
sequences corresponding to the portion of a subject hedgehog
protein to which antibodies are to be raised can be incorporated
into a fusion gene construct which includes coding sequences for a
late vaccinia virus structural protein to produce a set of
recombinant viruses expressing fusion proteins comprising hh
epitopes as part of the virion. It has been demonstrated with the
use of immunogenic fusion proteins utilizing the Hepatitis B
surface antigen fusion proteins that recombinant Hepatitis B
virions can be utilized in this role as well. Similarly, chimeric
constructs coding for fusion proteins containing a portion of an hh
protein and the poliovirus capsid protein can be created to enhance
immunogenicity of the set of polypeptide antigens (see, for
example, EP Publication No: 0259149; and Evans et al. (1989) Nature
339:385; Huang et al. (1988) J. Virol. 62:3855; and Schlienger et
al. (1992) J. Virol. 66:2).
[0146] The Multiple Antigen Peptide system for peptide-based
immunization can also be utilized to generate an immunogen, wherein
a desired portion of an hh polypeptide is obtained directly from
organo-chemical synthesis of the peptide onto an oligomeric
branching lysine core (see, for example, Posnett et al. (1988) JBC
263:1719 and Nardelli et al. (1992) J. Immunol. 148:914). Antigenic
determinants of hh proteins can also be expressed and presented by
bacterial cells.
[0147] In addition to utilizing fusion proteins to enhance
immunogenicity, it is widely appreciated that fusion proteins can
also facilitate the expression of proteins, and accordingly, can be
used in the expression of the vertebrate hh polypeptides of the
present invention. For example, hedgehog polypeptides can be
generated as glutathione-S-transferase (GST-fusion) proteins. Such
GST-fusion proteins can enable easy purification of the hedgehog
polypeptide, as for example by the use of glutathione-derivatized
matrices (see, for example, Current Protocols in Molecular Biology,
eds. Ausubel et al. (N.Y.: John Wiley & Sons, 1991)). In
another embodiment, a fusion gene coding for a purification leader
sequence, such as a poly-(His)/enterokinase cleavage site sequence,
can be used to replace the signal sequence which naturally occurs
at the N-terminus of the hh protein (e.g.of the pro-form, in order
to permit purification of the poly(His)-hh protein by affinity
chromatography using a Ni.sup.2+ metal resin. The purification
leader sequence can then be subsequently removed by treatment with
enterokinase (e.g., see Hochuli et al. (1987) J. Chromatography
411:177; and Janknecht et al. PNAS 88:8972).
[0148] Techniques for making fusion genes are known to those
skilled in the art. Essentially, the joining of various DNA
fragments coding for different polypeptide sequences is performed
in accordance with conventional techniques, employing blunt-ended
or stagger-ended termini for ligation, restriction enzyme digestion
to provide for appropriate termini, filling-in of cohesive ends as
appropriate, alkaline phosphatase treatment to avoid undesirable
joining, and enzymatic ligation. In another embodiment, the fusion
gene can be synthesized by conventional techniques including
automated DNA synthesizers. Alternatively, PCR amplification of
gene fragments can be carried out using anchor primers which give
rise to complementary overhangs between two consecutive gene
fragments which can subsequently be annealed to generate a chimeric
gene sequence (see, for example, Current Protocols in Molecular
Biology, eds. Ausubel et al. John Wiley & Sons: 1992).
[0149] Hedgehog polypeptides may also be chemically modified to
create hh derivatives by forming covalent or aggregate conjugates
with other chemical moieties, such as glycosyl groups, lipids,
phosphate, acetyl groups and the like. Covalent derivatives of
hedgehog proteins can be prepared by linking the chemical moieties
to functional groups on amino acid sidechains of the protein or at
the N-terminus or at the C-terminus of the polypeptide.
[0150] For instance, hedgehog proteins can be generated to include
a moiety, other than sequence naturally associated with the
protein, that binds a component of the extracellular matrix and
enhances localization of the analog to cell surfaces. For example,
sequences derived from the fibronectin "type-III repeat", such as a
tetrapeptide sequence R-G-D-S (Pierschbacher et al. (1984) Nature
309:30-3; and Komblihtt et al. (1985) EMBO 4:1755-9) can be added
to the hh polypeptide to support attachment of the chimeric
molecule to a cell through binding ECM components (Ruoslahti et al.
(1987) Science 238:491-497; Pierschbacher et al. (1987) J. Biol.
Chem. 262:17294-8.; Hynes (1987) Cell 48:549-54; and Hynes (1992)
Cell 69:11-25).
[0151] The present invention also makes available isolated hedgehog
polypeptides which are isolated from, or otherwise substantially
free of other cellular and extracellular proteins, especially
morphogenic proteins or other extracellular or cell surface
associated proteins which may normally be associated with the
hedgehog polypeptide. The term "substantially free of other
cellular or extracellular proteins" (also referred to herein as
"contaminating proteins") or "substantially pure or purified
preparations" are defined as encompassing preparations of hh
polypeptides having less than 20% (by dry weight) contaminating
protein, and preferably having less than 5% contaminating protein.
Functional forms of the subject polypeptides can be prepared, for
the first time, as purified preparations by using a cloned gene as
described herein. By "purified", it is meant, when referring to a
peptide or DNA or RNA sequence, that the indicated molecule is
present in the substantial absence of other biological
macromolecules, such as other proteins. The term "purified" as used
herein preferably means at least 80% by dry weight, more preferably
in the range of 95-99% by weight, and most preferably at least
99.8% by weight, of biological macromolecules of the same type
present (but water, buffers, and other small molecules, especially
molecules having a molecular weight of less than 5000, can be
present). The term "pure" as used herein preferably has the same
numerical limits as "purified" immediately above. "Isolated" and
"purified" do not encompass either natural materials in their
native state or natural materials that have been separated into
components (e.g., in an acrylamide gel) but not obtained either as
pure (e.g. lacking contaminating proteins, or chromatography
reagents such as denaturing agents and polymers, e.g. acrylamide or
agarose) substances or solutions. In preferred embodiments,
purified hedgehog preparations will lack any contaminating proteins
from the same animal from that hedgehog is normally produced, as
can be accomplished by recombinant expression of, for example, a
human hedgehog protein in a non-human cell.
[0152] As described above for recombinant polypeptides, isolated hh
polypeptides can include all or a portion of the amino acid
sequences represented in SEQ ID No:8, SEQ ID No:9, SEQ ID No:10,
SEQ ID No:11, SEQ ID No:12, SEQ ID No:13 or SEQ ID No:14, or a
homologous sequence thereto. Preferred fragments of the subject
hedgehog proteins correspond to the N-terminal and C-terminal
proteolytic fragments of the mature protein (see, for instance,
Examples 6 and 9). Bioactive fragments of hedgehog polypeptides are
described in great detail in U.S. Ser. No. 08/435,093, filed May 4,
1995, herein incorporated by reference.
[0153] Isolated peptidyl portions of hedgehog proteins can be
obtained by screening peptides recombinantly produced from the
corresponding fragment of the nucleic acid encoding such peptides.
In addition, fragments can be chemically synthesized using
techniques known in the art such as conventional Merrifield solid
phase f-Moc or t-Boc chemistry. For example, a hedgehog polypeptide
of the present invention may be arbitrarily divided into fragments
of desired length with no overlap of the fragments, or preferably
divided into overlapping fragments of a desired length. The
fragments can be produced (recombinantly or by chemical synthesis)
and tested to identify those peptidyl fragments which can function
as either agonists or antagonists of a wild-type (e.g.,
"authentic") hedgehog protein.
[0154] The recombinant hedgehog polypeptides of the present
invention also include homologs of the authentic hedgehog proteins,
such as versions of those protein which are resistant to
proteolytic cleavage, as for example, due to mutations which alter
potential cleavage sequences or which inactivate an enzymatic
activity associated with the protein. Hedgehog homologs of the
present invention also include proteins which have been
post-translationally modified in a manner different than the
authentic protein. Exemplary derivatives of vertebrate hedgehog
proteins include polypeptides which lack N-glycosylation sites
(e.g. to produce an unglycosylated protein), or which lack
N-terminal and/or C-terminal sequences.
[0155] Modification of the structure of the subject vertebrate hh
polypeptides can be for such purposes as enhancing therapeutic or
prophylactic efficacy, or stability (e.g., ex vivo shelf life and
resistance to proteolytic degradation in vivo). Such modified
peptides, when designed to retain at least one activity of the
naturally-occurring form of the protein, are considered functional
equivalents of the hedgehog polypeptides described in more detail
herein. Such modified peptides can be produced, for instance, by
amino acid substitution, deletion, or addition.
[0156] For example, it is reasonable to expect that an isolated
replacement of a leucine with an isoleucine or valine, an aspartate
with a glutamate, a threonine with a serine, or a similar
replacement of an amino acid with a structurally related amino acid
(i.e. isosteric and/or isoelectric mutations) will not have a major
effect on the biological activity of the resulting molecule.
Conservative replacements are those that take place within a family
of amino acids that are related in their side chains. Genetically
encoded amino acids are can be divided into four families: (1)
acidic aspartate, glutamate; (2) basic=lysine, arginine, histidine;
(3) nonpolar=alanine, valine, leucine, isoleucine, proline,
phenylalanine, methionine, tryptophan; and (4) uncharged
polar=glycine, asparagine, glutamine, cysteine, serine, threonine,
tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes
classified jointly as aromatic amino acids. In similar fashion, the
amino acid repertoire can be grouped as (1) acidic=aspartate,
glutamate; (2) basic=lysine, arginine histidine, (3)
aliphatic=glycine, alanine, valine, leucine, isoleucine, serine,
threonine, with serine and threonine optionally be grouped
separately as aliphatic-hydroxyl; (4) aromatic=phenylalanine,
tyrosine, tryptophan; (5) amide=asparagine, glutamine; and (6)
sulfur-containing=cysteine and methionine. (see, for example,
Biochemistry, 2nd ed., Ed. by L. Stryer, W H Freeman and Co.:
1981). Whether a change in the amino acid sequence of a peptide
results in a functional hedgehog homolog (e.g. functional in the
sense that it acts to mimic or antagonize the wild-type form) can
be readily determined by assessing the ability of the variant
peptide to produce a response in cells in a fashion similar to the
wild-type protein, or competitively inhibit such a response.
Polypeptides in which more than one replacement has taken place can
readily be tested in the same manner.
[0157] This invention further contemplates a method for generating
sets of combinatorial mutants of the subject hedgehog proteins as
well as truncation mutants, and is especially useful for
identifying potential variant sequences (e.g. homologs) that are
functional in binding to a receptor for hedgehog proteins. The
purpose of screening such combinatorial libraries is to generate,
for example, novel hh homologs which can act as either agonists or
antagonist, or alternatively, possess novel activities all
together. To illustrate, hedgehog homologs can be engineered by the
present method to provide more efficient binding to a cognate
receptor, yet still retain at least a portion of an activity
associated with hh. Thus, combinatorially-derived homologs can be
generated to have an increased potency relative to a naturally
occurring form of the protein. Likewise, hedgehog homologs can be
generated by the present combinatorial approach to act as
antagonists, in that they are able to mimic, for example, binding
to other extracellular matrix components (such as receptors), yet
not induce any biological response, thereby inhibiting the action
of authentic hedgehog or hedgehog agonists. Moreover, manipulation
of certain domains of hh by the present method can provide domains
more suitable for use in fusion proteins, such as one that
incorporates portions of other proteins which are derived from the
extracellular matrix and/or which bind extracellular matrix
components.
[0158] In one aspect of this method, the amino acid sequences for a
population of hedgehog homologs or other related proteins are
aligned, preferably to promote the highest homology possible. Such
a population of variants can include, for example, hh homologs from
one or more species. Amino acids which appear at each position of
the aligned sequences are selected to create a degenerate set of
combinatorial sequences. In a preferred embodiment, the variegated
library of hedgehog variants is generated by combinatorial
mutagenesis at the nucleic acid level, and is encoded by a
variegated gene library. For instance, a mixture of synthetic
oligonucleotides can be enzymatically ligated into gene sequences
such that the degenerate set of potential hh sequences are
expressible as individual polypeptides, or alternatively, as a set
of larger fusion proteins (e.g. for phage display) containing the
set of hh sequences therein.
[0159] As illustrated in FIG. 5A, to analyze the sequences of a
population of variants, the amino acid sequences of interest can be
aligned relative to sequence homology. The presence or absence of
amino acids from an aligned sequence of a particular variant is
relative to a chosen consensus length of a reference sequence,
which can be real or artificial. In order to maintain the highest
homology in alignment of sequences, deletions in the sequence of a
variant relative to the reference sequence can be represented by an
amino acid space (.circle-solid. or *), while insertional mutations
in the variant relative to the reference sequence can be
disregarded and left out of the sequence of the variant when
aligned. For instance, FIG. 5A includes the alignment of several
cloned forms of hh from different species. Analysis of the
alignment of the hh clones shown in FIG. 5A can give rise to the
generation of a degenerate library of polypeptides comprising
potential hh sequences.
[0160] In an illustrative embodiment, alignment of exons 1, 2 and a
portion of exon 3 encoded sequences (e.g. the N-terminal
approximately 221 residues of the mature protein) of each of the
Shh clones produces a degenerate set of Shh polypeptides
represented by the general formula:
[0161]
C-G-P-G-R-G-X(1)-G-X(2)-R-R-H-P-K-K-L-T-P-L-A-Y-K-Q-F-I-P-N-V-A-E-K-
-T-L-G-A-S-G-R-Y-E-G-K-I-X(3)-R-N-S-E-R-F-K-E-L-T-P-N-Y-N-P-D-I-I-F-K-D-E--
E-N-T-G-A-D-R-L-M-T-Q-R-C-K-D-K-L-N-X(4)-L-A-I-S-V-M-N-X(5)-W-P-G-V-X(6)-L-
-R-V-T-E-G-W-D-E-D-G-H-H-X(7)-E-E-S-L-H-Y-E-G-R-A-V-D-I-T-T-S-D-R-D-X(8)-S-
-K-Y-G-X(9)-L-X(10)-R-L-A-V-E-A-G-F-D-W-V-Y-Y-E-S-K-A-H-I-H-C-S-V-K-A-E-N--
S-V-A-A-K-S-G-G-C-F-P-G-S-A-X(11)-V-X(12)-L-X(13)-X(14)-G-G-X(15)-K-X-(16)-
-V-K-D-L-X(17)-P-G-D-X(18)-V-L-A-A-D-X(19)-X(20)-G-X(21)-L-X(22)-X(23)-S-D-
-F-X(24)-X(25)-F-X(26)-D-R (SEQ ID No: 40),
[0162] wherein each of the degenerate positions "X" can be an amino
acid which occurs in that position in one of the human, mouse,
chicken or zebrafish Shh clones, or, to expand the library, each X
can also be selected from amongst amino acid residue which would be
conservative substitutions for the amino acids which appear
naturally in each of those positions. For instance, Xaa(1)
represents Gly, Ala, Val, Leu, Ile, Phe, Tyr or Trp; Xaa(2)
represents Arg, His or Lys; Xaa(3) represents Gly, Ala, Val, Leu,
Ile, Ser or Thr; Xaa(4) represents Gly, Ala, Val, Leu, Ile, Ser or
Thr; Xaa(5) represents Lys, Arg, His, Asn or Gln; Xaa(6) represents
Lys, Arg or His; Xaa(7) represents Ser, Thr, Tyr, Trp or Phe;
Xaa(8) represents Lys, Arg or His; Xaa(9) represents Met, Cys, Ser
or Thr; Xaa(10) represents Gly, Ala, Val, Leu, Ile, Ser or Thr;
Xaa(11) represents Leu, Val, Met, Thr or Ser; Xaa(12) represents
His, Phe, Tyr, Ser, Thr, Met or Cys; Xaa(13) represents Gln, Asn,
Glu, or Asp; Xaa(14) represents His, Phe, Tyr, Thr, Gln, Asn, Glu
or Asp; Xaa(15) represents Gln, Asn, Glu, Asp, Thr, Ser, Met or
Cys; Xaa(16) represents Ala, Gly, Cys, Leu, Val or Met; Xaa(17)
represents Arg, Lys, Met, Ile, Asn, Asp, Glu, Gln, Ser, Thr or Cys;
Xaa(18) represents Arg, Lys, Met or Ile; Xaa(19) represents Ala,
Gly, Cys, Asp, Glu, Gln, Asn, Ser, Thr or Met; Xaa(20) represents
Ala, Gly, Cys, Asp, Asn, Glu or Gln; Xaa(21) represents Arg, Lys,
Met, Ile, Asn, Asp, Glu or Gln; Xaa(22) represent Leu, Val, Met or
Ile; Xaa(23) represents Phe, Tyr, Thr, His or Trp; Xaa(24)
represents Ile, Val, Leu or Met; .Xaa(25) represents Met, Cys, Ile,
Leu, Val, Thr or Ser; Xaa(26) represents Leu, Val, Met, Thr or Ser.
In an even more expansive library, each X can be selected from any
amino acid.
[0163] In similar fashion, alignment of each of the human, mouse,
chicken and zebrafish hedgehog clones (FIG. 5B), can provide a
degenerate polypeptide sequence represented by the general
formula:
[0164]
C-G-P-G-R-G-X(1)-X(2)-X(3)-R-R-X(4)-X(S)-X(6)-P-K-X(7)-L-X(8)-P-L-X-
(9)-Y-K-Q-F-X(10)-P-X(11)-X(12)-X(13)-E-X(14)-T-L-G-A-S-G-X(15)-X(16)-E-G--
X(17)-X(18)-X(19)-R-X(20)-S-E-R-F-X(21)-X(22)-L-T-P-N-Y-N-P-D-I-I-F-K-D-E--
E-N-X(23)-G-A-D-R-L-M-T-X(24)-R-C-K-X(25)-X(26)-X(27)-N-X(28)-L-A-I-S-V-M--
N-X(29)-W-P-G-V-X(30)-L-R-V-T-E-G-X(31)-D-E-D-G-H-H-X(32)-X(33)-X(34)-S-L--
H-Y-E-G-R-A-X(35)-D-I-T-T-S-D-R-D-X(36)-X(37)-K-Y-G-X(38)-L-X(39)-R-L-A-V--
E-A-G-F-D-W-V-Y-Y-E-S-X(40)-X(41)-H-X(42)-H-X(43)-S-V-K-X(44)-X(45)
(SEQ ID No: 41),
[0165] wherein, as above, each of the degenerate positions "X" can
be an amino acid which occurs in a corresponding position in one of
the wild-type clones, and may also include amino acid residue which
would be conservative substitutions, or each X can be any amino
acid residue. In an exemplary embodiment, Xaa(1) represents Gly,
Ala, Val, Leu, Ile, Pro, Phe or Tyr; Xaa(2) represents Gly, Ala,
Val, Leu or Ile; Xaa(3) represents Gly, Ala, Val, Leu, Ile, Lys,
His or Arg; Xaa(4) represents Lys, Arg or His; Xaa(5) represents
Phe, Trp, Tyr or an amino acid gap; Xaa(6) represents Gly, Ala,
Val, Leu, Ile or an amino acid gap; Xaa(7) represents Asn, Gln,
His, Arg or Lys; Xaa(8) represents Gly, Ala, Val, Leu, Ile, Ser or
Thr; Xaa(9) represents Gly, Ala, Val, Leu, Ile, Ser or Thr; Xaa(10)
represents Gly, Ala, Val, Leu, Ile, Ser or Thr; Xaa(11) represents
Ser, Thr, Gln or Asn; Xaa(12) represents Met, Cys, Gly, Ala, Val,
Leu, Ile, Ser or Thr; Xaa(13) represents Gly, Ala, Val, Leu, Ile or
Pro; Xaa(14) represents Arg, His or Lys; Xaa(15) represents Gly,
Ala, Val, Leu, Ile, Pro, Arg, His or Lys; Xaa(16) represents Gly,
Ala, Val, Leu, Ile, Phe or Tyr; Xaa(17) represents Arg, His or Lys;
Xaa(18) represents Gly, Ala, Val, Leu, Ile, Ser or Thr; Xaa(19)
represents Thr or Ser; Xaa(20) represents Gly, Ala, Val, Leu, Ile,
Asn or Gln; Xaa(21) represents Arg, His or Lys; Xaa(22) represents
Asp or Glu; Xaa(23) represents Ser or Thr; Xaa(24) represents Glu,
Asp, Gln or Asn; Xaa(25) represents Glu or Asp; Xaa(26) represents
Arg, His or Lys; Xaa(27) represents Gly, Ala, Val, Leu or Ile;
Xaa(28) represents Gly, Ala, Val, Leu, Ile, Thr or Ser; Xaa(29)
represents Met, Cys, Gln, Asn, Arg, Lys or His; Xaa(30) represents
Arg, His or Lys; Xaa(31) represents Trp, Phe, Tyr, Arg, His or Lys;
Xaa(32) represents Gly, Ala, Val, Leu, Ile, Ser, Thr, Tyr or Phe;
Xaa(33) represents Gln, Asn, Asp or Glu; Xaa(34) represents Asp or
Glu; Xaa(35) represents Gly, Ala, Val, Leu, or Ile; Xaa(36)
represents Arg, His or Lys; Xaa(37) represents Asn, Gln, Thr or
Ser; Xaa(38) represents Gly, Ala, Val, Leu, Ile, Ser, Thr, Met or
Cys; Xaa(39) represents Gly, Ala, Val, Leu, Ile, Thr or Ser;
Xaa(40) represents Arg, His or Lys; Xaa(41) represents Asn, Gln,
Gly, Ala, Val, Leu or Ile; Xaa(42) represents Gly, Ala, Val, Leu or
Ile; Xaa(43) represents Gly, Ala, Val, Leu, Ile, Ser, Thr or Cys;
Xaa(44) represents Gly, Ala, Val, Leu, Ile, Thr or Ser; and Xaa(45)
represents Asp or Glu.
[0166] There are many ways by which the library of potential hh
homologs can be generated from a degenerate oligonucleotide
sequence. Chemical synthesis of a degenerate gene sequence can be
carried out in an automatic DNA synthesizer, and the synthetic
genes then ligated into an appropriate expression vector. The
purpose of a degenerate set of genes is to provide, in one mixture,
all of the sequences encoding the desired set of potential hh
sequences. The synthesis of degenerate oligonucleotides is well
known in the art (see for example, Narang, S A (1983) Tetrahedron
39:3; Itakura et al. (1981) Recombinant DNA, Proc 3rd Cleveland
Sympos. Macromolecules, ed. A G Walton, Amsterdam: Elsevier
pp273-289; Itakura et al. (1984) Annu. Rev. Biochem. 53:323;
Itakura et al. (1984) Science 198:1056; Ike et al. (1983) Nucleic
Acid Res. 11:477. Such techniques have been employed in the
directed evolution of other proteins (see, for example, Scott et
al. (1990) Science 249:386-390; Roberts et al. (1992) PNAS
89:2429-2433; Devlin et al. (1990) Science 249: 404-406; Cwirla et
al. (1990) PNAS 87: 6378-6382; as well as U.S. Pat. Nos. 5,223,409,
5,198,346, and 5,096,815).
[0167] A wide range of techniques are known in the art for
screening gene products of combinatorial libraries made by point
mutations, and for screening cDNA libraries for gene products
having a certain property. Such techniques will be generally
adaptable for rapid screening of the gene libraries generated by
the combinatorial mutagenesis of hedgehog homologs. The most widely
used techniques for screening large gene libraries typically
comprises cloning the gene library into replicable expression
vectors, transforming appropriate cells with the resulting library
of vectors, and expressing the combinatorial genes under conditions
in which detection of a desired activity facilitates relatively
easy isolation of the vector encoding the gene whose product was
detected. Each of the illustrative assays described below are
amenable to high through-put analysis as necessary to screen large
numbers of degenerate hedgehog sequences created by combinatorial
mutagenesis techniques.
[0168] In one embodiment, the combinatorial library is designed to
be secreted (e.g. the polypeptides of the library all include a
signal sequence but no transmembrane or cytoplasmic domains), and
is used to transfect a eukaryotic cell that can be co-cultured with
embryonic cells. A functional hedgehog protein secreted by the
cells expressing the combinatorial library will diffuse to
neighboring embryonic cells and induce a particular biological
response, such as to illustrate, neuronal differentiation. Using
antibodies directed to epitopes of particular neuronal cells (e.g.
Islet-1 or Pax-1), the pattern of detection of neuronal induction
will resemble a gradient function, and will allow the isolation
(generally after several repetitive rounds of selection) of cells
producing active hedgehog homologs. Likewise, hh antagonists can be
selected in similar fashion by the ability of the cell producing a
functional antagonist to protect neighboring cells from the effect
of wild-type hedgehog added to the culture media.
[0169] To illustrate, target cells are cultured in 24-well
microtitre plates. Other eukaryotic cells are transfected with the
combinatorial hh gene library and cultured in cell culture inserts
(e.g. Collaborative Biomedical Products, Catalog #40446) that are
able to fit into the wells of the microtitre plate. The cell
culture inserts are placed in the wells such that recombinant hh
homologs secreted by the cells in the insert can diffuse through
the porous bottom of the insert and contact the target cells in the
microtitre plate wells. After a period of time sufficient for
functional forms of a hedgehog protein to produce a measurable
response in the target cells, the inserts are removed and the
effect of the variant hedgehog proteins on the target cells
determined. For example, where the target cell is a neural crest
cell and the activity desired from the hh homolog is the induction
of neuronal differentiation, then fluorescently-labeled antibodies
specific for Islet-1 or other neuronal markers can be used to score
for induction in the target cells as indicative of a functional hh
in that well. Cells from the inserts corresponding to wells which
score positive for activity can be split and re-cultured on several
inserts, the process being repeated until the active clones are
identified.
[0170] In yet another screening assay, the candidate hedgehog gene
products are displayed on the surface of a cell or viral particle,
and the ability of particular cells or viral particles to associate
with a hedgehog-binding moiety (such as an hedgehog receptor or a
ligand which binds the hedgehog protein) via this gene product is
detected in a "panning assay". Such panning steps can be carried
out on cells cultured from embryos. For instance, the gene library
can be cloned into the gene for a surface membrane protein of a
bacterial cell, and the resulting fusion protein detected by
panning (Ladner et al., WO 88/06630; Fuchs et al. (1991)
Bio/Technology 9:1370-1371; and Goward et al. (1992) TIBS
18:136-140). In a similar fashion, fluorescently labeled molecules
which bind hh can be used to score for potentially functional hh
homologs. Cells can be visually inspected and separated under a
fluorescence microscope, or, where the morphology of the cell
permits, separated by a fluorescence-activated cell sorter.
[0171] In an alternate embodiment, the gene library is expressed as
a fusion protein on the surface of a viral particle. For instance,
in the filamentous phage system, foreign peptide sequences can be
expressed on the surface of infectious phage, thereby conferring
two significant benefits. First, since these phage can be applied
to affinity matrices at very high concentrations, large number of
phage can be screened at one time. Second, since each infectious
phage displays the combinatorial gene product on its surface, if a
particular phage is recovered from an affinity matrix in low yield,
the phage can be amplified by another round of infection. The group
of almost identical E.coli filamentous phages M13, fd, and fl are
most often used in phage display libraries, as either of the phage
gIII or gVIII coat proteins can be used to generate fusion proteins
without disrupting the ultimate packaging of the viral particle
(Ladner et al. PCT publication WO 90/02909; Garrard et al., PCT
publication WO 92/09690; Marks et al. (1992) J. Biol. Chem.
267:16007-16010; Griffths et al. (1993) EMBO J 12:725-734; Clackson
et al. (1991) Nature 352:624-628; and Barbas et al. (1992) PNAS
89:4457-4461).
[0172] In an illustrative embodiment, the recombinant phage
antibody system (RPAS, Pharamacia Catalog number 27-9400-01) can be
easily modified for use in expressing and screening hh
combinatorial libraries. For instance, the pCANTAB 5 phagemid of
the RPAS kit contains the gene which encodes the phage gIII coat
protein. The hh combinatorial gene library can be cloned into the
phagemid adjacent to the gIII signal sequence such that it will be
expressed as a gIII fusion protein. After ligation, the phagemid is
used to transform competent E. coil TG1 cells. Transformed cells
are subsequently infected with M13KO7 helper phage to rescue the
phagemid and its candidate hh gene insert. The resulting
recombinant phage contain phagemid DNA encoding a specific
candidate hh, and display one or more copies of the corresponding
fusion coat protein. The phage-displayed candidate hedgehog
proteins which are capable of binding an hh receptor are selected
or enriched by panning. For instance, the phage library can be
applied to cultured embryonic cells and unbound phage washed away
from the cells. The bound phage is then isolated, and if the
recombinant phage express at least one copy of the wild type gIII
coat protein, they will retain their ability to infect E. coli.
Thus, successive rounds of reinfection of E. coli, and panning will
greatly enrich for hh homologs, which can then be screened for
further biological activities in order to differentiate agonists
and antagonists.
[0173] Combinatorial mutagenesis has a potential to generate very
large libraries of mutant proteins, e.g., in the order of 10.sup.26
molecules. Combinatorial libraries of this size may be technically
challenging to screen even with high throughput screening assays
such as phage display. To overcome this problem, a new technique
has been developed recently, recrusive ensembel mutagenesis (REM),
which allows one to avoid the very high proportion of
non-functional proteins in a random library and simply enhances the
frequency of functional proteins, thus decreasing the complexity
required to achieve a useful sampling of sequence space. REM is an
algorithm which enhances the frequency of functional mutants in a
library when an appropriate selection or screening method is
employed (Arkin and Yourvan, 1992, PNAS USA 89:7811-7815; Yourvan
et al., 1992, Parallel Problem Solving from Nature, 2., In Maenner
and Manderick, eds., Elsevir Publishing Co., Amsterdam, pp.
401-410; Delgrave et al., 1993, Protein Engineering
6(3):327-331).
[0174] The invention also provides for reduction of the vertebrate
hh protein to generate mimetics, e.g. peptide or non-peptide
agents, which are able to disrupt binding of a vertebrate hh
polypeptide of the present invention with an hh receptor. Thus,
such mutagenic techniques as described above are also useful to map
the determinants of the hedgehog proteins which participate in
protein-protein interactions involved in, for example, binding of
the subject vertebrate hh polypeptide to other extracellular matrix
components. To illustrate, the critical residues of a subject hh
polypeptide or hh ligand which are involved in molecular
recognition of an hh receptor can be determined and used to
generate hedgehog-derived peptidornimetics which competitively
inhibit binding of the authentic hedgehog protein with that moiety.
By employing, for example, scanning mutagenesis to map the amino
acid residues of each of the subject hedgehog proteins which are
involved in binding other extracellular proteins, peptidomimetic
compounds can be generated which mimic those residues of the
hedgehog protein which facilitate the interaction. Such mimetics
may then be used to interfere with the normal function of a
hedgehog protein. For instance, non-hydrolyzable peptide analogs of
such residues can be generated using benzodiazepine (e.g., see
Freidinger et al. in Peptides: Chemistry and Biology, G. R.
Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), azepine
(e.g., see Huffinan et al. in Peptides: Chemistry and Biology, G.
R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988),
substituted gama lactam rings (Garvey et al. in Peptides: Chemistry
and Biology, G. R. Marshall ed., ESCOM Publisher: Leiden,
Netherlands, 1988), keto-methylene pseudopeptides (Ewenson et al.
(1986) J Med Chem 29:295; and Ewenson et al. in Peptides: Structure
and Function (Proceedings of the 9th American Peptide Symposium)
Pierce Chemical Co. Rockland, Ill., 1985), .beta.-turn dipeptide
cores (Nagai et al. (1985) Tetrahedron Lett 26:647; and Sato et al.
(1986) J Chem Soc Perkin Trans 1:1231), and .beta.-aminoalcohols
(Gordon et al. (1985) Biochem Biophys Res Commun126:419; and Dann
et al. (1986) Biochem Biophys Res Commun 134:71).
[0175] Another aspect of the invention pertains to an antibody
specifically reactive with a vertebrate hedgehog protein. For
example, by using immunogens derived from hedgehog protein, e.g.
based on the cDNA sequences, anti-protein/anti-peptide antisera or
monoclonal antibodies can be made by standard protocols (See, for
example, Antibodies: A Laboratory Manual ed. by Harlow and Lane
(Cold Spring Harbor Press: 1988)). A mammal, such as a mouse, a
hamster or rabbit can be immunized with an immunogenic form of the
peptide (e.g., a vertebrate hh polypeptide or an antigenic fragment
which is capable of eliciting an antibody response). Techniques for
conferring immunogenicity on a protein or peptide include
conjugation to carriers or other techniques well known in the art.
An immunogenic portion of a hedgehog protein can be administered in
the presence of adjuvant. The progress of immunization can be
monitored by detection of antibody titers in plasma or serum.
Standard ELISA or other immunoassays can be used with the immunogen
as antigen to assess the levels of antibodies. In a preferred
embodiment, the subject antibodies are immunospecific for antigenic
determinants of a hedgehog protein of a vertebrate organism, such
as a mammal, e.g. antigenic determinants of a protein represented
by SEQ ID Nos:8-14 or a closely related homolog (e.g. at least 85%
homologous, preferably at least 90% homologous, and more preferably
at least 95% homologous). In yet a further preferred embodiment of
the present invention, in order to provide, for example, antibodies
which are immuno-selective for discrete hedgehog homologs, e.g. Shh
versus Dhh versus Ihh, the anti-hh polypeptide antibodies do not
substantially cross react (i.e. does not react specifically) with a
protein which is, for example, less than 85% homologous to any of
SEQ ID Nos:8-14; e.g., less than 95% homologous with one of SEQ ID
Nos:8-14; e.g., less than 98-99% homologous with one of SEQ ID
Nos:8-14. By "not substantially cross react", it is meant that the
antibody has a binding affinity for a non-homologous protein which
is at least one order of magnitude, more preferably at least 2
orders of magnitude, and even more preferably at least 3 orders of
magnitude less than the binding affinity of the antibody for one or
more of the proteins of SEQ ID Nos:8-14.
[0176] Following immunization of an animal with an antigenic
preparation of a hedgehog protein, anti-hh antisera can be obtained
and, if desired, polyclonal anti-hh antibodies isolated from the
serum. To produce monoclonal antibodies, antibody-producing cells
(lymphocytes) can be harvested from an immunized animal and fused
by standard somatic cell fusion procedures with immortalizing cells
such as myeloma cells to yield hybridoma cells. Such techniques are
well known in the art, an include, for example, the hybridoma
technique (originally developed by Kohler and Milstein, (1975)
Nature, 256: 495-497), the human B cell hybridoma technique (Kozbar
et al., (1983) Immunology Today, 4: 72), and the EBV-hybridoma
technique to produce human monoclonal antibodies (Cole et al.,
(1985) Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc.
pp. 77-96). Hybridoma cells can be screened immunochemically for
production of antibodies specifically reactive with a vertebrate hh
polypeptide of the present invention and monoclonal antibodies
isolated from a culture comprising such hybridoma cells.
[0177] The term antibody as used herein is intended to include
fragments thereof which are also specifically reactive with one of
the subject vertebrate hh polypeptides. Antibodies can be
fragmented using conventional techniques and the fragments screened
for utility in the same manner as described above for whole
antibodies. For example, F(ab).sub.2 fragments can be generated by
treating antibody with pepsin. The resulting F(ab).sub.2 fragment
can be treated to reduce disulfide bridges to produce Fab
fragments. The antibody of the present invention is further
intended to include bispecific and chimeric molecules having
affinity for a hedgehog protein conferred by at least one CDR
region of the antibody.
[0178] Both monoclonal and polyclonal antibodies (Ab) directed
against authentic hedgehog polypeptides, or hedgehog variants, and
antibody fragments such as Fab and F(ab).sub.2, can be used to
block the action of one or more hedgehog proteins and allow the
study of the role of these proteins in, for example, embryogenesis
and/or maintenance of differential tissue. For example, purified
monoclonal Abs can be injected directly into the limb buds of chick
or mouse embryos. It is demonstrated in the examples below that hh
is expressed in the limb buds of, for example, day 10.5 embryos.
Thus, the use of anti-hh Abs during this developmental stage can
allow assessment of the effect of hh on the formation of limbs in
vivo. In a similar approach, hybridomas producing anti-hh
monoclonal Abs, or biodegradable gels in which anti-hh Abs are
suspended, can be implanted at a site proximal or within the area
at which hh action is intended to be blocked. Experiments of this
nature can aid in deciphering the role of this and other factors
that may be involved in limb patterning and tissue formation.
