U.S. patent application number 09/897322 was filed with the patent office on 2002-08-01 for dorsal tissue affecting factor and compositions.
Invention is credited to Cudny, Henryk D., Harland, Richard M., Ip, Nancy Y., Knecht, Anne, Lamb, Teresa, Smith, William C., Valenzuela, David M., Yancopoulos, George D..
Application Number | 20020102643 09/897322 |
Document ID | / |
Family ID | 27420696 |
Filed Date | 2002-08-01 |
United States Patent
Application |
20020102643 |
Kind Code |
A1 |
Valenzuela, David M. ; et
al. |
August 1, 2002 |
Dorsal tissue affecting factor and compositions
Abstract
Novel dorsal growth inducing factors, complexes including the
factors, and DNA or RNA coding sequences for the factors are
described.
Inventors: |
Valenzuela, David M.;
(Franklin Square, NY) ; Ip, Nancy Y.; (Stamford,
CT) ; Cudny, Henryk D.; (Concord, CA) ;
Yancopoulos, George D.; (Yorktown Heights, NY) ;
Harland, Richard M.; (Moraga, CA) ; Smith, William
C.; (Santa Barbara, CA) ; Lamb, Teresa; (New
York, NY) ; Knecht, Anne; (Berkeley, CA) |
Correspondence
Address: |
Linda O. Palladino
Regeneron Pharmaceuticals, Inc.
777 Old Saw Mill River Road
Tarrytown
NY
10591
US
|
Family ID: |
27420696 |
Appl. No.: |
09/897322 |
Filed: |
July 2, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09897322 |
Jul 2, 2001 |
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09167874 |
Oct 7, 1998 |
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09167874 |
Oct 7, 1998 |
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08485721 |
Jun 7, 1995 |
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08485721 |
Jun 7, 1995 |
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08392935 |
Sep 22, 1995 |
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08392935 |
Sep 22, 1995 |
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PCT/US93/08326 |
Sep 2, 1993 |
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PCT/US93/08326 |
Sep 2, 1993 |
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07957401 |
Oct 6, 1992 |
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07957401 |
Oct 6, 1992 |
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07950410 |
Sep 23, 1992 |
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07950410 |
Sep 23, 1992 |
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07939954 |
Sep 3, 1992 |
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Current U.S.
Class: |
435/69.1 ;
435/320.1; 435/325; 514/17.1; 514/17.2; 514/17.7; 514/8.3; 530/350;
536/23.5 |
Current CPC
Class: |
C07K 16/22 20130101;
A61P 43/00 20180101; A61P 25/00 20180101; A61K 39/00 20130101; C07K
14/475 20130101; A61P 25/02 20180101; A61P 25/28 20180101; A61K
38/00 20130101 |
Class at
Publication: |
435/69.1 ;
435/325; 435/320.1; 514/12; 536/23.5; 530/350 |
International
Class: |
A61K 038/17; C07K
014/435; C12P 021/02; C12N 005/06 |
Goverment Interests
[0002] This invention was made, in part, with government support
under Grant Contract No. ROI-GM-42341, awarded by the National
Institutes of Health. The government has certain rights in this
invention.
Claims
What is claimed is:
1. An essentially purified and isolated noggin polypeptide having
an amino acid sequence as set forth in FIG. 1 (Sequence l.D. No. 2)
or a functionally equivalent amino acid sequence.
2. The human noggin polypeptide as claimed in claim 1, encoded by
the DNA of hnog.lambda.-9 (deposited with the American Type Culture
Collection under Accession No. 75310) or hnog.lambda.-10 (deposited
with the American Type Culture Collection under Accession No.
75308) and fragments and derivatives thereof exhibiting noggin
activity.
3. An isolated nucleic acid selected from a nucleic acid encoding a
noggin polypeptide as claimed in claim 1, and substantially similar
nucleotide sequences.
4. A nucleic acid as claimed in claim 3, which hybridizes to at
least one nucleotide probe selected from the sequence of
nucleotides 2 to 262 of Sequence I.D. No. 10 and the sequence:
5'GARGGIATGGTITGYAARCC (SEQ ID NO. 22).
5. A nucleic acid as claimed in claim 3, wherein said nucleic acid
is isolated from a .lambda. human placental genomic library.
6. A nucleic acid as claimed in claim 5, wherein said nucleic acid
is isolated from a phage selected from hnog.lambda.-9 (ATCC No.
75310) or hnog.lambda.-10 (ATCC No. 75308).
7. A substantially purified nucleic acid as claimed in claim 3,
encoding the human noggin polypeptide corresponding to Sequence
I.D. No. 2.
8. A mutated variant of a nucleic acid as claimed in claim 3, which
encodes a noggin agonist or antagonist.
9. A mutant noggin polypeptide which is a noggin agonist or
antagonist obtainable by expression of a nucleic acid as claimed in
claim 8.
10. An isolated nucleic acid which contains a nucleotide coding
sequence for a noggin polypeptide as claimed in claim 1, in the
anti-sense direction.
11. A phage selected from the group consisting of hnog.lambda.-9 as
deposited with the American Type Culture Collection and assigned
Accession Number 75310 and hnog.lambda.-10 as deposited with the
American Type Culture Collection and assigned Accession Number
75308.
12. An expression vector comprising expression regulatory sequences
operably linked to a nucleotide sequence which encodes noggin,
wherein said nucleotide sequence is selected from the group
consisting of: a) a nucleotide sequence which encodes the amino
acid sequence set forth in FIG. 1 (SEQ I.D. NO. 2), and b)
sequences which hybridize to the sequence of (a) and encode a
protein which promotes the induction of neural tissue.
13. An expression vector as claimed in claim 12, capable of
directing expression of a functional noggin polypeptide in a
eukaryotic host cell.
14. An expression vector as claimed in claim 13, wherein said host
cell is selected from the group consisting of COS cells and CHO
cells.
15. An expression vector as claimed in claim 12, capable of
directing the expression of a functional noggin polypeptide in a
prokaryotic host.
16. The expression vector as claimed in claim 15, wherein said host
is E. coli.
17. An expression vector as claimed in claim 12, capable of
directing the expression of a functional noggin polypeptide in a
baculovirus host.
18. Host cells transformed by an expression vector as claimed in
claim 12.
19. A method of producing a noggin polypeptide which comprises
culturing transformed host cells as claimed in claim 18, under
conditions suitable for expression of said polypeptide.
20. A method as claimed in claim 19, wherein human noggin is
produced in a form substantially free of proteins of non-human
origin.
21. A pharmaceutical composition comprising a therapeutically
effective amount of a polypeptide as claimed in claim 1, together
with a pharmaceutically acceptable carrier.
22. A culture medium suitable for use in culturing nerve cells
containing a noggin polypeptide as claimed in claim 1.
23. An isolated receptor which in vivo binds a noggin polypeptide
as claimed in claim 1, or a fragment thereof retaining the binding
site for said polypeptide.
24. An antibody which binds one or more noggin polypeptides as
claimed in claim 1, but not other growth factors.
25. A hybridoma capable of producing a monoclonal antibody as
claimed in claim 24.
26. The monoclonal antibody obtainable from hybridoma RP57-16.
27. Hybridoma RP57-16.
28. A hybridization probe suitable for detecting a nucleic acid as
claimed in claim 3, having the sequence: 5'GARGGIATGGTITGYAARCC
(SEQ I.D. NO. 22).
29. A noggin polypeptide as claimed in claim 1, for use in a method
of treatment of a human or animal.
30. A method of treatment of a human or animal comprising
administering a therapeutic dosage of a noggin polypeptide as
claimed in claim 1, wherein said treatment is selected from the
group consisting of regulation of cartilage and bone growth,
therapy of a congenital condition or degenerative disorder of the
nervous system, and treatment of damaged nerve cells.
Description
[0001] This application is a continuation of copending U.S. patent
application Ser. No. 08/392,935 filed on Mar. 3, 1995, which is the
U.S. National Stage of PCT/US93/08326 filed Sep. 2, 1993, which is
a continuation-in-part of U.S. Ser. No. 07/957,401 filed on Oct. 6,
1992, which is a continuation-in-part of U.S. patent application
Ser. No. 07/950,410 filed on Sep. 23, 1992, which is a
continuation-in-part of U.S. patent application Ser. No. 07/939,954
filed on Sep. 3, 1992.
FIELD OF THE INVENTION
[0003] The invention generally relates to growth factors and
neurotrophic factors, and more particularly to a soluble growth
factor with dorsal growth inducing activity, to complexes including
the factor, and to DNA or RNA coding sequences for the factor.
BACKGROUND OF THE INVENTION
[0004] Growth factors are substances, such as polypeptide hormones,
which affect the growth of defined populations of animal cells in
vivo or in vitro, but which are not nutrient substances. Proteins
involved in the growth and differentiation of tissues may promote
or inhibit growth, and promote or inhibit differentiation, and thus
the general term "growth factor" includes cytokines and trophic
factors. Among growth, or neurotrophic factors presently known are
those that can be classified into the insulin family [insulin,
insulin-like growth factors (e.g., IGF-I, IGF-II), mammary
stimulating factor (MSF), and nerve growth factor (NGF)]; those
classified into the epidermal growth factor family [epidermal
growth factor (EGF) and transforming growth factors (TGF.alpha.,
TGF.beta., TGF.gamma.)]; those classified 5 into the
platelet-derived growth factor family [platelet-derived growth
factor (PDGF), osteosarcoma-derived growth factor (ODGF), and
fibroblast growth factor (FGF)]; the neurotrophins [nerve growth
factor (NGF), brain derived neurotrophic factors (BDNF)
neurotrophins 3, 4, 5, (NT-3, NT-4, NT-5)]; and others [colony
stimulating factor (CSF), T-cell growth factor, tumor angiogenesis
factor (TAF), DNA synthesis promoting factor (DSF), tumor-derived
growth factors, fibroblast-derived growth factor (FDGF)].
[0005] Receptors that affect growth (that is, receptors for
growth-associated ligands) are proteins found associated with cell
surfaces that specifically bind their growth factors as
ligands.
[0006] Growth factor receptors are utilized in various clinical and
diagnostic applications.
[0007] U.S. Pat. No. 4,857,637, issued Aug. 15, 1989, inventors
Hammonds et al., describes a method for immunizing an animal
against its growth hormone receptor through use of vaccinating with
antibodies in order to stimulate growth of the animals.
[0008] U.S. Pat. No. 4,933,294, issued Jun. 12, 1990, inventors
Waterfield et al., describes studies of structural alterations of
the human EGF receptor and its gene and a relationship in
tumorigenesis for assays and therapies involving the human EGF
receptor. For example, such assays can involve detection of
structurally altered or abnormally expressed growth factor receptor
and the mRNA transcripts and genes which encode them. EGF may have
a role in cell proliferation and differentiation since it induces
early eyelid opening and incisor development in new born mice.
[0009] U.S. Pat. No. 5,030,576, issued Jul. 9, 1991, inventors Dull
et al., describes the role of receptors, such as receptors for
growth factors, in designing drugs by the pharmaceutical industry,
and discloses use of a receptor hybrid for screening drug purposes,
such as in studies of EGF binding domains. U.S. Pat. No. 5,087,616,
issued Feb. 11, 1992, inventors Myers and Bichon, describes a
method for destroying tumor cells with a composition including a
drug conjugate. The conjugate has a growth factor as one moiety and
a polymeric carrier with a cytotoxic compound as another moiety.
Thus, compositions of the Patent are described as binding
preferentially to tumor cells bearing EGF-binding receptors (when
an EGF growth factor, for example, is used as a first moiety).
[0010] U.S. Pat. No. 5,098,833, issued Mar. 24, 1992, inventors
Lasky, et al., describes a DNA isolant capable of hybridizing to
the epidermal growth factor domain. Expression systems for
recombinant production are said to be useful in therapeutic or
diagnostic compositions.
[0011] A good background review of a neurotrophic factor related to
NGF is provided by WO92/05254, published Apr. 2, 1992, which also
describes state of the art methods of: preparing amino acid
sequence variations, site-directed mutagenesis techniques, ligation
of coding DNA into a replicable vector for further cloning or for
expression, choice of promoters for expression vectors, suitable
host cells for expression, particularly mammalian cells, protein
purification upon recovery from culture medium as a secreted
protein, derivatization with bifunctional agents to cross-link
protein to a support matrix for use with antibodies, entrapment in
systems for drug delivery, preparation of therapeutic formulations,
and methods of administration. In addition, preparation of
polyclonal and monoclonal antibodies are described, such as are
useful in diagnostic assays. These various aspects of isolation,
preparation, and applications for a novel neurotrophic factor, as
illustrated by the W092/05254 publication, are incorporated herein
by reference.
[0012] Thus, growth factors, their receptors, and DNA or RNA coding
sequences, therefore, and fragments thereof are useful in a number
of therapeutic, clinical, research, diagnostic, and drug design
applications.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1. Nucleotide sequence (SEQ ID NO:1) for the human
noggin gene and deduced amino acid sequence (SEQ ID NO: 2).
[0014] FIG. 2 (A). Experimental design: competent animal cap(AC)
ectoderm was dissected from staged embryos as shown. St10.5 dorsal
and ventral AC and ventral marginal zones (VMXZ) also dissected as
shown. Explants were washed once in low Ca/Mg Ringers (LCMR)
solution and then placed in treatment medium containing factor
diluted in LCMR +0.5%BSA. Explants cultured to late stages
(St20.sub.+) were removed from treatment medium 6-16 hours after
the start of treatment and placed in LCMR. When explants reached
the desired stage they were either harvested for RNA, or they were
fixed for whole mount in situ hybridization or antibody
staining.
[0015] (B). Neural induction by noggin in the absence of muscle.