[0179] Antibodies which specifically bind hedgehog epitopes can
also be used in immunohistochemical staining of tissue samples in
order to evaluate the abundance and pattern of expression of each
of the subject hh polypeptides. Anti-hedgehog antibodies can be
used diagnostically in immuno-precipitation and immuno-blotting to
detect and evaluate hedgehog protein levels in tissue as part of a
clinical testing procedure. For instance, such measurements can be
useful in predictive valuations of the onset or progression of
neurological disorders, such as those marked by denervation-like or
disuse-like symptoms. Likewise, the ability to monitor hh levels in
an individual can allow determination of the efficacy of a given
treatment regimen for an individual afflicted with such a disorder.
The level of hh polypeptides may be measured in bodily fluid, such
as in samples of cerebral spinal fluid or amniotic fluid, or can be
measured in tissue, such as produced by biopsy. Diagnostic assays
using anti-hh antibodies can include, for example, immunoassays
designed to aid in early diagnosis of a neurodegenerative disorder,
particularly ones which are manifest at birth. Diagnostic assays
using anti-hh polypeptide antibodies can also include immunoassays
designed to aid in early diagnosis and phenotyping of a
differentiative disorder, as well as neoplastic or hyperplastic
disorders.
[0180] Another application of anti-hh antibodies of the present
invention is in the immunological screening of cDNA libraries
constructed in expression vectors such as .lambda.gt11,
.lambda.gt18-23, .lambda.ZAP, and .lambda.ORF8. Messenger libraries
of this type, having coding sequences inserted in the correct
reading frame and orientation, can produce fusion proteins. For
instance, .lambda.gt11 will produce fusion proteins whose amino
termini consist of .beta.-galactosidase amino acid sequences and
whose carboxy termini consist of a foreign polypeptide. Antigenic
epitopes of an hh protein, e.g. other orthologs of a particular
hedgehog protein or other homologs from the same species, can then
be detected with antibodies, as, for example, reacting
nitrocellulose filters lifted from infected plates with anti-hh
antibodies. Positive phage detected by this assay can then be
isolated from the infected plate. Thus, the presence of hedgehog
homologs can be detected and cloned from other animals, as can
alternate isoforms (including splicing variants) from humans.
[0181] Moreover, the nucleotide sequences determined from the
cloning of hh genes from vertebrate organisms will further allow
for the generation of probes and primers designed for use in
identifying and/or cloning hedgehog homologs in other cell types,
e.g. from other tissues, as well as hh homologs from other
vertebrate organisms. For instance, the present invention also
provides a probe/primer comprising a substantially purified
oligonucleotide, which oligonucleotide comprises a region of
nucleotide sequence that hybridizes under stringent conditions to
at least 10 consecutive nucleotides of sense or anti-sense sequence
selected from the group consisting of SEQ ID No:1, SEQ ID No:2, SEQ
ID No:3, SEQ ID No:4, SEQ ID No:5, SEQ ID No:6 and SEQ ID No:7, or
naturally occurring mutants thereof. For instance, primers based on
the nucleic acid represented in SEQ ID Nos:1-7 can be used in PCR
reactions to clone hedgehog homologs. Likewise, probes based on the
subject hedgehog sequences can be used to detect transcripts or
genomic sequences encoding the same or homologous proteins. In
preferred embodiments, the probe further comprises a label group
attached thereto and able to be detected, e.g. the label group is
selected from the group consisting of radioisotopes, fluorescent
compounds, enzymes, and enzyme co-factors.
[0182] Such probes can also be used as a part of a diagnostic test
kit for identifying cells or tissue which misexpress a hedgehog
protein, such as by measuring a level of a hedgehog encoding
nucleic acid in a sample of cells from a patient; e.g. detecting hh
mRNA levels or determining whether a genomic hh gene has been
mutated or deleted.
[0183] To illustrate, nucleotide probes can be generated from the
subject hedgehog genes which facilitate histological screening of
intact tissue and tissue samples for the presence (or absence) of
hedgehog-encoding transcripts. Similar to the diagnostic uses of
anti-hedgehog antibodies, the use of probes directed to hh
messages, or to genomic hh sequences, can be used for both
predictive and therapeutic evaluation of allelic mutations which
might be manifest in, for example, neoplastic or hyperplastic
disorders (e.g. unwanted cell growth) or abnormal differentiation
of tissue. Used in conjunction with immunoassays as described
above, the oligonucleotide probes can help facilitate the
determination of the molecular basis for a developmental disorder
which may involve some abnormality associated with expression (or
lack thereof) of a hedgehog protein. For instance, variation in
polypeptide synthesis can be differentiated from a mutation in a
coding sequence.
[0184] Accordingly, the present method provides a method for
determining if a subject is at risk for a disorder characterized by
aberrant control of differentiation or unwanted cell proliferation.
For instance, the subject assay can be used in the screening and
diagnosis of genetic and acquired disorders which involve
alteration in one or more of the hedgehog genes. In preferred
embodiments, the subject method can be generally characterized as
comprising: detecting, in a tissue sample of the subject (e.g. a
human patient), the presence or absence of a genetic lesion
characterized by at least one of (i) a mutation of a gene encoding
a hedgehog protein or (ii) the mis-expression of a hedgehog gene.
To illustrate, such genetic lesions can be detected by ascertaining
the existence of at least one of (i) a deletion of one or more
nucleotides from a hedgehog gene, (ii) an addition of one or more
nucleotides to a hedgehog gene, (iii) a substitution of one or more
nucleotides of a hedgehog gene, (iv) a gross chromosomal
rearrangement of a hedgehog gene, (v) a gross alteration in the
level of a messenger RNA transcript of an hh gene, (vi) the
presence of a non-wild type splicing pattern of a messenger RNA
transcript of a vertebrate hh gene, and (vii) a non-wild type level
of a hedgehog protein. In one aspect of the invention there is
provided a probe/primer comprising an oligonucleotide containing a
region of nucleotide sequence which is capable of hybridizing to a
sense or antisense sequence selected from the group consisting of
SEQ ID Nos:1-7, or naturally occurring mutants thereof, or 5' or 3'
flanking sequences or intronic sequences naturally associated with
a vertebrate hh gene. The probe is exposed to nucleic acid of a
tissue sample; and the hybridization of the probe to the sample
nucleic acid is detected. In certain embodiments, detection of the
lesion comprises utilizing the probe/primer in a polymerase chain
reaction (PCR) (see, e.g., U.S. Pat. Nos.: 4,683,195 and 4,683,202)
or, alternatively, in a ligation chain reaction (LCR) (see, e.g.,
Landegran et al. (1988) Science, 241:1077-1080; and NaKazawa et al.
(1944) PNAS 91:360-364) the later of which can be particularly
useful for detecting point mutations in hedgehog genes.
Alternatively, immunoassays can be employed to determine the level
of hh proteins, either soluble or membrane bound.
[0185] Yet another diagnostic screen employs a source of hedgehog
protein directly. As described herein, hedgehog proteins of the
present invention are involved in the induction of differentiation.
Accordingly, the pathology of certain differentiative and/or
proliferative disorders can be marked by loss of hedgehog
sensitivity by the afflicted tissue. Consequently, the response of
a tissue or cell sample to an inductive amount of a hedgehog
protein can be used to detect and characterize certain cellular
transformations and degenerative conditions. For instance,
tissue/cell samples from a patient can be treated with a hedgehog
agonist and the response of the tissue to the treatment determined.
Response can be qualified and/or quantified, for example, on the
basis of phenotypic change as result of hedgehog induction. For
example, expression of gene products induced by hedgehog treatment
can be scored for by immunoassay. The patched protein, for example,
is upregulated in drosophila in response to Dros-HH, and, in light
of the findings herein, a presumed vertebrate homolog will
similarly be upregulated. Thus, detection of patched expression on
the cells of the patient sample can permit detection of tissue that
is not hedgehog-responsive. Likewise, scoring for other phenotypic
markers provides a means for determining the response to
hedgehog.
[0186] Furthermore, by making available purified and recombinant
hedgehog polypeptides, the present invention facilitates the
development of assays which can be used to screen for drugs,
including hedgehog homologs, which are either agonists or
antagonists of the normal cellular function of the subject hedgehog
polypeptides, or of their role in the pathogenesis of cellular
differentiation and/or proliferation and disorders related thereto.
In one embodiment, the assay evaluates the ability of a compound to
modulate binding between a hedgehog polypeptide and a hedgehog
receptor. A variety of assay formats will suffice and, in light of
the present inventions, will be comprehended by skilled
artisan.
[0187] In many drug screening programs which test libraries of
compounds and natural extracts, high throughput assays are
desirable in order to maximize the number of compounds surveyed in
a given period of time. Assays which are performed in cell-free
systems, such as may be derived with purified or semi-purified
proteins, are often preferred as "primary" screens in that they can
be generated to permit rapid development and relatively easy
detection of an alteration in a molecular target which is mediated
by a test compound. Moreover, the effects of cellular toxicity
and/or bioavailability of the test compound can be generally
ignored in the in vitro system, the assay instead being focused
primarily on the effect of the drug on the molecular target as may
be manifest in an alteration of binding affinity with receptor
proteins. Accordingly, in an exemplary screening assay of the
present invention, the compound of interest is contacted with a
hedgehog receptor polypeptide which is ordinarily capable of
binding a hedgehog protein. To the mixture of the compound and
receptor is then added a composition containing a hedgehog
polypeptide. Detection and quantification of receptor/hedgehog
complexes provides a means for determining the compound's efficacy
at inhibiting (or potentiating) complex formation between the
receptor protein and the hedgehog polypeptide. The efficacy of the
compound can be assessed by generating dose response curves from
data obtained using various concentrations of the test compound.
Moreover, a control assay can also be performed to provide a
baseline for comparison. In the control assay, isolated and
purified hedgehog polypeptide is added to a composition containing
the receptor protein, and the formation of receptor/hedgehog
complex is quantitated in the absence of the test compound.
[0188] In an illustrative embodiment, the polypeptide utilized as a
hedgehog receptor can be generated from the drosophila patched
protein or a vertebrate homolog thereof. In light of the ability
of, for example, Shh to activate HH pathways in transgenic
drosophila (see Example 4), it may be concluded that vertebrate
hedgehog proteins are capable of binding to drosophila HH
receptors. Accordingly, an exemplary screening assay includes a
suitable portion of the patched protein (SEQ ID No. 42), such as
one or both of the substantial extracellular domains (e.g. residues
Lys-93 to His-426 and Arg-700 to Arg-966). For instance, the
patched protein can be provided in soluble form, as for example a
preparation of one of the extracellular domains, or a preparation
of both of the extracellular domains which are covalently connected
by an unstructured linker (see, for example, Huston et al. (1988)
PNAS 85:4879; and U.S. Pat. No. 5,091,513), or can be provided as
part of a liposomal preparation or expressed on the surface of a
cell.
[0189] Complex formation between the hedgehog polypeptide and a
hedgehog receptor may be detected by a variety of techniques. For
instance, modulation of the formation of complexes can be
quantitated using, for example, detectably labelled proteins such
as radiolabelled, fluorescently labelled, or enzymatically labelled
hedgehog polypeptides, by immunoassay, or by chromatographic
detection.
[0190] Typically, it will be desirable to immobilize either the
hedgehog receptor or the hedgehog polypeptide to facilitate
separation of receptor/hedgehog complexes from uncomplexed forms of
one of the proteins, as well as to accommodate automation of the
assay. In one embodiment, a fusion protein can be provided which
adds a domain that allows the protein to be bound to a matrix. For
example, glutathione-S-transferase/receptor (GST/receptor) fusion
proteins can be adsorbed onto glutathione sepharose beads (Sigma
Chemical, St. Louis, Mo.) or glutathione derivatized microtitre
plates, which are then combined with the hedgehog polypeptide, e.g.
an .sup.35S-labeled hedgehog polypeptide, and the test compound and
incubated under conditions conducive to complex formation, e.g. at
physiological conditions for salt and pH, though slightly more
stringent conditions may be desired. Following incubation, the
beads are washed to remove any unbound hedgehog polypeptide, and
the matrix bead-bound radiolabel determined directly (e.g. beads
placed in scintillant), or in the supernatant after the
receptor/hedgehog complexes are dissociated. Alternatively, the
complexes can dissociated from the bead, separated by SDS-PAGE gel,
and the level of hedgehog polypeptide found in the bead fraction
quantitated from the gel using standard electrophoretic
techniques.
[0191] Other techniques for immobilizing proteins on matrices are
also available for use in the subject assay. For instance, soluble
portions of the hedgehog receptor protein can be immobilized
utilizing conjugation of biotin and streptavidin. For instance,
biotinylated receptor molecules can be prepared from biotin-NHS
(N-hydroxy-succinimide) using techniques well known in the art
(e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.), and
immobilized in the wells of streptavidin-coated 96 well plates
(Pierce Chemical). Alternatively, antibodies reactive with the
hedgehog receptor but which do not interfere with hedgehog binding
can be derivatized to the wells of the plate, and the receptor
trapped in the wells by antibody conjugation. As above,
preparations of a hedgehog polypeptide and a test compound are
incubated in the receptor-presenting wells of the plate, and the
amount of receptor/hedgehog complex trapped in the well can be
quantitated. Exemplary methods for detecting such complexes, in
addition to those described above for the GST-immobilized
complexes, include immunodetection of complexes using antibodies
reactive with the hedgehog polypeptide, or which are reactive with
the receptor protein and compete for binding with the hedgehog
polypeptide; as well as enzyme-linked assays which rely on
detecting an enzymatic activity associated with the hedgehog
polypeptide. In the instance of the latter, the enzyme can be
chemically conjugated or provided as a fusion protein with the
hedgehog polypeptide. To illustrate, the hedgehog polypeptide can
be chemically cross-linked or genetically fused with alkaline
phosphatase, and the amount of hedgehog polypeptide trapped in the
complex can be assessed with a chromogenic substrate of the enzyme,
e.g. paranitrophenylphosphate. Likewise, a fusion protein
comprising the hedgehog polypeptide and glutathione-S-transferase
can be provided, and complex formation quantitated by detecting the
GST activity using 1-chloro-2,4-dinitrobenzene (Habig et al (1974)
J Biol Chem 249:7130).
[0192] For processes which rely on immunodetection for quantitating
one of the proteins trapped in the complex, antibodies against the
protein, such as the anti-hedgehog antibodies described herein, can
be used. Alternatively, the protein to be detected in the complex
can be "epitope tagged" in the form of a fusion protein which
includes, in addition to the hedgehog polypeptide or hedgehog
receptor sequence, a second polypeptide for which antibodies are
readily available (e.g. from commercial sources). For instance, the
GST fusion proteins described above can also be used for
quantification of binding using antibodies against the GST moiety.
Other useful epitope tags include myc-epitopes (e.g., see Ellison
et al. (1991) J Biol Chem 266:21150-21157) which includes a
10-residue sequence from c-myc, as well as the pFLAG system
(International Biotechnologies, Inc.) or the pEZZ-protein A system
(Pharamacia, N.J.).
[0193] Where the desired portion of the hh receptor (or other
hedgehog binding molecule) cannot be provided in soluble form,
liposomal vesicles can be used to provide manipulatable and
isolatable sources of the receptor. For example, both authentic and
recombinant forms of the patched protein can be reconstituted in
artificial lipid vesicles (e.g. phosphatidylcholine liposomes) or
in cell membrane-derived vesicles (see, for example, Bear et al.
(1992) Cell 68:809-818; Newton et al. (1983) Biochemistry
22:6110-6117; and Reber et al. (1987) J Biol Chem
262:11369-11374).
[0194] In addition to cell-free assays, such as described above,
the readily available source of vertebrate hedgehog proteins
provided by the present invention also facilitates the generation
of cell-based assays for identifying small molecule
agonists/antagonists and the like. Analogous to the cell-based
assays described above for screening combinatorial libraries, cells
which are sensitive to hedgehog induction can be contacted with a
hedgehog protein and a test agent of interest, with the assay
scoring for modulation in hedgehog inductive responses by the
target cell in the presence and absence of the test agent. As with
the cell-free assays, agents which produce a statistically
significant change in hedgehog activities (either inhibition or
potentiation) can be identified. In an illustrative embodiment,
motor neuron progenitor cells, such as from neural plate explants,
can be used as target cells. Treatment of such explanted cells
with, for example, Shh causes the cells to differentiate into motor
neurons. By detecting the co-expression of the LIM homeodomain
protein Islet-1 (Thor et al. (1991) Neuron 7:881-889; Ericson et
al. (1992) Science 256:1555-1560) and the immunoglobulin-like
protein SC1 (Tanaka et al. (1984) Dev Biol 106:26-37), the ability
of a candidate agent to potentiate or inhibit Shh induction of
motor neuron differentiation can be measured. The hedgehog protein
can be provided as a purified source, or in the form of
cells/tissue which express the protein and which are co-cultured
with the target cells.
[0195] In yet another embodiment, the method of the present
invention can be used to isolate and clone hedgehog receptors. For
example, purified hedgehog proteins of the present invention can be
employed to precipitate hedgehog receptor proteins from cell
fractions prepared from cells which are responsive to a hedgehog
protein. For instance, purified hedgehog protein can be derivatized
with biotin (using, for instance, NHS-Biotin, Pierce Chemical
catalog no. 21420G), and the biotinylated protein utilized to
saturate membrane bound hh receptors. The hedgehog bound receptors
can subsequently be adsorbed or immobilized on streptavidin. If
desired, the hedgehog-receptor complex can be cross-linked with a
chemical cross-linking agent. In such as manner, hh receptors can
be purified, preferably to near homogeneity. The isolated hh
receptor can then be partially digested with, for example, trypsin,
and the resulting peptides separated by reverse-phase
chromatography. The chromatography fragments are then analyzed by
Edman degradation to obtain single sequences for two or more of the
proteolytic fragments. From the chemically determined amino acid
sequence for each of these tryptic fragments, a set of
oligonucleotide primers can be designed for PCR. These primers can
be used to screen both genomic and cDNA libraries. Similar
strategies for cloning receptors have been employed, for example,
to obtain the recombinant gene for somatostatin receptors (Eppler
et al. (1992) J Biol Chem 267:15603-15612).
[0196] Other techniques for identifying hedgehog receptors by
expression cloning will be evident in light of the present
disclosure. For instance, purified hh polypeptides can be
immobilized in wells of micro titre plates and contacted with, for
example, COS cells transfected with a cDNA library (e.g., from
tissue expected to be responsive to hedgehog induction). From this
panning assay, cells which express hedgehog receptor molecules can
be isolated on the basis of binding to the immobilized hedgehog
protein. Another cloning system, described in PCT publications WO
92/06220 of Flanagan and Leder, involves the use of an expression
cloning system whereby a hedgehog receptor is stored on the basis
of binding to a hedgehog/alkaline phosphatase fusion protein (see
also Cheng et al. (1994) Cell 79:157-168)
[0197] Another aspect of the present invention relates to a method
of inducing and/or maintaining a differentiated state, enhancing
survival, and/or promoting proliferation of a cell responsive to a
vertebrate hedgehog protein, by contacting the cells with an hh
agonist or an hh antagonist as the circumstances may warrant. For
instance, it is contemplated by the invention that, in light of the
present finding of an apparently broad involvement of hedgehog
proteins in the formation of ordered spatial arrangements of
differentiated tissues in vertebrates, the subject method could be
used to generate and/or maintain an array of different vertebrate
tissue both in vitro and in vivo. The hh agent, whether inductive
or anti-inductive, can be, as appropriate, any of the preparations
described above, including isolated polypeptides, gene therapy
constructs, antisense molecules, peptidomimetics or agents
identified in the drug assays provided herein. Moreover, it is
contemplated that, based on the observation of activity of the
vertebrate hedgehog proteins in drosophila, hh agents, for purposes
of therapeutic and diagnostic uses, can include the Dros-HH protein
and homologs thereof. Moreover, the source of hedgehog protein can
be, in addition to purified protein or recombinant cells, cells or
tissue explants which naturally produce one or more hedgehog
proteins. For instance, as described in Example 2, neural tube
explants from embryos, particularly floorplate tissue, can provide
a source for Shh polypeptide, which source can be implanted in a
patient or otherwise provided, as appropriate, for induction or
maintenance of differentiation.
[0198] For example, the present method is applicable to cell
culture techniques. In vitro neuronal culture systems have proved
to be fundamental and indispensable tools for the study of neural
development, as well as the identification of neurotrophic factors
such as nerve growth factor (NGF), ciliary trophic factors (CNTF),
and brain derived neurotrophic factor (BDNF). Once a neuronal cell
has become terminally-differentiated it typically will not change
to another terminally differentiated cell-type. However, neuronal
cells can nevertheless readily lose their differentiated state.
This is commonly observed when they are grown in culture from adult
tissue, and when they form a blastema during regeneration. The
present method provides a means for ensuring an adequately
restrictive environment in order to maintain neuronal cells at
various stages of differentiation, and can be employed, for
instance, in cell cultures designed to test the specific activities
of other trophic factors. In such embodiments of the subject
method, the cultured cells can be contacted with an hh polypeptide,
or an agent identified in the assays described above, in order to
induce neuronal differentiation (e.g. of a stem cell), or to
maintain the integrity of a culture of terminally-differentiated
neuronal cells by preventing loss of differentiation. The source of
hedgehog protein in the culture can be derived from, for example, a
purified or semi-purified protein composition added directly to the
cell culture media, or alternatively, supported and/or released
from a polymeric device which supports the growth of various
neuronal cells and which has been doped with the protein. The
source of the hedgehog protein can also be a cell that is
co-cultured with the intended neuronal cell and which produces a
recombinant hh. Alternatively, the source can be the neuronal cell
itself which has been engineered to produce a recombinant hedgehog
protein. In an exemplary embodiment, a naive neuronal cell (e.g. a
stem cell) is treated with an hh agonist in order to induce
differentiation of the cells into, for example, sensory neurons or,
alternatively, motorneurons. Such neuronal cultures can be used as
convenient assay systems as well as sources of implantable cells
for therapeutic treatments. For example, hh polypeptides may be
useful in establishing and maintaining the olfactory neuron
cultures described in U.S. Pat. No. 5,318,907 and the like.
[0199] According to the present invention, large numbers of
non-tumorigenic neural progenitor cells can be perpetuated in vitro
and induced to differentiate by contact with hedgehog proteins.
Generally, a method is provided comprising the steps of isolating
neural progenitor cells from an animal, perpetuating these cells in
vitro or in vivo, preferably in the presence of growth factors, and
differentiating these cells into particular neural phenotypes,
e.g., neurons and glia, by contacting the cells with a hedgehog
agonist.
[0200] Progenitor cells are thought to be under a tonic inhibitory
influence which maintains the progenitors in a suppressed state
until their differentiation is required. However, recent techniques
have been provided which permit these cells to be proliferated, and
unlike neurons which are terminally differentiated and therefore
non-dividing, they can be produced in unlimited number and are
highly suitable for transplantation into heterologous and
autologous hosts with neurodegenerative diseases.
[0201] By "progenitor" it is meant an oligopotent or multipotent
stem cell which is able to divide without limit and, under specific
conditions, can produce daughter cells which terminally
differentiate such as into neurons and glia. These cells can be
used for transplantation into a heterologous or autologous host. By
heterologous is meant a host other than the animal from which the
progenitor cells were originally derived. By autologous is meant
the identical host from which the cells were originally
derived.
[0202] Cells can be obtained from embryonic, post-natal, juvenile
or adult neural tissue from any animal. By any animal is meant any
multicellular animal which contains nervous tissue. More
particularly, is meant any fish, reptile, bird, amphibian or mammal
and the like. The most preferable donors are mammals, especially
mice and humans.
[0203] In the case of a heterologous donor animal, the animal may
be euthanized, and the brain and specific area of interest removed
using a sterile procedure. Brain areas of particular interest
include any area from which progenitor cells can be obtained which
will serve to restore function to a degenerated area of the host's
brain. These regions include areas of the central nervous system
(CNS) including the cerebral cortex, cerebellum, midbrain,
brainstem, spinal cord and ventricular tissue, and areas of the
peripheral nervous system (PNS) including the carotid body and the
adrenal medulla. More particularly, these areas include regions in
the basal ganglia, preferably the striatum which consists of the
caudate and putamen, or various cell groups such as the globus
pallidus, the subthalamic nucleus, the nucleus basalis which is
found to be degenerated in Alzheimer's Disease patients, or the
substantia nigra pars compacta which is found to be degenerated in
Parkinson's Disease patients.
[0204] Human heterologous neural progenitor cells may be derived
from fetal tissue obtained from elective abortion, or from a
post-natal, juvenile or adult organ donor. Autologous neural tissue
can be obtained by biopsy, or from patients undergoing neurosurgery
in which neural tissue is removed, in particular during epilepsy
surgery, and more particularly during temporal lobectomies and
hippocampalectomies.
[0205] Cells can be obtained from donor tissue by dissociation of
individual cells from the connecting extracellular matrix of the
tissue. Dissociation can be obtained using any known procedure,
including treatment with enzymes such as trypsin, collagenase and
the like, or by using physical methods of dissociation such as with
a blunt instrument. Dissociation of fetal cells can be carried out
in tissue culture medium, while a preferable medium for
dissociation of juvenile and adult cells is artificial cerebral
spinal fluid (aCSF). Regular aCSF contains 124 mM NaCl, 5 mM KCl,
1.3 mM MgCl.sub.2, 2 mM CaCl.sub.2, 26 mM NaHCO.sub.3, and 10 mM
D-glucose. Low Ca.sup.2+ aCSF contains the same ingredients except
for MgCl.sub.2 at a concentration of 3.2 mM and CaCl.sub.2 at a
concentration of 0.1 mM.
[0206] Dissociated cells can be placed into any known culture
medium capable of supporting cell growth, including MEM, DMEM,
RPMI, F-12, and the like, containing supplements which are required
for cellular metabolism such as glutamine and other amino acids,
vitamins, minerals and useful proteins such as transferrin and the
like. Medium may also contain antibiotics to prevent contamination
with yeast, bacteria and fungi such as penicillin, streptomycin,
gentamicin and the like. In some cases, the medium may contain
serum derived from bovine, equine, chicken and the like. A
particularly preferable medium for cells is a mixture of DMEM and
F-12.
[0207] Conditions for culturing should be close to physiological
conditions. The pH of the culture media should be close to
physiological pH, preferably between pH 6-8, more preferably close
to pH 7, even more particularly about pH 7.4. Cells should be
cultured at a temperature close to physiological temperature,
preferably between 30.degree. C.-40.degree. C., more preferably
between 32.degree. C.-38.degree. C., and most preferably between
35.degree. C.-37.degree. C.
[0208] Cells can be grown in suspension or on a fixed substrate,
but proliferation of the progenitors is preferably done in
suspension to generate large numbers of cells by formation of
"neurospheres" (see, for example, Reynolds et al. (1992) Science
255:1070-1709; and PCT Publications WO93/01275, WO94/09119,
WO94/10292, and WO94/16718). In the case of propagating (or
splitting) suspension cells, flasks are shaken well and the
neurospheres allowed to settle on the bottom corner of the flask.
The spheres are then transferred to a 50 ml centrifuge tube and
centrifuged at low speed. The medium is aspirated, the cells
resuspended in a small amount of medium with growth factor, and the
cells mechanically dissociated and resuspended in separate aliquots
of media.
[0209] Cell suspensions in culture medium are supplemented with any
growth factor which allows for the proliferation of progenitor
cells and seeded in any receptacle capable of sustaining cells,
though as set out above, preferably in culture flasks or roller
bottles. Cells typically proliferate within 3-4 days in a
37.degree. C. incubator, and proliferation can be reinitiated at
any time after that by dissociation of the cells and resuspension
in fresh medium containing growth factors.
[0210] In the absence of substrate, cells lift off the floor of the
flask and continue to proliferate in suspension forming a hollow
sphere of undifferentiated cells. After approximately 3-10 days in
vitro, the proliferating clusters (neurospheres) are fed every 2-7
days, and more particularly every 2-4 days by gentle centrifugation
and resuspension in medium containing growth factor.
[0211] After 6-7 days in vitro, individual cells in the
neurospheres can be separated by physical dissociation of the
neurospheres with a blunt instrument, more particularly by
triturating the neurospheres with a pipette. Single cells from the
dissociated neurospheres are suspended in culture medium containing
growth factors, and differentiation of the cells can be induced by
plating (or resuspending) the cells in the presence of a hedgehog
agonist, and (optionally) any other factor capable of sustaining
differentiation, such as bFGF and the like.
[0212] To further illustrate other uses of hedgehog agonists and
antagonists, it is noted that intracerebral grafting has emerged as
an additional approach to central nervous system therapies. For
example, one approach to repairing damaged brain tissues involves
the transplantation of cells from fetal or neonatal animals into
the adult brain (Dunnett et al. (1987) J Exp Biol 123:265-289; and
Freund et al. (1985) J Neurosci 5:603-616). Fetal neurons from a
variety of brain regions can be successfully incorporated into the
adult brain, and such grafts can alleviate behavioral defects. For
example, movement disorder induced by lesions of dopaminergic
projections to the basal ganglia can be prevented by grafts of
embryonic dopaminergic neurons. Complex cognitive functions that
are impaired after lesions of the neocortex can also be partially
restored by grafts of embryonic cortical cells. The use of hedgehog
proteins or mimetics, such as Shh or Dhh, in the culture can
prevent loss of differentiation, or where fetal tissue is used,
especially neuronal stem cells, can be used to induce
differentiation.
[0213] Stem cells useful in the present invention are generally
known. For example, several neural crest cells have been
identified, some of which are multipotent and likely represent
uncommitted neural crest cells, and others of which can generate
only one type of cell, such as sensory neurons, and likely
represent committed progenitor cells. The role of hedgehog proteins
employed in the present method to culture such stem cells can be to
induce differentiation of the uncommitted progenitor and thereby
give rise to a committed progenitor cell, or to cause further
restriction of the developmental fate of a committed progenitor
cell towards becoming a terminally-differentiated neuronal cell.
For example, the present method can be used in vitro to induce
and/or maintain the differentiation of neural crest cells into
glial cells, schwann cells, chromaffin cells, cholinergic
sympathetic or parasympathetic neurons, as well as peptidergic and
serotonergic neurons. The hedgehog protein can be used alone, or
can be used in combination with other neurotrophic factors which
act to more particularly enhance a particular differentiation fate
of the neuronal progenitor cell. In the later instance, an hh
polypeptide might be viewed as ensuring that the treated cell has
achieved a particular phenotypic state such that the cell is poised
along a certain developmental pathway so as to be properly induced
upon contact with a secondary neurotrophic factor. In similar
fashion, even relatively undifferentiated stem cells or primitive
neuroblasts can be maintained in culture and caused to
differentiate by treatment with hedgehog agonists. Exemplary
primitive cell cultures comprise cells harvested from the neural
plate or neural tube of an embryo even before much overt
differentiation has occurred.
[0214] In addition to the implantation of cells cultured in the
presence of a functional hedgehog activity and other in vitro uses
described above, yet another aspect of the present invention
concerns the therapeutic application of a hedgehog protein or
mimetic to enhance survival of neurons and other neuronal cells in
both the central nervous system and the peripheral nervous system.
The ability of hedgehog protein to regulate neuronal
differentiation during development of the nervous system and also
presumably in the adult state indicates that certain of the
hedgehog proteins can be reasonably expected to facilitate control
of adult neurons with regard to maintenance, functional
performance, and aging of normal cells; repair and regeneration
processes in chemically or mechanically lesioned cells; and
prevention of degeneration and premature death which result from
loss of differentiation in certain pathological conditions. In
light of this understanding, the present invention specifically
contemplates applications of the subject method to the treatment of
(prevention and/or reduction of the severity of) neurological
conditions deriving from: (i) acute, subacute, or chronic injury to
the nervous system, including traumatic injury, chemical injury,
vasal injury and deficits (such as the ischemia resulting from
stroke), together with infectious/inflammatory and tumor-induced
injury; (ii) aging of the nervous system including Alzheimer's
disease; (iii) chronic neurodegenerative diseases of the nervous
system, including Parkinson's disease, Huntington's chorea,
amylotrophic lateral sclerosis and the like, as well as
spinocerebellar degenerations; and (iv) chronic immunological
diseases of the nervous system or affecting the nervous system,
including multiple sclerosis.
[0215] Many neurological disorders are associated with degeneration
of discrete populations of neuronal elements and may be treatable
with a therapeutic regimen which includes a hedgehog agonist. For
example, Alzheimer's disease is associated with deficits in several
neurotransmitter systems, both those that project to the neocortex
and those that reside with the cortex. For instance, the nucleus
basalis in patients with Alzheimer's disease have been observed to
have a profound (75%) loss of neurons compared to age-matched
controls. Although Alzheimer's disease is by far the most common
form of dementia, several other disorders can produce dementia.
Several of these are degenerative diseases characterized by the
death of neurons in various parts of the central nervous system,
especially the cerebral cortex. However, some forms of dementia are
associated with degeneration of the thalmus or the white matter
underlying the cerebral cortex. Here, the cognitive dysfunction
results from the isolation of cortical areas by the degeneration of
efferents and afferents. Huntington's disease involves the
degeneration of intrastraital and cortical cholinergic neurons and
GABAergic neurons. Pick's disease is a severe neuronal degeneration
in the neocortex of the frontal and anterior temporal lobes,
sometimes accompanied by death of neurons in the striatum.
Treatment of patients suffering from such degenerative conditions
can include the application of hedgehog polypeptides, or agents
which mimic their effects, in order to control, for example,
differentiation and apoptotic events which give rise to loss of
neurons (e.g. to enhance survival of existing neurons) as well as
promote differentiation and repopulation by progenitor cells in the
area affected. In preferred embodiments, a source of a hedgehog
agent is stereotactically provided within or proximate the area of
degeneration. In addition to degenerative-induced dementias, a
pharmaceutical preparation of one or more of the subject hedgehog
proteins can be applied opportunely in the treatment of
neurodegenerative disorders which have manifestations of tremors
and involuntary movements. Parkinson's disease, for example,
primarily affects subcortical structures and is characterized by
degeneration of the nigrostriatal pathway, raphe nuclei, locus
cereleus, and the motor nucleus of vagus. Ballism is typically
associated with damage to the subthalmic nucleus, often due to
acute vascular accident. Also included are neurogenic and myopathic
diseases which ultimately affect the somatic division of the
peripheral nervous system and are manifest as neuromuscular
disorders. Examples include chronic atrophies such as amyotrophic
lateral sclerosis, Guillain-Barre syndrome and chronic peripheral
neuropathy, as well as other diseases which can be manifest as
progressive bulbar palsies or spinal muscular atrophies. The
present method is amenable to the treatment of disorders of the
cerebellum which result in hypotonia or ataxia, such as those
lesions in the cerebellum which produce disorders in the limbs
ipsilateral to the lesion. For instance, a preparation of a
hedgehog homolog can used to treat a restricted form of cerebellar
cortical degeneration involving the anterior lobes (vermis and leg
areas) such as is common in alcoholic patients.
[0216] In an illustrative embodiment, the subject method is used to
treat amyotrophic lateral sclerosis. ALS is a name given to a
complex of disorders that comprise upper and lower motor neurons.
Patients may present with progressive spinal muscular atrophy,
progressive bulbar palsy, primary lateral sclerosis, or a
combination of these conditions. The major pathological abnormality
is characterized by a selective and progressive degeneration of the
lower motor neurons in the spinal cord and the upper motor neurons
in the cerebral cortex. The therapeutic application of a hedgehog
agonist, particularly Dhh, can be used alone, or in conjunction
with other neurotrophic factors such as CNTF, BDNF or NGF to
prevent and/or reverse motor neuron degeneration in ALS
patients.
[0217] Hedgehog proteins of the present invention can also be used
in the treatment of autonomic disorders of the peripheral nervous
system, which include disorders affecting the innervation of smooth
muscle and endocrine tissue (such as glandular tissue). For
instance, the subject method can be used to treat tachycardia or
atrial cardiac arrythmias which may arise from a degenerative
condition of the nerves innervating the striated muscle of the
heart.
[0218] Furthermore, a potential role for certain of the hedgehog
proteins, which is apparent from the appended examples, mainly the
data of respecting hedgehog expression in sensory and motor neurons
of the head and trunk (including limb buds), concerns the role of
hedgehog proteins in development and maintenance of dendritic
processes of axonal neurons. Potential roles for hedgehog proteins
consequently include guidance for axonal projections and the
ability to promote differentiation and/or maintenance of the
innervating cells to their axonal processes. Accordingly,
compositions comprising hedgehog agonists or other hedgehog agents
described herein, may be employed to support, or alternatively
antagonize the survival and reprojection of several types of
ganglionic neurons sympathetic and sensory neurons as well as motor
neurons. In particular, such therapeutic compositions may be useful
in treatments designed to rescue, for example, various neurons from
lesion-induced death as well as guiding reprojection of these
neurons after such damage. Such diseases include, but are not
limited to, CNS trauma infarction, infection (such as viral
infection with varicella-zoster), metabolic disease, nutritional
deficiency, toxic agents (such as cisplatin treatment). Moreover,
certain of the hedgehog agents (such as antagonistic form) may be
useful in the selective ablation of sensory neurons, for example,
in the treatment of chronic pain syndromes.
[0219] As appropriate, hedgehog agents can be used in nerve
prostheses for the repair of central and peripheral nerve damage.
In particular, where a crushed or severed axon is intubulated by
use of a prosthetic device, hedgehog polypeptides can be added to
the prosthetic device to increase the rate of growth and
regeneration of the dendridic processes. Exemplary nerve guidance
channels are described in U.S. Pat. Nos. 5,092,871 and 4,955,892.
Accordingly, a severed axonal process can be directed toward the
nerve ending from which it was severed by a prosthesis nerve guide
which contains, e.g. a semi-solid formulation containing hedgehog
polypeptide or mimetic, or which is derivatized along the inner
walls with a hedgehog protein.
[0220] In another embodiment, the subject method can be used in the
treatment of neoplastic or hyperplastic transformations such as may
occur in the central nervous system. For instance, certain of the
hedgehog proteins (or hh agonists) which induce differentiation of
neuronal cells can be utilized to cause such transformed cells to
become either post-mitotic or apoptotic. Treatment with a hedgehog
agent may facilitate disruption of autocrine loops, such as
TGF-.beta. or PDGF autostimulatory loops, which are believed to be
involved in the neoplastic transformation of several neuronal
tumors. Hedgehog agonists may, therefore, thus be of use in the
treatment of, for example, malignant gliomas, medulloblastomas,
neuroectodermal tumors, and ependymonas.
[0221] Yet another aspect of the present invention concerns the
application of the discovery that hedgehog proteins are morphogenic
signals involved in other vertebrate organogenic pathways in
addition to neuronal differentiation as described above, having
apparent roles in other endodermal patterning, as well as both
mesodermal and endodermal differentiation processes. As described
in the Examples below, Shh clearly plays a role in proper limb
growth and patterning by initiating expression of signaling
molecules, including Bmp-2 in the mesoderm and Fgf-4 in the
ectoderm. Thus, it is contemplated by the invention that
compositions comprising hedgehog proteins can also be utilized for
both cell culture and therapeutic methods involving generation and
maintenance of non-neuronal tissue.
[0222] In one embodiment, the present invention makes use of the
discovery that hedgehog proteins, such as Shh, are apparently
involved in controlling the development of stem cells responsible
for formation of the digestive tract, liver, lungs, and other
organs which derive from the primitive gut. As described in the
Examples below, Shh serves as an inductive signal from the endoderm
to the mesoderm, which is critical to gut morphogenesis. Therefore,
for example, hedgehog agonists can be employed in the development
and maintenance of an artificial liver which can have multiple
metabolic functions of a normal liver. In an exemplary embodiment,
hedgehog agonists can be used to induce differentiation of
digestive tube stem cells to form hepatocyte cultures which can be
used to populate extracellular matrices, or which can be
encapsulated in biocompatible polymers, to form both implantable
and extracorporeal artificial livers.
[0223] In another embodiment, therapeutic compositions of hedgehog
agonists can be utilized in conjunction with transplantation of
such artificial livers, as well as embryonic liver structures, to
promote intraperitoneal implantation, vascularization, and in vivo
differentiation and maintenance of the engrafted liver tissue.
[0224] In yet another embodiment, hedgehog agonists can be employed
therapeutically to regulate such organs after physical, chemical or
pathological insult. For instance, therapeutic compositions
comprising hedgehog agonists can be utilized in liver repair
subsequent to a partial hepatectomy. Similarly, therapeutic
compositions containing hedgehog agonists can be used to promote
regeneration of lung tissue in the treatment of emphysema.
[0225] In still another embodiment of the present invention,
compositions comprising hedgehog agonists can be used in the in
vitro generation of skeletal tissue, such as from skeletogenic stem
cells, as well as the in vivo treatment of skeletal tissue
deficiencies. The present invention particularly contemplates the
use of hedgehog agonists which maintain a skeletogenic activity,
such as an ability to induce chondrogenesis and/or osteogenesis. By
"skeletal tissue deficiency", it is meant a deficiency in bone or
other skeletal connective tissue at any site where it is desired to
restore the bone or connective tissue, no matter how the deficiency
originated, e.g. whether as a result of surgical intervention,
removal of tumor, ulceration, implant, fracture, or other traumatic
or degenerative conditions.
[0226] For instance, the present invention makes available
effective therapeutic methods and compositions for restoring
cartilage function to a connective tissue. Such methods are useful
in, for example, the repair of defects or lesions in cartilage
tissue which is the result of degenerative wear such as that which
results in arthritis, as well as other mechanical derangements
which may be caused by trauma to the tissue, such as a displacement
of torn meniscus tissue, meniscectomy, a laxation of a joint by a
torn ligament, malignment of joints, bone fracture, or by
hereditary disease. The present reparative method is also useful
for remodeling cartilage matrix, such as in plastic or
reconstructive surgery, as well as periodontal surgery. The present
method may also be applied to improving a previous reparative
procedure, for example, following surgical repair of a meniscus,
ligament, or cartilage. Furthermore, it may prevent the onset or
exacerbation of degenerative disease if applied early enough after
trauma.
[0227] In one embodiment of the present invention, the subject
method comprises treating the afflicted connective tissue with a
therapeutically sufficient amount of a hedgehog agonist,
particularly an Ihh agonist, to generate a cartilage repair
response in the connective tissue by stimulating the
differentiation and/or proliferation of chondrocytes embedded in
the tissue. Induction of chondrocytes by treatment with a hedgehog
agonist can subsequently result in the synthesis of new cartilage
matrix by the treated cells. Such connective tissues as articular
cartilage, interarticular cartilage (menisci), costal cartilage
(connecting the true ribs and the sternum), ligaments, and tendons
are particularly amenable to treatment in reconstructive and/or
regenerative therapies using the subject method. As used herein,
regenerative therapies include treatment of degenerative states
which have progressed to the point of which impairment of the
tissue is obviously manifest, as well as preventive treatments of
tissue where degeneration is in its earliest stages or imminent.
The subject method can further be used to prevent the spread of
mineralisation into fibrotic tissue by maintaining a constant
production of new cartilage.
[0228] In an illustrative embodiment, the subject method can be
used to treat cartilage of a diarthroidal joint, such as a knee, an
ankle, an elbow, a hip, a wrist, a knuckle of either a finger or
toe, or a temperomandibular joint. The treatment can be directed to
the meniscus of the joint, to the articular cartilage of the joint,
or both. To further illustrate, the subject method can be used to
treat a degenerative disorder of a knee, such as which might be the
result of traumatic injury (e.g., a sports injury or excessive
wear) or osteoarthritis. An injection of a hedgehog agonist into
the joint with, for instance, an arthroscopic needle, can be used
to treat the afflicted cartilage. In some instances, the injected
agent can be in the form of a hydrogel or other slow release
vehicle described above in order to permit a more extended and
regular contact of the agent with the treated tissue.
[0229] The present invention further contemplates the use of the
subject method in the field of cartilage transplantation and
prosthetic device therapies. To date, the growth of new cartilage
from either transplantation of autologous or allogenic cartilage
has been largely unsuccessful. Problems arise, for instance,
because the characteristics of cartilage and fibrocartilage varies
between different tissue: such as between articular, meniscal
cartilage, ligaments, and tendons, between the two ends of the same
ligament or tendon, and between the superficial and deep parts of
the tissue. The zonal arrangement of these tissues may reflect a
gradual change in mechanical properties, and failure occurs when
implanted tissue, which has not differentiated under those
conditions, lacks the ability to appropriately respond. For
instance, when meniscal cartilage is used to repair anterior
cruciate ligaments, the tissue undergoes a metaplasia to pure
fibrous tissue. By promoting chondrogenesis, the subject method can
be used to particularly addresses this problem, by causing the
implanted cells to become more adaptive to the new environment and
effectively resemble hypertrophic chondrocytes of an earlier
developmental stage of the tissue. Thus, the action of
chondrogensis in the implanted tissue, as provided by the subject
method, and the mechanical forces on the actively remodeling tissue
can synergize to produce an improved implant more suitable for the
new function to which it is to be put.
[0230] In similar fashion, the subject method can be applied to
enhancing both the generation of prosthetic cartilage devices and
to their implantation. The need for improved treatment has
motivated research aimed at creating new cartilage that is based on
collagen-glycosaminoglyc- an templates (Stone et al. (1990) Clin
Orthop Relat Red 252:129), isolated chondrocytes (Grande et al.
(1989) J Orthop Res 7:208; and Takigawa et al. (1987) Bone Miner
2:449), and chondrocytes attached to natural or synthetic polymers
(Walitani et al. (1989) J Bone Jt Surg 71B:74; Vacanti et al.
(1991) Plast Reconstr Surg 88:753; von Schroeder et al. (1991) J
Biomed Mater Res 25:329; Freed et al. (1993) J Biomed Mater Res
27:11; and the Vacanti et al. U.S. Pat. No. 5,041,138). For
example, chondrocytes can be grown in culture on biodegradable,
biocompatible highly porous scaffolds formed from polymers such as
polyglycolic acid, polylactic acid, agarose gel, or other polymers
which degrade over time as function of hydrolysis of the polymer
backbone into innocuous monomers. The matrices are designed to
allow adequate nutrient and gas exchange to the cells until
engraftment occurs. The cells can be cultured in vitro until
adequate cell volume and density has developed for the cells to be
implanted. One advantage of the matrices is that they can be cast
or molded into a desired shape on an individual basis, so that the
final product closely resembles the patient's own ear or nose (by
way of example), or flexible matrices can be used which allow for
manipulation at the time of implantation, as in a joint.
[0231] In one embodiment of the subject method, the implants are
contacted with a hedgehog agonist during the culturing process,
such as an Ihh agonist, in order to induce and/or maintain
differentiated chondrocytes in the culture in order as to further
stimulate cartilage matrix production within the implant. In such a
manner, the cultured cells can be caused to maintain a phenotype
typical of a chondrogenic cell (i.e. hypertrophic), and hence
continue the population of the matrix and production of cartilage
tissue.
[0232] In another embodiment, the implanted device is treated with
a hedgehog agonist in order to actively remodel the implanted
matrix and to make it more suitable for its intended function. As
set out above with respect to tissue transplants, the artificial
transplants suffer from the same deficiency of not being derived in
a setting which is comparable to the actual mechanical environment
in which the matrix is implanted. The activation of the
chondrocytes in the matrix by the subject method can allow the
implant to acquire characteristics similar to the tissue for which
it is intended to replace.
[0233] In yet another embodiment, the subject method is used to
enhance attachment of prosthetic devices. To illustrate, the
subject method can be used in the implantation of a periodontal
prosthesis, wherein the treatment of the surrounding connective
tissue stimulates formation of periodontal ligament about the
prosthesis, as well as inhibits formation of fibrotic tissue
proximate the prosthetic device.
[0234] In still further embodiments, the subject method can be
employed for the generation of bone (osteogenesis) at a site in the
animal where such skeletal tissue is deficient. Indian hedgehog is
particularly associated with the hypertrophic chondrocytes that are
ultimately replaced by osteoblasts. For instance, administration of
a hedgehog agent of the present invention can be employed as part
of a method for treating bone loss in a subject, e.g. to prevent
and/or reverse osteoporosis and other osteopenic disorders, as well
as to regulate bone growth and maturation. For example,
preparations comprising hedgehog agonists can be employed, for
example, to induce endochondral ossification, at least so far as to
facilitate the formation of cartilaginous tissue precursors to form
the "model" for ossification. Therapeutic compositions of hedgehog
agonists can be supplemented, if required, with other
osteoinductive factors, such as bone growth factors (e.g.
TGF-.beta. factors, such as the bone morphogenetic factors BMP-2
and BMP-4, as well as activin), and may also include, or be
administered in combination with, an inhibitor of bone resorption
such as estrogen, bisphosphonate, sodium fluoride, calcitonin, or
tamoxifen, or related compounds. However, it will be appreciated
that hedgehog proteins, such as Ihh and Shh are likely to be
upstream of BMPs, e.g. hh treatment will have the advantage of
initiating endogenous expression of BMPs along with other
factors.
[0235] In yet another embodiment of the present invention, a
hedgehog antagonist can be used to inhibit spermatogenesis. Thus,
in light of the present finding that hedgehog proteins are involved
in the differentiation and/or proliferation and maintenance of
testicular germ cells, hedgehog antagonist can be utilized to block
the action of a naturally-occurring hedgehog protein. In a
preferred embodiment, the hedgehog antagonist inhibits the
biological activity of Dhh with respect to spermatogenesis, by
competitively binding hedgehog receptors in the testis. In similar
fashion, hedgehog agonists and antagonists are potentially useful
for modulating normal ovarian function.
[0236] The hedgehog protein, or a pharmaceutically acceptable salt
thereof, may be conveniently formulated for administration with a
biologically acceptable medium, such as water, buffered saline,
polyol (for example, glycerol, propylene glycol, liquid
polyethylene glycol and the like) or suitable mixtures thereof. The
optimum concentration of the active ingredient(s) in the chosen
medium can be determined empirically, according to procedures well
known to medicinal chemists. As used herein, "biologically
acceptable medium" includes any and all solvents, dispersion media,
and the like which may be appropriate for the desired route of
administration of the pharmaceutical preparation. The use of such
media for pharmaceutically active substances is known in the art.
Except insofar as any conventional media or agent is incompatible
with the activity of the hedgehog protein, its use in the
pharmaceutical preparation of the invention is contemplated.
Suitable vehicles and their formulation inclusive of other proteins
are described, for example, in the book Remington's Pharmaceutical
Sciences (Remington's Pharmaceutical Sciences. Mack Publishing
Company, Easton, Pa., USA 1985). These vehicles include injectable
"deposit formulations". Based on the above, such pharmaceutical
formulations include, although not exclusively, solutions or
freeze-dried powders of a hedgehog homolog (such as a Shh, Dhh or
Mhh) in association with one or more pharmaceutically acceptable
vehicles or diluents, and contained in buffered media at a suitable
pH and isosmotic with physiological fluids. For illustrative
purposes only and without being limited by the same, possible
compositions or formulations which may be prepared in the form of
solutions for the treatment of nervous system disorders with a
hedgehog protein are given in U.S. Pat. No. 5,218,094. In the case
of freeze-dried preparations, supporting excipients such as, but
not exclusively, mannitol or glycine may be used and appropriate
buffered solutions of the desired volume will be provided so as to
obtain adequate isotonic buffered solutions of the desired pH.
Similar solutions may also be used for the pharmaceutical
compositions of hh in isotonic solutions of the desired volume and
include, but not exclusively, the use of buffered saline solutions
with phosphate or citrate at suitable concentrations so as to
obtain at all times isotonic pharmaceutical preparations of the
desired pH, (for example, neutral pH).
[0237] Pharmaceutical formulations of the present invention can
also include veterinary compositions, e.g., pharmaceutical
preparations of the hedgehog proteins, or bioactive fragments
thereof, suitable for veterinary uses, e.g., for the treatment of
live stock or domestic animals, e.g., dogs.
[0238] Methods of introduction of exogenous hh at the site of
treatment include, but are not limited to, intradermal,
intramuscular, intraperitoneal, intravenous, subcutaneous, oral,
intranasal and topical. In addition, it may be desirable to
introduce the pharmaceutical compositions of the invention into the
central nervous system by any suitable route, including
intraventricular and intrathecal injection. Intraventricular
injection may be facilitated by an intraventricular catheter, for
example, attached to a reservoir, such as an Ommaya reservoir.
[0239] Methods of introduction may also be provided by rechargeable
or biodegradable devices. Various slow release polymeric devices
have been developed and tested in vivo in recent years for the
controlled delivery of drugs, including proteinacious
biopharmaceuticals. A variety of biocompatible polymers (including
hydrogels), including both biodegradable and non-degradable
polymers, can be used to form an implant for the sustained release
of an hh at a particular target site. Such embodiments of the
present invention can be used for the delivery of an exogenously
purified hedgehog protein, which has been incorporated in the
polymeric device, or for the delivery of hedgehog produced by a
cell encapsulated in the polymeric device.
[0240] An essential feature of certain embodiments of the implant
can be the linear release of the hh, which can be achieved through
the manipulation of the polymer composition and form. By choice of
monomer composition or polymerization technique, the amount of
water, porosity and consequent permeability characteristics can be
controlled. The selection of the shape, size, polymer, and method
for implantation can be determined on an individual basis according
to the disorder to be treated and the individual patient response.
The generation of such implants is generally known in the art. See,
for example, Concise Encylopedia of Medical & Dental Materials,
ed. by David Williams (MIT Press: Cambridge, Mass., 1990); and the
Sabel et al. U.S. Pat. No. 4,883,666. In another embodiment of an
implant, a source of cells producing a hedgehog protein, or a
solution of hydogel matrix containing purified hh, is encapsulated
in implantable hollow fibers. Such fibers can be pre-spun and
subsequently loaded with the hedgehog source (Aebischer et al. U.S.
Pat. No. 4,892,538; Aebischer et al. U.S. Pat. No. 5,106,627;
Hoffman et al. (1990) Expt. Neurobiol. 110:39-44; Jaeger et al.
(1990) Prog. Brain Res. 82:4146; and Aebischer et al. (1991) J.
Biomech. Eng. 113:178-183), or can be co-extruded with a polymer
which acts to form a polymeric coat about the hh source (Lim U.S.
Pat. No. 4,391,909; Sefton U.S. Pat. No. 4,353,888; Sugamori et al.
(1989) Trans. Am. Artif. Intern. Organs 35:791-799; Sefton et al.
(1987) Biotehnol. Bioeng. 29:1135-1143; and Aebischer et al. (1991)
Biomaterials 12:50-55).
[0241] In yet another embodiment of the present invention, the
pharmaceutical hedgehog protein can be administered as part of a
combinatorial therapy with other agents. For example, the
combinatorial therapy can include a hedgehog protein with at least
one trophic factor. Exemplary trophic factors include nerve growth
factor, cilliary neurotrophic growth factor, schwanoma-derived
growth factor, glial growth factor, stiatal-derived neuronotrophic
factor, platelet-derived growth factor, and scatter factor
(HGF-SF). Antimitogenic agents can also be used, for example, when
proliferation of surrounding glial cells or astrocytes is
undesirable in the regeneration of nerve cells. Examples of such
antimitotic agents include cytosine, arabinoside, 5-fluorouracil,
hydroxyurea, and methotrexate.
[0242] Another aspect of the invention features transgenic
non-human animals which express a heterologous hedgehog gene of the
present invention, or which have had one or more genomic hedgehog
genes disrupted in at least one of the tissue or cell-types of the
animal. Accordingly, the invention features an animal model for
developmental diseases, which animal has hedgehog allele which is
mis-expressed. For example, a mouse can be bred which has one or
more hh alleles deleted or otherwise rendered inactive. Such a
mouse model can then be used to study disorders arising from
mis-expressed hedgehog genes, as well as for evaluating potential
therapies for similar disorders.
[0243] Another aspect of the present invention concerns transgenic
animals which are comprised of cells (of that animal) which contain
a transgene of the present invention and which preferably (though
optionally) express an exogenous hedgehog protein in one or more
cells in the animal. A hedgehog transgene can encode the wild-type
form of the protein, or can encode homologs thereof, including both
agonists and antagonists, as well as antisense constructs. In
preferred embodiments, the expression of the transgene is
restricted to specific subsets of cells, tissues or developmental
stages utilizing, for example, cis-acting sequences that control
expression in the desired pattern. In the present invention, such
mosaic expression of a hedgehog protein can be essential for many
forms of lineage analysis and can additionally provide a means to
assess the effects of, for example, lack of hedgehog expression
which might grossly alter development in small patches of tissue
within an otherwise normal embryo. Toward this and, tissue-specific
regulatory sequences and conditional regulatory sequences can be
used to control expression of the transgene in certain spatial
patterns. Moreover, temporal patterns of expression can be provided
by, for example, conditional recombination systems or prokaryotic
transcriptional regulatory sequences.
[0244] Genetic techniques which allow for the expression of
transgenes can be regulated via site-specific genetic manipulation
in vivo are known to those skilled in the art. For instance,
genetic systems are available which allow for the regulated
expression of a recombinase that catalyzes the genetic
recombination a target sequence. As used herein, the phrase "target
sequence" refers to a nucleotide sequence that is genetically
recombined by a recombinase. The target sequence is flanked by
recombinase recognition sequences and is generally either excised
or inverted in cells expressing recombinase activity. Recombinase
catalyzed recombination events can be designed such that
recombination of the target sequence results in either the
activation or repression of expression of one of the subject
hedgehog proteins. For example, excision of a target sequence which
interferes with the expression of a recombinant hh gene, such as
one which encodes an antagonistic homolog or an antisense
transcript, can be designed to activate expression of that gene.
This interference with expression of the protein can result from a
variety of mechanisms, such as spatial separation of the hh gene
from the promoter element or an internal stop codon. Moreover, the
transgene can be made wherein the coding sequence of the gene is
flanked by recombinase recognition sequences and is initially
transfected into cells in a 3' to 5' orientation with respect to
the promoter element. In such an instance, inversion of the target
sequence will reorient the subject gene by placing the 5' end of
the coding sequence in an orientation with respect to the promoter
element which allow for promoter driven transcriptional
activation.
[0245] In an illustrative embodiment, either the cre/loxP
recombinase system of bacteriophage P1 (Lakso et al. (1992) PNAS
89:6232-6236; Orban et al. (1992) PNAS 89:6861-6865) or the FLP
recombinase system of Saccharomyces cerevisiae (O'Gorman et al.
(1991) Science 251:1351-1355; PCT publication WO 92/15694) can be
used to generate in vivo site-specific genetic recombination
systems. Cre recombinase catalyzes the site-specific recombination
of an intervening target sequence located between loxP sequences.
loxP sequences are 34 base pair nucleotide repeat sequences to
which the Cre recombinase binds and are required for Cre
recombinase mediated genetic recombination. The orientation of loxP
sequences determines whether the intervening target sequence is
excised or inverted when Cre recombinase is present (Abremski et
al. (1984) J. Biol. Chem. 259:1509-1514); catalyzing the excision
of the target sequence when the loxP sequences are oriented as
direct repeats and catalyzes inversion of the target sequence when
loxP sequences are oriented as inverted repeats.
[0246] Accordingly, genetic recombination of the target sequence is
dependent on expression of the Cre recombinase. Expression of the
recombinase can be regulated by promoter elements which are subject
to regulatory control, e.g., tissue-specific, developmental
stage-specific, inducible or repressible by externally added
agents. This regulated control will result in genetic recombination
of the target sequence only in cells where recombinase expression
is mediated by the promoter element. Thus, the activation
expression of a recombinant hedgehog protein can be regulated via
control of recombinase expression.
[0247] Use of the cre/loxP recombinase system to regulate
expression of a recombinant hh protein requires the construction of
a transgenic animal containing transgenes encoding both the Cre
recombinase and the subject protein. Animals containing both the
Cre recombinase and a recombinant hedgehog gene can be provided
through the construction of "double" transgenic animals. A
convenient method for providing such animals is to mate two
transgenic animals each containing a transgene, e.g., an hh gene
and recombinase gene.
[0248] One advantage derived from initially constructing transgenic
animals containing a hedgehog transgene in a recombinase-mediated
expressible format derives from the likelihood that the subject
protein, whether agonistic or antagonistic, can be deleterious upon
expression in the transgenic animal. In such an instance, a founder
population, in which the subject transgene is silent in all
tissues, can be propagated and maintained. Individuals of this
founder population can be crossed with animals expressing the
recombinase in, for example, one or more tissues and/or a desired
temporal pattern. Thus, the creation of a founder population in
which, for example, an antagonistic hh transgene is silent will
allow the study of progeny from that founder in which disruption of
hedgehog mediated induction in a particular tissue or at certain
developmental stages would result in, for example, a lethal
phenotype.
[0249] Similar conditional transgenes can be provided using
prokaryotic promoter sequences which require prokaryotic proteins
to be simultaneous expressed in order to facilitate expression of
the hedgehog transgene. Exemplary promoters and the corresponding
trans-activating prokaryotic proteins are given in U.S. Pat. No.
4,833,080.
[0250] Moreover, expression of the conditional transgenes can be
induced by gene therapy-like methods wherein a gene encoding the
trans-activating protein, e.g. a recombinase or a prokaryotic
protein, is delivered to the tissue and caused to be expressed,
such as in a cell-type specific manner. By this method, a hedgehog
transgene could remain silent into adulthood until "turned on" by
the introduction of the trans-activator.
[0251] In an exemplary embodiment, the "transgenic non-human
animals" of the invention are produced by introducing transgenes
into the germline of the non-human animal. Embryonic target cells
at various developmental stages can be used to introduce
transgenes. Different methods are used depending on the stage of
development of the embryonic target cell. The zygote is the best
target for micro-injection. In the mouse, the male pronucleus
reaches the size of approximately 20 micrometers in diameter which
allows reproducible injection of 1-2pl of DNA solution. The use of
zygotes as a target for gene transfer has a major advantage in that
in most cases the injected DNA will be incorporated into the host
gene before the first cleavage (Brinster et al. (1985) PNAS
82:4438-4442). As a consequence, all cells of the transgenic
non-human animal will carry the incorporated transgene. This will
in general also be reflected in the efficient transmission of the
transgene to offspring of the founder since 50% of the germ cells
will harbor the transgene. Microinjection of zygotes is the
preferred method for incorporating transgenes in practicing the
invention.
[0252] Retroviral infection can also be used to introduce hedgehog
transgenes into a non-human animal. The developing non-human embryo
can be cultured in vitro to the blastocyst stage. During this time,
the blastomeres can be targets for retroviral infection (Jaenich,
R. (1976) PNAS 73:1260-1264). Efficient infection of the
blastomeres is obtained by enzymatic treatment to remove the zona
pellucida (Manipulating the Mouse Embryo, Hogan eds. (Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, 1986). The viral
vector system used to introduce the transgene is typically a
replication-defective retrovirus carrying the transgene (Jahner et
al. (1985) PNAS 82:6927-6931; Van der Putten et al. (1985) PNAS
82:6148-6152). Transfection is easily and efficiently obtained by
culturing the blastomeres on a monolayer of virus-producing cells
(Van der Putten, supra; Stewart et al. (1987) EMBO J. 6:383-388).
Alternatively, infection can be performed at a later stage. Virus
or virus-producing cells can be injected into the blastocoele
(Jahner et al. (1982) Nature 298:623-628). Most of the founders
will be mosaic for the transgene since incorporation occurs only in
a subset of the cells which formed the transgenic non-human animal.
Further, the founder may contain various retroviral insertions of
the transgene at different positions in the genome which generally
will segregate in the offspring. In addition, it is also possible
to introduce transgenes into the germ line by intrauterine
retroviral infection of the midgestation embryo (Jahner et al.
(1982) supra).
[0253] A third type of target cell for transgene introduction is
the embryonic stem cell (ES). ES cells are obtained from
pre-implantation embryos cultured in vitro and fused with embryos
(Evans et al. (1981) Nature 292:154-156; Bradley et al. (1984)
Nature 309:255-258; Gossler et al. (1986) PNAS 83: 9065-9069; and
Robertson et al. (1986) Nature 322:445-448). Transgenes can be
efficiently introduced into the ES cells by DNA transfection or by
retrovirus-mediated transduction. Such transformed ES cells can
thereafter be combined with blastocysts from a non-human animal.
The ES cells thereafter colonize the embryo and contribute to the
germ line of the resulting chimeric animal. For review see
Jaenisch, R. (1988) Science 240:1468-1474.
[0254] Methods of making hedgehog knock-out or disruption
transgenic animals are also generally known. See, for example,
Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y., 1986). Recombinase dependent
knockouts can also be generated, e.g. by homologous recombination
to insert recombinase target sequences flanking portions of an
endogenous hh gene, such that tissue specific and/or temporal
control of inactivation of a hedgehog allele can be controlled as
above.
Exemplification
[0255] The invention, now being generally described, will be more
readily understood by reference to the following examples, which
are included tnerely for purposes of illustration of certain
aspects and embodiments of the present invention and are not
intended to limit the invention.
EXAMPLE 1
Cloning and Expression of Chick Sonic Hedgehog
[0256] (i) Experimental Procedures
[0257] Using degenerate PCR primers, vHH5O (SEQ ID No:18), vHH3O
(SEQ ID No:19) and vHH3I (SEQ ID No:20) corresponding to a sequence
conserved between Drosophila hedgehog (SEQ ID No:34) (Lee, J. J. et
al. (1992) Cell 71: 33-50; Mohler, J. et al., (1992) Development
115: 957-971) and mouse Indian hedgehog (Ihh) (SEQ ID No:10), a 220
base pair (bp) fragment was amplified from chicken genomic DNA.
From 15 isolates, two distinct sequences were cloned, pCHA (SEQ ID
No:35) and pCHB (SEQ ID No:36), each highly homologous to mouse Ihh
(FIG. 1). A probe made from isolate pCHA did not detect expression
in embryonic tissues. Isolate pCHB, however, detected a 4 kb
message in RNA prepared from embryonic head, trunk, or limb bud
RNA. This cloned PCR fragment was therefore used as a probe to
screen an unamplified cDNA library prepared from Hamburger Hamilton
stage 22 (Hamburger, W. et al., (1951) J. Morph. 88: 49-92) limb
bud RNA as described below.
[0258] A single 1.6 kilobase (kb) cDNA clone, pHH-2, was selected
for characterization and was used in all subsequent analyses. The
gene encoding for this cDNA was named Sonic Hedgehog (after the
Sega computer game cartoon character). Sequencing of the entire
cDNA confirmed the presence of a single long open reading frame
potentially encoding for a protein of 425 amino acids (aa). The
clone extends 220 bp upstream of the predicted initiator methionine
and approximately 70 bp beyond the stop codon. No consensus
polyadenylation signal could be identified in the 3' untranslated
region. A second potential initiator methionine occurs at amino
acid residue 4. The putative translation initiation signals
surrounding both methionines are predicted to be equally efficient
(Kozak, M., (1987) Nuc. Acids Res. 15: 8125-8132). When the pHH-2
Sonic cDNA is used to probe a northern blot of stage 24 embryonic
chick RNA, a single mRNA species of approximately 4 kb is detected
in both limb and trunk tissue. The message size was predicted by
comparing it to the position of 18S and 28S ribosomal RNA.
Hybridized mRNA was visualized after a two day exposure to a
phosphoscreen. Because the Sonic cDNA clone pHH-2 is only 1.6 kb,
it is likely to be missing approximately 2.4 kb of untranslated
sequence.
[0259] PCR Cloning
[0260] All standard cloning techniques were performed according to
Ausubel et. al. (1989), and all enzymes were obtained from
Boehringer Mannheim Biochemicals. Degenerate oligonucleotides
corresponding to amino acid residues 161 to 237 of the Drosophila
hedgehog protein (SEQ ID No:34) (Lee, J. J. et. al., (1992) Cell
71: 33-50) were synthesized. These degenerate oligonucleotides,
vHH5O (SEQ ID No:18), vHH3O (SEQ ID No:19), and vHH3I (SEQ ID
No:20) also contained Eco RI, Cla I, and Xba I sites, respectively,
on their 5' ends to facilitate subcloning. The nucleotide sequence
of these oligos is given below:
[0261] vHH5O:
5'-GGAATTCCCAG(CA)GITG(CT)AA(AG)GA(AG)(CA)(AG)I(GCT)IAA-3'
[0262] vHH3O: 5'-TCATCGATGGACCCA(GA)TC(GA)AAICCIGC(TC)TC-3'
[0263] vHH3I: 5'-GCTCTAGAGCTCIACIGCIA(GA)IC(GT)IGC-3'
[0264] where I represents inosine. Nested PCR was performed by
first amplifying chicken genomic DNA using the vHH5O and vHH3O
primer pair and then further amplifying that product using the
vHH5O and vHH3I primer pair. In each case the reaction conditions
were: initial denaturation at 93.degree. C. for 2.5 mm., followed
by 30 cycles of 94.degree. C. for 45 s, 50.degree. C. for 1 min.,
72.degree. C. for 1, and a final incubation of 72.degree. C. for 5
min. The 220 bp PCR product was subcloned into pGEM7zf (Promega).
Two unique clones, pCHA (SEQ ID No:35) and pCHB (SEQ ID No:36) were
identified.
[0265] DNA Sequence Analysis
[0266] Nucleotide sequences were determined by the dideoxy chain
termination method (Sanger, F. et al., (1977) Proc. Natl. Acad.
Sci. USA 74: 5463-5467) using Sequenase v2.0 T7 DNA polymerase (US
Biochemicals). 5' and 3' nested deletions of pHH-2 were generated
by using the nucleases Exo III and S1 (Erase a Base, Promega) and
individual subclones sequenced. DNA and amino acid sequences were
analyzed using both GCG (Devereux, J. et al., (1984) Nuc. Acids
Res. 12: 387-394) and DNAstar software. Searches for related
sequences were done through the BLAST network service (Altschul, S.
F. et al., (1990) J. Mol. Biol. 215: 403-410) provided by the
National Center for Biotechnology Information.
[0267] Southern Blot Analysis
[0268] Five (5) .mu.g of chick genomic DNA was digested with Eco RI
and/or Bam HI, fractionated on a 1% agarose gel, and transferred to
a nylon membrane (Genescreen, New England Nuclear). The filters
were probed with .sup.32P-labeled hha or hhb at 42.degree. C. in
hybridization buffer (0.5% BSA, 500 mM NaHPO.sub.4, 7% SDS, 1 mM
EDTA, pH 7.2; Church, G. M. et al., (1984) Proc. Natl. Acad. Sci.
USA 81: 1991-1995). The blots were washed at 63.degree. C. once in
0.5% bovine serum albumin, 50 mM NaHPO.sub.4 (pH 7.2), 5% SDS, 1 mM
EDTA and twice in 40 mM NaHPO.sub.4 (pH 7.2), 1% SDS, 1 mM EDTA,
and visualized on Kodak XAR-5 film.
[0269] Isolation Of Chicken Sonic cDNA Clones
[0270] A stage 22 limb bud cDNA library was constructed in
.lambda.gt10 using Eco RI/NotI linkers. Unamplified phage plaques
(10.sup.6) were transferred to nylon filters (Colony/Plaque screen,
NEN) and screened with .alpha..sup.32P-labelled pooled inserts from
PCR clones pCHA (SEQ ID No:35) and pCHB (SEQ ID No:36).
Hybridization was performed at 42.degree. C. in 50% formamide
2.times.SSC, 10% dextran sulfate, 1% SDS and washing as described
in the Southern Blot procedure. Eight positive plaques were
identified, purified and their cDNA inserts excised with EcoRI and
subcloned into pBluescript SK+ (Stratagene). All eight had
approximately 1.7 kb inserts with identical restriction patterns.
One, pHH-2, was chosen for sequencing and used in all further
manipulations.
[0271] Preparation Of Digoxigenin-Labeled Riboprobes
[0272] Plasmid pHH-2 was linearized with Hind III and transcribed
with T3 RNA polymerase (for antisense probes) or with Bam HI and
transcribed with T7 RNA polymerase according to the manufacturers
instructions for the preparation of non-radioactive digoxigenin
transcripts. Following the transcription reaction, RNA was
precipitated, and resuspended in RNAse-free water.
[0273] Whole Mount in Situ Hybridization
[0274] Whole-mount in situ hybridization was performed using
protocols modified from Parr, B. A. et al. (1993) Development 119:
247-261; Sasaki, H. et al. (1993) Development 118: 47-59; Rosen, B.
et al. (1993) Trends Genet. 9: 162-167. Embryos from incubated
fertile White Leghorn eggs (Spafas) were removed from the egg and
extra-embryonic membranes dissected in calcium/magnesium-free
phosphate-buffered saline (PBS) at room temperature. Unless
otherwise noted, all washes are for five minutes at room
temperature. Embryos were fixed overnight at 4.degree. C. with 4%
paraformaldehyde in PBS, washed twice with PBT (PBS with 0.1%
Tween-20) at 4.degree. C., and dehydrated through an ascending
methanol series in PBT (25%, 50%, 75%, 2.times.100% methanol).
Embryos were stored at -20.degree. C. until further use.