Lanes 1-3 show specific fragments protected by N-CAM,
.beta.-tubulin, and XIF-3 probes respectively in whole St24 embryo
RNA. Lanes 4-8 show protection by the mixture of these three probes
while lanes 9-13 show protection by an actin probe on tRNA(t), St24
embryo RNA (E), and RNA collected from St9 AC treated with 50 pM
activin (A), 25% of 20 fold concentrated control CHO cell medium
(C) or 25% of 20 fold concentrated noggin conditioned CHO cell
medium (N). Ubiquitously expressed cytoskeletal actin used as a
loading control shows that RNA levels in all treatments are
comparable (lanes 11-13).
[0016] FIG. 3. 12% SDS-PAGE run under reducing conditions. Proteins
were visualized by silver staining. Lane 1 shows molecular size
standards. Lanes 2-7 show 0, 0.1, 0.2, 0.5, 1, and 2 .mu.g of
purified human noggin.
[0017] FIG. 4 (A). Time course of animal caps treated with purified
noggin vs. activin; direct vs indirect neural induction. Animal
caps were dissected as shown in FIG. 2A and treated with LCMR+0.5%
BSA (U), a 20% dilution of activin conditioned COS cell medium (A),
or 1 .mu.g/ml purified human noggin(N). RNA isolated from treated
animal caps (lanes 2-13) along with St22 whole embryo RNA (lane1)
and tRNA (lane 14) was probed for N-CAM, .beta.-tubulin, muscle and
cytoskeletal actins, collagen type II, and EF-1a.
[0018] (B). Expression of early mesoderm markers in activin but not
noggin induced animal caps. Animal caps were dissected from St8
embryos, treated as described in (A), and harvested at St11. Lanes
1 and 2 respectively show goosecoid and Xbra probe protection by
St10.5 whole embryo RNA. Lanes 3-6 show protection by. a mix of
these two probes. Relative RNA levels are demonstrated by separate
EF-1.alpha. probe protection.
[0019] (C). Plasmid directed gastrula stage noggin expression
directly induces neural tissue. One cell stage embryos were
injected with 20 pg of pCSKAlacZ or pCSKAnoggin into the animal
pole. Animal caps from injected embryos were dissected at St8-9 and
cultured until St20, when they were harvested for analysis by RNase
protection.
[0020] FIG. 5. Responsiveness of dorsal and ventral animal caps to
neural induction by noggin. St 105 ventral and dorsal animal caps
were dissected as shown in FIG. 2. Dorsal and ventral animal caps
were treated with activin medium (DA,VA) or 1 .mu.g/ml human noggin
(DN, VN) and harvested at St26 for RNase protection analysis using
N-CAM, .beta.-tubulin, and actin as probes.
[0021] FIG. 6. Dose response of ventral marginal zones and animal
caps to human noggin protein. St 10.5 VMZs and St9 animal caps were
dissected as shown in FIG. 2A., and treated with 0, 1, 10, 50, 200,
and 1000 ug/ml of human noggin (lanes 3-8 and 10-15 respectively).
RNA from treated explants and control whole embryos aged to St26
was then analyzed by RNase protection, using the probes N-CAM,
.beta.-tubulin, actin and collagen type II. In this experiment,
muscle induction at the dose of 1 ng/ml is stronger than at 10
ng/ml, and there is a low level of muscle actin expression in the
uninduced VMZs. This could be due to experimental variability since
in repeated experiments we saw muscle induction only at the doses
of 50 ng/ml and above (data not shown).
[0022] FIG. 7. In situ hybridization and antibody staining. Tailbud
embryos stained for NCAM showing side and dorsal views (a,b); NCAM
RNA is only detected in the neural tube, and not the somites. For
comparison, somites of a tailbud embryo stain for muscle actin,
dorsal view (c). Neural specific 6F11 antibody staining at St30
(d-f). Some cement gland pigment remained in these embryos after
bleaching as seen in (d), however this pigment is distinct from
antibody staining. The inner mass of staining in the noggin treated
animal caps is due to the 6F11 antibody detection. Cement gland
specific XAG-1 transcripts detected at St23 (g-i), and anterior
brain otxA transcripts detected at St35 (j-l) in whole embryos at
(d,g,j), human noggin treated (1 .mu.g/ml) animal caps (e,h,k), and
untreated animal caps (f,i,l).
[0023] FIG. 8. Reverse phase HPLC profile of two refolded isoforms
of noggin. The refolded noggin solution was applied onto a Brownlee
Aquapore AX-300, 0.46.times.22 cmHPLC column at a flow rate of 1
ml/min. The column was equilibrated with solvent A containing 0.1%
TFA in water. Solvent B was 0.1% TRA in acetonitrile. The column
was developed according to the following protocol: a) 2 min
isocratically at 95% of solvent A-5% of solvent B; 60 min linear
gradient to 65% of solvent B and 35% of solvent A. Correctly
refolded noggin elutes earlier at 44%-46% solvent B.
[0024] FIG. 9. Reverse-phase HPLC chromatography characterization
of recombinant noggin refolded and purified from E. coli.
Conditions as in the legend to FIG. 8.
[0025] FIG. 10. Recombinant noggin produced in E. coli and in
insect cells analyzed by 12.5% SDS-PAGE. Lanes H, L: High and low
molecular weight markers of the indicated size, respectively. Lanes
1,2: Recombinant noggin produced in E. coli and in insect cells
respectively, treated with 2-mercaptoethanol before
electrophoresis. The slower mobility of noggin from insect cells
corresponds to the size increase that would occur due to N-linked
glycosylation at the single consensus site. Lanes 2,3: Recombinant
noggin produced in E. coli and in insect cells respectively, not
treated with 2-mercaptoethanol before electrophoresis.
[0026] FIG. 11. Circular dichroism spectra of recombinant noggin
produced in E. coli (--), and in insect cells (-).
[0027] FIG. 12. Ventral marginal zone assay showing induction of
muscle actin mRNA after exposure to human noggin (0.01, 0.05, 0.2
.mu.g/ml) produced in baculovirus, a mock transfected culture of
baculovirus (0.02, 1 .mu.g/ml) or human noggin produced in E. coli
(0.1, 0.5, 2, or 10 .mu.g/ml).
[0028] FIG. 13. Nucleotide sequence (SEQ ID NO:25) for the mouse
noggin gene and deduced amino acid sequence (SEQ ID NO:26).
SUMMARY OF THE INVENTION
[0029] In one aspect of the present invention a peptide that can be
in substantially purified form is characterized by one or more of
the following, highly conserved amino acid sequences:
1 QMWLWSQTFCPVLY (SEQ ID NQ:3); RFWPRYVKVGSC (SEQ ID NO:4);
SKRSCSVPEGMVCK (SEQ ID NO:5); LRWRCQRR (SEQ ID NO:6); and, ISECKCSC
(SEQ ID NO:7).
[0030] Peptides of the invention induce dorsal growth in
vertebrates and can be prepared in soluble, physiologically active
form for a number of therapeutic, clinical, and diagnostic
applications.
[0031] In a preferred embodiment, human noggin protein as set forth
in FIG. 1 (SEQ ID NO: 2) is prepared for use in therapeutic,
clinical and diagnostic applications.
[0032] In another aspect of the present invention an
oligonucleotide, such as cDNA, is provided having substantial
similarity to (or being the same as) SEQ ID NO:8 (deduced amino
acid sequence, SEQ ID NO: 9), SEQ ID NO: 10 (deduced amino acid
sequence, SEQ ID NO: 11), or SEQ ID NO:1. This oligonucleotide can
be single or double stranded, be formed of DNA or RNA bases, and
can be in the antisense direction with respectto SEQ ID NOS: 8, 10
or 1. SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO:1 each code for a
functional polypeptide that we have designated "noggin," which is
capable of inducing dorsal development in vertebrates when
expressed.
[0033] Noggin or fragments thereof (which also may be synthesized
by in vitro methods) may be fused (by recombinant expression or in
vitro covalent methods) to an immunogenic polypeptide and this, in
turn, may be used to immunize an animal in order to raise
antibodies against a noggin epitope. Anti-noggin is recoverable
from the serum of immunized animals. Alternatively, monoclonal
antibodies may be prepared from cells to the immunized animal in
conventional fashion. Antibodies identified by routine screening
will bind to noggin but will not substantially cross-react with
"wnt" or other growth factors. Immobilized anti-noggin antibodies
are useful particularly in the diagnosis (in vitro or in vivo) or
purification of noggin.
[0034] Substitutional, deletional, or insertional mutants of noggin
may be prepared by in vitro or recombinant methods and screened for
immuno-crossreactivity with noggin and for noggin antagonist or
agonist activity. Noggin also may be derivatized in vitro in order
to prepare immobilized noggin and labelled noggin, particularly for
purposes of diagnosis of noggin or its antibodies, or for affinity
purification of noggin antibodies.
[0035] The present invention further provides for expression of
biologically active noggin molecules in prokaryotic and eukaryotic
expression systems.
[0036] The present invention further provides for the production of
noggin in quantities sufficient for therapeutic and diagnostic
applications. Likewise, anti-noggin antibodies may be utilized in
therapeutic and diagnostic applications. For most purposes, it is
preferable to use noggin genes or gene products from the same
species for therapeutic or diagnostic purposes, although cross-
species utility of noggin may be useful in specific embodiments of
the invention.
[0037] In additional embodiments, the noggin nucleic acids,
proteins, and peptides of the invention may be used to induce
neural tissue formation in mammals.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0038] We have discovered a structurally unique growth factor that
is readily available in substantially pure, soluble form. We have
named the inventive polypeptide "noggin." This newly isolated
neurotrophic factor induces dorsal development in vertebrates.
[0039] An earlier described family of proteins that also induces
dorsal development are the "wnt" proteins. These, however, in
contrast to noggin remain tenaciously bound to cell surfaces. Our
initial work with noggin has been in Xenopus embryos; however,
noggin is highly conserved among vertebrates, as our work with
mouse noggin has demonstrated. The prior known FGF growth factor
family is also known to be involved in early embryonic induction,
but both the FGF proteins and their receptors are distinctly
different from noggin. Noggin modifies the actions of FGF (and also
activin), for example by potentiating growth, and is thus
particularly suggested in therapeutic compositions for use in
combination with other growth factors (as therapeutic adjuvants),
such as to modify or potentiate their effects.
[0040] We have cloned cDNA for noggin. The noggin cDNA contains a
single reading frame encoding a 26 kDa protein with a hydrophobic
amino-terminal sequence. Noggin is secreted. Noggin's cDNA encodes
the protein as a 26 kDa protein, but we have determined that noggin
is secreted in vivo, apparently as a dimeric glycoprotein with a
starting apparent molecular weight of about 33 kDa (as the
wild-type subunit). When not glycosylated, the monomeric unit has
an apparent molecular weight on SDS PAGE of about 25-30 kDa.
[0041] We have cloned the gene for human noggin (FIG. 1; SEQ ID
NO:
[0042] 1). The sequence codes for a protein which has noggin
activity (SEQ ID NO: 2). The carboxy terminal region of noggin
shows homology to a Kunitz-type protease inhibitor, indicating that
noggin protein, or fragments thereof, may exhibit activities of a
protease inhibitor.
[0043] We have been able to express biologically active noggin in
both eukaryotic and prokaryotic host cells. Two expression systems
we have successfully used to express biologically active noggin
have been mammalian cell lines (COS and mouse 293). A third
expression system is injection of synthetic mRNA into Xenopus
oocytes. In addition, we have successfully expressed biologically
active human noggin in a prokaryotic system, E. coli, and in
baculovirus.
[0044] Expression in these several different systems also
illustrates the high degree of conservation for noggin. We have
found, for example, substantial sequence similarity between frog
noggin and mouse noggin with a number of completely conserved
stretches.
[0045] Thus, the following amino acid sequences represent
completely conserved portions as between frog noggin and mouse
noggin:
2 QMWLWSQTFCPVLY (SEQ ID NO:3); RFWPRYVKVGSC (SEQ ID NO:4);
SKRSCSVPEGMVCK (SEQ ID NO:5); LRWRCQRR (SEQ ID NO:6); and, ISECKCSC
(SEQ ID NO:7).
[0046] There is about 87% overall conservation between the mouse
and frog sequences, and we have also observed a unique cysteine
distribution between the two. Noggin nucleic acids, or
oligonucleotides, encode a noggin polypeptide or hybridize to such
DNA and remain stably bound to it under stringent conditions and
are greater than about 10 bases in length; provided, however, that
such hybridizing nucleic acid is novel and unobvious over any prior
art nucleic acid including that which encodes or is complementary
to nucleic acid encoding other growth factors.
[0047] By "stringent conditions" we mean are those which (1) employ
low ionic strength and high temperature for washing, for example,
0.15 M NaCl/0.015 M sodium citrate/0.1% NaDodSo.sub.4 at 50.degree.
C., or (2) use during hybridization a denaturing agent such as
formamide, for example, 50% (vol/vol) formamide with 0.1% bovine
serum albumin/0.1%Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium
phosphate buffer at pH 6.5 with 750 mM NaCI, 75 mM sodium citrate
at 42.degree. C.
[0048] By "substantial similarity," when we are referring to a
nucleotide sequence, is meant cross hybridization of sequences
under conditions of moderate stringency using a probe greater than
100 nucleotides long at 30.degree. C. in a standard buffer (Wahl et
al., PNAS,76, 3683) and washes at 37.degree. C. in 300 mM NaCl, 30
mM sodium citrate, 0.2% SDS at pH 7. Alternatively, one is able to
isolate, by polymerase chain reaction, a fragment of DNA coding for
noggin or noggin family members when using primers of degenerate
sequence that encode those SEQ ID NOS:3-7.
[0049] By "substantial similarity" when reference is made to
proteins is that noggin from different species, or noggin family
members within a species, will preserve the positions of cysteine
residues in at least 80% of positions throughout the protein. Like
the neurotrophin family, the sequence of the mature form of noggin
and noggin related polypeptides will be identical in at least 40%
of positions. Substantial similarity at the protein level includes
an ability of a subject protein to compete with noggin for binding
to receptors and some (but not all) monoclonal antibodies raised
against noggin epitopes.
[0050] The cloned cDNA for noggin (derived from frog) is designated
herein as SEQ ID NO: 8, partial sequence from mouse as SEQ ID NO:
10 or full sequence of mouse noggin as shown in FIG. 13 (SEQ ID NO:
25). The human sequence is designated herein as SEQ ID NO: 1. We
have used RNA transcripts from the SEQ ID NO: 8 clone to rescue
embryos and return them to substantially normal development when
the noggin RNA is injected into ventralized embryos. In high doses
this results in excessive head development and it is for this
reason we named the protein "noggin." In northern blot analysis the
noggin cDNA hybridizes to two mRNAs that are expressed both
maternally and zygotically.
[0051] When using nucleotide sequences coding for part or all of
noggin in accordance with this invention, the length of the
sequence should be at least sufficient in size to be capable of
hybridizing with endogenous mRNA for the vertebrate's own noggin.
Typically, sufficient sequence size (for example, for use as
diagnostic probes) will be about 15 consecutive bases (DNA or RNA).
In some diagnostic and therapeutic applications, one may wish to
use nucleotide noggin coding sequences (analogous to all or a
portion of SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 25 or SEQ ID
NO:1) in the anti-sense direction with respect to either SEQ ID
NOS: 8, 10, 25, or 1.
[0052] We suggest as a few preferred primers for amplifying noggin
from other species (e.g. human):
3 5' Primer 1 SEQ ID NO: 12 C A A/G A C N T T C/T T G C/T C C N G T
N 5' Primer 2 SEQ ID NO: 13 T T C/T T G G C C N C/A G N T A C/T G T
N A A A/G G T N G G 5' Primer 3 SEQ ID NO: 14 C C N G A A/G G G N A
T G G T N T G 3' Primer 1 SEQ ID NO: 15 C A N C/G T/A A/G C A C/T T
T A/G C A C/T T C 3' Primer 2 SEQ ID NO: 16 C A N A C C A T N C C
C/T T C N G G 3' Primer 3 SEQ ID NO: 17 C G/T N C G/T T/C T G G/A C
A N C G/T C C A
[0053] where N represents a mixture of all four nucleotides and
mixtures of two nucleotides are represented by alternates (e.g.
A/G).
[0054] Although noggin transcript is not localized in the oocyte
and cleavage stage embryo, zygotic transcripts are initially
restricted to the presumptive dorsal mesoderm, and reach their
highest levels at the gastrula stage in the dorsal lip of the
blastopore (Spemann's organizer). In the neurula, noggin is
transcribed in the notochord and prechordal mesoderm.
[0055] Without being bound by theory, we have formulated hypotheses
about the embryological effects of noggin based on where it is
expressed, and on the effects of RNA injection in embryos. Since
noggin is expressed in the Spemann organizer, we believe noggin to
be a mediator of the effects of the Spemann organizer, namely
neural induction and dorsalization of the mesoderm. We have shown
that noggin is able to directly induce neural tissue formation.
Since noggin is expressed in the notochord and head mesoderm, we
believe noggin to influence either the dorsal-ventral pattern or
anterior-posterior pattern of the neural plate. Since noggin is
expressed in the branchial arch neural crest, we believe it may
therefore influence whether neural crest cells deposit cartilage
and also to influence later branchial arch growth and remodelling.
Noggin is expressed in the tail fin neural crest, and since neural
crest is required for growth of the fin, noggin may act as a growth
factor for epidermis or mesenchyme.
[0056] Although much of our experimental work has involved rescue
of embryonic development, because expression in the notochord
persists in the growing tail bud and a discontinuous line of
stained cells (indicating expression of noggin initiated at new
sites) runs the length of the roof plate of the neural tube (and is
also apparent in the head mesoderm), we believe noggin is expressed
as an adult cell function also. A number of applications for noggin
are suggested from its properties.
[0057] The noggin CDNA should be useful as a diagnostic tool (such
as through use of antibodies in assays for proteins in cell lines
or use of oligonucleotides as primers in a PCR test to amplify
those with sequence similarities to the oligonucleotide primer, and
to see how much noggin is present, e.g. primers such as 5' Primers
1-3 and 3' Primers 1-3).
[0058] Because noggin has a pattern of expression that suggests it
is used to regulate cartilage production in the embryonic head,
clinical uses to regulate cartilage and bone growth are suggested
for noggin in therapeutic compositions and particularly in
combination with other growth factors due to a property of noggin
to potentiate at least some growth factors. Since neural crest
cells are required for the tadpole fin to grow, noggin seems to be
a growth factor for the tissue matrix and epidermis and should
prove useful, for example, in wound healing compositions.
[0059] Noggin, of course, provides the key to isolate its receptor
Since many receptors mutate to cellular oncogenes, the noggin
receptor should prove useful as a diagnostic probe for certain
tumor types. Thus, when one views noggin as ligand in complexes,
then complexes in accordance with the invention include antibody
bound to noggin, antibody bound to peptides derived from noggin,
noggin bound to its receptor, or peptides derived from noggin bound
to its receptor. Mutant forms of noggin, which are either more
potent agonists or antagonists, are believed to be clinically
useful. Such complexes of noggin and its binding protein partners
will find uses in a number of applications.
[0060] Practice of this invention includes use of an
oligonucleotide construct comprising a sequence coding for noggin
and for a promoter sequence operatively linked to noggin in a
mammalian, bacterial or a viral expression vector. Expression and
cloning vectors contain a nucleotide sequence that enables the
vector to replicate in one or more selected host cells. Generally,
in cloning vectors this sequence is one that enables the vector to
replicate independently of the host chromosomes, and includes
origins of replication or autonomously replicating sequences. The
well-known plasmid pBR322 is suitable for most gram negative
bacteria, the 2.mu. plasmid origin for yeast and various viral
origins (SV40, polyoma, adenovirus, VSV or BPV) are useful for
cloning vectors in mammalian cells.
[0061] Expression and cloning vectors should contain a selection
gene, also termed a selectable marker. Typically, this is a gene
that encodes a protein necessary for the survival or growth of a
host cell transformed with the vector. The presence of this gene
ensures that any host cell which deletes the vector will not obtain
an advantage in growth or reproduction over transformed hosts.
Typical selection genes encode proteins that (a) confer resistance
to antibiotics or other toxins, e.g. ampicillin, neomycin,
methotrexate or tetracycline, (b) complement auxotrophic
deficiencies.
[0062] Examples of suitable selectable markers for mammalian cells
are dihydrofolate reductase (DHFR) or thymidine kinase. Such
markers enable the identification of cells which were competent to
take up the noggin nucleic acid. The mammalian cell transformants
are placed under selection pressure in which only the transformants
are uniquely adapted to survive by virtue of having taken up the
marker. Selection pressure is imposed by culturing the
transformants under conditions in which the concentration of
selection agent in the medium is successively changed.
Amplification is the process by which genes in greater demand for
the production of a protein critical for growth are reiterated in
tandem within the chromosomes of successive generations of
recombinant cells. Increased quantities of noggin can therefore be
synthesized from the amplified DNA.
[0063] For example, cells transformed with the DHFR selection gene
are first identified by culturing all of the transformants in a
culture medium which contains methotrexate (Mtx), a competitive
antagonist of DHFR. An appropriate host cell in this case is the
Chinese hamster ovary (CHO) cell line deficient in DHFR activity,
prepared and propagated as described by Urlaub and Chasin, Proc.
Nat. Acad. Sci., 77, 4216 (1980). The transformed cells then are
exposed to increased levels of Mtx. This leads to the synthesis of
multiple copies of the DHFR gene and, concomitantly, multiple
copies of other DNA comprising the expression vectors, such as the
DNA encoding noggin. Alternatively, host cells transformed by an
expression vector comprising DNA sequences encoding noggin and
aminoglycoside 3' phosphotransferase (APH) protein can be selected
by cell growth in medium containing an aminoglycosidic antibiotic
such as kanamycin or neomycin or G418. Because eukarotic cells do
not normally express an endogenous APH activity, genes encoding APH
protein, commonly referred to as neo resistant genes, may be used
as dominant selectable markers in a wide range of eukaryotic host
cells, by which cells transformed by the vector can readily be
identified.
[0064] Expression vectors, unlike cloning vectors, should contain a
promoter which is recognized by the host organism and is operably
linked to the noggin nucleic acid. Promoters are untranslated
sequences located upstream from the start codon of a structural
gene (generally within about 100 to 1000 bp) that control the
transcription and translation of nucleic acid under their control.
They typically fall into two classes, inducible and constitutive.
Inducible promoters are promoters that initiate increased levels of
transcription from DNA under their control in response to some
change in culture conditions, e.g. the presence or absence of a
nutrient or a change in temperature. At this time a large number of
promoters recognized by a variety of potential host cells are well
known. These promoters can be operably linked to noggin encoding
DNA by removing them from their gene of origin by restriction
enzyme digestion, followed by insertion 5' to the start codon for
noggin.
[0065] Nucleic acid is operably linked when it is placed into a
functional relationship with another nucleic acid sequence. For
example, DNA for a presequence or secretory leader is operably
linked to DNA for a polypeptide if it is expressed as a preprotein
which participates in the secretion of the polypeptide; a promoter
or enhancer is operably linked to a coding sequence if it affects
the transcription of the sequence; or a ribosome binding site is
operably linked to a coding sequence if it is positioned so as to
facilitate translation. Generally, operably linked means that the
DNA sequences being linked are contiguous and, in the case of a
secretory leader, contiguous and in reading phase. Linking is
accomplished by ligation at convenient restriction sites. If such
sites do not exist then synthetic oligonucleotide adapters or
linkers are used in accord with conventional practice.
[0066] Transcription of noggin-encoding DNA in mammalian host cells
is controlled by promoters obtained from the genomes of viruses
such as polyoma, cytomegalovirus, adenovirus, retroviruses,
hepatitis-B virus, and most preferably Simian Virus 40 (SV40), or
from heterologous mammalian promoters, e.g. the actin promoter. Of
course, promoters from the host cell or related species also are
useful herein.
[0067] In particular embodiments of the invention expression of
noggin in E. coli is preferably performed using vectors which
comprise the following: a lac UV5 promoter which may be controlled
by the lactose operon repressor; a strong ribosome binding site,
for example, the ribosome binding site of bacteriophage T7; a
mutation in the replication control region of the plasmid which may
increase copy number; and a mutation which limits the expression of
the antibiotic resistance protein.
[0068] In a preferred embodiment, noggin is expressed in a high
copy number kanamycin resistant pBR322-derived plasmid under the
control of a lac UV5 promoter. In an additional preferred
embodiment, noggin is expressed in baculovirus under the control of
the polyhedrin promoter of Autrographa californica Multiple Nuclear
Polyhedrosis virus in insect host cells.
[0069] Noggin is believed to find use as an agent for enhancing the
survival or inducing the growth of nerve and muscle cells. It,
therefore, is useful in the therapy of congenital conditions or
degenerative disorders of the nervous system ("neurodegenerative
diseases"), including such diseases as Alzheimer's disease,
Parkinson's disease, Huntington's chorea, ALS, peripheral
neuropathies, and other conditions characterized by necrosis or
loss of neurons, whether central, peripheral, or motorneurons. In
addition, it may be useful for treating damaged nerve cells, e.g.,
nerves damaged by traumatic conditions such as burns and wounds,
diabetes, kidney dysfunction, and the toxic effects of
chemotherapeutics used to treat cancer and AIDS. It also is useful
as a component of culture media for use in culturing nerve cells in
vitro.
[0070] The capacity of noggin to induce neural tissue may be useful
in diseases where neural tissue is formed improperly or
incompletely during development. Thus, noggin and the noggin gene
are also useful in treating congenital malformations such as
anencephaly, or the loss of cerebral hemispheres which results from
failure of closure of the anterior neural tube during
development.
[0071] Practice of this invention includes preparation and uses of
a diagnostic or therapeutic agent comprising a nucleotide sequence
of at least about 15 DNA or RNA bases analogous to all or a portion
of either SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO:25, or SEQ ID NO:
1 or of the nucleic acid sequences contained in bacteriophages,
hnog.lambda.-9 or hnog.lambda.-10. That is, noggin preparations are
useful as standards in assays for noggin and in competitive-type
receptor binding assays when labelled with radioiodine, enzymes,
fluorophores, spin labels, and the like. Therapeutic formulations
of noggin are prepared for storage by mixing noggin having the
desired degree of purity with optional physiologically acceptable
carriers, excipients or stabilizers, in the form of lyophilized
cake or aqueous solutions. Acceptable carriers, excipients or
stabilizers are nontoxic to recipients at the dosages and
concentrations employed, and include buffers such as phosphate,
citrate, and other organic acids; antioxidants including ascorbic
acid; low molecular weight (less than about 10 residues)
polypeptides; proteins, such as serum albumin, gelatin or
immunoglobulins. Other components can include glycine, blutamine,
asparagine, arginine, or lysine; monosaccharides,disaccharides, and
other carbohydrates including glucose, mannose, or dextrins;
chelating agents such as EDTA; sugar alcohols such as mannitol or
sorbitol; salt-forming counterions such as sodium; and/or nonionic
surfactants such as Tween, Pluronics or PEG.
[0072] Noggin may be used according to the invention as described
supra. The concentration of the active ingredient used in the
formulation will depend upon the effective dose required and the
mode of administration used. The dose used should be sufficient to
achieve circulating plasma concentrations of active ingredient that
are efficacious. Effective doses may be extrapolated from dose-
response curves derived from in vitro or animal model test
systems.
[0073] By referring to noggin, the present invention also
contemplates the use of fragments, derivatives, agonists or
antagonists of noggin molecules.
[0074] Noggin may be administered in any pharmacologically
acceptable carrier. The administration route may be any mode of
administration known in the art, including but not limited to
intravenously, intrathecally, subcutaneously, by injection into
involved tissue, intraarterially, intranasally, orally, or via an
implanted device. The present invention provides for pharmaceutical
compositions comprising noggin in a pharmacologically acceptable
carrier.