[0275] Both pre-limb bud and limb bud stage embryos were rehydrated
through an descending methanol series followed by two washes in
PBT. Limb bud stage embryos were bleached in 6% hydrogen peroxide
in PBT, washed three times with PBT, permeabilized with proteinase
K (Boehringer, 2 .mu.g/ml) for 15 minutes, washed with 2 mg/ml
glycine in PBT for 10 minutes, and twice with PBT. Pre-limb bud
stage embryos were permealibized (without prior incubation with
hydrogen peroxide) by three 30 minute washes in RIPA buffer (150 mM
NaCl, 1% NP-40, 0.5% Deoxycholate, 0.1% SDS, 1 mM EDTA, 50 mM
Tris-HCl, pH 8.0). In all subsequent steps, pre-limb bud and limb
bud stage embryos were treated equivalently. Embryos were fixed
with 4% paraformaldehyde/0.2% gluteraldehyde in PBT, washed four
times with PBT, once with pre-hybridization buffer (50% formamide,
5.times.SSC, 1% SDS, 50 .mu.g/ml total yeast RNA, 50 .mu.g/ml
heparin, pH 4.5), and incubated with fresh pre-hybridization buffer
for one hour at 70.degree. C. The pre-hybridization buffer was then
replaced with hybridization buffer (pre-hybridization buffer with
digoxigenin labeled riboprobe at 1 .mu.g/ml) and incubated
overnight at 70.degree. C.
[0276] Following hybridization, embryos were washed 3.times.30
minutes at 70.degree. C. with solution 1 (50% formamide,
5.times.SSC, 1% SDS, pH 4.5), 3.times.30 minutes at 70.degree. C.
with solution 3 (50% formamide, 2.times.SSC, pH 4.5), and three
times at room temperature with TBS (Tris-buffered saline with 2 mM
levamisole) containing 0.1% Tween-20. Non-specific binding of
antibody was prevented by preblocking embryos in TBS/0.1% Tween-20
containing 10% heat-inactivated sheep serum for 2.5 hours at room
temperature and by pre-incubating anti-digoxigenin Fab
alkaline-phosphatase conjugate (Boehringer) in TBS/0.1% Tween-20
containing heat inactivated 1% sheep serum and approximately 0.3%
heat inactivated chick embryo powder. After an overnight incubation
at 4.degree. C. with the pre-adsorbed antibody in TBS/0.1% Tween-20
containing 1% sheep serum, embryos were washed 3.times.5 minutes at
room temperature with TBS/0.1% Tween-20, 5.times.1.5 hour room
temperature washes with TBS/1% Tween-20, and overnight with TBS/1%
Tween-20 at 4.degree. C. The buffer was exchanged by washing
3.times.10 minutes with NTMT (100 mM NaCl, 100 mM Tris-HCl, 50 mM
MgCl2, 0.1% Tween-20, 2 mM levamisole). The antibody detection
reaction was performed by incubating embryos with detection
solution (NTMT with 0.25 mg/ml NBT and 0.13 mg/ml X-Phos). In
general, pre-limb bud stage embryos were incubated for 5-15 hours
and limb bud stage embryos 1-5 hours. After the detection reaction
was deemed complete, embryos were washed twice with NTMT, once with
PBT (pH 5.5), postfixed with 4% paraformaldehyde/0.1%
gluteraldehyde in PBT, and washed several times with PBT. In some
cases embryos were cleared through a series of 30%, 50%, 70%, and
80% glycerol in PBT. Whole embryos were photographed under
transmitted light using a Nikon zoom stereo microscope with Kodak
Ektar 100 ASA film. Selected embryos were processed for frozen
sections by dehydration in 30% sucrose in PBS followed by embedding
in gelatin and freezing. 25 .mu.m cryostat sections were collected
on superfrost plus slides (Fisher), rehydrated in PBS, and mounted
with gelvatol. Sections were photographed with Nomarski optics
using a Zeiss Axiophot microscope and Kodak Ektar 25 ASA film.
[0277] (ii) Sequence Homolgy Comparison Between Chicken Sonic hh
and Drosophila hh and Other Vertebrate Sonic hh Proteins
[0278] The deduced Sonic amino acid sequence (SEQ ID No:8) is shown
and compared to the Drosophila hedgehog protein (SEQ ID No:34) in
FIG. 2. Over the entire open reading frame the two proteins are 48%
homologous at the amino acids level. The predicted Drosophila
protein extends 62 aa beyond that of Sonic at its amino terminus.
This N-terminal extension precedes the putative signal peptide
(residues 1-26) of the fly protein (SEQ ID No:34), and has been
postulated to be removed during processing of the secreted form of
Drosophila hedgehog (Lee, J. J. et al., (1992) Cell 71: 33-50). The
sequence of residues 1-26 of the Sonic protein (SEQ ID No:8)
matches well with consensus sequences for eukaryotic signal
peptides (Landry, S. J. et al., (1993) Trends. Biochem. Sci. 16:
159-163) and is therefore likely to serve that function for Sonic.
Furthermore, FIG. 3 shows a hydropathy plot (Kyte, J. et al.,
(1982) J. Mol. Bio. 57: 133-148) indicating that residues 1-26 of
the Sonic protein (SEQ ID No:8) exhibit a high hydrophobic moment
in accord with identified eukaryotic signal peptides. Cleavage of
the putative signal sequence should occur C-terminal to residue 26
according to the predictive method of von Henjie, G. (1986) Nuc.
Acid. Res. 11: 1986. A single potential N-linked glycosylation site
is located at amino acid residue 282 of the Sonic protein (SEQ ID
No:8). The predicted Sonic protein does not contain any other
strong consensus motifs, and is not homologous to any other
proteins outside of the Hedgehog family.
[0279] The mouse (SEQ ID No:11) and zebrafish (SEQ ID No:12)
homologs of Sonic have also been isolated. A comparison of these
and the Drosophila sequence is shown schematically in FIG. 4. All
of the vertebrate proteins have a similar predicted structure: a
putative signal peptide at their amino terminus, followed by an
extraordinarily similar 182 amino acid region (99% identity in
chicken versus mouse and 95% identity in chicken versus zebrafish)
and a less well conserved carboxy-terminal region.
[0280] (iii) At Least Three Hedgehog Homologues are Present in the
Chicken Genome
[0281] Since two distinct PCR products encoding for chicken
hedgehogs were amplified from genomic DNA, the total number of
genes in the chicken hedgehog family needed to be estimated. The
two PCR clones pCHA (SEQ ID No:35) and pCHB (SEQ ID No:36) were
used to probe a genomic Southern blot under moderately stringent
conditions as described in the above Experimental Procedures. The
blot was generated by digesting 5 .mu.g of chick chromosomal DNA
with EcoRI and BamHI alone and together. Each probe reacted most
strongly with a distinct restriction fragment. For example, the
blot probed with pCHA, shows three bands in each of the Bam HI
lanes, one strong at 6.6 kb and two weak at 3.4 and 2.7 kb. The
blot probed with pCHB, shows the 2.7 kb band as the most intense,
while the 3.4 and 6.6 kb bands are weaker. A similar variation of
intensities can also be seen in the Bam HI/Eco RI and EcoRI lanes.
Exposure times were 72 hr. This data indicates that each probe
recognizes a distinct chicken hedgehog gene, and that a third as
yet uncharacterized chicken hedgehog homolog exists in the chicken
genome.
[0282] (iv) Northern Analysis Defining Sites of Sonic
Transcription
[0283] Northern analysis was performed which confirmed that Sonic
is expressed during chick development. The spatial and temporal
expression of Sonic in the chick embryo from gastrulation to early
organogenesis was determined by whole mount in situ hybridization
using a riboprobe corresponding to the full-length Sonic cDNA (SEQ
ID No:1).
[0284] 20 .mu.g total RNA isolated from stage 24 chick leg buds or
bodies (without heads or limbs) was fractionated on a 0.8% agarose
formaldehyde gel and transferred to a nylon membrane (Hybond N,
Amersham). The blot was probed with the 1.6 kb EcoRI insert from
pHH-2. Random-primed .alpha..sup.32P-labelled insert was hybridized
at 42.degree. C. hybridization buffer (1% BSA, 500 mM NaHPO.sub.4,
7% SDS, 1 mM EDTA, pH 7.2) and washed at 63.degree. C. once in 0.5%
bovine serum albumin, 50 mM NaHPO.sub.4 (pH 7.2), 5% SDS, 1 mM EDTA
and once in 40 mM NaHPO.sub.4 (pH 7.2), 1% SDS, 1 mM EDTA. The
image was visualized using a phosphoimager (Molecular Dynamics) and
photographed directly from the video monitor.
[0285] (v) Expression of Sonic During Mid-Gastrulation
[0286] Sonic message is detected in the gastrulating blastoderm at
early stage 4, the earliest stage analyzed. Staining is localized
to the anterior end of the primitive streak in a region
corresponding to Hensen's node. As gastrulation proceeds, the
primitive streak elongates to its maximal cranial-caudal extent,
after which Hensen's node regresses caudally and the primitive
streak shortens. At an early point of node regression, Sonic mRNA
can be detected at the node and in midline cells anterior to the
node. By late stage 5, when the node has migrated approximately
one-third of the length of the fully elongated primitive streak,
prominent Sonic expression is seen at the node and in the midline
of the embryo, reaching its anterior limit at the developing head
process. Sections at a cranial level show that Sonic mRNA is
confined to invaginated axial mesendoderm, tissue which contributes
to foregut and notochord. More caudally, but still anterior to
Hensen's node, staining of axial mesoderm is absent and Sonic
expression is confined to the epiblast. At the node itself, high
levels of Sonic message are observed in an asymmetric distribution
extending to the left of and posterior to the primitive pit. This
asymmetric distribution is consistently observed (6/6 embryos from
stages 5-7) and is always located to the left of the primitive pit.
At the node, and just posterior to the node, Sonic expression is
restricted to the epiblast and is not observed in either mesoderm
or endoderm. The expression of Sonic in the dorsal epiblast layer
without expression in underlying axial mesoderm contrasts markedly
with later stages where Sonic expression in underlying mesoderm
always precedes midline neural tube expression.
[0287] (vi) Expression of Sonic During Head Fold Stages
[0288] During the formation and differentiation of the head
process, Sonic mRNA is detected in midline cells of the neural
tube, the foregut, and throughout most of the axial mesoderm. At
stage 7, Sonic message is readily detected asymmetrically at the
node and in ventral midline cells anterior to the node. The rostral
limit of Sonic expression extends to the anterior-most portions of
the embryo where it is expressed in the foregut and prechordal
mesoderm (Adelmann, H. B., (1932) Am. J. Anat. 31, 55-101). At
stage 8, expression of Sonic persists along the entire ventral
midline anterior to Hensen's node, while the node region itself no
longer expresses Sonic. Transverse sections at different axial
levels reveal that at stage 8 Sonic is coexpressed in the notochord
and the overlying ventromedial neuroectoderm from anterior to
Hensen's node to the posterior foregut. The levels of Sonic message
are not uniform in the neural tube: highest levels are found at the
presumptive mid- and hindbrain regions with progressively lower
levels anterior and posterior. The increasing graded expression in
the neural tube from Hensen's node to the rostral brain may reflect
the developmental age of the neuroectoderm as differentiation
proceeds from posterior to anterior. At the anterior-most end of
the embryo, expression is observed in midline cells of the dorsal
and ventral foregut as well as in prechordal mesoderm. Although the
prechordal mesoderm is in intimate contact with the overlying
ectoderm, the latter is devoid of Sonic expression.
[0289] (vii) Expression of Sonic During Early CNS
Differentiation
[0290] At stages 10 through 14, Sonic expression is detected in the
notochord, ventral neural tube (including the floor plate), and gut
precursors. By stage 10, there is a marked expansion of the
cephalic neuroectoderm, giving rise to the fore- mid- and
hind-brain. At stage 10, Sonic mRNA is abundantly expressed in the
ventral midline of the hindbrain and posterior midbrain. This
expression expands laterally in the anterior midbrain and posterior
forebrain. Expression does not extend to the rostral forebrain at
this or later stages. Sections reveal that Sonic is expressed in
the notochord, the prechordal mesoderm, and the anterior midline of
the foregut. Expression in the neuroepithelium extends from the
forebrain caudally. In the posterior-most regions of the embryo
which express Sonic, staining is found only in the notochord and
not in the overlying neurectoderm. This contrasts with earlier
expression in which the posterior domains of Sonic expression
contain cells are located in the dorsal epiblast, but not in
underlying mesoderm or endoderm. Midgut precursors at the level of
the anterior intestinal portal also show weak Sonic expression.
[0291] At stage 14, expression continues in all three germ layers.
The epithelium of the closing midgut expresses Sonic along with
portions of the pharyngeal endoderm and anterior foregut. Ectoderm
lateral and posterior to the tail bud also exhibits weak
expression. At this stage, Sonic is also expressed along entire
length of the notochord which now extends rostrally only to the
midbrain region and no longer contacts the neuroepithelium at the
anterior end of the embryo. Expression in head mesenchyme anterior
to the notochord is no longer observed. In the neural tube Sonic is
found along the ventral midline of the fore- mid- and hindbrain and
posteriorly in the spinal cord. In the forebrain, expression is
expanded laterally relative to the hindbrain. At midgut levels,
expression of Sonic in the neural tube appears to extend beyond the
floor plate into more lateral regions. As observed at stage 10,
Sonic at stage 14 is found in the notochord, but not in the ventral
neural tube in posterior-most regions of the embryo. When
neuroectodermal expression is first observed in the posterior
embryo, it is located in midline cells which appear to be in
contact with the notochord. At later stages, expression continues
in areas which show expression at stage 14, namely the CNS, gut
epithelium including the allantoic stalk, and axial mesoderm.
[0292] (viii) Sonic is Expressed in Posterior Limb Bud
Mesenchyme
[0293] The limb buds initially form as local thickenings of the
lateral plate mesoderm. As distal outgrowth occurs during stage 17,
Sonic expression becomes apparent in posterior regions of both the
forelimb and the hindlimb. Sections through a stage 21 embryo at
the level of the forelimbs reveal that expression of Sonic in limb
buds is limited to mesenchymal tissue. A more detailed expression
profile of Sonic during limb development is discussed below in
Example 3. Briefly, as the limb bud grows out, expression of Sonic
narrows along the anterior-posterior axis to become a thin stripe
along the posterior margin closely apposed to the ectoderm.
Expression is not found at more proximal regions of the bud. High
levels of Sonic expression are maintained until around stage 25/26
when staining becomes weaker. Expression of Sonic is no longer
observed in wing buds or leg buds after stage 28.
EXAMPLE 2
Mouse Sonic Hedgehog is Implicated in the Regulation of CNS and
Limb Polarity
[0294] (i) Experimental Procedures
[0295] Isolation of Hedgehog Phage Clones
[0296] The initial screen for mammalian hh genes was performed, as
above, using a 700 bp PCR fragment encompassing exons 1 and 2 of
the Drosophila hh gene. Approximately one million plaques of a
129/Sv Lambda Fix II genomic library (Stratagene) were hybridized
with an .alpha. .sup.32P-dATP labeled probe at low stringency
(55.degree. C. in 6.times.SSC, 0.5% SDS, 5.times.Denhardt's; final
wash at 60.degree. C. in 0.5.times.SSC, 0.1% SDS for 20'). Five
cross hybridizing phage plaques corresponding to the Dhh gene were
purified. Restriction enzyme analysis indicated that all clones
were overlapping. Selected restriction enzyme digests were then
performed to map and subclone one of these. Subclones in pGEM
(Promega) or Bluescript (Stratagene) which cross-hybridized with
the Drosophila hh fragment where sequenced using an ABI automatic
DNA sequencer.
[0297] Mouse Ihh and Shh were identified by low stringency
hybridization (as described above) with a chick Shh cDNA clone to
one million plaques of an 8.5 day .lambda.gt10 mouse embryo cDNA
library (Fahrner, K. et al., (1987) EMBO J. 6: 1265-1271). Phage
plaques containing a 1.8 kb Ihh and 0.64 and 2.8 kb Shh inserts
were identified. Inserts were excised and subcloned into Bluescript
(Stratagene) for dideoxy chain termination sequencing using
modified T7 DNA polymerase (USB). The larger Shh clone contained a
partially processed cDNA in which intron splicing at the exon 1/2
junction had not occurred.
[0298] To screen for additional Ihh and Shh cDNA clones, an 8.5 day
.lambda.ZAPII cDNA library was probed at high stringency (at
65.degree. C. in 6.times.SSC, 0.5% SDS, 5.times.Denhardt's; final
wash at 65 .degree. C. in 0.1.times.SSC, 0.1% SDS for 30') with the
Ihh and Shh mouse cDNA clones. No additional Ihh clones were
identified. However several 2.6 kb, apparently full length, Shh
clones were isolated. The DNA sequence of the additional 5' coding
region not present in the original 0.64 and 2.8 kb Shh clones was
obtained by analysis of one of the 2.6 kb inserts.
[0299] Northern Blot Analysis
[0300] Expression of Shh was investigated by RNA blot analysis
using 20 .mu.g of total RNA from adult brain, spleen, kidney,
liver, lung, 16.5 dpc brain, liver and lung; 9.5 dpc to 17.5 dpc
whole embryo; 9.5 dpc forebrain, midbrain and 10.5 dpc brain. RNA
samples were electrophoretically separated on a 1.2% agarose gel,
transferred and u.v. crosslinked to Genescreen (DuPont) and probed
with 2.times.10.sup.6 cpm/ml of an .alpha..sup.32P-dATP labeled
mouse Shh probe (2.8 kb insert from .lambda.gt 10 screen).
Hybridization was performed at 42.degree. C. in 50% formamide
5.times.Denhardt's, 5.times.SSPE, 0.1% SDS, 6.5% dextran, 200
.mu.g/ml salmon sperm DNA. Final wash was at 55.degree. C. in
0.1.times.SSC, 0.1% SDS. The blot was exposed for 6 days in the
presence of an intensifying screen.
[0301] In Situ Hybridization, .beta.-Galactosidase Staining and
Histological Analysis
[0302] Embryos from 7.25 to 14.5 dpc were analyzed for either Shh
or HNF-3.beta. expression by whole mount in situ hybridization to
digoxygenin labeled RNA probes as described in Wilkinson, (1992) In
situ Hybridization: A Practical Approach. Oxford; Parr et al.,
(1993) Development 119:247-261. The mouse Shh probe was either a
2.8 kb or 0.6 kb RNA transcript generated by T7 (2.8 kb) or T3 (0.6
kb) transcription of XbaI and HindIII digests of Bluescript
(Stratagene) subclones of the original Shh cDNA inserts. The
HNF-3.mu. probe was generated by HindIII linearization of a
HNF-3.beta. cDNA clone (Sasaki, H. et al., (1993) Development 118:
47-59) and T7 polymerase transcription of 1.6 kb transcript.
Embryos were photographed on an Olympus-SZH photomicroscope using
Kodak Ektachrome EPY 64T color slide film.
[0303] Sections through wild type and WEXP2-CShh transgenic embryos
were prepared and hybridized with .sup.35S-UIP labeled RNA probes
(Wilkinson, D. G. et al., (1987) Development 99: 493-500). Sections
were photographed as described in McMahon, A. P. et al., (1992)
Cell 69: 581-595.
[0304] .beta. Staining of WEXP2-lacZ embryos with Pwas performed
according to Whiting, J. et al., (1991) Genes & Dev. 5:
2048-2059. General histological analysis of wildtype and WEXP2-CShh
transgenic embryos was performed on paraffin sections of Bouin's
fixed embryos counterstained with haematoxylin and eosin.
Histological procedures were as described by Kaufman, M. H. (1992)
The Atlas of Mouse Development, London: Academic Press. Sections
were photographed on a Leitz Aristoplan compound microscope using
Kodak EPY 64T color slide film.
[0305] DNA Constructs for Transgenics
[0306] Genomic Wnt-l fragments were obtained by screening a
.lambda.GEM12 (Promega) 129/Sv mouse genomic library with a 375 bp
MluI-BglII fragment derived from the fourth exon of the murine
Wnt-l gene. One of the clones (W1-15.1) was used in this study.
[0307] As an initial step towards the generation of the pWEXP2
expression vector, W1-15.1 was digested to completion with
restriction enzymes AatII and ClaI, and a 2774 bp AatII-ClaI
fragment isolated. This fragment was ligated into AatII and ClaI
cut pGEM-7Zf vector (Promega), generating pW1-18. This plasmid was
digested with HindII and ligated to annealed oligonucleotides lacl
(SEQ ID No:21) and lac2 (SEQ ID No:22) generating pW1-18S* which
has a modified polylinker downstream of the ClaI restriction site.
This construct (pW1-18S*) was digested with ClaI and BglII and
ligated with both the 2.5 kb 3' ClaI-BglII exon-intron region and
5.5 kb 3' BglII-BglII Wnt-1 enhancer, generating pWRES4. This
construct contains a 10.5 kb genomic region which starts upstream
of the Wnt-1 translation initiation codon (at an AatII site
approximately 1.0 kb from the ATG) and extends to a BglII site 5.5
kb downstream of the Wnt-l polyadenylation signal. This plasmid
also contains a 250 bp region of the neomycin phosphotransferase
(neo) gene inserted in inverse orientation in the 3' transcribed
but untranslated region. Finally, to generate the WEXP2 expression
vector, a 2 kb Sfi I fragment was amplified from pWRES4 using Sf-1
(SEQ ID No:23) and Sf-2 (SEQ ID No:24) oligonucleotides. This
amplified fragment was digested with Sfi I and inserted into Sfi I
linearised pWRES4, generating pWEXP2. This destroys the Wnt-l
translation initiation codon, and replaces it by a polylinker
containing Nru I, Eco RV, Sac II, and Bst BI restriction sites,
which are unique in pWEXP2.
[0308] The WEXP2-lacZ construct was obtained by inserting an
end-filled Bgl II-Xho I lacZ fragment isolated from the pSDKlacZpA
vector in the Nru I cut pWEXP2 expression vector. Similarly, the
WEXP2-CShh construct was obtained by inserting an end-filled XbaI
cDNA fragment containing the full Chick Shh coding sequence (SEQ ID
No:1) into the Nru I cut WEXP2 expression vector.
[0309] Oligonucleotide sequences are as follows:
[0310] lac1:
5'-AGCTGTCGACGCGGCCGCTACGTAGGTTACCGACGTCAAGCTTAGATCTC-3'
[0311] lac2:
5'-AGCTGAGATCTAAGCTTGACGTCGGTAACCTACGTAGCGGCCGCGTCGAC-3'
[0312] Sf-1:
5'-GATCGGCCAGGCAGGCCTCGCGATATCGTCACCGCGGTATTCGAA-3'
[0313] Sf-2: 5'-AGTGCCAGTCGGGGCCCCCAGGGCCGCGCC-3'
[0314] Production and Genotyping of Transgenic Embryos
[0315] Transgenic mouse embryos were generated by microinjection of
linear DNA fragments into the male pronucleus of B6CBAF1/J
(C57BL/6J X CBA/J) zygotes. CD-1 or B6CBAF1/J females were used as
recipients for injected embryos. G.sub.O mice embryos were
collected at 9.5, 10.5, and 11.5 dpc, photographed using an Olympus
SZH stereophoto-microscope on Kodak EPY-64T color slide film, then
processed as described earlier.
[0316] WEXP2-lacZ and WEXP2-CShh transgenic embryos were identified
by PCR analysis of proteinase-K digests of yolk sacs. Briefly, yolk
sacs were carefully dissected free from maternal and embryonic
tissues, avoiding cross-contamination between littermates, then
washed once in PBS. After overnight incubation at 55.degree. C. in
50 .mu.l of PCR proteinase-K digestion buffer (McMahon, A. P. et
al., (1990) Cell 62: 1073-1085). 1 .mu.l of heat-inactivated digest
was subjected to polymerase chain reaction (PCR) in a 20 .mu.l
volume for 40 cycles as follows: 94.degree. C. for 30 seconds,
55.degree. C. for 30 seconds, 72.degree. C. for 1 minute, with the
reaction ingredients described previously (McMahon, A. P. et al.,
(1990) Cell 62: 1073-1085)). In the case of the WEXP2-lacZ
transgenic embryos, oligonucleotides 137 (SEQ ID No:25) and 138
(SEQ ID No:26) amplify a 352 bp lacZ specific product. In the case
of the WEXP2-CShh embryos, oligonucleotides WPR2 (Wnt-1-specific)
(SEQ ID No:27) and 924 (Chick Shh-specific) (SEQ ID No:28) amplify
a 345 bp fragment spanning the insertion junction of the Chick-Shh
cDNA in the WEXP2 expression vector. Table 2 summarizes the results
of WEXP2-C-Shh transgenic studies.
[0317] Oligonucleotide sequences are as follows:
[0318] 137: 5'-TACCACAGCGGATGGTTCGG-3'
[0319] 138: 5'-GTGGTGGTTATGCCGATCGC-3'
[0320] WPR2: 5'-TAAGAGGCCTATAAGAGGCGG-3'
[0321] 924: 5'-AAGTCAGCCCAGAGGAGACT-3'
[0322] (ii) Mouse hh Genes
[0323] The combined screening of mouse genomic and 8.5 day post
coitum (dpc) cDNA libraries identified three mammalian hh
counterparts (FIG. 5A) which herein will be referred to as Desert,
Indian and Sonic hedgehog (Dhh, Ihh and Shh, respectively).
Sequences encoding Dhh (SEQ ID No:2) were determined from analysis
of clones identified by low stringency screening of a mouse genomic
library. DNA sequencing of one of five overlapping lambda phage
clones identified three homologous regions encoding a single open
reading frame interrupted by introns in identical position to those
of the Drosophila hh gene (FIG. 5A). Splicing across the exon 1/2
boundary was confirmed by polymerase chain reaction (PCR)
amplification of first strand cDNA generated from adult testicular
RNA. The partial sequence of Ihh (SEQ ID No:3) and the complete
sequence of Shh (SEQ ID No:4) coding regions were determined from
the analysis of overlapping cDNA clones isolated from 8.5 dpc cDNA
libraries. The longest Shh clone, 2.6 kb, appears to be full length
when compared with the Shh transcript present in embryonic RNAs.
The 1.8 kb partial length Ihh cDNA is complete at the 3' end, as
evidenced by the presence of a polyadenylation consensus sequence
and short poly A tail.
[0324] Alignment of the predicted Drosophila hh protein sequence
(SEQ ID No:34) with those of the mouse Dhh (SEQ ID No:9), Ihh (SEQ
ID No:10) and Shh (SEQ ID No:11), and chick Shh (SEQ ID No:8) and
zebrafish Shh (SEQ ID No:12), reveals several interesting features
of the hh-family (FIG. 5A). All the vertebrate hh-proteins contain
an amino terminal hydrophobic region of approximately 20 amino
acids immediately downstream of the initiation methionine. Although
the properties of these new hh proteins have not been investigated,
it is likely that this region constitutes a signal peptide and
vertebrate hhs are secreted proteins. Signal peptide cleavage is
predicted to occur (von Heijne, G., (1986) Nucleic Acids Research
14: 4683-4690) just before an absolutely conserved six amino acid
stretch, CGPGRG (SEQ ID No:29) (corresponding to residues
85-90)(FIG. 5A), in all hh proteins. This generates processed mouse
Dhh (SEQ ID No:9) and Shh (SEQ ID No:11) proteins of 41 and 44 kd,
respectively. Interestingly, Drosophila hh (SEQ ID No:34) is
predicted to contain a substantial amino terminal extension beyond
the hydrophobic domain suggesting that the Drosophila protein
enters the secretory pathway by a type II secretory mechanism. This
would generate a transmembrane tethered protein which would require
subsequent cleavage to release a 43 kd secreted form of the
protein. In vitro analysis of Drosophila hh is consistent with this
interpretation (Lee, J. J. et al., (1992) Cell 71: 33-50). However,
there also appears to be transitional initiation at a second
methionine (position 51 of SEQ ID No:34) just upstream of the
hydrophobic region (Lee, J. J. et al., (1992) Cell 71: 33-50),
suggesting that Drosophila hh, like its vertebrate counterparts,
may also be secreted by recognition of a conventional amino
terminal signal peptide sequence.
[0325] Data base searches for protein sequences related to
vertebrate hh's failed to identify any significant homologies,
excepting Drosophila hh. In addition, searching the "PROSITE" data
bank of protein motifs did not reveal any peptide motifs which are
conserved in the different hh proteins. Thus, the hhs represent a
novel family of putative cell signaling molecules.
[0326] One feature of the amino acid alignment is the high
conservation of hh sequences. Vertebrate hhs share 47 to 51% amino
acid identity with Drosophila hh throughout the predicted processed
polypeptide sequence (FIG. 6). Dhh has a slightly higher identity
than that of Ihh and Shh suggesting that Dhh may be the orthologue
of Drosophila hh. Conservation is highest in the amino terminal
half of the proteins, indeed, from position 85 (immediately after
the predicted shared cleavage site) to 249, 62% of the amino acids
are completely invariant amongst the Drosophila and vertebrate
proteins. Comparison of mouse Dhh, Ihh and Shh where their
sequences overlap in this more conserved region, indicates that Ihh
and Shh are more closely related (90% amino acid identity; residues
85 to 266) than with the Dhh sequence (80% amino acid identity;
residues 85 to 266). Thus, Ihh and Shh presumably resulted from a
more recent gene duplication event.
[0327] Comparison of cross species identity amongst Shh proteins
reveals an even more striking sequence conservation. Throughout the
entire predicted processed sequence mouse and chick Shh share 84%
of amino acid residues (FIG. 6). However, in the amino terminal
half (positions 85 to 266) mouse and chick are 99% and mouse and
zebrafish 94% identical in an 180 amino acid stretch. Conservation
falls off rapidly after position 266 (FIG. 5A). SEQ ID No:40 shows
the consensus sequence in the amino terminal half of all vertebrate
Shh genes (human, mouse, chicken and zebrafish) identified to date.
SEQ ID No:41 shows the consensus sequence in the amino terminal
half of vertebrate hedgehog genes (Shh, Ihh, and Dhh) identified to
date in different species (mouse, chicken, human and
zebrafish).
[0328] In summary, hh family members are likely secreted proteins
consisting of a highly conserved amino terminal and more divergent
carboxyl terminal halves. The extreme interspecies conservation of
the vertebrate Shh protein points to likely conservation of Shh
function across vertebrate species.
[0329] (iii) Expression of Mouse Shh at the Axial Midline
[0330] Expression of Shh in the mouse was examined in order to
explore the role of mouse Shh (SEQ ID No:11) in vertebrate
development. Northern blots of embryonic and adult RNA samples were
probed with a radiolabelled mouse Shh cDNA probe. An Shh transcript
of approximately 2.6 kb was detected in 9.5 dpc whole embryo RNA,
and 9.5 and 10.5 dpc brain RNA fractions. No expression was
detected in total RNA samples from later embryonic stages. Of the
late fetal and adult tissue RNAs examined Shh expression was only
detected in 16.5 dpc and adult lung.
[0331] To better define the precise temporal and spatial expression
of Shh an extensive series of whole mount and serial section in
situ hybridizations were performed using digoxygenin and
.sup.35S-radiolabelled RNA probes, respectively, to mouse embryo
samples from 7.25 dpc (mid streak egg cylinder stage of
gastrulation) to 13.5 dpc. No Shh expression is detected at
mid-gastrulation stages (7.25 dpc) prior to the appearance of the
node, the mouse counterpart of the amphibian organizer and chick
Hensens node. When the primitive streak is fully extended and the
midline mesoderm of the head process is emerging from the node (7.5
to 7.75 dpc), Shh is expressed exclusively in the head process. At
late head fold stages, Shh is expressed in the node and midline
mesoderm of the head process extending anteriorly under the
presumptive brain. Just prior to somite formation, Shh extends to
the anterior limit of the midline mesoderm, underlying the
presumptive midbrain. As somites are formed, the embryonic axis
extends caudally. The notochord, which represents the caudal
extension of the head process, also expresses Shh, and expression
is maintained in the node.
[0332] Interestingly, by 8 somites (8.5 dpc) strong Shh expression
appears in the CNS. Expression is initiated at the ventral midline
of the midbrain, above the rostral limit of the head process. By 10
somites CNS expression in the midline extends rostrally in the
forebrain and caudally into the hindbrain and rostral spinal cord.
Expression is restricted in the hindbrain to the presumptive
floorplate, whereas midbrain expression extends ventro-laterally.
In the forebrain, there is no morphological floor plate, however
ventral Shh expression here is continuous with the midbrain. By 15
somites ventral CNS expression is continuous from the rostral limit
of the diencephalon to the presumptive spinal cord in somitic
regions. Over the next 18 to 24 hrs, to the 25-29 somite stage, CNS
expression intensifies and forebrain expression extends rostral to
the optic stalks. In contrast to all other CNS regions, in the
rostral half of the diencephalon, Shh is not expressed at the
ventral midline but in two strips immediately lateral to this area
which merge again in the floor of the forebrain at its rostral
limit. Expression of Shh in both the notochord and floorplate is
retained until at least 13.5 dpc.
[0333] Several groups have recently reported the cloning and
expression of vertebrate members of a family of transcription
factors, related to the Drosophila forkhead gene. One of these,
HNF-3.beta. shows several similarities in expression to Shh
(Sasaki, H. et al., (1993) Development 118: 47-59) suggesting that
HNF-3.beta. may be a potential regulator of Shh. To investigate
this possibility, direct comparison of HNF-3.beta. and Shh
expression was undertaken. HVF-3.beta. transcripts are first
detected in the node (as previously reported by Sasaki, H. et al.,
(1993) supra), prior to the emergence of the head process and
before Shh is expressed. From the node, expression proceeds
anteriorly in the head process, similar to Shh expression.
Activation of HNF-3.beta. within the CNS is first observed at 2-3
somites, in the presumptive mid and hindbrain, prior to the onset
of Shh expression. By 5 somites, expression in the midbrain
broadens ventro-laterally, extends anteriorly into the forebrain
and caudally in the presumptive floor plate down much of the
neuraxis in the somitic region. Strong expression is maintained at
this time in the node and notochord. However, by 10 somites
expression in the head process is lost and by 25-29 somites
notochordal expression is only present in the most extreme caudal
notochord. In contrast to the transient expression of HNF-3.beta.
in the midline mesoderm, expression in the floor plate is stably
retained until at least 11.5 dpc. Thus, there are several spatial
similarities between the expression of HNF-3.beta. and Shh in both
the midline mesoderm and ventral CNS and it is likely that both
genes are expressed in the same cells. However, in both regions,
HNF-3.beta. expression precedes that of Shh. The main differences
are in the transient expression of HNF-3.beta. in the head process
and notochord and Shh expression in the forebrain. Whereas
HNF-3.beta. and Shh share a similar broad ventral and ventral
lateral midbrain and caudal diencephalic expression, only Shh
extends more rostrally into the forebrain. In general, these
results are consistent with a model in which initial activation of
Shh expression may be regulated by HNF-3.beta..
[0334] The similarity in Shh and HNF-3.beta. expression domains is
also apparent in the definitive endoderm which also lies at the
midline. Broad HNF-3.beta. expression in the foregut pocket is
apparent at 5 somites as previously reported by Sasaki, H. et al.,
(1993) supra. Shh is also expressed in the endoderm, immediately
beneath the forebrain. Both genes are active in the rostral and
caudal endoderm from 8 to 11 somites. Whereas HNF-3.beta. is
uniformly expressed, Shh expression is initially restricted to two
ventro-lateral strips of cells. Ventral restricted expression of
Shh is retained in the most caudal region of the presumptive gut
until at least 9.5 dpc whereas HNF-3.beta. is uniformly expressed
along the dorso-ventral axis. Both genes are expressed in the
pharyngeal ectoderm at 9.5 dpc and expression is maintained in the
gut until at least 11.5 dpc. Moreover, expression of Shh in the
embryonic and adult lung RNA suggests that endodermal expression of
Shh may continue in, at least some endoderm derived organs.
[0335] (iv) Expression of Shh in the Limb
[0336] Expression of Shh is not confined to midline structures. By
30-35 somites (9.75 dpc), expression is detected in a small group
of posterior cells in the forelimb bud. The forelimb buds form as
mesenchymal outpocketings on the flanks, opposite somites 8 to 12,
at approximately the 17 to 20 somite stage. Shh expression is not
detectable in the forelimbs until about 30-35 somites, over 12
hours after the initial appearance of the limbs. Expression is
exclusively posterior and restricted to mesenchymal cells. By 10.5
dpc, both the fore and hindlimbs have elongated substantially from
the body flank. At this time Shh is strongly expressed in the
posterior, distal aspect of both limbs in close association with
the overlying ectoderm. Analysis of sections at this stage detects
Shh expression in an approximately six cell wide strip of posterior
mesenchymal cells. In the forelimb, Shh expression ceases by 11.5
dpc. However, posterior, distal expression is still detected in the
hindlimb. No limb expression is detected beyond 12.5 dpc.