[0075] Administration may result in the distribution of noggin
throughout the body or in a localized area. For example, in some
conditions which involve distant regions of the nervous system,
intravenous or intrathecal administration of noggin may be
desirable. Alternatively, and not by way of limitation, when
localized regions of the nervous system are involved, local
administration may be desirable. In such situations, an implant
containing noggin may be placed in or near the lesioned area.
Suitable implants include, but are not limited to, gelfoam, wax, or
microparticle-based implants.
[0076] Depending upon the mode of administration, the active
ingredient may be formulated in a liquid carrier such as saline,
incorporated into liposomes, microcapsules, polymer or wax-based
and controlled release preparations, or formulated into tablet,
pill or capsule forms.
[0077] Inventive complexes comprise a ligand characterized by one
or more of the SEQ ID NOS:3-7. The ligand can be bound to a
protein, such as antibody. Such antibodies can be polygonal or
monoclonal.
[0078] Polyclonal antibodies to noggin generally are raised in
animals by multiple subcutaneous (sc) or intraperitoneal (ip)
injections of noggin and an adjuvant. It may be useful to conjugate
noggin or a fragment containing the target amino acid sequence to a
protein which is immunogenic in the species to be immunized, e.g.,
keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or
soybean trypsin inhibitor using a bifunctional or derivatizing
agent, for example, maleimidobenzoyl sulfosuccinimide ester
(coniugation through cysteine residues), N-hydroxy-succinimide
(through lysine residues), glutaraldehyde, succinic anhydride,
SOCl.sub.2, or R.sup.1N.dbd.C.dbd.NR.
[0079] Animals can be immunized against the immunogenic conjugates
or derivatives by combining 1 mg or 1 .mu.g of conjugate (for
rabbits or mice, respectively) with 3 volumes of Freund's complete
adjuvant and injecting the solution intradermally in multiple
sites. One month later the animals are boosted with 1/5 to 1/10 the
original amount of conjugate in Fruend's complete adjuvant by
subcutaneous injection at multiple sites. Seven to 14 days later
animals are bled and the serum is assayed for anti-noggin titer.
Animals are boosted until the titer plateaus. Preferably, the
animal is boosted with the conjugate of the same noggin
polypeptide, but conjugated to a different protein and/or through a
different cross-linking agent. Conjugates also can be made in
recombinant cell culture as protein fusions. Also, aggregating
agents such as alum are used to enhance the immune response.
[0080] Monoclonal antibodies are prepared by recovering spleen
cells from immunized animals and immortalizing the cells in
conventional fashion, e.g. by fusion with myeloma cells or by EB
virus transformation and screening for clones expressing the
desired antibody.
[0081] In a preferred embodiment, a rat monoclonal antibody such as
RP57-16, prepared after immunization of a rat with recombinant
human noggin, reacts specifically with both Xenopus and human
noggin, but not with the neurotrophins BDNF, NT-3 and NT-4.
[0082] Noggin antibodies are useful in diagnostic assays for noggin
or its antibodies. In one embodiment of a receptor binding assay,
an antibody composition which binds to all of a selected plurality
of members of the noggin family is immobilized on an insoluble
matrix, the test sample is contacted with the immobilized antibody
composition in order to adsorb all noggin family members, and then
the immobilized family members are contacted with a plurality of
antibodies specific for each member, each of the antibodies being
individually identifiable as specific for a predetermined family
member, as by unique labels such as discrete fluorophores or the
like. By determining the presence and/or amount of each unique
label, the relative proportion and amount of each family member can
be determined.
[0083] Noggin antibodies also are useful for the affinity
purification of noggin from recombinant cell culture or natural
sources. Noggin antibodies that do not detectably cross-react with
other growth factors can be used to purify noggin free from these
other family members.
[0084] Aspects of the invention will now be illustrated by the
following examples.
Experimental Procedures
[0085] Production of Xenopus Embryos
[0086] Xenopus embryos were prepared by the protocol described by
Condie and Harland (Development, 101, 93-105, 1987). Embryos were
staged according to the table of Nieuwkoop and Faber ("Normal Table
of Xenopus laevis" (Daubin), Amsterdam: North Holland, 1967).
Ventralized embryos were produced by irradiation with a Statalinker
(Stratagene), and dorsalized embryos were produced by treatment
with LiCl as described by us in our paper on certain "wnt" proteins
(designated "Xwnt-8"), Smith and Harland, Cell, Vol. 67, pp.
753-765 (1991) (incorporated by reference and occasionally referred
to hereinafter as "S&H, supra").
EXAMPLE 1
[0087] Isolation and Sequencing of Noggin cDNA
[0088] The construction of the size-selected plasmid cDNA library
from stage 11 LiCl-treated embryos was as follows. Sixty micrograms
of poly(A) RNA from stage 11 LiCl-treated embryos was size
fractionated on a 10% to 30% sucrose gradient in the presence of
methylmercuric hydroxide. First strand cDNA was synthesized from 2
.mu.g of the size-fractionated poly(A) RNAs primed with oligo(dT)
oligonucleotide containing the recognition site for NotI.
[0089] After synthesis of the second strand, cDNAs were treated
with EcoRI methylase, ligated with EcoRI linkers, digested with
EcoRI and NotI, and finally ligated to 125 ng of modified
pGEM-5Zf(-) (Promega). The pGEM-5Zf(-) used here was modified by
the addition of an oligonucleotide into the Nsil site to create an
EcoRi site. The vector was not treated with alkaline phosphatase,
but the excised polylinker sequence was removed on a sepharose 4BCL
column. The ligated products were used to transform XL-I Blue cells
(Stratagene), and plated to give 100,000 colonies per 10 cm plate.
Plasmid DNAs were isolated from plate cultures by the
alkaline-lysis/polyethylene glycol precipitation protocol.
[0090] Dorsalizing activity in the library was assayed by injecting
RNA transcripts made from pooled plasmid DNA. Single clones were
isolated by a process of sib selection. In this procedure the
plasmid library was replated on 12 plates with 10-fold fewer
colonies per plate. RNA was synthesized from pooled plasmid DNAs
isolated from each plate and tested for dorsalizing activity by
injection into UV- ventralized embryos. Those pools with
dorsalizing activity were replated and screened as described above.
This process was repeated until single clones were isolated.
[0091] In vitro RNA synthesis, injection assay for dorsal axis
rescue and sib-selections were also done, as described by us in
S&H, supra.
[0092] The nucleotide sequence of both strands of the isolated
noggin cDNA clone was determined by the dideoxy termination method
using modified T7 DNA polymerase (US Biochem). Deletions were
prepared in sequencing templates by both restriction enzyme and
exonuclease ill digestion (Henikoff, Meth. Enzymol, 155, 156-165,
1987).
[0093] In vitro Translation
[0094] One-half .mu.g of in vitro synthesized noggin, Xwnt-8, and
goosecoid mRNAs were translated in a nuclease treated rabbit
reticulocyte lysate (Promega) with added 35S-methionine according
to the manufacturer's instructions. The translation products were
visualized by SDS-polyacrylamide gel electrophoresis (12% gels)
followed by fluorography. Noggin protein had the molecular weight
predicted by the open reading frame.
[0095] RNA Isolation and Analysis
[0096] Total RNA was isolated from embryos and oocytes by a small
scale protocol as described by Condie and Harland, supra. Dorsal
lips were dissected from 30 unfixed stage 10.5 embryos and pooled
for total RNA preparation. Samples containing either the total RNA
equivalent of 2.5 embryos or approximately 2 .mu.g of poly A+ RNA
were analyzed by northern blotting. Random primed DNA probes were
prepared from a 1,323 bp fragment of noggin cDNA from the EcoRI
site at nucleotide -83 to an EcoRV site that lies in the vector
immediately 3' to the end of the cDNA.
[0097] RNAse protection assays were done using a protocol as
detailed by Melton et al. (Nuc. Acids Res., 12, 7035-7056, 1984)
with minor modifications (C. Kintner, Salk institute, La Jolla,
California). A noggin cDNA exonuclease Ill deletion clone,
illustrated by SEQ ID NO: 8 but having a deletion from the 3' end
to nucleotide 383, was used as a template for synthesizing RNA
probes. The template DNA was linearized by EcoRl restriction enzyme
digestion and a 463 base antisense RNA incorporating 32p was
synthesized with T7 RNA polymerase. A 387 base antisense EFI.alpha.
RNA probe was used as a control for amount of RNA per sample.
Probes were gel purified prior to use.
[0098] In situ Hybridization
[0099] After fixation and storage, the embryos were checked to
ensure the blastocoel and archenteron were punctured. Care was
taken to puncture the residual blastocoel of neurulae and tadpoles
as well as the archenteron. Embryos were rewashed at room
temperature in 100% ethanol for two hours to remove residual lipid.
After hybridization, staining was allowed to develop overnight and
the embryos were then fixed in Bouin's. Newly stained embryos have
a high background of pink stain but most of this washes out,
leaving the specific stain. Following overnight fixation, the
embryos were washed well with 70% ethanol, 70% ethanol buffered
with PBS and methanol. Embryos were cleared in Murray's mix and
photographed with Kodak Ektar 25 film, using a Zeiss axioplan
microscope (2.5 or 5.times. objective with 3.times.12B telescope to
assist with focusing).
[0100] Lineage Tracing
[0101] Lineage tracing with mRNA that encodes nuclear localized
B-galactosidase was as we described in S&H, supra. Ventralized
embryos were coinjected at the 32 cell stage with 0.5 ng
B-galactosidase and 25 pg noggin.DELTA.5' mRNAs. Embryos were fixed
and stained with X-gal at approximately stage 22.
[0102] Results
[0103] Noggin cDNA Encodes a Novel Polypeptide
[0104] The 1833 nucleotide sequence of the selected clone is shown
by SEQ ID NO: 8 and sometimes also referred to as "clone A3." The
sequence contains a single long open reading frame encoding a 222
amino acid polypeptide with a predicted molecular weight of 26 kDa.
At the amino terminus, the hydrophobic stretch of amino acids
suggests that the polypeptide enters the secretory pathway. There
is a single potential site for N-linked glycosylation at amino acid
61. Extensive untranslated regions are located both 5' and 3' to
the reading frame (593 and 573 bp, respectively). The 3'
untranslated region is particularly rich in repeated dA and dT
nucleotides, and contains, in addition to a polyadenylation signal
sequence located 24 bp upstream from the start of the poly A tail,
a second potential polyadenylation sequence 147 bp further
upstream.
[0105] Sense RNA synthesized from clone A3 with SP6 RNA polymerase
was translated in a rabbit reticulocyte lysate system. The
3S-labeled products were fractionated on a 12% SDS-polyacrylamide
gel and visualized by fluorography. The major protein product had
the expected molecular weight of approximately 26 kDa.
[0106] Comparison of the amino acid sequence of the predicted
polypeptide to the National Center for Biotechnology Information
BLAST network (non-redundant data base) did not identify any
similar sequence. Thus, this clone encodes the new type of protein
we have named "noggin" which is secreted, and which has dorsal
inducing activity in Xenopus.
[0107] Noggin mRNA Can Rescue a Complete Dorsal-ventral Axis
[0108] Injection of noggin RNA into a single blastomere of a four
cell stage UV-ventralized embryo can restore the complete spectrum
of dorsal structures. The degree of axis rescue was dependent upon
the amount of RNA injected, with embryos receiving low doses having
only posterior dorsal structures, while embryos receiving higher
doses had excess dorsal-anterior tissue. RNA transcripts from two
noggin plasmids were tested. The first contained the full cDNA. The
second (pNogginA5') had a deletion removing the first 513
nucleotides of the 5' untranslated region up to the EcoRI site. The
resulting embryos from injection of RNA transcripts of these two
plasmids, as well as Xwnt-8 RNA for comparison, were scored
according to the dorsoanterior index (DAI) scale of Rao and Elinson
(Dev. Biol., 127, 64-77, 1988). In this scale, a completely
ventralized embryo is scored as zero, a normal embryo is scored as
5, and the most severely dorsoanteriorized embryos, those having
radial dorsoanterior structures, were scored as 10. RNA synthesized
from pNoggin.DELTA.5' (noggin.DELTA.5' mRNA) repeatedly gave a
higher DAI than the equivalent amount of mRNA synthesized from the
complete cDNA. The dose-dependency of axis rescue by
noggin.DELTA.5' mRNA was very similar to that of Xwnt-8 mRNA.
[0109] UV treated embryos were also injected with a higher doses
(1,000 pg) of the noggin mRNAs. Injection of this dose of noggin
mRNA into one blastomere at the four cell stage resulted in embryos
with very severe hyperdorsalization (DAI>7). However, most of
these embryos died at the late gastrulalearly neurula stage.
Apparently excessively strong gastrulation movements resulted in
the thinning and rupture of the blastocoel roof. We have also
observed this effect with high doses of injected Xwnt-8 mRNA.
[0110] The rescue of dorsal development by both nogginA5' and
Xwnt-8 mRNAs followed a consistent pattern in which increasing
amounts of the mRNAs lead to progressively more anterior structures
being rescued. For example, embryos that received 1 pg of the RNAs
had primarily the posterior and trunk dorsal structures rescued,
and for the most part lacked head structures. Higher doses (10 or
100 pg) of both of the RNAs resulted in embryos with more anterior
development, and many had either nearly normal or hyperdorsalized
phenotypes.
[0111] Noggin Injected Blastomeres Act as a Nieuwkoop Center
[0112] The effect of varying the site of noggin mRNA injection was
investigated. Thirty-two cell stage UV-treated embryos were
injected with either 0.5 ng of B-galactosidase mRNA alone or 0.5 ng
B-galactosidase mixed with 25 pg noggin.DELTA.5' mRNA. Injection of
noggin mRNA into blastomeres of the vegetal tier gave the most
strongly dorsoanteriorized embryos. In both of the vegetal injected
embryos the nuclear X-Gal staining was found almost exclusively in
the endoderm (the mRNA encodes a B-galactosidase that translocates
to the nucleus, allowing distinction from the diffuse background
stain). One of the embryos shown was strongly hyperdorsalized (DAI
approximately 7) as a result of the noggin mRNA injection, and had
a severely truncated tail and enlarged head structures. Embryos
were also rescued by noggin mRNA injections into the marginal
zone.