[0337] (v) Ectopic Expression of Shh
[0338] Grafting studies carried out principally in the chick
demonstrate that cell signals derived from the notochord and floor
plate pattern the ventral aspect of the CNS (as described above).
In the limb, a transient signal produced by a group of posterior
cells in both limb buds, the zone of polarizing activity (ZPA), is
thought to regulate patterning across the anterior-posterior axis.
Thus, the sequence of Shh, which predicts a secreted protein and
the expression profile in midline mesoderm, the floor plate and in
the limb, suggest that Shh signaling may mediate pattern regulation
in the ventral CNS and limb.
[0339] To determine whether Shh may regulate ventral development in
the early mammalian CNS, a Wnt-l enhancer was used to alter its
normal domain of expression. Wnt-l shows a dynamic pattern of
expression which is initiated in the presumptive midbrain just
prior to somite formation. As the neural folds elevate and fuse to
enclose the neural tube, Wnt-1 expression in the midbrain becomes
restricted to a tight circle, just anterior of the midbrain, the
ventral midbrain and the dorsal midline of the diencephalon,
midbrain, myelencephalon and spinal cord (Wilkinson, D. G. et al.,
(1987) Cell 50: 79-88; McMahon, A. P. et al., (1992) Cell 69:
581-595; Parr, B. A. et al., (1993) Development 119: 247-261).
[0340] It was determined that essentially normal expression of lacZ
reporter constructs within the Wnt-l expression domain is dependent
upon a 5.5 kb enhancer region which lies downstream of the Wnt-1
polyadenylation sequence. A construct was generated for ectopic
expression of cDNA clones in the Wnt-l domain and tested in
transgenics using a lacZ reporter (pWEXP-lacZ; FIG. 9). Two of the
four G.sub.O transgenic embryos showed readily detectable
.beta.-galactosidase activity, and in both expression occurred
throughout the normal Wnt-l expression domain. More extensive
studies with a similar construct also containing the 5.5 kb
enhancer gave similar frequencies. Some ectopic expression was seen
in newly emerging neural crest cells, probably as a result of
perdurance of .beta.-galactosidase RNA or protein in the dorsally
derived crest. Thus, the Wnt-l expression construct allows the
efficient ectopic expression of cDNA sequences in the midbrain and
in the dorsal aspect of much of the CNS.
[0341] An Shh ectopic expression construct (pWEXP-CShh) containing
two tandem head to tail copies of a chick Shh cDNA was generated
(FIG. 7). By utilizing this approach, ectopic expression of the
chick Shh is distinguishable from that of the endogenous mouse Shh
gene. Chick Shh shows a high degree of sequence identity and
similar expression to the mouse gene. Thus, it is highly likely
that Shh function is widely conserved amongst vertebrates, a
conclusion further supported by studies of the same gene in
zebrafish.
[0342] Table 2 shows the results of several transgenic experiments
in which the G.sub.O population was collected at 9.5 to 11.5 dpc.
Approximately half of the transgenic embryos identified at each
stage of development had a clear, consistent CNS phenotype. As we
expect, on the basis of control studies using the 5.5 kb Wnt-l
enhancer, that only half the transgenics will express the
transgene, it is clear that in most embryos ectopically expressing
chick Shh, an abnormal phenotype results.
2TABLE 2 Summary of WEXP2-Chick Shh transgenic studies Number of
Number of Number of Embryos with Age (dpc) Embryos Transgenics CNS
phenotype.sup.a 9.5 37 11 6 (54.5%) 10.5 59 16 8 (50%) 11.5 33 7 3
(42.9%) Figures in parentheses, refer to the percentage of
transgenic embryos with a CNS phenotype .sup.aIn addition one 9.5
pc and two 10.5 pc transgenic embryos showed non-specific growth
retardation, # as occurs at low frequency in transgenic studies.
These embryos were excluded from further analysis.
[0343] At 9.5 dpc, embryos with a weaker phenotype show an open
neural plate from the mid diencephalon to the myelencephalon. In
embryos with a stronger phenotype at the same stage, the entire
diencephalon is open and telencephalic and optic development is
morphologically abnormal. As the most anterior diencephalic
expression of Wnt-l is lower than that in more caudal regions, the
differences in severity may relate to differences in the level of
chick Shh expression in different G.sub.O embryos. At the lateral
margins of the open neural folds, where Wnt-l is normally
expressed, there is a thickening of the neural tissue extending
from the diencephalon to myelencephalon. The cranial phenotype is
similar at 10.5 and 11.5 dpc. However, there appears to be a
retardation in cranial expansion of the CNS at later stages.
[0344] In addition to the dorsal cranial phenotype, there is a
progressive dorsal phenotype in the spinal cord. At 9.5 dpc, the
spinal cord appears morphologically normal, except at extreme
rostral levels. However by 10.5 dpc, there is a dorsal
dysmorphology extending to the fore or hindlimbs. By 11.5 dpc, all
transgenic embryos showed a dorsal phenotype along almost the
entire spinal cord. Superficially, the spinal cord had a rippled,
undulating appearance suggestive of a change in cell properties
dorsally. This dorsal phenotype, and the cranial phenotype were
examined by histological analysis of transgenic embryos.
[0345] Sections through a 9.5 dpc embryo with an extreme CNS
phenotype show a widespread dorsal perturbation in cranial CNS
development. The neural/ectodermal junction in the diencephalon is
abnormal. Neural tissue, which has a columnar epithelial morphology
quite distinct from the squamous epithelium of the surface
ectoderm, appears to spread dorsolaterally. The myelencephalon,
like the diencephalon and midbrain, is open rostrally.
Interestingly, there are discontinuous dorso-lateral regions in the
myelencephalon with a morphology distinct from the normal roof
plate regions close to the normal site of Wnt-l expression. These
cells form a tight, polarized epithelium with basely located
nuclei, a morphology similar to the floor plate and distinct from
other CNS regions. Differentiation of dorsally derived neural crest
occurs in transgenic embryos as can be seen from the presence of
cranial ganglia. In the rostral spinal cord, the neural tube
appeared distended dorso-laterally which may account for the
superficial dysmorphology.
[0346] By 11.5 dpc, CNS development is highly abnormal along the
entire dorsal spinal cord to the hindlimb level. The dorsal half of
the spinal cord is enlarged and distended. Dorsal sensory
innervation occurs, however, the neuronal trajectories are highly
disorganized. Most obviously, the morphology of dorsal cells in the
spinal cord, which normally are elongated cells with distinct
lightly staining nuclei and cytoplasm, is dramatically altered.
Most of the dorsal half of the spinal cord consists of small
tightly packed cells with darkly staining nuclei and little
cytoplasm. Moreover, there appears to be many more of these densely
packed cells, leading to abnormal outgrowth of the dorsal CNS. In
contrast, ventral development is normal, as are dorsal root
ganglia, whose origins lie in neural cells derived from the dorsal
spinal cord.
[0347] (vi) Ectopic Shh Expression Activates Floor Plate Gene
Expression
[0348] To determine whether ectopic expression of chick Shh results
in inappropriate activation of a ventral midline development in the
dorsal CNS, expression of two floor plate expressed genes,
HNF-3.beta. and mouse Shh, were examined. Whole mounts of 9.5 dpc
transgenic embryos show ectopic expression of HNF-3.beta.
throughout the cranial Wnt-l expression domain. In addition to
normal expression at the ventral midline, HNF-3.beta. transcripts
are expressed at high levels, in a circle just rostral to the
mid/hindbrain junction, along the dorsal (actually lateral in
unfused brain folds) aspects of the midbrain and, more weakly, in
the roof plate of the myelencephalon. No expression is observed in
the metencephalon which does not express Wnt-l. Thus, ectopic
expression of Shh leads to the activation of HNF-3.beta. throughout
the cranial Wnt-l expression domain.
[0349] The relationship between chick Shh expression and the
expression of HNF-3.beta. in serial sections was also examined.
Activation of HNF-3.beta. in the brain at 9.5 and 10.5 dpc is
localized to the dorsal aspect in good agreement with the observed
ectopic expression of chick Shh. Interestingly mouse Shh is also
activated dorsally. Thus, two early floor plate markers are induced
in response to chick Shh.
[0350] From 9.5 dpc to 11.5 dpc, the spinal cord phenotype becomes
more severe. The possibility that activation of a floor plate
pathway may play a role in the observed phenotype was investigated.
In contrast to the brain, where ectopic HNF-3.beta. and Shh
transcripts are still present, little or no induction of these
floor plate markers is observed. Thus, although the dorsal spinal
cord shows a widespread transformation in cellular phenotype, this
does not appear to result from the induction of floor plate
development.
EXAMPLE 3
Chick Sonic Hedgehog Mediates ZPA Activity
[0351] (i) Experimental Procedures
[0352] Retinoic Acid Bead Implants
[0353] Fertilized white Leghorn chicken eggs were incubated to
stage 20 and then implanted with AG1-X2 ion exchange beads (Biorad)
soaked in 1 mg/ml retinoic acid (RA, Sigma) as described by Tickle,
C. et al., (1985) Dev. Biol 109: 82-95. Briefly, the beads were
soaked for 15 min in 1 mg/ml RA in DMSO, washed twice and implanted
under the AER on the anterior margin of the limb bud. After 24 or
36 hours, some of the implanted embryos were harvested and fixed
overnight in 4% paraformaldehyde in PBS and then processed for
whole mount in situ analysis as previously described. The remainder
of the animals were allowed to develop to embryonic day 10 to
confirm that the dose of RA used was capable of inducing mirror
image duplications. Control animals were implanted with DMSO soaked
beads and showed no abnormal phenotype or gene expression.
[0354] Plasmids
[0355] Unless otherwise noted, all standard cloning techniques were
performed according to Ausubel, F. M. et al., (1989) Current
Protocols in Molecular Biology (N.Y.: Greene Publishing Assoc. and
Wiley Inerscience), and all enzymes were obtained from Boehringer
Mannheim Biochemicals. pHH-2 is a cDNA contain the entire coding
region of chicken Sonic hedgehog (SEQ ID No:1). RCASBP(A) and
RCASBP(E) are replication-competent retroviral vectors which encode
viruses with differing host ranges. RCANBP(A) is a variant of
RCASBP(A) from which the second splice acceptor has been removed.
This results in a virus which can not express the inserted gene and
acts as a control for the effects of viral infection (Hughes, S. H.
et al., (1987) J. Virol. 61: 3004-3012; Fekete, D. et al., (1993)
Mol. Cell. Biol. 13: 2604-2613). RCASBP/AP(E) is version of
RCASBP(E) containing a human placental alkaline phosphatase cDNA
(Fekete, D. et al., (1993b) Proc. Natl. Acad. Sci. USA 90:
2350-2354). SLAX13 is a pBluescript SK+ derived plasmid with a
second Cla I restriction site and the 5' untranslated region of
v-src (from the adaptor plasmid CLA12-Nco, Hughes, S. H. et al.,
(1987) J. Virol. 61: 3004-3012) cloned 5' of the EcoRI (and ClaI)
site in the pBluescript polylinker. RCASBP plasmids encoding Sonic
from either the first (M1) or second (M2) methionine (at position
4) were constructed by first shuttling the 1.7 kb Sonic fragment of
pHH-2 into SLAX-13 using oligonucleotides to modify the 5' end of
the cDNA such that either the first or second methionine is in
frame with the NcoI site of SLAX-13. The amino acid sequence of
Sonic is not mutated in these constructs. The M1 and M2 Sonic ClaI
fragments (v-src 5'UTR:Sonic) were each then subcloned into
RCASBP(A), RCANBP(A) and RCASBP(E), generating Sonic/RCAS-A1,
Sonic/RCAS-A2, Sonic/RCAN-A1, Sonic/RCAN-A2, Sonic/RCAS-E1 and
Sonic/RCAS-E2.
[0356] Chick Embryos, Cell Lines and Virus Production
[0357] All experimental manipulations were performed on standard
specific-pathogen free White Leghorn chick embryos (S-SPF) from
closed flocks provided fertilized by SPAFAS (Norwich, Conn.). Eggs
were incubated at 37.5.degree. C. and staged according to
Hamburger, V. et al., (1951) J. Exp. Morph. 88: 49-92. All chick
embryo fibroblasts (CEF) were provided by C. Cepko. S-SPF embryos
and CEFs have previously been shown to be susceptible to RCASBP(A)
infection but resistant to RCASBP(E) infection (Fekete, D. et al.,
(1993b) Proc. Natl. Acad. Sci. USA 90: 2350-2354). Line 15b CEFs
are susceptible to infection by both RCASBP(A) and (E). These viral
host ranges were confirmed in control experiments. CEF cultures
were grown and transfected with retroviral vector DNA as described
(Morgan, B. A. et al., (1993) Nature 358: 236-239; Fekete, D. et
al., (1993b) Proc. Natl. Acad. Sci. USA 90: 2350-2354). All viruses
were harvested and concentrated as previously described (Morgan, B.
A. et al., (1993) Nature 358: 236-239; Fekete, D. et al., (1993b)
Proc. Natl. Acad. Sci. USA 90: 2350-2354) and had titers of
approximately 10.sup.8 cfu/ml.
[0358] Cell Implants
[0359] A single 60 mm dish containing line 15 b CEFs which had been
infected with either RCASBP/AP(E), Sonic/RCAS-E1 or Sonic/RCAS-E2
were grown to 50-90% confluence, lightly trypsinized and then spun
at 1000 rpm for 5 min in a clinical centrifuge. The pellet was
resuspended in 1 ml media, transferred to a microcentrifuge tube
and then microcentrifuged for 2 min at 2000 rpm. Following a 30 min
incubation at 37.degree. C., the pellet was respun for 2 min at
2000 rpm and then lightly stained in media containing 0.01% nile
blue sulfate. Pellet fragments of approximately 300 .mu.m.times.100
.mu.m.times.50 .mu.m were implanted as a wedge to the anterior
region of hh stage 19-23 wing buds (as described by Riley, B. B. et
al., (1993) Development 118: 95-104). At embryonic day 10, the
embryos were harvested, fixed in 4% paraformaldehyde in PBS,
stained with alcian green, and cleared in methyl salicylate
(Tickle, C. et al., (1985) Dev. Biol 109: 82-95).
[0360] Viral Infections
[0361] Concentrated Sonic/RCAS-A2 or Sonic/RCAN-A2 was injected
under the AER on the anterior margin of stage 20-22 wing buds. At
24 or 36 hours post-infection, the embryos were harvested, fixed in
4% paraformaldehyde in PBS and processed for whole mount in situ
analysis as previously described.
[0362] (ii) Co-Localization of Sonic Expression And Zpa
Activity
[0363] ZPA activity has been carefully mapped both spatially and
temporally within the limb bud (Honig, L. S. et al., (1985) J.
Embryol. exp. Morph. 87: 163-174). In these experiments small
blocks of limb bud tissue from various locations and stages of
chick embryogenesis (Hamburger, V et al., (1951) J. Exp. Morph. 88:
49-92) were grafted to the anterior of host limb buds and the
strength of ZPA activity was quantified according to degrees of
digit duplication. Activity is first weakly detected along the
flank prior to limb bud outgrowth. The activity first reaches a
maximal strength at stage 19 in the proximal posterior margin of
the limb bud. By stage 23 the activity extends the full length of
the posterior border of the limb bud. The activity then shifts
distally along the posterior margin so that by stage 25 it is no
longer detectable at the base of the flank. The activity then fades
distally until it is last detected at stage 29.
[0364] This detailed map of endogenous polarizing activity provided
the opportunity to determine the extent of the correlation between
the spatial pattern of ZPA activity and Sonic expression over a
range of developmental stages. Whole mount in situ hybridization
was used to assay the spatial and temporal pattern of Sonic
expression in the limb bud. Sonic expression is not detected until
stage 17, at the initiation of limb bud formation, at which time it
is weakly observed in a punctate pattern reflecting a patchy
expression in a few cells. From that point onwards the Sonic
expression pattern exactly matches the location of the ZPA, as
determined by Honig, L. S. et al., (1985) J. Embryol. exp. Morph.
87: 163-174, both in position and in intensity of expression.
[0365] (iii) Induction of Sonic Expression by Retinoic Acid
[0366] A source of retinoic acid placed at the anterior margin of
the limb bud will induce ectopic tissue capable causing
mirror-image duplications (Summerbell, D. et al., (1983) In Limb
Development and Regeneration (N.Y.: Ala R. Liss) pp. 109-118;
Wanek, N. et al., (1991) Nature 350: 81-83). The induction of this
activity is not an immediate response to retinoic acid but rather
takes approximately 18 hours to develop (Wanek, N. et al., (1991)
Nature 350: 81-83). When it does develop, the polarizing activity
is not found surrounding the implanted retinoic acid source, but
rather is found distal to it in the mesenchyme along the margin of
the limb bud (Wanek, N. et al., (1991) Nature 350: 81-83).
[0367] If Sonic expression is truly indicative of ZPA tissue, then
it should be induced in the ZPA tissue which is ectopically induced
by retinoic acid. To test this, retinoic acid-soaked beads were
implanted in the anterior of limb buds and the expression of Sonic
after various lengths of time using whole-mount in situ
hybridization was assayed. As the limb bud grows, the bead remains
imbedded proximally in tissue which begins to differentiate.
Ectopic Sonic expression is first detected in the mesenchyme 24
hours after bead implantation. This expression is found a short
distance from the distal edge of the bead. By 36 hours Sonic is
strongly expressed distal to the bead in a stripe just under the
anterior ectoderm in a mirror-image pattern relative to the
endogenous Sonic expression in the posterior of the limb bud.
[0368] (iv) Effects of Ectopic Expression of Sonic on Limb
Patterning
[0369] The normal expression pattern of Sonic, as well as that
induced by retinoic acid, is consistent with Sonic being a signal
produced by the ZPA. To determine whether Sonic expression is
sufficient for ZPA activity, the gene was ectopically expressed
within the limb bud. In most of the experiments we have utilized a
variant of a replication-competent retroviral vector called RCAS
(Hughes, S. H. et al., (1987) J. Virol. 61: 3004-3012)) both as a
vehicle to introduce the Sonic sequences into chick cells and to
drive their expression. The fact that there exists subtypes of
avian retroviruses which have host ranges restricted to particular
strains of chickens was taken advantage of to control the region
infected with the Sonic/RCAS virus (Weiss, R. (et al.) (1984) RNA
Tumor Viruses, Vol. 1 Weiss et al. eds., (N.Y.: Cold Spring Harbor
Laboratories) pp. 209-260); Fekete, D. et al., (1993a) Mol. Cell.
Biol. 13: 2604-2613). Thus a vector with a type E envelope protein
(RCAS-E, Fekete, D. et al., (1993b) Proc. Natl. Acad. Sci. USA 90:
2350-2354) is unable to infect the cells of the SPAFAS outbred
chick embryos routinely used in our lab. However, RCAS-E is able to
infect cells from chick embryos of line 15b. In the majority of
experiments, primary chick embryo fibroblasts (CEFs) prepared from
line 15b embryos in vitro were infected. The infected cells were
pelleted and implanted into a slit made in the anterior of S-SPF
host limb buds. Due to the restricted host range of the vector, the
infection was thus restricted to the graft and did not spread
through the host limb bud.
[0370] To determine the fate of cells implanted and to control for
any effect of the implant procedure, a control RCAS-E vector
expressing human placental alkaline phosphatase was used. Alkaline
phosphatase expression can be easily monitored histochemically and
the location of infected cells can thus be conveniently followed at
any stage. Within 24 hours following implantation the cells are
dispersed proximally and distally within the anterior margin of the
limb bud. Subsequently, cells are seen to disperse throughout the
anterior portion of the limb and into the flank of the embryo.
[0371] Limb buds grafted with alkaline phosphatase expressing cells
or uninfected cells give rise to limbs with structures
indistinguishable from unoperated wild type limbs. Such limbs have
the characteristic anterior-to-posterior digit pattern 2-3-4. ZPA
grafts give rise to a variety of patterns of digits depending on
the placement of the graft within the bud (Tickle, C. et al.,
(1975) Nature 254: 199-202) and the amount of tissue engrafted
(Tickle, C. (1981) Nature 289: 295-298). In some instances the
result can be as weak as the duplication of a single digit 2.
However, in optimal cases the ZPA graft evokes the production of a
full mirror image duplication of digits 4-3-2-2-3-4 or 4-3-2-3-4
(see FIG. 8). A scoring system has been devised which rates the
effectiveness of polarizing activity on the basis of the most
posterior digit duplicated: any graft which leads to the
development of a duplication of digit 4 has been defined as
reflecting 100% polarizing activity (Honig, L. S. et al., (1985) J.
Embryol. Exp. Morph. 87:163-174).
[0372] Grafts of 15b fibroblasts expressing Sonic resulted in a
range of ZPA-like phenotypes. In some instances the resultant limbs
deviate from the wild type solely by the presence of a mirror-image
duplication of digit 2. The most common digit phenotype resulting
from grafting Sonic-infected CEF cells is a mirror-image
duplication of digits 4 and 3 with digit 2 missing: 4-3-3-4. In
many such cases the two central digits appear fused in a 4-3/3-4
pattern. In a number of the cases the grafts induced full
mirror-image duplications of the digits equivalent to optimal ZPA
grafts 4-3-2-2-3-4. Besides the digit duplications, the ectopic
expression of Sonic also gave rise to occasional duplications of
proximal elements including the radius or ulna, the humerus and the
coracoid. While these proximal phenotypes are not features of ZPA
grafts, they are consistent with an anterior-to-posterior
respecification of cell fate. In some instances, most commonly when
the radius or ulna was duplicated, more complex digit patterns were
observed. Typically, an additional digit 3 was formed distal to a
duplicated radius.
[0373] The mirror-image duplications caused by ZPA grafts are not
limited to skeletal elements. For example, feather buds are
normally present only along the posterior edge of the limb. Limbs
exhibiting mirror-image duplications as a result of ectopic Sonic
expression have feather buds on both their anterior and posterior
edges, similar to those observed in ZPA grafts.
[0374] While ZPA grafts have a powerful ability to alter limb
pattern when placed at the anterior margin of a limb bud, they have
no effect when placed at the posterior margin (Saunders, J. W. et
al., (1968) Epithelial-Mesenchymal Interaction, Fleischmayer and
Billingham, eds. (Baltimore: Williams and Wilkins) pp. 78-97).
Presumably, the lack of posterior effect is a result of polarizing
activity already being present in that region of the bud.
Consistent with this, grafts of Sonic expressing cells placed in
the posterior of limb buds never result in changes in the number of
digits. Some such grafts did produce distortions in the shape of
limb elements, the most common being a slight posterior curvature
in the distal tips of digits 3 and 4 when compared to wild type
wings.
[0375] (v) Effect of Ectopic Sonic Expression on Hoxd Gene
Activity
[0376] The correct expression of Hoxd genes is part of the process
by which specific skeletal elements are determined (Morgan, B. A.
et al., (1993) Nature 358: 236-239). A transplant of a ZPA into the
anterior of a chick limb bud ectopically activates sequential
transcription of Hoxd genes in a pattern which mirrors the normal
sequence of Hoxd gene expression (Nohno, T. et al., (1991) Cell 64:
1197-1205; Izpisua-Belmonte, J. C. et al., (1991) Nature 350:
585-589). Since ectopic Sonic expression leads to the same pattern
duplications as a ZPA graft, we reasoned that Sonic would also lead
to sequential activation of Hoxd genes.
[0377] To test this hypothesis, anterior buds were injected with
Sonic/RCAS-A2, a virus which is capable of directly infecting the
host strains of chicken embryos. This approach does not strictly
limit the region expressing Sonic (being only moderately controlled
by the timing, location and titer of viral injection), and thus
might be expected to give a more variable result. However,
experiments testing the kinetics of viral spread in infected limb
buds indicate that infected cells remain localized near the
anterior margin of the bud for at least 48 hours. Hoxd gene
expression was monitored at various times post infection by whole
mount in situ hybridization. As expected, these genes are activated
in a mirror-image pattern relative their expression in the
posterior of control limbs. For example, after 36 hours Hoxd-13 is
expressed in a mirror-image symmetrical pattern in the broadened
distal region of infected limb buds. Similar results were obtained
with other Hoxd genes (manuscript in preparation).
EXAMPLE 4
A Functionally Conserved Homolog of Drosophila Hedgehog is
Expressed in Tissues With Polarizing Activity in Zebrafish
Embryos
[0378] (i) Experimental Procedures
[0379] Cloning and Sequencing
[0380] Approximately 1.5.times.10.sup.6 plaques of a 33 h zebrafish
embryonic .lambda.gt11 cDNA library were screened by plaque
hybridization at low stringency (McGinnis, W. et al., (1984) Nature
308: 428-433) using a mix of two hh sequences as a probe: a
Drosophila hh 400 bp EcoRI fragment and a murine Ihh 264 bp
BamHI-EcoRI exon 2 fragment. Four clones were isolated and
subcloned into the EcoRI sites of pUC18 T3T7 (Pharmacia). Both
strands of clone 8.3 were sequenced using nested deletions
(Pharmacia) and internal oligonucleotide primers. DNA sequences and
derived amino acid sequences were analyzed using "Geneworks"
(Intelligenetics) and the GCG software packages.
[0381] PCR Amplification
[0382] Degenerate oligonucleotides hh5.1 (SEQ ID No:30) and hh3.3
(SEQ ID No:31) were used to amplify genomic zebrafish DNA
[0383] hh 5.1: AG(CA)GITG(CT)AA(AG)GA(AG)(CA)(AG)I(GCT)IAA
[0384] hh 3.3: CTCIACIGCIA(GA)ICK=(GT)IGCIA
[0385] PCR was performed with an initial denaturation at 94.degree.
C. followed by 35 cycles of 47.degree. C. for 1 min, 72.degree. C.
for 2 min and 94.degree. C. for 1 min with a final extension at
72.degree. C. Products were subcloned in pUC18 (Pharmacia).
[0386] In Situ Hybridization
[0387] In situ hybridizations of zebrafish embryos were performed
as described in Oxtoby, E. et al., (1993) Nuc. Acids REs. 21:
1087-1095 with the following modifications: Embryos were rehydrated
in ethanol rather than methanol series; the proteinase K digestion
was reduced to 5 min and subsequent washes were done in PBTw
without glycine; the antibody was preadsorbed in PBTw, 2 mg/ml BSA
without sheep serum; and antibody incubation was performed in PBTw,
2 mg/ml BSA. Drosophila embryos were processed and hybridized as
previously described.
[0388] Histology
[0389] Stained embryos were dehydrated through ethanol:butanol
series, as previously described (Godsave, S. F. et al., (1988)
Development 102: 555-566), and embedded in Fibrowax. 8 .mu.m
sections were cut on an Anglian rotary microtome
[0390] RNA Probe Synthesis
[0391] For analysis of Shh expression, two different templates were
used with consistent results; (i) phh[c] 8.3 linearized with Bgl II
to transcribe an antisense RNA probe that excludes the conserved
region, and (ii) phh[c] 8.3 linearized with Hind III to transcribe
an antisense RNA that covers the complete cDNA. All in situ
hybridizations were performed with the latter probe which gives
better signal. Other probes were as follows: Axial DraI-linearised
p6TIN (Strahle, U. et al., (1993) Genes & Dev. 7: 1436-1446)
using T3 RNA polymerase. gsc linearized with EcoR1 and transcribed
with T7: pax 2 Bam HI-linearized pcF16 (Krauss, S. et al., (1991)
Development 113: 1193-1206) using T7 RNA polymerase. In situ
hybridizations were performed using labelled RNA at a concentration
of 1 ng/ml final concentration. Antisense RNA probes were
transcribed according to the manufacturer's protocol (DIG RNA
Labelling Kit, BCL).
[0392] Zebrafish Strains
[0393] Wild type fish were bred from a founder population obtained
from the Goldfish Bowl, Oxford. The mutant cyclops strain b16 and
the mutant notail strains b160 and b195 were obtained from Eugene,
Oreg. Fish were reared at 28.degree. C. on a 14 h light/10 h dark
cycle.
[0394] RNA Injections
[0395] The open reading frame of Shh was amplified by PCR, using
oligonucleotides 5'-CTGCAGGGATCCACCATGCGGCTTTTGACGAG-3' (SEQ ID
No:32), which contains a consensus Kozak sequence for translation
initiation, and 5'-CTGCAGGGATC-CTTATTCCACACGAGGGATT-3' (SEQ ID
No:33), and subcloned into the BglII site of pSP64T (Kreig, P. A.
et al., (1984) Nuc. Acids Res. 12: 7057-7070). This vector includes
5' and 3' untranslated Xenopus .beta.-Globin sequences for RNA
stabilization and is commonly used for RNA injections experiments
in Xenopus. In vitro transcribed Shh RNA at a concentration of
approximately 100 .mu.g/ml was injected into a single cell of
naturally spawned zebrafish embryos at one-cell to 4-cell stages
using a pressure-pulsed Narishige microinjector. The injected
volume was within the picolitre range. Embryos were fixed 20 to 27
hrs after injection in BT-Fix (Westerfield, M. (1989) The Zebrafish
Book, (Eugene: The University of Oregon Press)) and processed as
described above for whole-mount in situ hybridizations with the
axial probe.
[0396] Transgenic Drosophila
[0397] An EcoR1 fragment, containing the entire Shh ORF, was
purified from the plasmid phh[c]8.3 and ligated with phosphatased
EcoR1 digested transformation vector pCaSpeRhs (Thummel, C. S. et
al., (1988) Gene 74: 445456). The recombinant plasmid, pHS Shh
containing the Shh ORF in the correct orientation relative to the
heat shock promoter, was selected following restriction enzyme
analysis of miniprep DNA from transformed colonies and used to
transform Drosophila embryos using standard microinjection
procedures (Roberts, D. B. (1986), Drosophila, A Practical
Approach, Roberts, D. B., ed., (Oxford: IRL Press) pp. 1-38).
[0398] Ectopic Expression in Drosophila Embryos
[0399] Embryos carrying the appropriate transgenes were collected
over 2 hr intervals, transferred to thin layers of 1% agarose on
glass microscope slides and incubated in a plastic Petri dish
floating in a water bath at 37.degree. C. for 30 min intervals.
Following heat treatment, embryos were returned to 25.degree. C.
prior to being fixed for in situ hybridization with DIG labelled
single stranded Shh, wg or ptc RNA probes as previously described
(Ingham et al., (1991) Curr. Opin. Genet. Dev. 1: 261-267).
[0400] (ii) Molecular Cloning of Zebrafish Hedgehog Homologues
[0401] In an initial attempt to isolate sequences homologous to
Drosophila hh, a zebrafish genomic DNA library was screened at
reduced stringency with a partial cDNA, hhPCR4.1, corresponding to
the first and second exons of the Drosophila gene (Mohler, J. et
al., (1992) Development 115: 957-971). This screen proved
unsuccessful; however, a similar screen of a mouse genomic library
yielded a single clone with significant homology to hh.,
subsequently designated Ihh. A 264 bp BamHI-EcoRI fragment from
this lambda clone containing sequences homologous to the second
exon of the Drosophila gene was subcloned and, together with the
Drosophila partial cDNA fragment, used to screen a .lambda.gt11
zebrafish cDNA library that was prepared from RNA extracted from 33
h old embryos. This screen yielded four clones with overlapping
inserts the longest of which is 1.6 kb in length, herein referred
to as Shh (SEQ ID No:5).
[0402] (iii) A Family of Zebrafish Genes Homologous to the
Drosophila Segment Polarity Gene, Hedgehog
[0403] Alignment of the predicted amino acid sequences of Shh (SEQ
ID No:12) and hh (SEQ ID No:34) revealed an identity of 47%,
confirming that Shh is a homolog of the Drosophila gene. A striking
conservation occurs within exon 2: an 80 amino acid long domain
shows 72% identity between Shh and Drosophila hh. (FIG. 9A). This
domain is also highly conserved in all hh-related genes cloned so
far and is therefore likely to be essential to the function of hh
proteins. A second domain of approximately 30 amino acids close to
the carboxy-terminal end, though it shows only 61% amino-acid
identity, possesses 83% similarity between Shh and hh when allowing
for conservative substitutions and could also, therefore, be of
functional importance (FIG. 9B). Although putative sites of
post-translational modification can be noted, their position is not
conserved between Shh and hh.
[0404] Lee, J. J. et al., (1992) Cell 71: 33-50, identified a
hydrophobic stretch of 21 amino acids flanked downstream by a
putative site of signal sequence cleavage (predicted by the
algorithm of von Heijne, G. (1986) Nuc. Acids Res. 11) close to the
amino-terminal end of hh. Both the hydrophobic stretch and the
putative signal sequence cleavage sites of hh, which suggest it to
be a signaling molecule, are conserved in Shh. In contrast to hh,
Shh does not extend N-terminally to the hydrophobic stretch.
[0405] Using degenerate oligonucleotides corresponding to
amino-acids flanking the domain of high homology between Drosophila
hh and mouse Ihh exons 2 described above, fragments of the expected
size were amplified from zebrafish genomic DNA by PCR. After
subcloning and sequencing, it appeared that three different
sequences were amplified, all of which show high homology to one
another and to Drosophila hh (FIG. 10). One of these corresponds to
Shh therein referred to as 2-hh(a) (SEQ ID No:16) and 2hh(b) (SEQ
ID No:17), while the other two represent additional zebrafish hh
homologs (SEQ ID No:5). cDNAs corresponding to one of these
additional homologs have recently been isolated, confirming that it
is transcribed. Therefore, Shh represents a member of a new
vertebrate gene family.
[0406] (iv) Shh Expression in the Developing Zebrafish Embryo
[0407] Gastrula Stages
[0408] Shh expression is first detected at around the 60% epiboly
stage of embryogenesis in the dorsal mesoderm. Transcript is
present in a triangular shaped area, corresponding to the embryonic
shield, the equivalent of the amphibian organizer, and is
restricted to the inner cell layer, the hypoblast. During
gastrulation, presumptive mesodermal cells involute to form the
hypoblast, and converge towards the future axis of the embryo,
reaching the animal pole at approximately 70% epiboly. At this
stage, Shh-expressing cells extend over the posterior third of the
axis, and the signal intensity is not entirely homogeneous,
appearing stronger at the base than at the apex of the elongating
triangle of cells.
[0409] This early spatial distribution of Shh transcript is
reminiscent of that previously described for axial, a
forkhead-related gene; however, at 80% epiboly, axial expression
extends further towards the animal pole of the embryo and we do not
see Shh expression in the head area at these early developmental
stages.
[0410] By 100% epiboly, at 9.5 hours of development, the posterior
tip of the Shh expression domain now constitutes a continuous band
of cells that extends into the head. To determine the precise
anterior boundary of Shh expression, embryos were simultaneously
hybridized with probes of Shh and pax-2 (previously pax[b]), the
early expression domain of which marks the posterior midbrain
(Krauss, S. et al. (1991) Development 113: 1193-1206). By this
stage, the anterior boundary of the Shh expression domain is
positioned in the centre of the animal pole and coincides
approximately with that of axial. At the same stage, prechordal
plate cells expressing the homeobox gene goosecoid (gsc) overlap
and underlay the presumptive forebrain (Statchel, S. E. et al.,
(1993) Development 117: 1261-1274). Whereas axial is also thought
to be expressed in head mesodermal tissue at this stage, we cannot
be certain whether Shh is expressed in the same cells. Sections of
stained embryos suggest that in the head Shh may by this stage be
expressed exclusively in neuroectodermal tissue.
[0411] (v) Somitogenesis
[0412] By the onset of somitogenesis (approximately 10.5 h of
development), Shh expression in the head is clearly restricted to
the ventral floor of the brain, extending from the tip of the
diencephalon caudally through the hindbrain. At this stage,
expression of axial has also disappeared from the head mesoderm and
is similarly restricted to the floor of the brain; in contrast to
Shh, however, it extends only as far as the anterior boundary of
the midbrain. At this point, gsc expression has become very weak
and is restricted to a ring of cells that appear to be migrating
away from the dorsal midline.
[0413] As somitogenesis continues, Shh expression extends in a
rostral-caudal progression throughout the ventral region of the
central nervous system (CNS). Along the spinal cord, the expression
domain is restricted to a single row of cells, the floor plate, but
gradually broadens in the hindbrain and midbrain to become 5-7
cells in diameter, with a triangular shaped lateral extension in
the ventral diencephalon and two strongly staining bulges at the
tip of the forebrain, presumably in a region fated to become
hypothalamus.
[0414] As induction of Shh in the floor plate occurs, expression in
the underlying mesoderm begins to fade away, in a similar manner to
axial (Strhle, U. et al., (1993) Genes & Dev. 7: 1436-1446).
This downregulation also proceeds in a rostral to caudal sequence,
coinciding with the changes in cell shape that accompany notochord
differentiation. By the 22 somite stage, mesodermal Shh expression
is restricted to the caudal region of the notochord and in the
expanding tail bud where a bulge of undifferentiated cells continue
to express Shh at relatively high levels. Expression in the
midbrain broadens to a rhombic shaped area; cellular rearrangements
that lead to the 90.degree. kink of forebrain structures, position
hypothalamic tissue underneath the ventral midbrain. These
posterior hypothalamic tissues do not express Shh. In addition to
Shh expression in the ventral midbrain, a narrow stripe of
expressing cells extends dorsally on either side of the third
ventricle from the rostral end of the Shh domain in the ventral
midbrain to the anterior end of, but not including, the epiphysis.
The most rostral Shh expressing cells are confined to the
hypothalamus. In the telencephalon, additional Shh expression is
initiated in two 1-2 cell wide stripes.
[0415] By 36 hours of development, Shh expression in the ventral
CNS has undergone further changes. While expression persists in the
floor plate of the tailbud, more rostrally located floor plate
cells in the spinal cord cease to express the gene. In contrast, in
the hindbrain and forebrain Shh expression persists and is further
modified.
[0416] At 26-28 h, Shh expression appears in the pectoral fin
primordia, that are visible as placode like broadenings of cells
underneath the epithelial cell layer that covers the yolk. By 33
hrs of development high levels of transcript are present in the
posterior margin of the pectoral buds; at the same time, expression
is initiated in a narrow stripe at the posterior of the first gill.
Expression continues in the pectoral fin buds in lateral cells in
the early larva. At this stage, Shh transcripts are also detectable
in cells adjacent to the lumen of the foregut.
[0417] (vi) Expression of Shh in Cyclops and Notail Mutants
[0418] Two mutations affecting the differentiation of the Axial
tissues that express Shh have been described in zebrafish embryos
homozygous for the cyclops (cyc) mutation lack a differentiated
floorplate (Hatta, K. et al., (1991) Nature 350: 339-341). By
contrast, homozygous notail (ntl) embryos are characterized by a
failure in notochord maturation and a disruption of normal
development of tail structures (Halpern, M. E. et al., (1993) Cell
75: 99-111).
[0419] A change in Shh expression is apparent in cyc embryos as
early as the end of gastrulation; at this stage, the anterior limit
of expression coincides precisely with the two pax-2 stripes in the
posterior midbrain. Thus, in contrast to wild-type embryos, no Shh
expression is detected in midline structures of the midbrain and
forebrain. By the 5 somite stage, Shh transcripts are present in
the notochord which at this stage extends until rhombomere 4;
however, no expression is detected in more anterior structures.
Furthermore, no Shh expression is detected in the ventral neural
keel, in particular in the ventral portions of the midbrain and
forebrain.
[0420] At 24 hours of development, the morphologically visible cyc
phenotype consists of a fusion of the eyes at the midline due to
the complete absence of the ventral diencephalon. As at earlier
developmental stages, Shh expression is absent from neural tissue.
Shh expression in the extending tail bud of wild-type embryos is
seen as a single row of floor plate cells throughout the spinal
cord. In a cyc mutant, no such Shh induction occurs in cells of the
ventral spinal cord with the exception of some scattered cells that
show transient expression near the tail. Similarly, no Shh
expression is seen rostrally in the ventral neural tube. However, a
small group of Shh expressing cells is detected underneath the
epiphysis which presumably correspond to the dorsal-most group of
Shh expressing cells in the diencephalon of wild-type embryos.
[0421] In homozygous notail (ntl) embryos, no Shh staining is seen
in mesodermal tissue at 24 hours of development, consistent with
the lack of a notochord in these embryos; by contrast, expression
throughout the ventral CNS is unaffected. At the tail bud stage,
however, just prior to the onset of somitogenesis, Shh expression
is clearly detectable in notochord precursor cells.
[0422] (vii) Injection of Synthetic Shh Transcripts Into Zebrafish
Embryos Induces Expression of a Floor Plate Marker
[0423] To investigate the activity of Shh in the developing embryo,
an over-expression strategy, similar to that employed in the
analysis of gene function in Xenopus, was adopted. Newly fertilized
zebrafish eggs were injected with synthetic Shh RNA and were fixed
14 or 24 hours later. As an assay for possible changes in cell fate
consequent upon the ectopic activity of Shh, we decided to analyze
Axial expression, since this gene serves as a marker for cells in
which Shh is normally expressed. A dramatic, though highly
localized ectopic expression of Axial in a significant proportion
(21/80) of the injected embryos fixed after 24 hours of development
is observed. Affected embryos show a broadening of the Axial
expression domain in the diencephalon and ectopic Axial expression
in the midbrain; however, in no case has ectopic expression in the
telencephalon or spinal cord been observed. Many of the injected
embryos also showed disturbed forebrain structures, in particular
smaller ventricles and poorly developed eyes. Arnongst embryos
fixed after 14 h, a similar proportion (8/42) exhibit the same
broadening and dorsal extension of the Axial stripe in the
diencephalon as well as a dorsal extension of Axial staining in the
midbrain; again, no changes in Axial expression were observed
caudal to the hindbrain with the exception of an increased number
of expressing cells at the tip of the tail.
[0424] (viii) Overexpression of Shh in Drosophila Embryos Activates
the hh-Dependent Pathway
[0425] In order to discover whether the high degree of structural
homology between the Drosophila and zebrafish hh genes also extends
to the functional level, an overexpression system was used to test
the activity of Shh in flies. Expression of Drosophila hh driven by
the HSP70 promoter results in the ectopic activation of both the
normal targets of hh activity; the wg transcriptional domain
expands to fill between one third to one half of each parasegment
whereas ptc is ectopically activated in all cells except those
expressing en (Ingham, P. W. (1993) Nature 366:560-562). To compare
the activities of the fly and fish genes, flies transgenic for a HS
Shh construct were generated described above and subjected to the
same heat shock regime as H Shh transgenic flies. HS Shh embryos
fixed immediately after the second of two 30 min heat shocks
exhibit ubiquitous transcription of the Shh cDNA. Similarly treated
embryos were fixed 30 or 90 min after the second heat shock and
assayed for wg or ptc transcription. Both genes were found to be
ectopically activated in a similar manner to that seen in heat
shocked H Shh embryos; thus, the zebrafish Shh gene can activate
the same pathway as the endogenous hh gene.
EXAMPLE 5
Cloning, Expression and Localization of Human Hedgehogs
[0426] (i) Experimental Procedures
[0427] Isolation of Human Hedgehog cDNA Clones
[0428] Degenerate nucleotides used to clone chick Shh (Riddle et
al., (1993) Cell 75:1401-1416) were used to amplify by nested PCR
human genomic DNA. The nucleotide sequence of these oligos is as
follows:
[0429]
vHH5O:5'-GGAATTCCCAG(CA)GITG(CT)AA(AG)GA(AG)(CA)(AG)I(GCT)TIAA-3'
(SEQ ID NO:18);
[0430] vHH3O:5'-TCATCGATGGACCCA(GA)TC(GA)AAICCIGC(TC)TC-3' (SEQ ID
NO:19);
[0431] vHH3I:5'-GCTCTAGAGCTCIACIGCIA(GA)IC(GT)IGGIA-3' (SEQ ID
NO:20)
[0432] The expected 220 bp PCR product was subcloned into pGEM7zf
(Promega) and sequenced using Sequenase v2.0 (U.S. Biochemicals).
One clone showed high nucleotide similarity to mouse Ihh and mouse
Shh sequence (Echelard et al., (1993) Cell 75:1417-1430) and it was
used for screening a human fetal lung 5'-stretch plus cDNA library
(Clontech) in .lambda.gt10 phage. The library was screened
following the protocol suggested by the company and two positive
plaques were identified, purified, subcloned into pBluescript SK+
(Stratagene) and sequenced, identifying them as the human
homologues of Shh (SEQ ID NO:6) and Ihh (SEQ ID NO:7).
[0433] One clone contained the full coding sequence of a human
homolog of Shh as well as 150 bp of 5' and 36 bp of 3' untranslated
sequence. The other clone, which is the human homolog of Ihh,
extends from 330 bp 3' of the coding sequence to a point close to
the predicted boundary between the first and second exon. The
identity of these clones was determined by comparison to the murine
and chick genes. The protein encoded by human Shh has 92.4% overall
identity to the mouse Shh, including 99% identity in the
amino-terminal half. The carboxyl-terminal half is also highly
conserved, although it contains short stretches of 16 and 11 amino
acids not present in the mouse Shh. The human Ihh protein is 96.8%
identical to the mouse Ihh. The two predicted human proteins are
also highly related, particularly in their amino-terminal halves
where they are 91.4% identical. They diverge significantly in their
carboxyl halves, where they show only 45.1% identity. The high
level of similarity in the amino portion of all of these proteins
implies that this region encodes domains essential to the activity
of this class of signaling molecules.
[0434] Northern Blotting
[0435] Multiple Tissue Northern Blot (Clontech) prepared from poly
A+RNA isolated from human adult tissues was hybridized with either
full length .sup.32P-labeled human Shh clone or .sup.32P-labeled
human Ihh clone following the protocol suggested by the
company.
[0436] Digoxigenin in Situ Hybridization.
[0437] Sections: tissues from normal human second trimester
gestation abortus specimens were washed in PBS and fixed overnight
at 4.degree. C. paraformaldehyde in PBS, equilibrated 24 hours at
4.degree. C. in 50% sucrose in PBS and then placed in 50% sucrose
in oct for one hour before embedding in oct. Cryostat sections
(10-25 mm) were collected on superfrost plus slides (Fisher) and
frozen at -80.degree. C. until used. Following a postfixation in 4%
paraformaldehyde the slides were processed as in Riddle et al.,
(1993) Cell 75:1401-1416 with the following alterations: proteinase
K digestion was performed at room temperature from 1-15 minutes
(depending on section thickness), prehybridization, hybridization
and washes time was decreased to 1/10 of time.
[0438] Whole-mounts: tissues from normal second trimester human
abortus specimens were washed in PBS, fixed overnight at 4.degree.
C. in 4% paraformaldehyde in PBS and then processed as in Riddle et
al., (1993) Cell 75:1401-1416.
[0439] Isolation of an Shh P1 Clone.
[0440] The human Shh gene was isolated on a P1 clone from a P1
library (Pierce and Sternberg, 1992) by PCR (polymerase chain
reaction) screening. Two oligonucleotide primers were derived from
the human Shh sequence. The two olignucleotide primers used for PCR
were:
[0441] SHHF5'-ACCGAGGGCTGGGACGAAGATGGC-3' (SEQ ID NO:43)
[0442] SHR5'-CGCTCGGTCGTACGGCATGAACGAC-3' (SEQ ID NO:44)
[0443] The PCR reaction was carried using standard conditions as
described previously (Thierfelder et al., 1994) except that the
annealing temperature was 65.degree. C. These primers amplified a
119 bp fragment from human and P1 clone DNA. The P1 clone was
designated SHHP1. After the P1 clone was isolated these
oligonucleotides were used as sequencing primers. A 2.5 Kb EcoRI
fragment that encoded a CA repeat was subcloned from this P1 clone
using methods described previously (Thierfelder et al. 1994).
Oligonucleotide primers that amplified this CA repeat sequence were
fashioned from the flanking sequences:
[0444] SHHCAF5'-ATGGGGATGTGTGTGGTCAAGTGTA-3' (SEQ ID NO:45)
[0445] SHHCAR5'-TTCACAGACTCTCAAAGTGTATTTT-3' (SEQ ID NO:46)
[0446] Mapping the Human Ihh and Shh Genes.
[0447] The human Ihh gene was mapped to chromosome 2 using somatic
cell hybrids from NIGMS mapping pannel 2 (GM10826B).
[0448] The Shh gene was mapped to chromosome 7 using somatic cell
hybrids from NIGMS mapping panel 2 (GM10791 and GM10868).
[0449] Linkage between the limb deformity locus on chromosome 7 and
the Shh gene was demonstrated using standard procedures. Family LD
has been described previously (Tkukurov et al., (1994) Nature
Genet. 6:282-286). A CA repeat bearing sequence near the Shh gene
was amplified from the DNA of all members of Family LD by PCR using
the SHHCAF and SHHCAR primers. Linkage between the CA repeat and
the LD disease gene segregating in Family LD was estimated by the
MLINK program (October 1967). Penetrance was set at 100% and the
allele frequencies were determined using unrelated spouses in the
LD family.
[0450] Interspecific Backcross Mapping
[0451] Interspecific backcross progeny were generated by mating
(C57BL/6J.times.M. spretus) F1 females and C57BL/6J males as
described (Copeland and Jenkins, (1991) Trends Genet. 7:113-118). A
total of 205 N2 mice were used to map the Ihh and Dhh loci. DNA
isolation, restriction enzyme digestions, agarose gel
electrophoresis, Southern blot transfer and hybridization were
performed essentially as described (Jenkins et al., (1982) J.
Virol. 43:26-36). All blots were prepared with Hybond-N+ nylon
membrane (Amersham). The probe, an .about.1.8 kb EcoRI fragment of
mouse cDNA, detected a major fragment of 8.5 kb in C57BL/6j (B) DNA
and a major fragment 6.0 kb in M. spretus (S) DNA following
digestion with BgIII. The Shh probe, an .about.900 bp SmaI fragment
of mouse cDNA, detected HincII fragments of 7.5 and 2.1 kb (B) as
well as 4.6 and 2.1 (S). The Dhh probe, and .about.800 bp
BamHi/EcoRi fragment of mouse genomic DNA, detected major fragments
of 4.7 and 1.3 kb (B) and 8.2 and 1.3 kb (S) following digestion
with SphI. The presence or absence of M. spretus specific fragments
was followed in backcross mice.
[0452] A description of the probes and RFLPs for loci used to
position the Ihh, Shh and Dhh loci in the interspecific backcross
has been reported. These include: Fn1, Vil and Acrg, chromosome 1
(Wilkie et al., (1993) Genomics 18:175-184), Gnai1, En2, Il6,
chromosomes 5 (Miao et al., (1994) PNAS USA 91:11050-11054) and
Pdgfb, Gdc1 and Rarg, chromosome 15 (Brannan et al., (1992)
Genomics 13:1075-1081). Recombination distances were calculated as
described (Green, (1981) Linkage, recombination and mapping. In
"Genetics and Probability in Animal Breeding Experiments", pp.
77-113, Oxford University Press, NY) using the computer program
SPRETUS MADNESS. Gene order was determined by minimizing the number
of recombination events required to explain the allele distribution
patterns.
[0453] (ii) Expression of Human Shh and Ihh
[0454] To investigate the tissue distribution of Shh and Ihh
expression, poly(A)+RNA samples from various adult human tissues
were probed with the two cDNA clones. Of the tissues tested, an
Ihh-specific message of .about.2.7 kb is only detected in liver and
kidney. Shh transcripts was not detected in the RNA from any of the
adult tissues tested. All the samples contained approximately equal
amounts of intact RNA, as determined by hybridization with a
control probe.
[0455] The hedgehog family of genes were identified as mediators of
embryonic patterning in flies and vertebrates. No adult expression
of these genes had previously been reported. These results indicate
that Ihh additionally plays a role in adult liver and kidney. Since
the hedgehog genes encode intercellular signals, Ihh may function
in coordinating the properties of different cell types in these
organs. Shh may also be used as a signaling molecule in the adult,
either in tissues not looked at here, or at levels too low to be
detected under these conditions.
[0456] In situ hybridization was used to investigate the expression
of Shh in various mid-gestational human fetal organs. Shh
expression is present predominantly in endoderm derived tissues:
the respiratory epithelium, collecting ducts of the kidney,
transitional epithelium of the ureter, hepatocytes, and small
intestine epithelium. Shh was not detectable in fetal heart or
placental tissues. The intensity of expression is increased in
primitive differentiating tissues (renal blastema, base villi,
branching lung buds) and decreased or absent in differentiated
tissues (e.g. glomeruli). Shh expression is present in the
mesenchyme immediately abutting the budding respiratory tubes. The
non-uniform pattern of Shh expression in hepatocytes is consistent
with expression of other genes in adult liver (Dingemanse et al.,
(1994) Differentiation 56:153-162). The base of villi, the renal
blastema, and the lung buds are all regions expressing Shh and they
are areas of active growth and differentiation, suggesting Shh is
important in these processes.
[0457] (iii) The Chromosomal Map Location of Human Shh and Ihh
[0458] Since Shh is known to mediate patterning during the
development of the mouse and chick and the expression of Shh and
Ihh are suggestive of a similar role in humans, mutations in these
genes would be expected to lead to embryonic lethality or
congenital defects. One way of investigating this possibility is to
see whether they are genetically linked to any known inherited
disorders.
[0459] Shh- and Ihh-specific primers were designed from their
respective sequences and were used in PCR reactions on a panel of
rodent-human somatic cell hybrids. Control rodent DNA did not
amplify specific bands using these primers. In contrast, DNA from
several rodent-human hybrids resulted in PCR products of the
appropriate size allowing us to assign Shh to chromosome 7q and Ihh
to chromosome 2.
[0460] One of the central roles of chick Shh is in regulating the
anterior-posterior axis of the limb. A human congenital
polysyndactyly has recently been mapped to chromosome 7q36
(Tsukurov et al., (1994) Nature Genet. 6:282-286; Heutink et al.,
(1994) Nature Genet. 6:287-291). The phenotype of this disease is
consistent with defects that might be expected from aberrant
expression of Shh in the limb. Therefore, the chromosomal location
of Shh was mapped more precisely, in particular in relation to the
polysyndactyly locus.
[0461] A P1 phage library was screened using the Shh specific
primers for PCR amplification and clone SHHP1 was isolated. Clone
SHHP1 contained Shh sequence. A Southern blot of an EcoRi digest of
this phage using [CA]/[GT] probe demonstrated that a 2.5 Kb EcoRi
fragment contained a CA repeat. Nucleotide sequence analysis of
this subcloned EcoRI fragment demonstrated that the CA repeat lay
near the EcoRI sites. Primers flanking the CA repeat were designed
and used to map the location of Shh relative to other markers on 7q
in individuals of a large kindred with complex polysyndactyly
(Tsukurov et al., (1994) Nature Genet. 6:282-286). Shh maps close
to D75550 on 7q36, with no recombination events seen in this study.
It is also extremely close to, but distinct from, the
polysyndactyly locus with one recombination event observed between
them (maximum lod score=4.82, .THETA.=0.05). One unaffected
individual (pedigree ID V-10 in Tsukurov et al., (1994) Nature
Genet. 6:282-286) has the Shh linked CA repeat allele found in all
affected family members. No recombination was observed between the
locus En2 and the Shh gene (maximum lod score=1.82,
.THETA.=0.0).
[0462] (iv) Chromosomal Mapping of the Murine Ihh, Shh and Dhh
Genes
[0463] The murine chromosomal location of Ihh, Shh and Dhh was
determined using an interspecific backcross mapping panel derived
from crosses of [(C57BL/6J.times.M spetrus)F1.times.C57BL/J)] mice.
cDNA fragments from each locus were used as probes in Southern blot
hybridization analysis of C57BL/6J and M. spretus genomic DNA that
was separately digested with several different restriction enzymes
to identify informative restriction fragment length polymorphisms
(RFLPs) useful for gene mapping. The strain distribution pattern of
each RFLP in the interspecific backcross was then determined by
following the presence or absence of RFLPs specific for M spretus
in backcross mice.
[0464] Ihh mapped to the central region of mouse chromosome 1, 2.7
cM distal of Fn1 and did not recombine with Vil in 190 animals
typed in common, suggesting that the two loci are within 1.6 cM
(upper 95% confidence level) (FIG. 16). Shh mapped to the proximal
region of mouse chromosome 5, 0.6 cM distal of En2 and 1.9 cM
proximal of I16 in accordance to Chang et al., (1994) Development
120:3339-3353. Dhh mapped to the very distal region of mouse
chromosome 15, 0.6 cM distal of Gdc1 and did not recombine with
Rarg in 160 animals typed in common, suggesting that the two loci
are within 1.9 cM of each other (upper 95% confidence level) (FIG.
16).
[0465] Interspecific maps of chromosome 1, 5 and 15 were compared
with composite mouse linkage maps that report the map location of
many uncloned mouse mutations (compiled by M. T. Davisson, T. H.
Roderick, A. L. Hillyard and D. P. Doolittle and provided from
GBASE, a computerized database maintained at The Jackson
Laboratory, Bar Harbor, Me.). The hemimelic extra-toe (Hx) mouse
mutant maps 1.1 cM distal to En2 on chromosome 5 (Martin et al.,
(1990) Genomics 6:302-308), a location in close proximity to where
Shh has been positioned. Hx is a dominant mutation which results in
preaxial polydactyly and hemimelia affecting all four limbs
(Dickie, (1968) Mouse News Lett 38:24; Knudsen and Kochhar, (1981)
J. Embryol. Exp. Morph. 65: Suppl. 289-307). Shh has previously
been shown to be expressed in the limb (Echelard et al., (1993)
Cell 75:1417-1430). To determine whether Shh and Hx are tightly
linked we followed their distribution in a backcross panel in which
Hx was segregating. Two recombinants between Shh and Hx were
identified, thus excluding the possibility that the two loci are
allelic and these observations are again consistent with those of
Chang et al., (1994) Development 120:3339-3353. While there are
several other mutations in the vicinity of Ihh and Dhh, none is an
obvious candidate for an alteration in the corresponding gene.
[0466] The central region of mouse chromosome 1 shares homology
with human chromosome 2q (summarized in FIG. 16). Placement of Ihh
in this interval suggests the human homolog of Ihh will reside on
2q, as well. Similarly, it is likely that human homolog of Dhh will
reside on human chromosome 12q.
EXAMPLE 6
Proteolytic Processing Yields Two Secreted Forms of Sonic
Hedgehog
[0467] (i) Experimental Procedures
[0468] In Vitro Translation and Processing
[0469] Mouse and chick sonic hedgehog coding sequences were
inserted into the vector pSP64T (kindly provided by D. Melton)
which contains an SP6 phage promoter and both 5' and 3'
untranslated sequences derived from the Xenopus laevis
.beta.-Globin gene. After restriction endonuclease digestion with
Sal I to generate linear templates, RNA was transcribed in vitro
using SP6 RNA polymerase (Promega, Inc.) in the presence of 1 mM
cap structure analog (m.sup.7G(5')ppp(5')Gm; Boehringer-Mannheim,
Inc.) Following digestion with RQ1 DNase I (Promega, Inc.) to
remove the DNA template, transcripts were purified by
phenol:choloroform extraction and ethanol precipitation.
[0470] Rabbit reticulocyte lysate (Promega, Inc.) was used
according to the manufacturer's instructions. For each reaction,
12.5 .mu.l of lysate was programmed with 0.5-2.0 .mu.g of in vitro
transcribed RNA. The reactions contained 20 .mu.Ci of Express
labeling mix (NEN/DuPont, Inc.) were included. To address
processing and secretion in vitro, 1.0-2.0 .mu.l of canine
pancreatic microsomal membranes (Promega, Inc.) were included in
the reactions. The final reaction volume of 25 .mu.l was incubated
for one hour at 30.degree. C. Aliquots of each reaction (between
0.25 and 3.0 .mu.l) were boiled for 3 minutes in Lacmmli sample
buffer (LSB: 125 mM Tris-Hcl [pH 6.8]; 2% SDS; 1%
2-mercaptoethanol; 0.25 mg/ml bromophenol blue) before separating
on a 15% polyacrylamide gel. Fixed gels were processed for
fluorography using EnHance (NEN/DuPont, Inc.) as described by the
manufacturer.
[0471] Glycosylation was addressed by incubation with
Endoglycosidase H (Endo H; New England Biolabs, Inc.) according to
the manufacturer's directions. Reactions were carried out for 1-2
hr at 37.degree. C. before analyzing reaction products by
polyacrylamide gel electrophoresis (PAGE).
[0472] Xenopus Oocyte Injection and Labeling
[0473] Oocytes were enzymatically defolliculated and rinsed with
OR2 (50 mM HEPES [pH 7.2], 82 mM NaCl, 2.5 mM KCl, 1.5 mM Na2HPO4).
Healthy stage six oocytes were injected with 30 ng of in vitro
transcribed, capped mouse Shh RNA (prepared as described above).
Following a 2 hr recovery period, healthy injected oocytes and
uninjected controls were cultured at room temperature in groups of
ten in 96-well dishes containing 0.2 ml of OR2 (supplemented with
0.1 mg/ml Gentamicin and 0.4 mg/ml BSA) per well. The incubation
medium was supplemented with 50 .mu.Ci of Express labeling mix.
Three days after injection, the culture media were collected and
expression of Shh protein analyzed by immunoprecipitation. Oocytes
were rinsed several times in OR2 before lysing in TENT (20 mM
Tris-HCl [pH 8.0]; 150 mM NaCl, 2mM EDTA; 1% Triton-X-100; 10
.mu.l/oocyte) supplemented with 1 .mu.g/ml aprotinin, 2 .mu.g/ml
leupeptin and 1 mM phenylmethylsufonylfluoride (PMSF). After
centrifugation at 13000.times.g for 10 minutes at 4.degree. C.,
soluble protein supernatants were recovered and analyzed by
immunoprecipitation (see below).
[0474] Cos Cell Transfection and Labeling
[0475] Cos cells were cultured in Dulbecco's Modified Eagle Medium
(DMEM; Sigma, Inc.) supplemented with 10% fetal bovine serum
(Gibco/BRL), 2 mM L-Glutamine (Gibco/BRL) and 50 mU/ml penicillin
and 50 .mu.g/ml streptomycin (Gibco/BRL). Subconfluent cos cells in
35 mm or 60 mm dishes (Falcon, Inc.) were transiently transfected
with 2 mg or 6 mg supercoiled plasmid DNA, respectively. Between 42
and 44 hr post-transfection, cells were labeled for 4-6 hr in 0.5
ml (35 mm dishes) or 1.5 ml (60 mm dishes) serum-free DMEM lacking
Cysteine and Methionine (Gibco/BRL) and supplemented with 125
.mu.Ci/ml each of Express labeling mix and L-35S-Cysteine
(NEN/DuPont). After labeling, media were collected and used for
immunoprecipitation. Cells were rinsed with cold PBS and lysed in
the tissue culture dishes by the addition of 0.5 ml (35 mm dishes)
or 1.5 ml (60 mm dishes) TENT (with protease inhibitors as
described above) and gentle rocking for 30 minutes at 4.degree. C.
Lysates were cleared by centrifugation (13000.times.g for 5 min. at
4.degree. C.) and the supernatants were analyzed by
immunoprecipitation (see below).
[0476] Baculovirus Production and Infection
[0477] A recombinant baculovirus expressing mouse sonic hedgehog
with a myc epitope tag inserted at the carboxy terminus was
generated using the Baculogold kit (Pharmingen, Inc.). The initial
virus production used Sf 9 cells, followed by two rounds of
amplification in High Five cells (Invitrogen, Inc.) in serum-free
medium (ExCell 401; Invitrogen, Inc.). A baculovirus lacking Shh
coding sequences was also constructed as a control. For protein
induction, High Five cells were infected at a multiplicity of
approximately 15. Three days later, medium and cells were collected
by gentle pipetting. Cells were collected by centrifugation
(1000.times.g) and the medium was recovered for Western blot
analysis. Cell pellets were washed twice in cold PBS and lysed in
TENT plus protease inhibitors (see above) by rotating for 30
minutes at 4.degree. C. in a microcentrifuge tube. The lysate was
cleared as described above prior to Western blotting.
[0478] Western Blotting
[0479] For Western blotting, 0.25 ml samples of media (1% of the
total) were precipitated with TCA and redissolved in 15 .mu.l of
LSB. Cell lysate samples (1% of total) were brought to a final
volume of 15 .mu.l with water and concentrated (5.times.) LSB .
Samples were boiled S minutes prior to separation on a 15%
acrylamide gel. Proteins were transferred to PVDF membrane
(Immobilon-P; Millipore, Inc.) and blocked in BLOTTO (5% w/v
non-fat dried milk in PBS) containing 0.2% Tween-20. Hybridoma
supernatant recognizing the human c-myc epitope (9E10; Evan, G. I.
et al., (1985) Mol. Cell. Biol. 5:3610-3616) was added at a
dilution of 1:200 for one hour followed by a 1:5000 dilution of
Goat anti-Mouse-Alkaline phosphatase conjugate (Promega, Inc.) for
30 minutes. Bands were visualized using the Lumi-Phos 530 reagent
(Boehringer-Mannheim) according to the manufacturer's
directions.
[0480] For Western blotting of COS cell material, cleared media
(see above) were precipitated with TCA in the presence of 4.mu.g of
BSA per ml as a carrier. the protein pellets were dissolved in 20
.mu.l of LSB. Dissolved medium protein and cell lysates (see above)
were boiled for 5 min, and 10 .mu.l (50%) of each medium sample and
10 .mu.l (10%) of each cell lysate were separated on a 15%
acrylamide gel. The gel was blotted to a polyvinylidene difluoride
membrane as described above. The membrane was blocked as described
above and incubated in a 1:200 dilution of affinity-purified Shh
antiserum (see below) and then in a 1:5,000 dilution of horseradish
peroxidase-conjugated donkey anti-rabbit immunoglobulin g (IgG;
Jackson Immuno research, Inc.). Bands were visualized with the
Enhanced Chemiluminescence kit (Amersham, Inc.) according to the
manufacturer's instructions.
[0481] For Western blotting of mouse and chicken embryonic tissue
lysates, 60 .mu.g of each sample was separated on 15% acrylamide
gels. Blotting and probing with affinity-purified Shh antiserum as
well as chemiluminescence detection were carried out as described
above for the COS cell material.
[0482] Immunoprecipitation
[0483] Cell lysates (Xenopus oocytes or cos cells) were brought to
0.5 ml with TENT (plus protease inhibitors as above). Media samples
(OR2 or DMEM) were cleared by centrifugation at 13000.times.g for 5
min. (4.degree. C.) and 10.times.TENT was added to a final
concentration of 1.times. (final volume: 0.5-1.5 ml). The c-myc
monoclonal antibody hybridoma supernatant was added to 1/20 of the
final volume. Samples were rotated for 1 hr at 40.degree. C., then
0.1 ml of 10% (v/v) protein A-Sepharose CL-4B (Pharmacia, Inc.) was
added. Samples were rotated an additional 14-16 h. Immune complexes
were washed 4 times with 1.0 ml TENT. Immunoprecipitated material
was eluted and denatured by boiling for 10 minutes in 25 .mu.l
1.times.LSB. Following centrifugation, samples were separated on
15% acrylamide gels and processed for fluorography as described
previously. Samples for Endo H digestion were eluted and denatured
by boiling for 10 minutes in the provided denaturation buffer
followed by digestion with Endo H for 1-2 hr at 37.degree. C.
Concentrated (SX) LSB was added and the samples were processed for
electrophoresis as described.
[0484] For immunoprecipitation with the anti-mouse Shh serum,
samples (Cos cell lysates and DMEM) were precleared by incubating 1
hr on ice with 3 .mu.l pre-immune serum, followed by the addition
of 0.1 ml 10% (v/v) Protein A-Sepharose. After rotating for 1 hr at
4 C., supernatants were recovered and incubated for 1 hr on ice
with 3 .mu.l depleted anti-mouse Shh serum (see below). Incubation
with Protein A-Sepharose, washing, elution and electrophoresis were
then performed as described above.
[0485] Immunofluorescent Staining of Cos Cells
[0486] Twenty-four hours after transfection, cells were transferred
to 8-chamber slides (Lab-Tek, Inc.) and allowed to attach an
additional twenty-four hours. Cells were fixed in 2%
paraformaldehyde/0.1% glutaraldehyde, washed in PBS and
permeabilized in 1% Triton-X-100 (Munro, S. and Pelham, H. R. B.,
(1987) Cell 48:899-907). After washing in PBS, cells were treated
for 10 minutes in 1 mg/ml sodium borohydride. Cells were incubated
with the c-myc monoclonal antibody hybridoma supernatant (diluted
1:10) and the affinity purified mouse Sonic hedgehog antiserum
(diluted 1:4) for 45 minutes followed by incubation in 1:100
Goat-anti Mouse IgG-RITC plus 1:100 Goat anti Rabbit IgG FlTC
(Southern Biotechnology Associates, Inc.) for 45 minutes. DAPI
(Sigma, Inc.) was included at 0.3 .mu.g/ml The slides were mounted
in Slo-Fade (Molecular Probes, Inc.) and photographed on a Leitz
DMR compound microscope.
[0487] Embryonic Tissue Dissection and Lysis
[0488] Mouse forebrain, midbrain, hindbrain, lung, limb, stomach,
and liver tissues form 15.5-day-postcoitum Swiss Webster embryos
were dissected into cold PBS, washed several times in PBS, and then
lysed by trituraton and gentle sonication in LSB lacking
bromophenol blue. Lysates were cleared by brief centrifugation, and
protein concentrations were determined by the Bradford dye-binding
assay.
[0489] To obtain chicken CNS and limb bud tissue, fertilized eggs
(Spafas, Inc.) were incubated at 37.degree. C. until the embryos
reached stages 20 and 25, respectively (Hamburg and Hamilton (1951)
J. Exp. Morphol. 88:49-92). By using sharp tungsten needles, dorsal
and ventral pieces of the anterior CNS were obtained from the stage
15 embryos, and limb buds from the stage 25 embryos were cut into
anterior and posterior halves. Tissues were lysed, and protein
concentrations were determined as described above. Prior to
electrophoresis of the mouse and chicken proteins (see above),
samples were brought to 20 .mu.l with LSB containing bromophenol
blue and boiled for 5 minutes.
[0490] Antibody Production and Purification
[0491] A PCR fragment encoding amino acids 44-143 of mouse Sonic
hedgehog was cloned in frame into the Eco RI site of pGEX-2T
(Pharmacia, Inc.). Transformed bacteria were induced with IPTG and
the fusion protein purified on a Glutathione-Agarose affinity
column (Pharmacia, Inc.) according to the manufacturer's
instructions. Inoculation of New Zealand White rabbits, as well as
test and production bleeding were carried out at Hazelton Research
Products, Inc.
[0492] To deplete the serum of antibodies against
Glutathione-S-transferas- e (GST) and bacterial proteins, a lysate
of E. coli transformed with pGEX-2T and induced with IPTG was
coupled to Affi-Gel 10 (Bio-Rad, Inc.) The serum was incubated in
batch for two hours with the depletion matrix before centrifugation
(1000.times.g for 5 min.) and collection of the supernatant. To
make an affinity matrix, purified bacterially expressed protein
corresponding to the amino terminal two-thirds of mouse Sonic
hedgehog was coupled to Affi-Gel 10 (Bio-Rad, Inc.). The depleted
antiserum was first adsorbed to this matrix in batch, then
transferred to a column. The matrix was washed with TBST (25 mM
Tris-HCl [pH 7.5], 140 mM NaCl, 5 mM KCl, 0.1% Triton-X-100), and
the purified antibodies were eluted with ten bed volumes of 0.15 M
Glycine [pH 2.5]. The solution was neutralized with one volume of 1
M Tris-HCl [pH 8.0], and dialyzed against 160 volumes of PBS.
[0493] Other antibodies have been generated against hedgehog
proteins and three polyclonal rabbit antisera obtained to hh
proteins can be characterized as follows: Ab77-reacts only with the
carboxyl processed chick Shh peptide (27 kd); Ab79-reacts with
amino processed chick, mouse and human Shh peptide (19 kd). Weakly
reacts with 27 kd peptide from chick and mouse. Also reacts with
mouse Ihh; and Ab80-reacts with only amino peptide (19 kd) of
chick, mouse and human.
[0494] (ii) In Vitro Translated Sonic Hedgehog is Proteolytically
Processed and Glycosylated
[0495] The open reading frames of chick and mouse Shh encode
primary translation products of 425 and 437 amino acids,
respectively, with predicted molecular masses of 46.4 kilodaltons
(kDa) and 47.8 kDa (Echelard, Y. et al., (1993) Cell 75:1417-1430;
Riddle, R. D. et al., (1993) Cell 75:1401-1416). Further
examination of the protein sequences revealed a short stretch of
amino terminal residues (26 for chick, 24 for mouse) that are
highly hydrophobic and are predicted to encode signal peptides.
Removal of these sequences would generate proteins of 43.7 kDa
(chick Shh) and 45.3 kDa (mouse Shh). Also, each protein contains a
single consensus site for N-linked glycosylation (Tarentino, A. L.
et al., (1989) Methods Cell Biol. 32:111-139) at residue 282
(chick) and 279 (mouse). These features of the Shh proteins are
summarized in FIG. 11.