[0113] In these embryos B-galactosidase staining was observed
primarily in the axial and head mesoderm. Injection of noggin mRNA
into the animal pole had very little effect on axis formation.
Likewise, B-galactosidase mRNA alone was without effect.
[0114] Noggin mRNA is Expressed Both Maternally and Zygotically
[0115] In northern blot analysis of RNA from Xenopus embryos two
noggin mRNA species of approximate sizes 1.8 and 1.4 kb were
observed. A relatively low level of noggin mRNA was detected in
oocytes. By stage 11 the level of noggin mRNA was significantly
higher, reflecting zygotic transcription (as opposed to the
maternally deposited transcripts seen in oocytes). Noggin mRNA
remained at the elevated level up to the latest stage examined
(stage 45).
[0116] We expect that the primary dorsalizing RNA in our library to
be elevated in LiCl-treated embryos relative to normal or
UV-treated embryos. Lithium ion treatment resulted in a large
increase in the amount of noggin mRNA expressed, relative to
untreated embryos.
[0117] UV treatment had the opposite effect. Noggin mRNA expression
was essentially undetectable in total RNA samples from these
embryos. Thus, the abundance of noggin mRNA in manipulated embryos
parallels the rescuing activity.
[0118] We analyzed the distribution of noggin in oocytes and
cleavage stage embryos. Since the amount of maternally deposited
noggin RNA is too low for in situ hybridization to detect above
background, we used an RNAse protection assay. Oocytes were
dissected into animal and vegetal halves. No enrichment of noggin
mRNA was seen in either hemisphere relative to total oocyte RNA.
Four-cell stage embryos were dissected into dorsal and ventral
halves, as well as animal and vegetal halves. Noggin transcripts
were found to be distributed evenly between dorsal and ventral
hemispheres as well as animal and vegetal hemispheres. The same
result was obtained with embryos that were tilted 90.degree.
immediately following fertilization and then marked with a vital
dye on their uppermost side to indicate the future dorsal side.
Older (32 cell stage) blastula embryos were also dissected into
dorsal-ventral and animal-vegetal halves. No enrichment of noggin
mRNA in any of the hemispheres was seen relative to the total
embryo. In addition, treatment did not alter the abundance of
maternally deposited noggin RNA, indicating no preferential
degradation in ventral tissues. Samples with known amounts of in
vitro synthesized noggin mRNA were included in the RNAase
protection assay. From these and other data we estimate that there
is approximately 0.1 pg of noggin mRNA per blastula stage embryo
and 1 pg per gastrula stage embryo.
[0119] The localization of noggin transcripts was investigated in
early gastrula stage embryos. Dorsal lips were dissected from stage
10.5 embryos. A northern blot of equal amounts of total RNA from
intact embryos, dissected dorsal lips, and from the remaining
embryo after dissection of the dorsal lip was hybridized with a
noggin probe and then re-hybridized with an EFla probe, as a
control for amount of RNA loaded per sample. The autoradiograph of
the blot showed that noggin mRNA at this stage is enriched in the
dorsal lip.
[0120] In situ Hybridization: Zygotic Expression of Noggin in the
Spemann Organizer
[0121] The localization of noggin transcripts in developing embryos
was examined in greater detail using whole mount in situ
hybridization. Whole fixed embryos were hybridized with digoxigenin
containing RNA probes.
[0122] Hybridized RNA probe was then visualized with an alkaline
phosphatase-conjugated anti-digoxigenin antibody. The specificity
of hybridization seen with antisense noggin probes was tested both
by hybridizing embryos with sense noggin probes, and by using two
non-overlapping antisense probes. Due both to the low level of
expression, and to background staining, noggin mRNA could not be
detected unequivocally before the late blastula stage. The
increased level of noggin mRNA that was detected by northern blot
following activation of zygotic transcription was apparent in in
situ hybridization at stage 9 as a patch of staining cells on the
dorsal side of the embryo. Viewed from the vegetal pole, this patch
of cells was restricted to a sector of about 600. A side view of
the same embryo shows that the staining cells were located within
the marginal zone (i.e., between the animal and vegetal poles and
within the presumptive dorsal mesoderm forming region). Transcripts
are largely restricted to the nucleus at this stage.
[0123] A side view of an early gastrula stage embryo 30
(approximate stage 10.5) shows specific hybridization primarily in
the involuting mesoderm at the dorsal lip. A vegetal view of the
same embryo (blastopore lip arrowed) shows that noggin mRNA is most
abundant on the dorsal side, but expression extends at the lower
level to the ventral side of the embryo. This method of in situ
hybridization does not detect transcripts in the most yolky
endodermal region of embryos, therefore we cannot rule out
expression in more vegetal regions than those seen in the Figure.
Treatments which are known to affect the size of the dorsal lip
(LiCl treatment, UV irradiation) had a profound effect on the
pattern of noggin in situ hybridization. In LiCI treated embryos
the staining is intense throughout the marginal zone. UV treatment
reduced the hybridization signal to low levels. This result is
consistent with amounts of noggin mRNA seen by northern blot
analysis. The UV treated embryo also is a negative control for
specificity of hybridization.
[0124] As gastrulation proceeds, noggin mRNA staining follows the
involuting dorsal mesoderm, and is highest in the presumptive
notochord. By the late neurula stage (approximately 18) noggin mRNA
expressing cells are clearest in the most dorsal mesoderm,
primarily in the notochord but also extending more anteriorly into
the pre-chordal mesoderm. The anterior tip of the notochord is
arrowed. During tailbud stages expression of noggin in the dorsal
mesoderm declines, through expression in the notochord persists in
the growing tailbud. Expression of noggin initiates at several new
sites, which become progressively clearer as the tadpole matures. A
discontinuous line of stained cells runs the length of the roof
plate of the neural tube. Staining is also apparent in the head
mesoderm, primarily in the mandibular and gill arches. We suspect
that this expression corresponds to skeletogenic neural crest
cells. Furthermore, subsets of these cells express homeobox genes
that mark different anterior-posterior levels of the head neural
crest, for example En-2 in the mandibular arch is seen by antibody
staining. Cells with stellate morphology stained from noggin mRNA
in the tail fin. These stellate cells are also likely to be derived
from the neural crest. None of these patterns were seen with the
sense probe, or with a number of other probes.
EXAMPLE 2
[0125] Noggin cDNA Transfected into COS Cells Produces Active
Conditioned Medium
[0126] For COS cells the noggin cDNA was inserted into a COS cell
expression vector. COS cells were transfected, and medium harvested
after allowing expression of the introduced noggin genes. This
medium has been tested in an animal cap assay for mesoderm inducing
or dorsalizing activity. We have tested two transfection protocols,
a standard one, where cells recover and then are transferred to
serum-free medium, and an alternate where cells are transferred to
a defined medium lacking serum but containing transferrin, insulin,
and BSA. Cells remain healthy in the supplemented medium and a
cotransfected .beta.-galactosidase gene gives 100 fold more
activity than in the unsupplemented medium. The results of treating
cells with these media is shown below in Table 1. Animal caps were
taken from ventralized animals, treated and at the end of
neurulation they were scored for elongation, usually a sign that
notochord or neural tissues have been induced. Elongation is
indicated in Table 1 by a "+" and even greater elongation a "++."
In addition, they are scored for a molecular marker by Northern
blotting.
[0127] As shown by the data of Table 1, the noggin cDNA has a large
effect on the COS cell conditioned medium. However, noggin is
probably interacting with something else in the medium, since
COS-cell conditioned medium alone has some activity. It is possible
that noggin is causing the cells to secrete something that they
normally would not, but the experiments do indicate that noggin is
secreted and is responsible for some of the activity.
4TABLE 1 Cos Cell Conditioned Medium: Effects on Animal Caps
expression Elongation N-CAM Transferred to serum free medium +
transferrin, BSA, and insulin 1. Vector only +/- + 2. Noggin cDNA
++ ++ Transferred to serum free medium without supplements 1.
Vector only - - 2. Noggin cDNA - -
[0128] Noggin mRNA Injected into Oocytes Produces Active Secreted
Noggin Protein
[0129] A second approach to studying whether protein can be
secreted in active form is to inject oocytes with mRNA and take
material secreted by the oocyte. A particular advantage of this
method is that the injected mRNA is efficiently translated, and
most of the translation of the oocyte can be taken up by the
injected mRNA. A new protein, whose synthesis is directed by
injected noggin mRNA is secreted into the medium. Noggin clearly
synergizes with activin to produce elongated explants that express
elevated levels of muscle actin.
[0130] Biochemical Properties of Noggin
[0131] Injected oocytes are injected with mRNA, and labelled with
.sup.35S methionine. Most of the radioactive protein secreted into
the medium is from the injected mRNA. The noggin protein, which is
almost isotopically pure, can then be analyzed. From this analysis
we have determined that noggin is a dimeric glycoprotein. When run
under reducing conditions, and treated with N-glycanase to remove
sugar residues, noggin migrates only slightly slower than its
predicted molecular weight of 26 kDa. The removal of sugar side
chains results in a loss of about 4 kDa from a starting apparent
molecular weight of 33 kDa. When run under non-reducing conditions
it migrates at double this value.
[0132] We do not yet know if the dimer of the protein is the active
species, or if there is a proteolytically processed form which is
active. In a control experiment with activin mRNA, oocytes produce
activin activity, but the bulk of the radiolabelled protein
migrates as the precursor form. Only a small amount of processed
protein (15 kDa) was detected. It is possible that noggin injected
oocytes secrete predominantly unprocessed protein and a trace of
extremely active processed protein that we have not detected.
Despite the caveats, the main point from analysis of injected
oocytes and transfected COS cells is that active noggin can be
obtained as a freely soluble secreted polypeptide. This sets it
apart from the other group of genes with dorsalizing activity, the
wnts. Wnt proteins have not been available in soluble form and this
has greatly hampered the analysis of their biological activities,
and of the receptor that binds to them.
EXAMPLE 3
[0133] Cloning of the Mouse Noggin Homolog
[0134] It is currently impossible to eliminate zygotic noggin
transcription from developing Xenopus embryos. In contrast, it
should be possible to generate homozygous null mutations in the
mouse. We have cloned the mouse noggin cDNA (SEQ ID NO: 10). This
is useful to generate mutant mice. In addition to generating the
probes and tools to make mutant mice, a comparison of the noggin
sequences should be a useful predictor of conserved domains and
functions. The C-terminal 80 amino acids are 87% identical between
SEQ ID NOS: 8 and 10.
[0135] Mouse noggin was isolated from an embryonic cDNA library by
probing with a radiolabelled frog noggin cDNA under conditions of
moderate stringency (as defined earlier). Subsequently a genomic
clone was isolated by probing a genomic library with the mouse
noggin cDNA 15 under conditions of high stringency (as defined, but
hybridized at 42.degree. C. and washed at 50.degree. C. in 15 mM
NaCl, 1.5 mM sodium citrate). The full nucleotide sequence of mouse
noggin cDNA (SEQ ID NO: 25) as well as the deduced amino acid
sequence (SEQ ID NO: 26) are shown in FIG. 13. There are only two
amino acid differences between mouse noggin and human noggin.
EXAMPLE 4
[0136] Cloning of the Human Noggin Homolog
[0137] Materials and Methods
[0138] Probe Preparation
[0139] Two oligonucleotides were synthesized based on the mouse
noggin sequence (supra). The sequence of the oligonucleotides is
noggin 5': 5'-CAG ATG TGG CTG TGG TCA-3' (SEQ ID NO: 18)
corresponding to amino acids QMWLWS (SEQ ID NO: 19) and noggin 3':
5'-GCAGGAACACTTACACTC-3' (SEQ ID NO: 20) corresponding to amino
acids ECKCSC (SEQ ID NO: 21) of the mouse noggin protein .
[0140] The oligonucleotides were used for PCR amplification of a
segment of DNA of 260 nucleotides using as a template a mouse cDNA
clone prepared as set forth in Example 3. The amplified fragment
had a nucleotide sequence that corresponds to nucleotides 2 through
262 of the mouse sequence as set forth in SEQ ID NO: 10. After
amplification, the PCR reaction was electrophosed in agarose gels,
the DNA band of 260 nts purified by Magic PCR (Promega), and used
as template for the probe labeling reaction. The probe was labeled
using a standard PCR reaction (Perkin-Elmer) on 20 ng of DNA
template and 0.2 m Curie of alpha 32P-dCTP (Du Pont 3000 Ci/mmol)
instead of dCTP. Unincorporated label was separated from the probes
on a G50 NICK column (Pharmacia). The excluded volume of the
reaction contained a total of 1.8.times.108 cpm.
[0141] In addition, one degenerated oligonucleotide, named noggin
D, corresponding to conserved mouse and Xenopus noggin sequences,
was synthesized as follows: Noggin D: 5'-GARGGIATGGTITGYAARCC-3'
(SEQ ID NO: 22). Noggin D (SEQ ID NO: 22) was labeled by kinase
reaction using T4 polynucleotide kinase and gamma 32P ATP. The
labeled oligonucleotide was purified by NAP5 (Pharmacia) column and
used for library hybridization.