[0496] A rabbit reticulocyte lysate programmed with in vitro
translated messenger RNA encoding either chick or mouse Shh
synthesizes proteins with molecular masses of 46 kDa and 47 kDa,
respectively. These values are in good agreement with those
predicted by examination of the amino acid sequences. To examine
posttranslational modifications of Shh proteins, a preparation of
canine pancreatic microsomal membranes was included in the
translation reactions. This preparation allows such processes as
signal peptide cleavage and core glycosylation. When the Shh
proteins are synthesized in the presence of these membranes, two
products with apparent molecular masses of approximately 19 and 28
kDa (chick), or 19 and 30 kDa (mouse) are seen in addition to the
46 kDa and 47 kDa forms. When the material synthesized in the
presence of the membranes is digested with Endoglycosidase H (Endo
H), the mobilities of the two larger proteins are increased. The
apparent molecular masses of the Endo H digested forms are 44 kDa
and 26 kDa for chick Shh, and 45 kDa and 27 kDa for mouse Shh. The
decrease in the molecular masses of the largest proteins
synthesized in the presence of the microsomal membranes after Endo
H digestion is consistent with removal of the predicted signal
peptides. The mobility shift following Endo H treatment indicates
that N-linked glycosylation occurs, and that the 26 kDa (chick) and
27 kDa (mouse) proteins contain the glycosylation sites.
[0497] The appearance of the two lower molecular weight bands
(hereafter referred to as the "processed forms") upon translation
in the presence of microsomal membranes suggests that a proteolytic
event in addition to signal peptide cleavage takes place. The
combined molecular masses of the processed forms (19 kDa and 26 kDa
for chick; 19 kDa and 27 kDa for mouse) add up to approximately the
predicted masses of the signal peptide cleaved proteins (44 kDa for
chick and 45 kDa for mouse) suggesting that only a single
additional cleavage occurs.
[0498] The mouse Shh protein sequence is 12 amino acid residues
longer than the chick sequence (437 versus 425 residues). Alignment
of the chick and mouse Shh protein sequences reveals that these
additional amino acids are near the carboxy terminus of the protein
(Echelard, Y. et al., (1993) Cell 75:1417-1430). Since the larger
of the processed forms differ in molecular mass by approximately 1
kDa between the two species, it appears that these peptides contain
the carboxy terminal portions of the Shh proteins. The smaller
processed forms, whose molecular masses are identical, presumably
consist of the amino terminal portions.
[0499] (iii) Secretion of Shh Peptides
[0500] To investigate the synthesis of Shh proteins in vivo, the
mouse protein was expressed in several different eukaryotic cell
types. In order to detect synthesized protein, and to facilitate
future purification, the carboxy terminus was engineered to contain
a twenty-five amino acid sequence containing a recognition site for
the thrombin restriction protease followed by a ten amino acid
sequence derived from the human c-myc protein and six consecutive
histidine residues. The c-myc sequence serves as an epitope tag
allowing detection by a monoclonal antibody (9E10; Evan, G. I. et
al., (1985) Mol. Cell Biol. 5:3610-3616). The combined molecular
mass of the carboxy terminal additions is approximately 3 kDa.
[0501] Xenopus laevis oocytes
[0502] Immunoprecipitation with the c-myc antibody detects several
proteins in lysates of metabolically labeled Xenopus laevis oocytes
injected with Shh mRNA. Cell lysates and medium from .sup.35S
labeled oocytes injected with RNA encoding mouse Shh with the c-myc
epitope tag at the at the carboxy terminus, or from control oocytes
were analyzed by immunoprecipitation with c-myc monoclonal
antibody. A band of approximately 47 kDa is seen, as is a doublet
migrating near 30 kDa. Treatment with Endo H increases the mobility
of the largest protein, and resolves the doublet into a single
species of approximately 30 kDa. These observations parallel the
behaviors seen in vitro. Allowing for the added mass of the carboxy
terminal additions, the largest protein would correspond to the
signal peptide cleaved form, while the doublet would represent the
glycosylated and unglycosylated larger processed form. Since the
epitope tag was placed at the carboxy terminus of the protein, the
identity of the 30 kDa peptide as the carboxy terminal portion of
Shh is confirmed. Failure to detect the 19 kDa species supports its
identity as an amino terminal region of the protein.
[0503] To test whether Shh is secreted by Xenopus oocytes, the
medium in which the injected oocytes were incubated was probed by
immunoprecipitation with the c-myc antibody. A single band
migrating slightly more slowly than the glycosylated larger
processed form was observed. This protein is insensitive to Endo H.
This result is expected since most secreted glycoproteins lose
sensitivity to Endo H as they travel through the Golgi apparatus
and are modified by a series of glycosidases (Kornfeld, R. and
Kornfeld, S., (1985) Annu. Rev. Biochem. 54:631-664). The enzymatic
maturation of the Asn-linked carbohydrate moiety could also explain
the slight decrease in mobility of the secreted larger protein
versus the intracellular material. Following Endo H digestion, a
band with a slightly lower mobility than the signal peptide cleaved
protein is also apparent, suggesting that some Shh protein is
secreted without undergoing proteolytic processing. Failure to
detect this protein in the medium without Endo H digestion suggests
heterogeneity in the extent of carbohydrate modification in the
Golgi preventing the material from migrating as a distinct band.
Resolution of this material into a single band following Endo H
digestion suggests that the carbohydrate structure does not mature
completely in the Golgi apparatus. Structural differences between
the unprocessed protein and the larger processed form could account
for this observation (Kornfeld, R. and Kornfeld, S., (1985) Annu.
Rev. Biochem. 54:631-664).
[0504] Cos Cells
[0505] The behavior of mouse Shh in a mammalian cell type was
investigated using tansfected cos cells. Synthesis and secretion of
the protein was monitored by immunoprecipitation using the c-myc
antibody. Transfected cos cells express the same Sonic hedgehog
species that were detected in the injected Xenopus oocytes, and
their behavior following Endo H digestion is also identical.
Furthermore, secretion of the 30 kDa glycosylated form is observed
in cos cells, as well as the characteristic insensitivity to Endo H
after secretion. Most of the secreted protein co-migrates with the
intracellular, glycosylated larger processed form, but a small
amount of protein with a slightly lower mobility is also detected
in the medium. As in the Xenopus oocyte cultures, some Shh which
has not undergone proteolytic processing is evident in the medium,
but only after Endo H digestion.
[0506] Baculovirus Infected Cells
[0507] To examine the behavior of the mouse Shh protein in an
invertebrate cell type, and to potentially purify Shh peptides, a
recombinant baculovirus was constructed which placed the Shh coding
sequence, with the carboxy terminal tag, under the control of the
baculoviral Polyhedrin gene promoter. When insect cells were
infected with the recombinant baculovirus, Shh peptides could be
detected in cell lysates and medium by Western blotting with the
c-myc antibody.
[0508] The Shh products detected in this system were similar to
those described above. However, virtually no unprocessed protein
was seen in cell lysates, nor was any detected in the medium after
Endo H digestion. This suggests that the proteolytic processing
event occurs more efficiently in these cells than in either of the
other two cell types or the in vitro translation system. A doublet
corresponding to the glycosylated and unglycosylated 30 kDa forms
is detected, as well as the secreted, Endo I resistant peptide as
seen in the other expression systems. Unlike the other systems,
however, all of the secreted larger processed form appears to
comigrate with the glycosylated intracellular material.
[0509] (iv) Secretion of a Highly Conserved Amino Terminal
Peptide
[0510] To determine the behavior of the amino terminal portion of
the processed Sonic hedgehog protein, the c-myc epitope tag was
positioned 32 amino acids after the putative signal peptide
cleavage site (FIG. 12). Cos cells were transfected with Shh
expression constructs containing the c-myc tag at the carboxy
terminus or near the amino terminus. When this construct was
expressed in cos cells, both the full length protein and the
smaller processed form (approximately 20 kDa due to addition of the
c-myc tag) were detected by immunoprecipitation of extracts from
labeled cells. However, the 20 kDa product is barely detected in
the medium. In cells transfected in parallel with the carboxy
terminal c-myc tagged construct, the full length and 30 kDa
products were both precipitated from cell lysates and medium as
described earlier.
[0511] As the amino terminal c-myc tag may affect the secretion
efficiency of the smaller processed form, the expression of this
protein was examined in cos cells using an antiserum directed
against amino acids 44 through 143 of mouse Shh (FIG. 12). After
transfection with the carboxy-terminal c-myc tagged construct,
immunoprecipitation with the anti-Shh serum detected a very low
level of the smaller processed form in the medium despite a strong
signal in the cell lysate. This recapitulates the results with the
myc antibody.
[0512] To examine the subcellular localization of Shh proteins, cos
cells were transfected with the carboxy terminal tagged Shh
construct and plated on multi-chamber slides, fixed and
permeabilized. The cells were incubated simultaneously with the
anti-Shh serum and the c-myc antibody followed by FITC conjugated
Goat anti-Rabbit-IgG and RITC conjugated Goat anti-Mouse-IgG. DAPI
was included to stain nuclei. Strong perinuclear staining
characteristic of the Golgi apparatus was observed with the
anti-Shh serum. The same subcellular region was also stained using
the c-myc antibody. The coincidence of staining patterns seen with
the two antibody preparations suggest that the low level of the
smaller processed form detected in the medium is not due to its
retention in the endoplasmic reticulum, since both processed forms
traffic efficiently to the Golgi apparatus.
[0513] One explanation for the failure to detect large amounts of
the smaller processed form in the culture medium could that this
protein associates tightly with the cell surface or ECM. To examine
this, cells were treated with the polyanionic compounds herparin
and suramin. These compounds have been shown to increase the levels
of some secreted proteins in culture medium, possibly by displacing
them from cell surface or ECM components or by directly binding the
proteins and perhaps protecting them from proteolytic degradation
(Bradley and Brown (1990) EMBO J. 9:1569-1575; Middaugh et al.
(1992) Biochem. 31:9016-9024; Smolich et al. (1993) Mol. Biol. Cell
4:1267-1275). The 19-kDa amino-terminal form of Shh is barely
detectable in the medium of transfected COS cells, despite its
obvious presence in the cell lysate. However, in the presence of 10
mg of heparin per ml, this peptide is readily detected in the
medium. The addition of 10 mM suramin to the medium has an even
greater effect. Since the concentrations used where those
previously determined to elicit maximal responses, it is clear that
suramin is more active than heparin in this assay.
[0514] The ability of heparin and suramin to increase the amount of
the smaller processed form in the medium of transfected cells
implies that this peptide may be tightly associated with the cell
surface of ECM. As a first step toward determining which region(s)
of the Shh protein may be responsible for this retention, a
truncated form of mouse Shh deleted of all sequence downstream of
amino acid 193 was expressed in COS cells. This protein contains
all of the sequences encode by exons one and two, as well as five
amino acids derived for exon three. Since its predicted molecular
mass (19.2 kDa) is very close to the observed molecular mass of the
smaller processed form, the behavior of this protein would be
expected to mimic that of the smaller processed form. This protein
is detected at a very high level in the medium, even in the absence
of heparin or suramin, and migrates at a position indistinguishable
form that of the amino-terminal cleavage product generated from the
full-length protein. In fact, virtually no protein is seen in the
cell lysates, suggesting nearly quantitative release of the protein
into the medium. This raises the possibility that the actual amino
terminally processed form may extend a short distance beyond amino
acid 193 and that these additional amino acids contain a cell
surface-ECM retention signal.
[0515] The influence of sequences located at the extreme amino and
carboxy termini of mouse Shh on the behavior of the protein in
transfected cells was examined using the amino terminus-specific
antiserum. Expression of a mouse Shh construct lacking a signal
peptide results in the accumulation of approximately 28-kDa
protein, as well as a small amount of protein which comigrates with
the smaller processed form. This implies that correct cleavage of
Shh requires targeting of the protein to the endoplasmic reticulum,
since the bulk of the processed form of Shh expressed in the
cytoplasm is cleaved at a new position that is approximately 9 kDa
carboxy terminal to the normal cleavage site. Expression of a mouse
Shh protein engineered to terminate after amino acid 428 (lacking
nine carboxy-terminal amino acids [.DELTA.Ct]) results in the
expected amino-terminal cleavage product; however, the efficiency
of cleavage is significantly decreased compared with that seen with
the wild-type protein. Therefore, sequences located at a distance
from the proteolytic processing site are able to affect the
efficiency of processing.
[0516] (v) Sonic Hedgehog Processing in Embryonic Tissues
[0517] In order to determine whether the proteolytic processing of
Shh observed in the different expression systems reflects the
behavior of the protein in embryos, the amino terminus-specific
mouse Shh antiserum was used to probe Western blots of various
chicken and mouse embryonic tissues. A protein with an
electrophoretic mobility identical to that of COS cell-synthesized
amino terminally processed form is detected at a substantial level
in the stomach and lung tissue and at a markedly lower level in the
forebrain, midbrain, and hindbrain tissues of 15.5-day-postcoitum
mouse embryos. These tissues have all been shown to express Shh
RNA. The 19 kDa peptide is not detected in liver or late limb
tissues, which do not express Shh RNA. Thus, the proteolytic
processing of Shh observed in cell culture also occurs in embryonic
mouse tissue.
[0518] The cross-reactivity of the amino terminus-specific mouse
Shh antiserum with chicken Shh protein allowed for examination of
expression of Shh in chicken embryonic tissue. The antiserum
detects the 19-kDa amino terminally processed form of chicken Shh
in transfected COS cells, as well as in two tissues which have been
shown by whole-mount in situ hybridization and antiserum staining
to express high levels of Shh RNA and protein, i.e., the posterior
region of the limb bud and the ventral region of the anterior CNA
(Riddle et al. (1993) Cell 75:1401-1416). Therefore, the expected
proteolytic processing of Shh occurs in chicken embryonic tissues,
and diffusion of the 19-kDa protein does not extend into the
anterior limb buds and dorsal CNS.
[0519] (v) Hedgehog Processing
[0520] In summary, the results discussed above demonstrate that the
mouse and chick Shh genes encode secreted glycoproteins which
undergo additional proteolytic processing. Data indicate that this
processing occurs in an apparently similar fashion in a variety of
cell types suggesting that it is a general feature of the Shh
protein, and not unique to any particular expression system. For
mouse Shh, data indicate that both products of this proteolytic
processing are secreted. These observations are summarized in FIG.
13.
[0521] It was observed that the 19 kDa amino peptide accumulates to
a lower level in the medium than the 27 kDa carboxyl peptide. This
may reflect inefficient secretion or rapid turnover of this species
once secreted. Alternatively, the smaller form may associate with
the cell surface or extracellular matrix components making it
difficult to detect in the medium. The insensitivity of the
secreted, larger form to Endo H is a common feature of secreted
glycoproteins. During transit through the Golgi apparatus, the
Asn-linked carbohydrate moiety is modified by a series of specific
glycosidases (reviewed in Kornfeld, R. and Kornfeld, S., (1985)
Annu. Rev. Biochem 54:631-664; Tarentino, A. L. et al., (1989)
Methods Cell Biol 32:111-139). These modifications convert the
structure from the immature "high mannose" to the mature "complex"
type. At one step in this process, a Golgi enzyme,
.alpha.-mannosidase II, removes two mannose residues from the
complex rendering it insensitive to Endo H (Kornfeld, R. and
Kornfeld, S., (1985) Annu. Rev. Biochem 54:631-664).
[0522] The biochemical behavior of mouse Shh appears to be quite
similar to that described for the Drosophila Hedgehog (Dros-HH)
protein (Lee, J. L. et al., (1992) Cell 71:33-50; Tabata, T. et
al., (1992) Genes & Dev. 6:2635-2645). In vitro translation of
Drosophila hh mRNA, in the presence of microsomes, revealed
products with molecular masses corresponding to full length
protein, as well as to the product expected after cleavage of the
predicted internal (Type II) signal peptide (Lee, J. L. et al.,
(1992) Cell 71:33-50); Interestingly, no additional, processed
forms were observed. However, such forms could have been obscured
by breakdown products migrating between 20 and 30 kDa. When an RNA
encoding a form of the protein lacking the carboxy-terminal 61
amino acids was translated, no breakdown products were seen, but
there is still no evidence of the proteolytic processing observed
with mouse Shh. A similar phenomenon has been observed in these
experiments. A reduction in the extent of proteolytic processing is
seen when a mouse Shh protein lacking 10 carboxy-terminal amino
acids is translated in vitro or expressed in cos cells (data not
shown). This suggests that sequences at the carboxy termini of Hh
proteins act at a distance to influence the efficiency of
processing.
[0523] Recently, Lee et al. (Science 266:1528-1537, 1994) described
the biochemical behavior of the Drosophila HH protein. Using
region-specific antisera, they detected similar processed forms of
HH in embryonic tissues, thus confirming studies in which
processing of HH was observed in embryos forced to express high
levels of HH from a heat shock promoter (Tabata and Kornberg (1994)
Cell 76:89-102). Thus, Drosophila HH is processed to yield a 19 kDa
amino-terminal peptide and a 25 kDa carboxy-terminal peptide.
Furthermore, Lee et al. concluded that the production of the
processed forms occurs via an autocatalytic mechanism and
identified a conserved histidine residue (at position 329,
according to Lee et al. (Science 266:1528-1537, 1994)) which is
required for self-cleavage of HH protein in vitro and in vivo. The
significance of the proteolytic processing is demonstrated by the
inability of self-processing-either because of mutation of this
histidine residue or because of truncation of sequences at the
extreme carboxy terminus-to carry out HH functions in Drosophila
embryos.
[0524] Their studies of the biochemical behavior of mouse and
chicken Shh and mouse Ihh proteins correlate well with the
Drosophila studies of Lee et al. (Science 266:1528-1537, 1994) in
that the similar proteolytic processing of endogenous vertebrate
proteins in embryonic tissues was demonstrated. Furthermore, it was
demonstrated that the efficiency of processing depends on sequences
located at the extreme carboxy terminus of mouse Shh.
Interestingly, it has also been shown that he specificity of mouse
Shh cleavage may depend on targeting of the protein to the
secretory pathway, since a form lacing a signal peptide is
processed into an approximately 28-kDa amino-terminal form. A
similar protein is observed as the predominant species when it was
attempted to express full-length mouse Shh in bacteria (data no
shown). Lee et al. (Science 266:1528-1537, 1994) have demonstrated
that two zebra fish hedgehog proteins undergo proteolytic
processing when translated in vitro, even in the absence of
microsomal membranes. The electrophoretic mobilities of the
processed peptides are consistent with cleavage occurring at a
position similar to that of the Drosophila HH cleavage site.
Furthermore, they showed that the cleavage fails to occur if the
conserved histidine residue is mutated, arguing for an
autoproteolytic mechanism similar to that of the Drosophila
protein. However, the processing of mouse or chicken Shh protein
translated in vitro was not detected unless microsomal membrane are
included. Therefore, it is possible that correct proteolytic
processing of vertebrate hedgehog proteins is dependent on specific
incubation conditions or may require cellular factors in addition
to Shh itself.
[0525] An additional correlation between the work presented here
and that of Lee et al. (Science 266:1528-1537, 1994) concerns the
different behaviors of the amino (smaller) and carboxy (larger)
terminally processed forms of the hedgehog proteins. The evidence
is presented that the 27 kDa carboxy-terminal form diffuses more
readily from expressing cells than the 19 kDa amino-terminal form,
which seems to be retained near the cell surface. The polyanions
heparin and suramin appear capable of releasing the amino peptide
into the medium. Similarly, the amino-terminal form of Drosophila
HH is more closely associated with the RNA expression domain in
embryonic segments than is the carboxy-terminal form, and the
amino-terminal form binds to heparin agarose beads. Therefore, the
distinct behaviors of the different hedgehog peptides have been
conserved across phyla.
[0526] The observed molecular masses of the amino terminally
processed forms of mouse and chicken Shh, mouse Ihh proteins, and
Drosophila HH are between 19 and 20 kDa. Therefore, the predicted
secondary proteolytic cleavage site would be located near the
border of the sequences encoded by the second and third exons.
Interestingly, the region marks the end of the most highly related
part of the hedgehog proteins. The amino terminal (smaller) form
would contain the most highly conserved portion of the protein. In
fact, the amino acids encoded by exons one and two (exclusive of
sequences upstream of the putative signal peptide cleavage sites)
share 69% identity between Drosophila Hh and mouse Shh, and 99%
identity between chick and mouse Shh. Amino acid identity in the
region encoded by the third exon is much lower 30% mouse to
Drosophila and 71% mouse to chick (Echelard, Y. et al., (1993) Cell
75:1417-1430).
[0527] However, the boundary between sequences encoded by exons 2
and 3 is unlikely to be the actual proteolytic processing site,
because a Drosophila HH protein containing a large deletion which
extends three amino acids beyond this boundary is still cleaved at
the expected position in vitro (Lee et al. (1994) Science
266:1528-1537). Moreover, the analysis of an amino-terminal mouse
Shh peptide truncated at amino acid 193 (the fourth amino acid
encoded by exon 3, described below) suggests that normal cleavage
must occur downstream of this position. Close examination of
hedgehog protein sequences reveals that strong sequence
conservation between the Drosophila and vertebrate proteins
continues for only a short distance into the third exon. If it is
assumed that cleavage will generate an amino terminal product of no
greater than 20 kDa, given the resolution of analysis, all of the
data would indicate that cleavage occurs at 1 of the 10 amino acids
within the mouse Shh positions 194-203, according to Echelard et
al. (Cell 75:1417-1430, 1993).
[0528] (vi) Hedgehog Signalling
[0529] In order to satisfy the criteria for intercellular
signaling, hedgehog proteins must be detected outside of their
domains of expression. This has been clearly demonstrated for
Drosophila HH. Using an antiserum raised against nearly full length
Dros-HH protein, Tabata and Kornberg (Tabata, T. and Kornberg, T.
B., (1992) Cell 76:89-102) detect the protein in stripes that are
slightly wider than the RNA expression domains in embryonic
segments, and just anterior to the border of the RNA expression
domain in wing imaginal discs. Similarly, Taylor, et. al., (1993)
Mech. Dev. 42:89-96, detected HH protein in discrete patches within
cells adjacent to those expressing hh RNA in embryonic segments
using an antiserum directed against an amino-terminal portion of Hh
which, based on the proteolytic processing data (Tabata, T. et al.,
(1992) Genes & Dev. 6:2635-2645), is not likely to recognize
the carboxyl cleavage product.
[0530] The detection of Hh beyond cells expressing the hh gene is
consistent with the phenotype of hh mutants. In these animals,
cellular patterning in each embryonic parasegment in disrupted
resulting in an abnormal cuticular pattern reminiscent of that seen
in wg mutants. Further analysis has revealed that the loss of hh
gene function leads to loss of wg expression in a thin stripe of
cells just anterior to the hh expression domain (Ingham, P. W. and
Hidalgo, A., (1993) Development 117:283-291). This suggests that Hh
acts to maintain wg expression in neighboring cells. The
observation that ubiquitously expressed Hh leads to ectopic
activation of wg supports this model (Tabata, T. and Kornberg, T.
B., (1992) Cell 76:89-102). In addition to these genetic studies,
there is also indirect evidence that Hh acts at a distance from its
site of expression to influence patterning of the epidermis
(Heemskerk, J. and DiNardo, S., (1994) Cell 76:449-460).
[0531] The apparent effect of Drosophila Hh on neighboring cells,
as well as on those located at a distance from the site of hh
expression is reminiscent of the influence of the notochord and
floor plate on the developing vertebrate CNS, and of the ZPA in the
limb. The notochord (a site of high level Shh expression) induces
the formation of the floor plate in a contact dependent manner,
while the notochord and floor plate (another area of strong Shh
expression) are both capable of inducing motorneurons at a distance
(Placzek, M. et al., (1993) Development 117:205-218; Yamada, T. et
al., (1993) Cell 73:673-686).
[0532] Moreover ZPA activity is required not only for patterning
cells in the extreme posterior of the limb bud where Shh is
transcribed, but also a few hundred microns anterior of this zone.
Several lines of evidence indicate that Shh is able to induce floor
plate (Echelard, Y. et al., (1993) Cell 75:1417-1430; Roelink, H.
et al., (1994) Cell 76:761-775) and mediate the signaling activity
of the ZPA (Riddle, R. D. et al., (1993) Cell 75:1401-1416). Since
it has been shown that Shh is cleaved, it can be speculated that
the processed peptides may have distinct activities. The smaller
amino terminal form, which appears to be more poorly secreted, less
stable or retained at the cell surface or in the extracellular
matrix, may act locally. In contrast, the larger carboxy terminal
peptide could possibly function at a distance. In this way, Shh
peptides may mediate distinct signaling functions in the vertebrate
embryo. Alternatively, the carboxy-terminal peptide may be
necessary only for proteolytic processing, with all signaling
activity residing in the amino-terminal peptide.
EXAMPLE 7
Sonic Hedgehog and Fgf-4 Act Through a Signaling Cascade and
Feedback Loop to Integrate Growth and Patterning of the Developing
Limb Bud
[0533] (i) Experimental Procedures
[0534] Cloning of Chicken Fgf-4 and Bmp-2
[0535] A 246 bp fragment of the chicken Fgf-4 gene was cloned by
PCR from a stage 22 chicken limb bud library. Degenerate primers
were designed against previously cloned Fgf-4 and Fgf-6 genes:
fgf5' (sense) AAA AGC TTT AYT GYT AYG TIG GIA THG G (SEQ ID No:38)
and fgf3' (antisense) AAG AAT TCT AIG CRT TRT ART TRT TIG G (SEQ ID
No:39). Denaturation was at 94.degree. C. for 2 min, followed by 30
cycles of 94.degree. C. for 30 sec, 50.degree. C. for 60 sec, and
72.degree. C. for 30 sec, with a final extension at 72.degree. C.
for 5 min. The PCR product was subcloned into the Bluescript SK+
vector. A clone was sequenced and confirmed as Fgf-4 by comparison
with previously published Fgf-4 genes and a chicken Fgf-4 gene
sequence kindly provided by Lee Niswander.
[0536] BMP-related sequences were amplified from a stage 22
posterior limb bud cDNA library prepared in Bluescript using
primers and conditions as described by Basler, et al. (1993).
Amplified DNAs were cloned and used to screen a stage 22 limb bud
library prepared in .lambda.-Zap (Stratagene). Among the cDNAs
isolated was chicken Bmp-2. Its identity was confirmed by sequence
comparison to the published clones (Francis, et al., (1994)
Development 120:209-218) and by its expression patterns in chick
embryos.
[0537] Chick Surgeries and Recombinant Retroviruses
[0538] All experimental manipulations were performed on White
Leghorn chick embryos (S-SPF) provided by SPAFAS (Norwich, Conn.).
Eggs were staged according to Hamburger and Hamilton (1951) J. Exp.
Morph. 88:49-92.
[0539] Viral supernatants of Sonic/RCAS-A2 or a variant containing
an influenza hemaglutinin epitope tag at the carboxyl terminus of
the hedgehog protein (Sonic7.1/RCAS-A2, functionally
indistinguishable from Sonic/RCAS-A2), were prepared as described
(Hughes, et al., (1987) J. Virol. 61:3004-13; Fekete and Cepko,
(1993) Mol. & Cell. Biol. 13:2604-13; Riddle, et al., (1993)
Cell 75:1401-16). For focal injections the right wings of stage
18-21 embryos were transiently stained with nile blue sulfate (0.01
mg/ml in Ringer's solution) to reveal the AER. A trace amount of
concentrated viral supernatant was injected beneath the AER.
[0540] The AER was removed using electrolytically sharpened
tungsten wire needles. Some embryos had a heparin-acrylic bead
soaked in FGF-4 solution (0.8 mg/ml; a gift from Genetics
Institute) or PBS stapled to the limb bud with a piece of 0.025 mm
platinum wire (Goodfellow, Cambridge UK) essentially as described
by Niswander et al, (1993) Cell 75:579-87.
[0541] Limbs which were infected with Sonic/RCAS virus after AER
removal were infected over a large portion of the denuded mesoderm
to ensure substantial infection. Those embryos which received both
an Fgf-4 soaked bead and virus were infected only underneath the
bead.
[0542] In Situ Hybridizations and Photography
[0543] Single color whole mount in situ hybridizations were
performed as described (Riddle, et al., (1993) Cell 75:1401-16).
Two color whole mount in situ hybridizations were performed
essentially as described by Jowett and Lettice (1994) Trends Genet.
10:73-74. The second color detection was developed using 0.125
mg/ml magenta-phos (Biosynth) as the substrate. Radioactive in situ
hybridizations on 5 .mu.m sections was performed essentially as
described by Tessarollo, et al. (1992) Development 115:11-20.
[0544] The following probes were used for whole mount and section
in situ hybridizations: Sonic: 1.7 kb fragment of pHH2 (Riddle, et
al., (1993) Cell 75:1401-16). Bmp-2: 1.5 kb fragment encoding the
entire open reading frame. Fgf-4: 250 bp fragment described above.
Hox d-11: a 600 bp fragment, Hoxd-13: 400 bp fragment both
including 5' untranslated sequences and coding sequences upstream
of the homeobox. RCAS: 900 bp SalI-ClaI fragment of RCAS (Hughes et
al., (1987) J Virol. 61:3004-12).
[0545] (ii) Relationship of Sonic to Endogenous Bmp-2 and Hoxd Gene
Expression
[0546] The best candidates for genes regulated by Sonic in vivo are
the distal members of the Hoxd gene cluster, Hoxd-9 through -13,
and Bmp-2. Therefore, the relationships of the expression domains
of these genes in a staged series of normal chick embryos were
analyzed. Hoxd-9 and Hoxd-10 are expressed throughout the
presumptive wing field at stage 16 (Hamburger and Hamilton, (1951)
J. Exp. Morph. 88:49-92), prior to the first detectable expression
of Sonic at early stage 18. Hoxd-11 expression is first detectable
at early stage 18, the same time as Sonic, in a domain coextensive
with Sonic. Expression of Hoxd-12 and Hoxd-13 commence shortly
thereafter. These results suggest that Sonic might normally induce,
directly or indirectly, the expression of only the latter three
members of the cluster, even though all five are nested within the
early limb bud.
[0547] As limb outgrowth proceeds Sonic expression remains at the
posterior margin of the bud. In contrast the Hoxd gene expression
domains, which are initially nested posteriorly around the Sonic
domain, are very dynamic and lose their concentric character. By
stage 23 the Hoxd-11 domain extends anteriorly and distally far
beyond that of Sonic, while Hoxd-13 expression becomes biased
distally and displaced from Sonic.
[0548] While it is not clear whether Bmp-2 is expressed before
Sonic (see Francis et. al., (1994) Development 120:209-218) Bmp-2
is expressed in a mesodermal domain which apparently overlaps and
surrounds that of Sonic at the earliest stages of Sonic expression.
As the limb bud develops, the mesodermal expression of Bmp-2
remains near the posterior limb margin, centered around that of
Sonic, but in a larger domain than Sonic. This correspondence
between Sonic and Bmp-2 expression lasts until around stage 25,
much longer than the correspondence between Sonic and Hoxd gene
expression. After stage 25 Bmp-2 expression shifts distally and is
no longer centered on Sonic.
[0549] (iii) Relationship of Sonic to Induced Bmp-2 and Hoxd Gene
Expression
[0550] The fact that the expression domains of the Hoxd genes
diverge over time from that of Sonic hedgehog implies that Sonic
does not directly regulate their later patterns of expression. This
does not preclude the possibility that the later expression domains
are genetically downstream of Sonic. If this were the case,
exogenously expressed Sonic would be expected to initiate a program
of Hoxd gene expression which recapitulates that seen endogenously.
Therefore, the spatial distribution of Hoxd gene expression at
various times following Sonic misexpression was compared. The
anterior marginal mesoderm of early bud (Stage 18-20) wings was
injected at a single point under the AER with a replication
competent virus that expresses a chicken Sonic cDNA. Ectopic Sonic
expressed by this protocol leads to both anterior mesodermal
outgrowth and anterior extension of the AFR.
[0551] The Sonic and Hoxd gene expression domains in the infected
limbs were analyzed in sectioned and intact embryos. Viral Sonic
message is first detected approximately 18 hours after infection at
the anterior margin, at the same time as, and approximately
coextensively with, induced Hoxd-11. This suggests that Sonic can
rapidly induce Hoxd-11 expression and that the lag after injection
represents the time required to achieve Sonic expression. By 35
hours post infection distal outgrowth of infected cells combined
with lateral viral spread within the proliferating cells leads to
viral expression in a wedge which is broadest at the distal margin
and tapers proximally. By this time, Hoxd-11 expression has
expanded both antero-proximally and distally with respect to the
wedge of Sonic-expressing cells, into a domain which appears to
mirror the more distal aspects of the endogenous Hoxd-11 domain.
Weak Hoxd-13 expression is also detected at 35 hours in a subset of
the Sonic expressing domain at its distal margin. 51 hours after
infection the relationship of Sonic and Hoxd-11 expression is
similar to that seen at 35 hours, while the induced Hoxd-13
expression has reached wild type levels restricted to the distal
portions of the ectopic growth. Thus the ectopic Hoxd expression
domains better reflect the endogenous patterns of expression than
they do the region expressing Sonic. This suggests that there are
multiple factors regulating Hoxd expression but their actions lie
downstream of Sonic.
[0552] Since the endogenous Bmp-2 expression domain correlates well
with that of Sonic, and Bmp-2 is induced by ZPA grafts, it was
looked to see if Bmp-2 is also induced by Sonic. Bmp-2 is normally
expressed in two places in the early limb bud, in the posterior
mesoderm and throughout the AER (Francis, et al., (1994)
Development 120:209-218). In injected limb buds additional Bmp-2
expression is seen in both the anterior mesoderm and in the
anteriorly extended AER. The domain of Bmp-2 expression is slightly
more restricted than that of viral expression, suggesting a delay
in Bmp-2 induction. Bmp-2 expression in both the mesoderm and
ectoderm is thus a downstream target of Sonic activity in the
mesoderm. In contrast to the expression domains of the Hoxd genes,
the endogenous and ectopic Bmp-2 expression domains correlate well
with that of Sonic. This suggests that Bmp-2 expression is
regulated more directly by Sonic than is expression of the Hoxd
genes.
[0553] (iv) The AER and Competence to Respond to Sonic
[0554] Ectopic activation of Hoxd gene expression is biased
distally in virally infected regions, suggesting that ectodermal
factors, possibly from the AER, are required for Hoxd gene
induction by Sonic. To test this, Sonic virus was injected into the
proximal, medial mesoderm of stage 21 limb buds, presumably beyond
the influence of the AER. Although the level of Sonic expression
was comparable to that observed in distal injections, proximal
misexpression of Sonic did not result in ectopic induction of the
Hoxd genes or Bmp-2, nor did it result in any obvious morphological
effect (data not shown). The lack of gene induction following
proximal misexpression of Sonic suggests that exposure to Sonic
alone is insufficient to induce expression of these genes.
[0555] This was tested more rigorously by injection of Sonic virus
into the anterior marginal mesoderm of stage 20/21 limb buds after
the anterior half of the AER had been surgically removed. Embryos
were allowed to develop for a further 36 to 48 hours before
harvesting. During this time the AER remaining on the posterior
half of the limb bud promotes almost wild type outgrowth and
patterning of the bud. Gene expression was monitored both in
sectioned and intact embryos. In the presence of the AFR, Sonic
induces both anterior mesodermal proliferation and expression of
Hoxd-11, Hoxd-13 and Bmp-2. In the absence of the overlying AER,
Sonic does not induce either mesodermal proliferation or expression
of these genes above background. Signals from the AER are thus
required to allow both the proliferative and patterning effects of
Sonic on the mesoderm.
[0556] Since application of FGF protein can rescue other functions
of the AER such as promoting PD outgrowth and patterning, it was
sought to determine whether FGFs might also promote mesodermal
competence to respond to Sonic. FGF-4-soaked beads were stapled to
AER-denuded anterior mesoderm which was infected with Sonic virus.
Gene expression and mesodermal outgrowth were monitored as
described previously. In the presence of both Sonic virus and FGF-4
protein, Hoxd-11, Hoxd-13 and Bmp-2 expression are all induced. The
expression levels of the induced genes are similar to or greater
than the endogenous expression levels, and are equivalent in
magnitude to their induction in the presence of the AER. Thus Fgf-4
can induce the competence of the mesoderm to respond to Sonic.
[0557] Sonic alone is insufficient to induce either gene expression
or mesodermal proliferation in the absence of the AER, while the
combination of Sonic and FGF-4 induces both proliferation and gene
expression. It was than asked whether FGF-4 alone has any effect on
gene induction or mesodermal proliferation. Application of FGF-4 in
the absence of Sonic virus does not induce Hoxd or Bmp-2 gene
expression above control levels, however FGF-4 alone induces
mesodermal outgrowth. These results suggest that mesodermal gene
activation requires direct action of Sonic on the mesoderm and that
proliferative response to Sonic is indirect, due to the induction
of FGFs.