[0142] Library Screening
[0143] A human placental genomic library (Clontech Cat#HL1067J,
average insert size 15 kb) in vector EMBL-3 was plated according to
manufacturer specifications in NM 538 E. coli. Approximately 3
million plaques were transferred to nitrocellulose filters (BA-85
Schleicher and Schuell) in three replicas (named A, B and C) and
screened according to Maniatis, et al.[Sambrook, et a., Molecular
cloning a laboratory manual, CSH Lab Press, New York (1989)]. The
replica filters A and C were hybridized in a buffer containing 0.5
M sodium phosphate, pH 7.2, 7% sodium dodecyl sulphate, 1%
crystalline BSA, 1 mM EDTA, 40 m g/ml denaturated salmon sperm DNA
and about 1.times.106 cpm/ml of the PCR probe (supra). After
hybridization for 12 h at 65.degree. C., the filters were washed
twice at room temperature in 2.times. SSC (30 mM sodium citrate,
0.3 M NaCl), 0.1% SDS and then at 65.degree. C. in 2.times. SSC,
0.1% SDS for 20 min and exposed to Kodak X-OMAT AR film. The filter
replica B were hybridized with the labeled oligonucleotide noggin D
in 6.times. SSC, 0.1% SDS at 51.degree. C. for 12 h followed by
wash at 2.times. SCC, 0.1% SDS at room temperature, and in 6.times.
SSC, 0.1% SDS at 50.degree. C. and exposed to Kodak X-OMAT AR film.
Positive plaques from all replicas were isolated and purified by
re-screening as above. Purified positive plaques were suspended in
500 .mu.l SM (100 mM NaCl, 10 mM MgSO4.times.7H2O, 50 mM Tris HCl
pH 7.5, 0.01% gelatin). 160 .mu.l of phage suspension was mixed
with 0.5 ml saturated NM538 culture, incubated for 20 min at
37.degree. C. and then inoculated into 250 ml LB containing 10 mM
Mg SO4, 0.2% maltose. The cultures were incubated until cell lysis
(7-8 hr) at 37.degree. C. The phage lysates were used for phage DNA
purification by the Qiagen procedure according to the manufacturers
recommendations (Qiagen).
[0144] Sequencing
[0145] Sequencing was performed by using the Applied Biosystems
Model 373A automatic sequencer and Applied Biosystems Taq
DyeDeoxy.TM. Terminator Cycle Sequencing Kit.
[0146] Results
[0147] Filters hybridized to the PCR mouse noggin probes (SEQ ID
NOS: 18 and 20) showed two strong signals corresponding to phage
plaques named hnog.lambda.-9 and hnog.lambda.-10. These plaques
also hybridized to degenerate oligonucleotide probe nogginD (SEQ ID
NO: 22) revealed that these clones correspond to the human noggin
gene. In addition, two other plaques named hnog.lambda.-5 and
hnog.lambda.-7 produced slightly weaker signals when hybridized to
the PCR probes. These clones correspond to either human noggin or
related gene(s). All of the human DNA inserts can be excised from
the vectors using known restriction sites as described in the
literature regarding each particular library.
[0148] A 1.6 kb SacI fragment from clone hnog.lambda.-9 containing
the human noggin gene was subcloned and the nucleotide sequence
determined as set forth in FIG. 1. The amino acid sequence for
human noggin, as deduced from the nucleotide sequence, is also set
forth in FIG. 1. The gene or cDNA may be expressed in various
eukaryotic or prokaryotic expression systems to produce
biologically active human noggin protein. It is expected that the
human protein will exhibit neurotrophic activity similar to that
exhibited by Xenopus noggin protein.
EXAMPLE 5
[0149] Tissue Localization of Message for Human Noggin
[0150] Materials and Methods
[0151] Probe Preparation
[0152] Probes were prepared as set forth in Example 4. The oligos
used are as follows:
5 SEQ ID NO:23: 5' GAC.TCG.AGT.CGA.CAT.CGC.AGA.TGT.GGC.TGT.-
GGT.CAC SEQ ID NO:24: 5' CCA.AGC.TTC.TAG.AAT.TCG.CA-
G.GAA.CAC.TTA.CAC.TCG.G
[0153] (The underlined sequence represent mouse noggin sequence;
the rest of the sequence are tails containing restriction sites for
cloning.)
[0154] A DNA fragment of approximately 300 bp was obtained by PCR
amplification of a mouse cDNA clone prepared as described in
Example 3.
[0155] RNA Preparation and Northern Blots
[0156] Selected tissues were dissected from Sprague-Dawley rats and
immediately frozen in liquid nitrogen. RNAs were isolated by
homogenization of tissues in 3 M LiCl, 6 M urea, as described in
Bothwell, et al. 1990 (Methods of Cloning and Analysis of
Eukaryotic Genes, Boston, MS, Jones and Bartlett). RNAs (10 .mu.g)
were fractionated by electrophoresis through quadruplicate 1%
agarose-formaldehyde gels (Bothwell, et al., 1990, Methods of
Cloning and Analysis of Eukaryotic Genes, Boston, MS, Jones and
Bartlett) followed by capillary transfer to nylon membranes
(MagnaGraph, Micron Separations Inc.) with 10.times. SSC (pH7).
RNAs were UV-cross-linked to the membranes by exposure to
ultraviolet light (Stratalinker, Stratagen, Inc.) and hybridized at
68.degree. C. with radiolabled probes in the presence of 0.5 M
NaPO.sub.4 (pH 7), 1% bovine serum albumin (fraction V, Sigma,
Inc.) 7% SDS, 1 mM EDTA [Mahoudi, et al., Biotechniques Z:331-333
(1989)], 100 .mu.g/ml sonicated, denatured salmon sperm DNA.
Filters were washed at 68.degree. C. with 3.times. SSC, 0.1% SDS
and subjected to autoradiography for 1 day to 2 weeks with one or
two intensifying screens (Cronex, DuPont) and X-ray film (AR-5,
Kodak) at 70.degree. C. Ethidium bromide staining of the gels
demonstrated that equivalent levels of total RNA were being assayed
for the different samples [as in Maisonpierre, et al., Science
247:1446-1451 (1990)].
[0157] RNA was prepared from the following human cell lines:
6 Neuroblastoma Neuroepithelioma CHP-134 SK-N-MC LA-N-1 CHP-100
LA-N-5 IARC-EWI IMR-32 SK-N-LO SHSY5Y SK-ES SKNSH DADY SHEP
[0158]
7 Small Cell Hematopoetic Lung Carcinoma Cervical Carcinoma K562
Calu 3 HeLa U937 SKLu M1 NCI-H69 TF1 SKMES BAF B9 Sympathoadrenal
Precursor Hepatoblastoma Medulloblastoma MAH HEPG2 Madsen Med U266
Pheochromocytoma PC12
[0159] Results
[0160] We have amplified a DNA fragment from the mouse noggin
plasmid, corresponding to the region conserved between Xenopus and
mouse noggin.
[0161] The amplified fragment of approximately 300bp was used as
probe to hybridize to northerns, with RNAs prepared from adult and
embryonic tissues, as well as from various cell lines. Noggin
transcript of about 2kb in size was detected in adult rat brain,
and in a cell line, SKMES, a small cell lung carcinoma.
[0162] Expression of noggin transcripts was examined in various
tissues from rat and mouse at different stages of development and
in adult. In the mouse, noggin transcripts can be detected in
embryos or head from E9 to E12, as well as in newborn brain and
adult brain. There was no detectable signal in peripheral tissues
examined except in skeletal muscle. Abundant level of expression
was also found in hippocampai astrocytes isolated from postnatal
mouse. In the rat, noggin transcripts were detectable in embryos or
head from E9 to E18, as well as in brain from P1, P19 and adult
brain. In the cerebellum, expression of noggin appeared to be
higher in E18 and P1; in the spinal cord, expression of noggin mRNA
peaked at P1. Examination of noggin expression in all of the CNS
regions, especially the olfactory bulb, midbrain, hindbrain and
cerebellum. In the adult, noggin mRNA could be detected in all CNS
regions, especially the olfactory bulb and cerebellum. There also
appeared to be low levels in the skin.
EXAMPLE 6
[0163] Neural Induction by Noggin
[0164] Materials and Methods
[0165] Preparation of Xenopus Noggin CHO Cell Conditioned
Medium
[0166] Xenopus noggin CHO conditioned medium was made by selecting
for stably transfected CHO cells. Dihydrofolate reductase (DHFR)
deficient CHO parental cells (J. Papkoff, Syntex Research) were
transfected with a Xenopus noggin expression plasmid containing
noggin in tandem with the dihydrofolate reductase gene. Growth in
nucleoside free medium was used to select for successfully
transfected cells. Nine colonies of transfectants were picked and
grown up individually. The noggin gene in these cells was amplified
by slowly increasing the dose of methotrexate, an inhibitor of
DHFR.
[0167] The presence of noggin transcripts was first tested by
Northern analysis. Subsequently, two clones, B3 and C3, were shown
to secrete noggin protein, since conditioned medium from these
lines was capable of dorsalizing ventral marginal zones.
Furthermore, by labeling B3 cellular proteins with 35S-methionine,
noggin protein could be identified as a band of about 30 kD on
reducing SDS-PAGE, and a band of 6OkD on non-reducing SDS-PAGE
indicating it forms the expected dimer. These properties matched
those of the noggin protein previously produced in Xenopus oocytes
supra, (Smith et al., Nature 361, 547-49, 1993). B3 conditioned
medium was collected in a mixture of 1 part alpha MEM and 9 parts
CHO-S-SFMII (Gibco-BRL). The cells were allowed to condition the
medium for 3 days. Control medium from parental cells (CHO dhfr-)
was collected identically. Twenty fold concentrated medium was made
using Centriprep 10 concentrators, where the 20 fold change is
measured by volume.
[0168] Purification of Human Noggin from COS Cells
[0169] Human noggin protein was purified by a cationic exchange
column. COS/M5 cells were transiently transfected with a human
noggin expression plasmid, pCAE11. Cells were allowed to condition
DMEM (Specialty Media) for two to three days, after which the
medium was removed. Particulates from the medium were removed by a
centrifugation step and subsequent passage through a 0.2 um
cellulose acetate filter. This cleared medium was pumped onto a
MonoS (Pharmacia) column which was washed with several volumes 40
mM sodium phosphate (pH 7.3), 150 mM NaCl, 1 mM EDTA. Proteins were
then eluted in a linear gradient with 40 mM sodium phosphate (pH
8.5), 1.8M NaCl, 1 mM EDTA. Noggin protein elutes at 0.8M NaCl and
is .gtoreq.90% pure by SDS-PAGE.
[0170] Xenopus Otx Isolation
[0171] To isolate Xenopus Otx clones a tadpole head cDNA library
(Hemmati-Brivanlou, et al., Development 106, 611-617, 1989) was
screened with a mouse otx cDNA (S-L Ang and Rossant, Toronto) at
low stringency. The clones that were picked fell into two classes.
One class, which we have designated otxA, included pXOT21.2, the
probe used here. By in situ hybridization, transcripts are first
detected prior to gastrulation in the superficial layer on the
dorsal side. During neurulation a large anterior domain expressed
the gene, and includes both neural and non-neural tissues. After a
decline in expression in the tailbud tadpole, the gene is
reexpressed specifically in the brain and eyes.
[0172] Ventral Marginal Zone Assay
[0173] Embryo Preparation
[0174] Xenopus laevis embryos are fertilized and de-jellied as
described (Condie and Harland, 1987. Development 101, 93-105),
routinely the evening before dissections. Embryos are cultured
overnight at 15.degree. C. The vitelline membrane surrounding each
developing embryo is manually removed the following morning at the
late blastula stage. Until dissection, the embryos are maintained
in 1/3.times. modified ringers in agarose coated dishes.
[0175] Ventral Marginal Zone Dissection
[0176] Embryos are oriented with their yolky vegetal hemisphere up
so the dorsal side can be identified. The dorsal side of the early
gastrula is marked by the presence of a small arc of dense pigment
called the "dorsal lip" which marks the start of involution of
dorsal mesoderm. The ventral marginal zone (VMZ) is found directly
opposite the dorsal lip, and is dissected. Since the vitelline
membrane has been removed, the embryo tends to flatten. Using a
specially constructed knife made of an eyebrow, mounted onto a
glass pipet with wax, two cuts are made through the flattened
embryo from the top facing vegetal pole through to the animal pole.
The cuts are made such that they isolate approximately 30-60
degrees of the ventral side away from more lateral tissues. A third
cut which is perpendicular to the first two cuts completely
isolates the ventral marginal zone tissue away from the rest of the
embryo. This third cut is at the level of approximately two thirds
of the radius of the embryo from the center. Prior to treatment the
VMZ is washed 1.times. in the culture medium.
[0177] Assay
[0178] Approximately between 5 to 10 VMZs are used per assay. The
washed VMZs are dropped gently (trying to minimize transfer of
liquid) into eppendorf tubes containing the desired treatment
protein medium for assay. The VMZs are allowed to develop to the
late neurula or early tailbud stage as assessed by control whole
embryo development. At this time RNA is isolated from the VMZs and
control whole embryos as described (Condie and Harland, ibid). The
expression of muscle actin in VMZs indicates a dorsalization event
(Lettice and Slack, 1933. Development, 117, 263-72). RNA from each
sample is run on a formaldehyde-agarose gel and blotted to gene
screen. The blot is then hybridized with a Xenopus muscle actin
probe (Dworkin-Rastl et al., 1986. J. Embryol. exp. Morph. 91,
153-68). Quantitation of dorsalization can be carried out by
normalizing muscle actin signal to that of the ubiquitously
expressed EF-la (Krieg et al., 1989. Devl. Biol. 133, 93-100).
Quantitation is done using phosphor imaging.
[0179] RNase Protection Assay
[0180] RNase protection was carried out as described (D. A. Melton
et al., Nucleic Acids Res 12,7035-56, 1984), with the modification
that digestion was carried out at room temperature (22.degree. C.)
using RNase T1 only (Calbiochem 556785) at 10 units/ml. 20-30
animal 1.5 caps were harvested for each lane, of this 80% was used
for neural markers and 10% for muscle actin and collagen type 11.
For goosecoid and brachyury 20 caps were used. Exposures ranged
from 12 hours to 5 days. In all cases, films were preflashed. In
cases where a marker was not expressed, the result was confirmed
with greater sensitivity using phosphor imaging.