[0558] (v) Sonic Induces Polarized Fgf-4 Expression in the AER
[0559] Fgf-4 is expressed in a graded fashion in the AER of the
mouse limb bud, with maximal expression at the posterior region of
the AER tapering to undetectable levels in the anterior ridge
(Niswander and Martin, (1992) Development 114:755-68). Therefore,
it was appropriate to investigate whether Fgf-4 is asymmetrically
expressed in the chick AER, and whether its expression is induced
by Sonic. A fragment of the chicken Fgf-4 gene was cloned from a
stage 22 chicken limb library by PCR using degenerate primers
designed from mouse Fgf-4 and Xenopus e-Fgf sequence; based on
information provided by L. Niswander and G. Martin. Assignment of
gene identity was based on primary sequence as well as comparison
of expression patterns with that of murine Fgf-4 (Niswander and
Martin, (1992) Development 114:755-68). Whole mount in situ
hybridization analysis showed strong limb expression of chick Fgf-4
in the AER. Fgf-4, like Bmp-2, is expressed all the way to the
posterior border of the AER, but its anterior domain ends before
the morphological end of the AER creating a posterior bias that has
also been observed by Niswander et al., (1994) Nature (in press).
Expression is first detected in the distal AER at about stage 18.
As outgrowth proceeds the posterior bias develops. Expression peaks
around stage 24/25 and then fades by stage 28/29.
[0560] The expression domain of Fgf-4 becomes posteriorly biased as
Sonic is expressed in the posterior mesoderm. This observation is
consistent with Sonic influencing the expression of Fgf-4 in the
posterior AER. To test the effect of Sonic on Fgf-4 expression in
the AER, stage 18-20 embryos were infected with Sonic virus in a
single point at their anterior margin beyond the anterior limit of
the AER. The embryos were harvested one to two days later, when an
extension of the anterior AER became apparent. The expression of
Fgf-4 was analyzed by in situ hybridization. Fgf-4 expression is
induced in the anteriormost segment of the AER, in a region which
is discontinuous with the endogenous expression domain, and
overlies the domain of viral Sonic infection. This result contrasts
with the Bmp-2 expression induced in the extended AER, which is
always continuous with the endogenous expression domain. The
asymmetry of the induced Fgf-4 expression indicates that Sonic
polarizes the extended AER, much as a ZPA graft does (Maccabe and
Parker, (1979) J. Embryol. Exp. Morph. 53:67-73). Since FGFs by
themselves are mitogenic for limb mesoderm, these results are most
consistent with Sonic inducing distal proliferation indirectly,
through the induction of mitogens in the overlying AER.
[0561] (vi) Reciprocal Regulation of Sonic by Fgf-4
[0562] Sonic thus appears to be upstream of Fgf-4 expression in the
AER. However, since the AER is required to maintain polarizing
activity in the posterior mesoderm (Vogel and Tickle, (1993)
Development 19:199-206; Niswander et al., (1993) Cell 75:579-87),
Sonic may also be downstream of the AER. If Sonic is regulated by
the AER and the AER by Sonic, this would imply that they are
reinforcing one another through a positive feedback loop.
[0563] To test whether the AER dependence of ZPA activity is
controlled at the level of transcription of the Sonic gene, Sonic
expression following removal of the AER from the posterior half of
the limb bud was assayed. Sonic expression is reduced in an
operated limb compared to the contralateral control limb within ten
hours of AER removal, indicating that Sonic expression is indeed
AER dependent. The dependence of Sonic expression on signals from
the AER suggests that one of the functions of the AER is to
constrain Sonic expression to the more distal regions of the
posterior mesoderm.
[0564] In addition to their mitogenic and competence-inducing
properties, FGFs can also substitute for the AER to maintain the
ZPA. In order to test whether FGFs can support the expression of
Sonic, beads soaked in FGF-4 protein were stapled to the
posterior-distal tips of limb buds after posterior AER removal.
Embryos were assayed for Sonic expression approximately 24 hours
later, when Sonic expression is greatly reduced in operated limb
buds which had not received an FGF-4 bead. Strong Sonic expression
is detectable in the posterior mesoderm, slightly proximal to the
bead implant, and reflecting the normal domain of Sonic expression
seen in the contralateral limb. With the finding that FGF-4 can
maintain Sonic expression, the elements required for a positive
feedback loop between Sonic expression in the posterior mesoderm
and Fgf-4 expression in the posterior AER are established (see also
Niswander et al. (1994) Nature (in press)).
[0565] The induction of Bmp-2 expression by Sonic requires signals
from the AER, and its domain correlates over time with that of
Sonic. Therefore, it was interesting to learn if the continued
expression of Bmp-2 also requires signals from the AER, and if so,
whether they could be replaced by FGF-4. To test this, Bmp-2
expression following posterior AER removal, and following its
substitution with an FGF-4 bead was assayed. Bmp-2 expression fades
within hours of AER removal, and can be rescued by FGF-4. These
data indicate that the maintenance of Bmp-2 expression in the
posterior mesoderm, like that of Sonic, is dependent on signals
from the AER, which are likely to be FGFs.
[0566] (vii) The Mesodermal Response to Sonic
[0567] It has been found that only mesoderm underlying the AER is
responsive to Sonic, apparently because the AER is required to
provide competence signals to the limb mesoderm. Fgf-4, which is
expressed in the AER, can substitute for the AER in this regard,
and thus might act in combination with Sonic to promote Hoxd and
Bmp-2 gene expression in the mesoderm. FGFs may be permissive
factors in a number of instructive pathways, as they are also
required for activins to pattern Xenopus axial mesoderm (Cornell
and Kimelman, (1994) Development 120:2187-2198; LaBonne and
Whitman, (1994) Development 120:463-472).
[0568] The induction of Hoxd and Bmp-2 expression in response to
Sonic and FGF-4 in the absence of an AER suggests that the mesoderm
is a direct target tissue of Sonic protein. Since Sonic can induce
Fgf-4 expression in the AER, it follows -that Sonic also acts
indirectly on the mesoderm through the induction of competence
factors in the AER.
[0569] (viii) Downstream Targets and a Cascade of Signals Induced
by Sonic
[0570] The five AbdB-like Hoxd genes, Hoxd-9 through -13, are
initially expressed in a nested pattern centered on the posterior
of the limb bud, a pattern which suggests they might be controlled
by a common mechanism (Dolle, et al., (1989) Cell 75:431-441;
Izpisua-Belmonte, et al., (1991) Nature 350:585-9). The analysis of
the endogenous and induced domains of Hoxd gene expression suggests
that Sonic normally induces expression of Hoxd-11, -12 and -13. In
contrast it was found that Hoxd-9 and -10 expression initiate
before Sonic mRNA is detectable. This implies that at least two
distinct mechanisms control the initiation of Hoxd gene expression
in the wing bud, only one of which is dependent on Sonic.
[0571] Several observations suggest that the elaboration of the
Hoxd expression domains is not controlled directly by Sonic, but
rather by signals which are downstream of Sonic. The Hoxd
expression domains rapidly diverge from Sonic, and evolve into
several distinct subdomains. Moreover these subdomains appear to be
separately regulated, as analysis of the murine Hoxd-11 gene
promoter suggests that it contains independent posterior and distal
elements (Gerard, et al., (1993) Embo. J. 12:3539-50). In addition,
although initiation of Hoxd-11 through -13 gene expression is
dependent on the AER, their expression is maintained following AER
removal (Izpisua-Belmonte, et al., (1992) Embo. J. 11:1451-7). As
Sonic expression fades rapidly under similar conditions, this
implies that maintenance of Hoxd gene expression is independent of
Sonic. Since ectopic Sonic can induce a recapitulation of the Hoxd
expression domains in the limb, it can be concluded that although
indirect effectors appear to regulate the proper patterning of the
Hoxd expression domains, they are downstream of Sonic. Potential
mediators of these indirect effects include Bmp-2 in the mesoderm
and Fgf-4 from the AER.
[0572] In contrast to the Hoxd genes, Bmp-2 gene expression in the
posterior limb mesoderm appears to be continually regulated by
Sonic. It was found that both endogenous and ectopic Bmp-2
expression correspond to that of Sonic. Furthermore, continued
Bmp-2 expression is dependent on the AER and can be rescued by
FGF-4. It is likely that this is an indirect consequence of the
fact that Sonic expression is also maintained by the AER and can be
rescued by FGF-4. In fact, Bmp-2 expression might be a direct
response of cells to secreted Sonic protein. The differences
between Bmp-2 and Hoxd gene expression suggest that multiple
pathways downstream of Sonic regulate gene expression in the
mesoderm.
[0573] Bmp-2 itself is a candidate for a secondary signaling
molecule in the cascade of patterning events induced by Sonic.
Bmp-2 is a secreted molecule of the TGF-.beta. family and its
expression can be induced by Sonic. This appears to be an
evolutionarily conserved pathway, as HH, the Drosophila homolog of
Sonic, activates the expression of dpp, the homolog of Bmp-2, in
the eye and wing imaginal discs (Heberlein, et al., (1993) Cell
75:913-26; Ma, et al., (1993) Cell 75:927-38; Tabata and Kornberg,
(1994) Cell 76:89-102). Expression of HH is normally confined to
the posterior of the wing disc. Ectopic expression of HH in the
anterior of the disc results in ectopic expression of dpp and
ultimately in the duplication of wing structure with mirror image
symmetry (Bassler and Struhl, (1994) Nature 368:208-214). This
effect is strikingly parallel to the phenotypic results of ectopic
expression of Sonic in the chick limb.
[0574] (ix) Regulation of Sonic Expression
[0575] Sonic expression is activated in the posterior of the limb
bud very early during mesodermal outgrowth (Riddle et al., (1993)
Cell 75:1401-16). The factors which initiate this localized
expression are not yet identified but ectopic expression of Hoxb-8
at the anterior margin of the mouse limb bud results in the
activation of a second domain of Sonic expression under the
anterior AER (Charite el al., (1994) Cell 78:589-601). Since
retinoic acid is known to be able to induce the expression of
Hoxb-8 and other Hox genes in vitro (Mavilio et al., (1988)
Differentiation 37:73-79) it is possible that endogenous retinoic
acid acts to make cells competent to express Sonic by inducing
expression of upstream Hox genes, either in the very early limb bud
or in the flank prior to the limb bud formation.
[0576] Several lines of evidence suggest that once induced Sonic
expression is dependent on signals from the posterior AER.
Following its initiation in the posterior limb mesoderm, the Sonic
expression domain moves distally as the limb bud grows out, always
remaining subjacent to the AER. Similarly, Sonic expression can
also be induced on the anterior margin of the limb bud by
implantation of a retinoic acid bead, but the induced ectopic
expression is limited to the mesoderm directly underlying the AER
(Riddle, et al., (1993) Cell 75:1401-16). In addition, ZPA activity
fades rapidly following removal of the AER (Niswander, et al.,
(1993) Cell 75:579-87; Vogel and Tickle, (1993) Development
119:199-206), and ZPA grafts only function when placed in close
proximity to the AER (Tabin, (1991) Cell 66:199-217; Tickle, (1991)
Development Supp. 1:113-21). The observation that continued Sonic
expression depends on signals from the posterior AER reveals the
mechanism underlying these observations.
[0577] The reliance of Sonic expression on AER-derived signals
suggests an explanation for the distal shift in Sonic expression
during limb development (Riddle et al., (1993) Cell 75:1401-16).
Signals from the AER also promote distal outgrowth of the
mesodermal cells of the progress zone, which in turn results in the
distal displacement of the AER. Hence, as maintenance of Sonic
expression requires signals from the AER, its expression domain
will be similarly displaced.
[0578] It was found that replacement of the AER with FGF-4 soaked
beads results in the maintenance of Sonic expression. This result
is consistent with the previous findings that ZPA activity can be
maintained in vivo and in vitro by members of the FGF family
(Anderson, et al., (1993) Development 117:1421-33; Niswander et
al., (1993) Cell 75:1401-16; Vogel and Tickle, (1993) Development
119:199-206). Since Fgf-4 is normally expressed in the posterior
AER, these results suggest that Fgf-4 is the signal from the
ectoderm involved in maintaining Sonic expression.
[0579] (x) Sonic and Regulation and Maintenance of the AER
[0580] Sonic can induce anterior extensions of the AER which have
an inverted polarity relative to the endogenous AER. This polarity
is demonstrated by examining the expression of two markers in the
AER. In normal limbs Bmp-2 is expressed throughout the AER, while
Fgf-4 is expressed in the posterior two thirds of the AER. In the
extended AER resulting from ectopic Sonic expression, Bmp-2 is
again found throughout the AER, while Fgf-4 expression is biphasic,
found at either end of the AER, overlying the anterior and
posterior mesodermal domains expressing Sonic. These results are
consistent with previous observations that antero-posterior
polarity of the AER appears to be regulated by the underlying
mesoderm, and that ZPA grafts lead to the induction of ectopic,
polarized AER tissue (Maccabe and Parker, (1979) J. Embryol. Exp.
Morph. 53:67-73). Our results also suggest that the normal AP
polarity of the AER is a reflection of endogenous Sonic expression.
The induced AER is sufficient to promote complete PD outgrowth of
the induced structures (Riddle et al., (1993) Cell 75:1401-16).
Hence whatever factors are necessary to maintain the AER are also
downstream of Sonic.
[0581] (xi) A Positive Feedback Loop Between Sonic and Fgf-4
[0582] The induction of Fgf-4 expression by Sonic in the ectopic
AER, and the maintenance of Sonic expression by FGF-4 suggest that
Sonic and Fgf-4 expression are normally sustained by a positive
feedback loop. Such a feedback loop would allow the coordination of
mesodermal outgrowth and patterning. This coordination is possible
because Sonic patterns mesodermal tissue and regulates Fgf-4
expression, while. FGF-4 protein induces mesodermal proliferation
and maintains Sonic expression. Moreover mesodermal tissue can only
be patterned by Sonic in the context of a competence activity
provided by F8f-4. Thus patterning is always coincident with
proliferation.
[0583] It remains possible that exogenously applied Fgf-4 might be
mimicking the activity of a different member of the FGF family. For
example, Fgf-2 is expressed in the limb mesoderm and the AER
(Savage et al., (1993) Development Dynamics 198:159-70) and has
similar effects on limb tissue as Fgf-4 (Niswander and Martin,
(1993) Nature 361:68-71; Niswander, et al., (1993) Cell 75:579-87;
Riley, et al., (1993) Development 118:95-104; Fallon, et al.,
(1994) Science 264:104-7).
[0584] (xii) Coordinated Regulation of Limb Outgrowth and
Patterning
[0585] Patterning and outgrowth of the developing limb are known to
be regulated by two major signaling centers, the ZPA and AER. The
identification of Sonic and FGFs as molecular mediators of the
activities of the ZPA and AER has allowed for dissociation of the
activities of these signaling centers from their regulation, and
investigation of the signaling pathways through which they
function.
[0586] The results presented above suggest that the ability of
cells to respond to Sonic protein is dependent on FGFs produced by
the AER. It was also found that Sonic induces a cascade of
secondary signals involved in regulating mesodermal gene expression
patterns. In addition evidence was found for a positive feedback
loop initiated by Sonic, which maintains expression of Sonic in the
posterior mesoderm and Fgf-4 in the AER. The feedback loop
described suggests a mechanism whereby outgrowth and patterning
along the AP and PD axes of the limb can be coordinately
regulated.
[0587] The results described above further suggest that Sonic acts
as a short range signal which triggers a cascade of secondary
signals whose interplay determines the resultant pattern of
structures. The data suggest a number of inductive pathways that
can be combined to generate a model (FIG. 14) which describes how
Sonic, in coordination with the AER, acts to pattern mesodermal
tissues along the anterior-posterior limb axis, while
simultaneously regulating proximal-distal outgrowth.
[0588] Following its induction, Sonic signals to both the limb
ectoderm and mesoderm. Sonic imposes a distinct polarity on the
forming AER, including the posteriorly biased expression of Fgf-4,
and the AER becomes dependent on continued Sonic expression. The
mesoderm, as long as it is receiving permissive signals from the
overlying ectoderm, responds to the Sonic signal by expressing
secondary signaling molecules such as Bmp-2 and by activating Hoxd
genes. Bmp-2 expression is directly dependent on continued Sonic
expression, while the continued expression of the Hoxd genes,
rapidly becomes Sonic. independent. In a reciprocal fashion,
maintenance of Sonic hedgehog expression in the posterior mesoderm
becomes dependent on signals from the AER. Since the factors
expressed by the AER are not only required for the maintenance of
Sonic expression and activity, but are also mitogenic, growth and
patterning become inextricably linked. Coordination of limb
development through interdependent signaling centers forces the AP
and PD structures to be induced and patterned in tandem. The
pathways elucidated herein thus provide a molecular framework for
the controls governing limb patterning
EXAMPLE 8
Sonic, BMP-4, and Hox Gene Expression Suggest a Conserved Pathway
in Patterning the Vertebrate and Drosophila Gut
[0589] (i) Experimental Procedure
[0590] In Situ Hybridization and Photography
[0591] BMP probes were isolated using primers designed to amplify
members of the TGF- and BMP families (Basler, K. et al., (1993)
Cell 73:687-702, eight independent 120 bp BMP fragments were
amplified from a stage 22 chicken posterior limb bud plasmid cDNA
library. These fragments were pooled and used to screen an
unamplified stage 22 limb bud lambda zap cDNA library constructed
as in Riddle et al., (1993) Cell 75:1401-16. Among the BMP related
clones isolated were an approximately 1.9 kb cDNA clone
corresponding to chicken BMP-2 and an approximately 1.5 kb cDNA
clone corresponding to chicken BMP-4. Both clones contain the
entire coding regions. The Sonic clone was obtained as described in
Riddle et al, (1993)Cell 75:1401-16. Digoxigenin-UTP labeled RNA
probes were transcribed as per Riddle et al., (1993) Cell
75:1401-16. Briefly, harvested chick embryos were fixed overnight
in 4% paraformaldehyde, washed in PBS then processed for whole
mount in situ hybridization methods are per Riddle et al., (1993)
Cell 75:1401-16. Embryos were photographed from either ventral or
dorsal surfaces under transmitted light using a Nikon zoom stereo
microscope with Kodak Ektar 100 ASA film. Whole mount in situ
hybridization embryos and viscera were processed for sectioning as
described in Riddle et al., (1993) Cell 75:1401-16. 15-25 .mu.m
transverse sections were air dried and photographed with
brightfield or numarski optics using a Zeiss Axiophot microscope
and Kodak Ektar 25 ASA film.
[0592] Chick Embryos and Recombinant Retroviruses
[0593] A retroviral vector engineered to express a full length cDNA
of chicken Sonic, as in Riddle et al. (1993)Cell 75:1401-16, was
injected unilaterally into stage 8-13 chicken embryos targeting the
definitive endoderm at the mid-embryo level. At this stage the CIP
has not formed and neither Sonic nor BMP-4 are expressed in the
region injected. Injections were performed on the ventral surface
on embryos cultured with their ventral surface facing up (New, D.
A. T. (1955) Embryol. Exp. Morph. 3:320-31. Embryos were harvested
18-28 hours after injection and prepared for whole mount in situ
hybridization (see above description of in situ experiment),
hybridized with Sonic or BMP-4 digoxigenin labeled probes.
[0594] In Situ Hybridization with Hox Genes
[0595] Cloned cDNA of the chicken homologues of Hoxa-9, -10, -11,
-13; b-9, c-9, -10, -11; d-9, -10, -11, -12, and -13 were used to
transcribe digoxigenen-UTP labeled riboprobes for whole mount in
situ hybridization. Domestic chick embryos were harvested into PBS
and eviscerated. The visceral organ block was fixed in 4%
paraformaldehyde overnight and processed for whole mount in situ
hybridization. Methods and photographic technique as described
above.
[0596] (ii) Expression of Sonic and BMP-4 in Stage 13 Chick Embryos
Determined by Whole Mount In Situ Hybridization
[0597] Chick gut morphogenesis begins at stage 8 (Hamberger and
Hamilton, (1987) Nutr; 6:14-23 with a ventral in-folding of the
anterior definitive endoderm to form the anterior intestinal portal
(AIP) (Romanoff, A. L., (1960) The Avian Embryo, The Macmillan Co.,
NY. This lengthens posteriorly forming the foregut. A second wave
of endodermal invagination is initiated posteriorly at stage 13,
creating the caudal intestinal portal (CIP). The CIP extends
anteriorly forming the hindgut. Sonic expression, previously noted
in the endodern of the vertebrate gut (Riddle et al., (1993) Cell
75:1401-16; Echelard et al., (1993) Cell 75:1417-1430), is
expressed early in a restricted pattern in the endodermal lips of
the AIP and CIP. Sonic expression is detected in the endoderm of
the AIP and CIP in pre gut closure stages. At later stages, stage
28 embryos, Sonic is expressed in the gut in all levels (fore-,
mid-, and hind-gut) restricted to the endoderm. Sonic is known to
be an important inductive signal in other regions of the embryo
including the limb bud (Riddle et al., (1993) Cell 75:1401-16) and
neural tube (Echelard et al., (1993) Cell 75:1417-1430; Kraus et
al., (1994) Cell 75:1437-1444; Roelink et al., (1994) Cell
76:761-775). Since primitive gut endoderm is known to cause
gut-specific mesodermal differentiation when combined with non-gut
mesenchyme (Haffen et al., (1987) Nutr. 6:14-23), we speculated
that Sonic might function as an inductive signal to the visceral
mesoderm. A potential target gene for the action of Sonic was
suggested by analogy to the Drosophila imaginal discs where HH, the
homologue of vertebrate Sonic, activates the expression of the
TGF-.beta. related gene dpp in adjacent cells (Tabata abd Kornberg,
(1994) Cell 76:89-102; Heberlein et al., (1993) Cell 75:913-926; Ma
et al., (1993) Cell 75:913-926; Basler et al., (1993) Cell
73:687-702). There are two vertebrate homologues of dpp, BMP-2 and
BMP-4. The earliest detectable expression of BMP-4 occurs
simultaneously with the first observable expression of Sonic in the
developing gut. BMP-4 is expressed in a domain abutting Sonic at
the AIP and the CIP, but is restricted to the adjacent ventral
mesoderm. BMP-4 gut expression persists into later stage embryos,
stage 33 embryos, in the visceral mesoderm only. The tissue
restricted expression of both genes is maintained in all stages
studied. BMP-2 is not expressed in the gut at the AIP or CIP, but
is expressed in clusters of cells in the gut mesoderm in later
stages, a pattern distinct from that of BMP-4.
[0598] (iii) Ectopic Expression of Sonic Induces Ectopic Expression
of BMP-4 in Mesodermal Tissues of the Developing Chick
[0599] To test whether Sonic is capable of inducing BMP-4 in the
mesoderm we an ectopic expression system previously used to study
the role of Sonic in limb development was utilized (Riddle et al.,
(1993) Cell 75:1401-16). A replication competent retrovirus
engineered to express Sonic was injected unilaterally into the
presumptive endoderm and visceral mesoderm at mid-embryo positions
in stage 8-13 chick embryos in vitro (New, D. A. T. (1955) Embryol.
Exp. Morph. 3:320-321). When embryos were examined by in situ
hybridization 18-26 hours later, the normal wild type expression of
Sonic is detected at the AIP, CIP, and in the midline (neural tube
and notochord). Ectopic Sonic expression is present unilaterally on
the left ventral surface. Also, wild type Sonic expression is seen
in the floor plate of the neural tube and notochord. Ectopic
expression is seen unilaterally in the visceral endoderm, its
underlying splanchnic mesoderm, and somatic mesoderm. BMP-4
expression can be seen induced in the mesoderm at the site of
injection, in addition to its normal expression in the mesoderm of
the CIP. Wild type BMP-4 expression is seen in the most dorsal
aspects of the neural tube and symmetrical lateral regions adjacent
to the neural tube. Induced BMP-4 expression is present
unilaterally in the splanchnic mesoderm at the site of Sonic viral
injection, and not in the visceral endoderm.
[0600] Since BMP-4 is, itself, a secreted protein, it could
function as a secondary signal in an inductive cascade, similar to
the signal cascades from HH to dpp in Drosophila imaginal discs
(Tabata abd Kornberg, (1994) Cell 76:89-102; Heberlein et al.,
(1993) Cell 75:913-926; Ma et al., (1993) Cell 75:913-926; Basler
et al., (1993) Cell 73:687-702) and from Sonic to BMP-2 in the limb
bud. In the gut, BMP-4 could act as a secondary signal either as
part of a feedback loop to the endoderm or within the visceral
mesoderm. This latter possibility is consistent with the finding
that in mice homozygous for a deletion in the BMP-4 gene, the
ventral mesoderm fails to close.
[0601] (iv) Expression of Hox Genes in the Developing Chick Gut
[0602] There is a striking parallel between the apparent role of
Sonic as an endoderm-to-mesoderm signal in early vertebrate gut
morphogenesis and that of its Drosophila homologue, HH. HH (like
Sonic) is expressed in the Drosophila gut endoderm from the
earliest stages of morphogenesis (Taylor et al., (1993) Mech. Dev.
42:89-96). Its putative receptor, patched, is found in the visceral
mesoderm implicating HH (like Sonic) in endodermal-mesodermal
inductive interactions. This led to consideration whether other
genes known to be involved in regulating Drosophila gut development
might also play a role in regulating chick gut morphogenesis.
Regionally specific pattern in Drosophila gut endoderm is regulated
by a pathway involving restricted expression of homeotic genes in
the mesoderm (McGinnis and Krumlauf, (1992) Cell 68:283-302).
Although the basis for patterning the vertebrate gut is poorly
understood, in several other regions of the embryo Hox genes have
been implicated as key regulators of patterns. Vertebrate Hox genes
are expressed in overlapping anteroposterior domains which
correlate with structural boundaries in the developing hindbrain,
vertebrae, and limbs (McGinnis and Krumlauf, (1992) Cell
68:283-302). Whole mount in situ hybridization was used to test
whether these genes are also expressed in the developing vertebrate
hindgut and whether their domains of expression correlate with
morphologic borders of the chick gut.
[0603] Lumenal gut differentiation creates three morphologically
and physiologically distinct regions: fore-, mid-, and hind-gut.
The fore-gut and hind-gut are the derivatives of the primitive gut
tubes initiated at the AIP and CIP respectively. Ultimately these
tubes meet and fuse at the yolk stalk around stage 24-28. The
midgut is formed from both foregut and hindgut primordia, just
anterior and posterior to the yolk stalk.
[0604] The most posterior derivative of the hindgut is the cloaca,
the common gut-urogenital opening. The rest of the hindgut develops
into the large intestine. The midgut/hindgut border is demarcated
by a paired tubal structure, the ceca (analogous to the mammalian
appendix), which forms as budding expansions at the midgut/hindgut
border at stage 19-20. Anterior to the ceca, the midgut forms the
small intestine.
[0605] The expression pattern of the 5' members of the Hox gene
clusters in the chick hindgut by whole mount in situ hybridization
was studied. Hox gene expression patterns in the gut are dynamic.
They are initially expressed (by stage 10) in broad mesodermal
domains extending anteriorly and laterally. Later they become
restricted. By stage 25, the Abd-B like genes of the Hoxa and Hoxd
cluster are regionally restricted in their expression in hindgut
mesoderm. The most anteriorly expressed gene, Hoxa-9, has an
anterior border of expression within the mesoderm of the distal
midgut (to a point approximating the distal third of the midgut
length). Each successive gene within the A and D Hox clusters has a
more posterior domain of expression. Hoxa-10, Hoxd-9 and Hoxd-10
are restricted in their expression to the ceca Hoxa-11 and Hoxd-11
have an anterior limit of expression in the mid-ceca at the
approximate midgut/hindgut boundary (Romanoff, A. L. (1960) The
Avian Embryo, The Macmillan Co. NY). Hoxd-12 has an anterior limit
at the posterior border of the ceca and extends posteriorly
throughout the hindgut to the cloaca. Hoxa-13 and Hoxd-13 are
expressed in the most posteriorly restricted domain, in the ventral
mesoderm surrounding the cloaca. Hoxa-13 and Hoxd-13 are the only
Abd-B like genes which are also expressed within the gut endoderm,
from the ceca to the cloaca.
[0606] The only member of the B or C Hox clusters which we found to
be expressed in the hindgut is Hoxc-9. The expression of Hoxc-9
overlaps with its paralogues Hoxa-9 and Hoxd-9 in the midgut
mesoderm, but has a sharp posterior boundary, complementary to
Hoxa-11 and Hoxd-11 in the mid-ceca.
[0607] The restricted expression of the Abd-B like Hox genes appear
to demarcate the successive regions of the gut which will form the
cloaca, the large intestine, the ceca, the mid-ceca at the
midgut/hindgut border, and the lower portion of the midgut (perhaps
identifying that portion of the midgut derived from the posterior
gut tube3). Moreover, these molecular events presage regional
distinctions. Expression of all Hox genes could be detected by
stage 14, well before the hindgut lumen is closed (by stage 28) and
is maintained in subsequent stages studied. Cytodifferentiation of
the hindgut mesoderm and epithelium begins later, at stages 29-31
(Romanoff, A. L. (1960) The Avian Embryo, The Macmillan Co.
NY).
[0608] These results suggest that specific Hox genes might be
responsible for regulating morphogenesis of the gut. Consistent
with this, there is an apparent homeotic alteration in the gut of a
transgenic mouse in which the anterior limit of expression of
Hoxc-8 is shifted rostrally: a portion of foregut epithelium
mis-differentiates as midgut (Pollock and Bieberich, (1992) Cell
71:911-923).
[0609] (v) Conservation in the Expression of Regulatory Genes
Involved in the Formation of Vertebrate and Drosophila Gut
[0610] There is an intriguing parallel between the expression
patterns of Sonic, BMP-4, and the Hox genes in the vertebrate gut
and those of their homologues during Drosophila gut morphogenesis
(FIG. 15). This conservation is of particular interest because in
Drosophila the role played by these genes has been clarified
genetically. HH (like its vertebrate homologue, Sonic) is expressed
at the earliest stages in the gut endoderm and may be a signal to
visceral mesoderm (Taylor et al., (1993) Mech. Dev. 42:89-96).
Nothing is known directly of the relationship between HH expression
and activation of expression of other genes in the Drosophila gut.
However, in Drosophila imaginal discs, HH is known to activate the
expression of dpp in a signaling cascade (Kraus et al., (1994) Cell
75:1437-1444; Heberlein et al., (1993) Cell 75:913-926; Ma et al.,
(1993) Cell 75:913-926; Basler et al., (1993) Cell 73:687-702).
Later in gut development, the production of dpp in the mesoderm
contributes to the regulation of the expression of homeotic genes
in both the mesoderm and the endoderm (Bienz, M. (1994) TIG
10:22-26). Drosophila homeotic genes are expressed in the gut
visceral mesoderm and their expression is known to determine the
morphologic borders of the midgut. This involves proper induction
of gene expression in the adjacent endoderm, one of the mediators
of the interaction is dpp (Bienz, M. (1994) TIG 10:22-26). If HH is
required for the ultimate activation of the homeotic genes in the
Drosophila midgut, this would parallel the situation in the
vertebrate limb bud where Sonic functions as an upstream activator
of the Hox genes (Riddle et al., (1993) Cell 75:1401-1416), perhaps
in a signaling cascade involving BMP-2.
[0611] The extraordinary conservation in the expression of
regulatory genes in the vertebrate and Drosophila gut strongly
suggests a conservation of patterning mechanisms. Pathways
established by genetic studies in Drosophila provide direct
insights into the molecular basis for the regionalization and
morphogenesis of the vertebrate gut.
EXAMPLE 9
Bacterially Expressed Hedgehog Proteins Retain Motorneuron-inducing
Activity
[0612] Various fragments of the mouse Shh gene were cloned into the
pET11D vector as fusion proteins with a poly(His) leader sequence
to facilitate purification. Briefly, fusion genes encoding the
mature M-Shh protein (corresponding to Cys-25 through Ser-437 of
SEQ ID No. 11) or N-terminal containing fragments, and an
N-terminal exogenous leader having the sequence
M-G-S-S-H-H-H-H-H-H-L-V-P-R-G-S-H-M were cloned in pET11D and
introduced into E. coli. The poly(His)-Shh fusion proteins were
purified using nickel chelate chromatography according to the
vendor's instructions (Qiagen catalog 30210), and the poly(His)
leader cleaved from the purified proteins by treatment with
thrombin.
[0613] Preparations of the purified Shh proteins were added to
tissue explants (neural tube) obtained from chicken embryos and
cultured in a defined media (e.g., no serum). M-Shh protein was
added to final concentrations of between 0.5 pM to 5 nM, and
differentiation of the embryonic explant tissue to motorneuron
phenotype was detected by expression of Islet-1 antigen. The
bacterially produced protein was demonstrated to be active in the
explant cultures at concentrations as low as 5 to 50 pM. An Shh
polypeptide containing all 19 kd of the amino terminal fragment and
approximately 9 kd of the carboxyl terminal fragment (see Example
6) displayed both motor neuron inducing activity and weak floor
plate inducing activity, indicating that these activities likely
reside with the N-terminal fragment.
EXAMPLE 10
Induction of Dopaminergic Neuron Phenotype with Sonic Hedgehog
[0614] Hamburger-Hamilton stage 8-10 chick embryos were dissected
free of the vitelline membranes and the areas opaca and pellucida.
The embryos were then incubated in Dulbecco's Modified Eagle's
Medium containing 0.5% dispase (Boehringer), 10 .mu.g/ml
hyaluronidase (Sigma), and 0.04% DNAse I (Sigma). The neural plate
was then separated from its underlying mesoderm and notochord. The
presumptive midbrain was identified and located according to its
fate map (Couly and Le Douarin, 1987, Developmental Biol.
120:198-214) and isolated. The ventral one-third of the
mesencephalic neural plate, comprising the presumptive floor plate
and adjacent prospective dopaminergic neurons was then removed and
discarded. The dorsal one-third was likewise dissected and removed.
The remaining intermediate region was then incubated in vitro on a
2% agarose (Sigma) containing substrate made with alpha medium
(Gibco). Recombinant Shh hedgehog, both human and mouse (full
length cDNA), was then introduced to the tissue in one of two ways:
(1) Bound to nickel-agarose beads (Qiagen) via the 6-histidine tag
engineered onto the amino terminus of the protein, or (2) was
incorporated in a soluble form directly into the agarose substrate.
Dihydrofolate reductase was used as the control protein for these
experiments. The tissue was then incubated at 37.degree. C. for
periods ranging from 36-48 hours. For analysis, tissue was fixed at
4.degree. C. in 4% paraformaldehyde and stored in 50% MeOH until
staining. Staining was done for both tyrosine hydroxylase (TH)
(Boehringer), L-DOPA (Chemicon), and dopamine (DA) (Chemicon).
[0615] The data indicate that both mouse and human recombinant Shh
hedgehog were active in the above described experiments.
Furthermore, results indicate that addition of Shh induces both
islet-1 (a motor neuron marker) and TH (a catecholaminergic neuron)
as well as the accumulation of L-DOPA in the mesencephalon, which
is indicative of a dopaminergic phenotype.
EXAMPLE 11
Sonic Hedgehog Induces Bone Formation
[0616] The ectopic bone formation assay was essentially done as
described in Sampath and Reddi, 1983, PNAS USA 80:6591-6595. The
mouse Shh protein was frozen and lyophilized, and the powder was
enclosed in no. 5 gelatin capsule. Alternatively, 0.9-2.0 mg of
collagen sponge (Collastat) was used as matrix. The Shh protein
(12.5 .mu.g) was added directly to the washed sponge, the sponge
lyophilized, and the sponge implanted. The capsules or collagen
sponges were implanted subcutaneously in the abdominal thoracic
area of 21- to 49-day female Long-Evans rats and routinely removed
at 11 days. Samples were processed for histological analysis, with
1-.mu.m glycolmethacrylate sections stained with Von Kossa and acid
fuschin or toluidine blue. Von Kossa staining shows mineral
(hydroxyapatite) formation. The collagen sponge by itself was used
as a control in these experiments. The results indicate that the
addition of mouse Shh protein induced bone formation in these
rats.
[0617] All of the above-cited references and publications are
hereby incorporated by reference.
Equivalents
[0618] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, numerous
equivalents to the specific polypeptides, nucleic acids, methods,
assays and reagents described herein. Such equivalents are
considered to be within the scope of this invention and are covered
by the following claims.
Sequence CWU 1
1
* * * * *