[0181] Results
[0182] The development of vertebrate embryos requires several
inductive interactions. Mesoderm, which eventually forms tissues
such as notochord, muscle, heart, mesenchyme and blood, is induced
in the equatorial region of the embryo (Nieuwkoop, Wilhelm Roux'
Arch. EntwMech. Org, 162, 341-373, 1969). This inductive event is
well studied, and there are several candidates for the endogenous
inducer(s) including members of the fibroblast growth factor(FGF)
family and activin (Jessell and Melton, Cell 68, 257-70 1992; Sive,
Genes Dev 7, 1-12, 1993) and TGFb family (Asashima, et al., Roux's
Arch. Dev. Biol. 198, 330-335, 1990; Asashima, et al.,
Naturwissenschaften 77, 8, 389-91, 1990; Green and Smith, Nature
347, 391-394, 1990; Smith, et al., Nature 345, 6277, 729-31, 1990;
Thomsen, et al., Cell 63, 485-493, 1990; van, et al., Nature 345,
6277, 732-4, 1990). The use of dominant negative receptors for both
FGF (Amaya, et al., Cell 66, 257-270, 1991) and activin
(Hemmati-Brivanlou and Melton, Nature 359, 609-614, 1992) in
Xenopus embryos strongly suggests that the signaling pathways
activated by these molecules are essential for proper mesoderm
formation . Molecules such as wnts (Christian, et al., Development
111, 1045-1055, 1991; McMahon and Moon, Cell 58, 1075-84, 1989;
Smith and Harland, Cell 67, 753-765, 1991; Sokol, et al., Cell 67,
741-752, 1991) and noggin (Smith, et al., Nature 361, 547-49, 1993)
modify the kinds of mesoderm made without inducing mesoderm
directly.
[0183] In a subsequent induction, the dorsal mesoderm of the
Spemann organizer signals nearby lateral mesoderm to take on a more
dorsal fate (Dale and Slack, Development 100, 2, 279-95, 1987;
Lettice and Slack, Development, 117, 263-271, 1993; Spemann and
Mangold, Arch. Mikrosk. Anat. EntwMech. 100, 599-638, 1924; Stewart
and Gerhart, Development 109, 363-372, 1990). The only known factor
which is expressed in the organizer and can mimic its dorsalizing
activity is noggin.
[0184] Dorsal mesoderm of the Spemann organizer also signals nearby
ectoderm to become neural tissue. Neural induction by dorsal
mesoderm has been demonstrated in amphibians (Dixon and Kintner,
Development 106, 749-757, 1989; Doniach, et al., Science 257, 5069,
542-5, 1992; Hamburger, The Heritage of Experimental Embryology:
Hans Spemann and the Organizer, 1988; Kintner and Melton,
Development 99, 311-25, 1987; Spemann, Arch. mikrosk. Anat.
EntwMech. 100, 599-638, 1938), birds (Kintner and Dodd, Development
113, 1495-1506, 1991; Tsung, et al., Acta Biol exp Sinica 10,
69-80, 1965), and recently in mice (Ang and Rossant, Development
118, 139-149, 1993). Despite decades of effort, little is known
about the molecular nature of the factors responsible for this
induction. Among known inducers, activin can promote formation of
neural tissue, but this is due to a secondary induction by the
dorsal mesoderm that activin induces (Green, et al., Development
108, 1, 173-83, 1990; Green and Smith, Nature 347, 391-394, 1990;
Kintner and Dodd, Development 113, 1495-1506, 1991). Thus, activin
cannot promote formation of neural tissue when added to gastrula
ectoderm; however, such ectoderm remains competent to be neuralized
by dorsal mesoderm until the end of gastrulation (Sharpe and
Gurdon, Development 109, 765-74, 1990).
[0185] Direct Neural Induction by Noggin
[0186] Candidates for the endogenous inducer are expected to induce
neural tissue in the absence of dorsal mesoderm. Competent animal
cap ectoderm from late blastula stage embryos (St9) was used to
test noggin's neural inducing capacity. Xenopus noggin protein
conditioned medium was collected from stably transfected CHO cells
and twenty fold concentrated medium was used to treat St 9 animal
caps. Markers used in an RNase protection assay were N-CAM
(Jacobson and Rutishauser, Developmental Biology 116, 524-31, 1986;
Kintner and Melton, Development 99, 311-25, 1987), a neural cell
adhesion molecule, a neural specific isoform of b-tubulin (Good, et
al. Nucleic Acids Res 17, 8000, 1989; Good, et al., Dev Biol 137,
414-8, 1990; Richter, et al., Proc Natl Acad Sci USA 85, 8086-90,
1988) that is expressed in the hind brain and spinal cord, and
XIF3, a neurally expressed intermediate filament gene (Sharpe, et
al., Development 107, 701-14, 1989) to assay for neural induction.
All these markers are restricted to neural tissue, however, only
NCAM is expressed throughout the nervous system. We found that
Xenopus-noggin conditioned medium induces high levels of N-CAM and
XIF3 expression[FIG. 2.; lane8] in treated animal caps, without
inducing muscle actin(lane 13) (Dworkin-Rastl, et al., J. Embryol.
exp. Morph. 91, 153-168, 1986; Mohun, et al., Nature 311, 716-721,
1984). Control CHO cell medium induces neither muscle nor neural
tissues (lanes 7, 12). St 9 activin treated animal caps express
muscle actin(lanell) and all three neural markers(lane 6),
demonstrating activin's ability to generate neural tissue
indirectly. It is interesting to note that noggin induces very
little, if any b-tubulin expression, while inducing high levels of
N-CAM, but activin induction has nearly the converse effect.
[0187] To determine whether noggin protein is sufficient to induce
neural tissue, COS cells were transfected with pCAE11, a human
noggin expression plasmid, and the conditioned medium was purified
by cation exchange chromatography resulting in noggin preparations
that were 90% pure [FIG. 3.]. Such purified human noggin protein is
also able to induce neural tissue in animal caps [FIG. 4a., see
below].
[0188] We have shown that noggin does not induce muscle in late
blastula stage animal caps, however, it is possible that noggin
induces other types of dorsal mesoderm. To address this concern, we
asked whether noggin could induce the expression of the early
mesoderm markers goosecoid (Blumberg, et al., Science 253, 194-6,
1991; Cho, et al., Cell 67, 1111-20, 1991), a marker of organizer
tissue and subsequently head mesoderm or X-brachyury (Smith, et
al., Cell 67, 79-87, 1991), which appears to be expressed in all
mesodermal precursors early, and subsequently is expressed in
posterior mesoderm and notochord. Animal caps were treated at stage
9 and collected at stage 11, when expression of goosecoid and
brachyury in the normal embryo is high. Neither marker is turned on
by purified human noggin (FIG. 4b. lane 5) at a dose with
demonstrated neural inducing activity (FIG. 6 lane 15); in contrast
animal caps treated in the same fashion with activin show both
goosecoid and X-bra expression (FIG. 4b. lane 4) as expected for
this mesoderm inducing factor (Cho, et al., Cell 67, 1111-20, 1991;
Smith, et al., Cell 67, 79-87, 1991). Untreated animal caps show no
expression of these mesodermal markers (lane 3), and RNA levels in
the collected animal caps are shown to be comparable using EF-1a
levels (Krieg, et al., Dev Biol 133, 93-100, 1989).
[0189] Since purified human noggin is capable of driving neural
induction, no additional factors which may have been present in the
crude conditioned medium are required. Furthermore, Xenopus and
human noggin, with 80% amino acid identity, can both act to induce
neural tissue in Xenopus, suggesting a conserved function for these
two proteins. However, for noggin to be a candidate endogenous
neural inducer it must be able to induce neural tissue at a stage
when neural induction occurs in normal whole embryos. It is unclear
when the first instructive signals are sent from dorsal mesoderm to
ectoderm in embryos. However, it is known that by early gastrula
stages, dorsal ectoderm has already been specified to become neural
tissue (Jones and Woodland, Development 107, 785-91, 1989). The
neural inducing signal is therefore likely to start before this
stage. The latest stage at which animal caps have been shown to be
competent to respond to neural-inducing mesoderm is the early
neuruia (St13-14) (Sharpe and Gurdon, Development 109, 765-74,
1990). Thus, a candidate endogenous neural inducer must be able to
induce neural tissue from gastrula stage competent ectoderm.
[0190] Neural Induction at the Gastrula Stage
[0191] In order to assess the competence of ectoderm to respond to
noggin we treated animal caps taken from blastula (St8), late
blastula (St9), early gastrula (St10) and ventral animal caps from
mid-gastrula (St10.5) stage embryos with purified human noggin[FIG.
2.]. We also treated similarly staged animal caps with activin to
demonstrate its mesoderm inducing and secondary neural inducing
activities, and to contrast activin's effects with those of noggin
[FIG. 4a.]. Activin treated animal caps show neural induction only
in conjunction with induction of dorsal mesoderm, such as muscle
and notochord (lanes 3,6,9). In a number of experiments, we
confirmed that activin's ability to induce dorsal mesoderm, and
consequently neural tissue, declines rapidly at the gastrula stage
(lane 12) (Green, et al., Development 108, 173-83, 1990; Kintner
and Dodd, Development 113, 1495-1506, 1991) In the experiment shown
here a larger than usual dose of activin was given. Under these
conditions, only a small amount of neural tissue is made, perhaps
because so much mesoderm is induced that there is not much
competent ectoderm left in the explant to be neuralized. In
contrast noggin can induce neural tissue in animal caps taken from
all of these stages without inducing the notochord and somite
marker, collagen type II (Amaya, et al., Development 118, 477-87,
1993; Bieker and Yazdani-Buicky, J Histochem Cytochem 40, 1117-20,
1992), or muscle actin (lanes 4,7,10,13). This gives additional
support to the proposal that noggin is a direct neural inducer,
since it can act in the absence of both early and late mesoderm
markers. Furthermore, we have shown that noggin can induce neural
tissue in competent ectoderm at a time when mesoderm inducers are
inactive.
[0192] In some experiments, noggin addition to gastrula (but not
blastula) animal caps resulted in induction of muscle (data not
shown). This occurred at stages when activin could no longer induce
muscle. We interpret this as a result of a dorsalizing action by
noggin on tissues that have received a weak mesoderm-inducing
signal. The mesoderm-inducing signal which spreads into the
gastrula animal cap is not enough to induce mesoderm, but in the
presence of Xwnt-8 or noggin, muscle is formed. One interesting
corollary of the induction of muscle is that the kinds of neural
tissue seen in the explant are modified. Induction in explants that
contain no muscle usually yields N-CAM expression, but if muscle is
present, expression of both N-CAM and b-tubulin is seen. This
phenomenon is demonstrated in the secondary neural induction by
activin in St. 9 animal caps[FIG. 2.] and in the comparison of
neural tissue induced by noggin in ventral marginal zones versus
animal caps [FIG. 6.]. In the ventral marginal zones and animal
caps in which muscle is present, both N-CAM and b-tubulin are
expressed, whereas induced animal caps without muscle, show only
N-CAM expression.
[0193] Neural Induction After Injection of DNA Coding for
Noggin
[0194] To confirm our conclusions using a different experimental
approach, we have directed noggin expression to gastrula stage
animal caps by injecting the plasmid pCSKA-noggin into the animal
pole of a one cell stage embryo. This plasmid, in which noggin is
under the control of the cytoskeletal actin promoter, turns on the
expression of noggin mRNA at the onset of gastrulation (Smith, et
al., Nature 361, 547-49, 1993). At the blastula stage, the animal
caps are dissected and then matured to tailbud stages for molecular
analysis. Animal caps injected with the noggin plasmid show
expression of N-CAM in the absence of muscle or notochord markers
(FIG. 4c. lane 2). A control plasmid directing the expression of
lac Z showed no neural or mesodermal induction as expected (lane
1). This experiment demonstrates that ectopic noggin expression can
directly induce neural tissue in gastrula stage ectoderm, a stage
when neural induction is taking place in whole embryos.
[0195] Differences in Competence Between Dorsal and Ventral Animal
Caps.
[0196] Animal caps taken from the dorsal side of gastrula stage
embryos show greater competence to form neural tissue than ventral
animal caps (Otte and Moon, Cell 68, 1021-29, 1992; Sharpe, et al.,
Cell 50, 749-58, 1987), when involuted anterior mesoderm is used as
the inducer. This type of mesoderm, however, has weaker inducing
capacity than the rest of the involuted mesoderm (Sive, et al.,
Cell 58, 171-180, 1989). Furthermore, the ventral side of an embryo
can support the formation of a complete secondary axis when the
organizer is placed on that side (Gimlich and Cooke, Nature 306,
471-3, 1983; Smith and Slack, J. Embryol. Exp. Morph. 78, 299-317,
1983; Spemann, Arch. Mikjrosk. Anat. EntwMech. 100, 599-638, 1938),
indicating that there is no qualitative difference in competence.
Thus, while a weak inducer might unmask slight differences in
competence of the ectoderm, it has been suggested that a robust
neural inducer would show little difference in its effects on
dorsal and ventral ectoderm (Servetnick and Grainger, Development
112, 177-88, 1991). Therefore we tested noggin's effects on dorsal
and ventral ectoderm from the early gastrula. No difference in
N-CAM expression is detected (FIG. 5, lanes 4,6), while the ventral
animal caps treated with noggin show a greater amount of muscle
actin expression (presumably through dorsalization of tissues that
received a low-grade mesoderm induction). Activin treated dorsal
caps show induction of roughly the same level of muscle actin
expression (lane 5) as the ventral noggin treated caps, however,
activin treatment did not induce detectable neural specific
transcripts (lanes 3, 5). This indicates that muscle tissue induced
at this stage is not sufficient to secondarily induce neural
tissue, and that noggin must be present to induce neural
tissue.
[0197] We conclude that there is no dorsal-ventral difference in
noggin mediated neural induction, suggesting that noggin behaves
like the robust neural inducing signal of the Spemann organizer,
not like the weaker signal from early anterior mesoderm.
[0198] Dose Dependence
[0199] To determine what levels of noggin protein are required for
neural inducing activity, we carried out a dose response
experiment.
[0200] In addition to determining the doses required for neural
induction in animal caps, we have also carried out a dose response
of the dorsalization of ventral marginal zones in order to compare
the doses required for these two types of inductions. Stage 9
animal caps or St. 10.5 VMZ were treated with purified human
noggin, and N- CAM and .beta.-tubulin were used to assay neural
induction, while muscle actin was used as a marker of dorsal
mesoderm. This experiment shows that neural induction occurs at a
dose of 1 .mu.g/ml, which is a twenty fold higher dose than
required for dorsaiization of VMZ [FIG. 5]. There are several
observations that may account for the apparently high dose
requirement. First, to get a maximal neural response from dorsal
mesoderm, the tissues must be left in contact through most of
neurulation (Sharpe and Gurdon, Development 109, 765-74, 1990); in
contrast, the animal caps treated with noggin close up rapidly,
this inhibits factor access, and consequently they receive only a
brief effective dose. Second, it is likely that noggin is not the
only neural inducer active in the embryo; it has been shown in a
variety of amphibians that the somites (Hemmati-Brivanlou, et al.,
Science 250, 800-802, 1990; Jones and Woodland, Development 107,
785-91, 1989) and the neural plate have neural inducing activity
(Hamburger, The Heritage of Experimental Embryology: Hans Spemann
and the Organizer, 1988; Servetnick and Grainger, Dev Biol 147,
73-82, 1991) and noggin transcripts are not detected there. Thus it
is plausible that noggin is one of several neural-inducing
activities. In this connection it is worth noting that noggin is
equally potent in inducing neural tissue in ventral marginal zones
as in dorsalizing them to generate muscle. Numerous other
experiments (see FIG. 5) show that induction of a similar amount of
muscle at this stage by activin does not result in neural
induction. Fourth, it may be that only a small fraction of the
purified protein is active, and that the experiment overestimates
the amount of protein needed for neural induction. Finally, it is
possible that the accessibility of exogenously added soluble noggin
is significantly lower than noggin protein being secreted
endogenously.
[0201] Patterning
[0202] Embryonic neural tissue develops an anteroposterior (A-P)
pattern, with various brain structures, eyes, and the spinal cord.
It is thought that A-P neural pattern requires the presence of
dorsal mesoderm, whether it be adjacent to the responding ectoderm
in a planar configuration (Dixon and Kintner, Development 106,
749-757, 1989; Doniach, et al., Science 257, 542-5, 1992; Kintner
and Melton, Development 99, 311-25, 1987; Ruiz i Attaba,
Development 108,595-604, 1990), or directly beneath it in a
vertical interaction (Dixon and Kintner, Development 106, 749-757,
1989) (Hemmati-Brivanlou, et al., Science 250, 800-802, 1990;
Sharpe and Gurdon, Development 109, 765-74, 1990; Sive, et al.,
Cell 58, 171-180, 1989). Both of these types of interactions occur
in normal development, and both probably contribute to the
resulting pattern. To determine if noggin induces patterned neural
tissue, and if so, what neural regions are represented, we used
Xenopus otx as a marker of forebrain and mid brain; En-2
(Hemmati-Brivanlou, et al., Development 111, 715-724, 1991) as a
marker of the mid brain-hind brain boundary, and Krox-20
(Wilkinson, et al., Nature 337, 461-4, 1989) as a marker of the
third and fifth rhombomeres of the hind brain in in situ
hybridization (Harland, Methods in Cell Biology, 36, 675-685,
1991). Antibodies directed against XIHbox 6 (Wright, et al.,
Development 109, 225-34, 1990) mark posterior hind brain and spinal
cord structures. Prior to the use of these markers, we observed the
formation of cement glands in noggin treated animal caps. Since
cement glands are induced organs of ectodermal origin found
anterior to the neural plate, this result suggests that noggin
induces anterior structures. In situ hybridization confirms this by
showing the presence of a cement gland specific transcript, XAG-1
(Sive, et al., Cell 58, 171-180, 1989) in noggin treated animal
caps, but not in control treated animal caps [FIG. 7.]. In situ
hybridization with the region specific neural markers [FIG. 7.]
show that noggin induces forebrain type tissue as seen by the
expression of otx in noggin treated animal caps. We have not
detected En-2, Krox20, or XIHbox, suggesting that these more
posterior markers are not induced by noggin.
[0203] Expression of Neural Antigens
[0204] We have demonstrated that noggin directly induces the
expression of neural specific transcripts. A further demonstration
is to use antibodies against neural specific antigens to show that
the noggin induced tissue is phenotypically neural. To this end, we
have treated animal cap tissue with noggin and cultured them to a
late stage (St 35) for antibody staining. We have used the 6F11
anti-N-CAM antibody, which stains the entire neural tube of a
normal embryo. Noggin treated animal caps express this antigen
[FIG. 7.] while control untreated animal caps do not. This
indicates that noggin can induce the production of neural specific
proteins in treated animal caps. We have failed to detect the
expression of numerous other antigens that are characteristic of
various subclasses of differentiated neural cells. These included
2G9, which stains most neural tissue, including peripheral neurons,
Tor 24.55, which stains sensory neurons, and Tor 23, which stains a
variety of neurons including motor neurons.
EXAMPLE 7
[0205] Production of Recombinant Human Noggin from E. coli and
Baculovirus
[0206] Materials and Methods
[0207] Genetic Engineering and Cell Culture
[0208] A lactose inducible expression plasmid was constructed by
replacing the Swa1/Bsm1 region of pRPN40 (Masiakowski et al, J.
Neurochem. 57, 1003-1012, 1991 ) with the Swa1/Bsm1 region of the
human noggin gene obtained by PCR and spanned by the same
restriction sites, resulting in plasmid pRG301. pRG301 is a high
copy number kanamycin resistant plasmid derived from pBR322 with
the human noggin gene under the control of the lacUV5 promoter. A
plasmid containing the high copy number kanamycin resistant gene
was deposited with the Agricultural Research Collection (NRRL),
Peoria, Illinois, and bears accession number B-18600. This plasmid
was described in U.S. patent application Ser. No. 07/478,338, which
is incorporated by reference herein in its entirety. E. coli
W3110laclq cells transformed with pRG301 were grown at 37.degree.
C., induced with lactose, harvested by centrifugation, washed once
with 100 mM Tris-HCI, 50 mM EDTA pH 8 and stored frozen,
essentially as described (Masiakowski et al, ibid. ).
[0209] Recovery from Inclusion Bodies
[0210] E. coli cell paste (32 g) was suspended in ten volumes (v/w)
of 50 mM TrisHCl-pH 8.0-5 mM EDTA, lysed in a French Press at 8,000
psi and 8.degree. C. and centrifuged at 8,000.times. g for 30 min
at 4.degree. C. The pellet containing noggin was suspended in the
original volume of 2 M urea-20 mM TrisHCl, pH 8.0 and stirred for
30 min. The suspension was centrifuged at 8,000.times. g at
4.degree. C. for 30 min and the pellet consisting mostly of
inclusion bodies (IB) was suspended in 20 volumes (v/w) of 6 M
guanidine HCl, 50 mM Tris HCl, 1 mM EDTA, 50 mM DTT and stirred for
one hour at room temperature. After centrifugation at 8,000.times.
g for 30 min, the supernatant containing 0.45-0.50 g denatured and
reduced noggin was diafiltered against 10 volumes of 6 M urea-50 mM
sodium acetate pH 4.5-1 mM EDTA-0.1 mM DTT using Omega 10,000 MW
cut-off membranes. The diafiltrate containing 0.4-0.44 g noggin was
loaded at a flow rate of 30 ml/min onto a 2.6.times.10 cm column of
S-Sepharose (Pharmacia), equilibrated in 6 M urea-50 mM sodium
acetate-1 mM EDTA-0.1 mM DTT pH 4.5. The column was first washed
with the same buffer and then with a one liter gradient (0-1M NaCl)
at a flow rate of 30 ml/min. Fractions containing noggin were
identified by gel electrophoresis and pooled. The yield was
0.2-0.25 g noggin.
[0211] Refolding.
[0212] The denatured and reduced noggin solution was adjusted to
0.05-0.2 mg/ml protein concentration and brought to 1.5-2.5 M
guanidineHCl-0.1 M TrisHCl pH 8.0-0.1 mM EDTA-0.2-2 mM reduced
glutathione-0.02-0.2 mM oxidized glutathione (preferably at a ratio
of 10:1 reduced to oxidized glutathione) at 4.degree. C. under slow
stirring. After 24-72 hours, two refolded noggin isoforms were
identified by RP-HPLC chromatography (FIG. 8). The refolded noggin
solution was diafiltered against 20 volumes of 0.05 M sodium
acetate pH 4.5, brought to 50 mM potassium phosphate pH 7.2 and
stirred slowly at 4.degree. C. for 1 hour minimum. Misfolded noggin
precipitated and was removed by centrifugation for 30 min at
8,000.times. g.
[0213] Reverse Phase HPLC Chromatography.
[0214] Refolded noggin can be purified by chromatography on a 12 mm
C8, 1.times.25 cm Dynamax 300 A column equilibrated in solvent A
(0.1% TFA in water). After loading, the column was washed with
solvent A and was developed at a flow rate of 4 ml/min according to
the following protocol: (a) 10 min isocratically at 70% of solvent
A, 30 % of solvent B (0.1% TFA in acetonitrile); 30 min linear
gradient to 60% solvent B and 40% solvent A. Correctly refolded
noggin elutes earlier at 44%-46% solvent B. The yield was 0.07-0.1
g noggin.
[0215] Production of Human Noggin in Baculovirus Cell Culture,
[0216] The SF21 line of Spodoptera frugiperda was routinely
maintained as cell monolayers in Grace's Insect Cell medium
supplemented with lactalbumin hydrolysate and yeastolate (Gibco).
This medium completed with 10% v/v heat-inactivated fetal calf
serum (Irvine Scientific) is identified as TMNFH-10. Cells were
also cultured in serum-free medium (SF-900-II; Gibco) after
adaptation. Suspension cultures in either medium were raised in
microcarrier culture flasks (Bellco) using a stirring speed of 80
rpm. All cultures were maintained at >96% viability, as judged
by trypan blue exclusion.
[0217] Construction of Recombinant Baculovirus.
[0218] Sequences corresponding to human noggin were excised as a
5'-BamH1-Pst1-3' fragment from an expression plasmid containing the
human noggin gene. This fragment was inserted into BamH1-Pst1
digested pVL1393 (Invitrogen). The resulting plasmid, pTR 1009, has
the human noggin sequence immediately downstream of the polyhedrin
promoter of Autrographa caiifornica Multiple Nuclear Polyhedrosis
Virus (AcMNPV), and this promoter-heterologous gene fusion is
flanked in turn by recombination targets derived from the AcMNPV
polyhedrin region. Recombinant plasmid DNA was purified by alkaline
lysis and CsCI centrifugation. SF21 cells were co-transfected with
plasmid and viral DNA by the following method: Plasmid DNA (3 mg)
was mixed with 0.5 mg linearized, deleted viral DNA (Baculo
Gold.TM., Pharminigen), and precipitated with ethanol. Dried DNA
was then resuspended in water (50 ml), mixed with 1.5ml Grace's
medium, and 30 ml Lipofectin.TM. cationic liposomes (BRL). The
DNA-liposome mixture was vortexed, allowed to stand at room
temperature for 15 minutes and added dropwise to a monolayer of
SF21 cells (2.times.106 cells/60 mm plate). After incubation at
27.degree. C. for four hours, 2 ml TMNFH-10 was added and the
culture returned to incubation for 5 days. Tissue culture medium
was harvested and used as a source of virus for plaque
isolation.
[0219] Recombinant virus was isolated by multiple rounds of plaque
purification on SF21 cells Diluted virus (0.5 ml) was adsorbed to
cell monolayers (2x106 cells/60 mm plate) for a period of one hour
at 27.degree. C., aspirated, and virus plaques were allowed to
develop with an overlay of 0.5% agarose in TMNFH-10 medium for a
period of 6 days. Virus plaques were picked after microscopic
inspection, and eluted into 2ml SF900-II medium. Virus stocks were
amplified by low multiplicity (0.1 pfu/cell) infection. Virus
clones expressing noggin were identified by metabolic labeling of
infected cultures with 35S-methionine and 35S-cysteine and
analyzing total labeled protein by polyacrylamide gel
electrophoresis and autoradiography. A labeled protein of the
expected apparent Mr of 20,000-30,000 was detected by this method
in candidate clones but not in control cultures.
[0220] Expression and Purification of Baculovirus-derived
Noggin.
[0221] SF21 cells were cultured in suspension flasks to a density
of approximately 1.8.times.106/ml in SF900-II medium. Cultures (500
ml) were collected by centrifugation at 1000.times. g for 10 min
and resuspended in 20ml of growth medium containing 5-10 pfu/cell
recombinant virus. Virus was allowed to adsorb for 1 hour at room
temperature with gentle mixing. Infected cells were then diluted to
their original volume with fresh growth medium, and incubated at
27.degree. C. for 3 days. Cells and debris were subsequently
clarified from the growth medium by centrifugation at 1000.times. g
for 20 min.
[0222] Cell supernatants were brought to pH 8.0, passed through a
0.45 mm Millipak 60 filter and applied to a Fast S column that had
been equilibrated in 25 mM HEPES pH 8.0. The column was washed with
the same buffer and developed with a linear NaCl gradient to remove
other medium components. Noggin eluted from this column at 1 M
NaCl.
[0223] Results
[0224] Characterization of Human Noggin Produced in E. coli and in
Baculovirus
[0225] Reverse-phase HPLC chromatography shows that recombinant
noggin refolded and purified from E. coli elutes in a single sharp
peak, indicating the presence of one predominant isoform (FIG.
9).
[0226] Electrophoresis on 15% polyacrylamide-SDS-reducing gels
shows that noggin from either E. coli or insect cells is better
than 95% pure and migrates in a single band corresponding to a
protein of 20-30 kD. Noggin from insect cells shows slightly slower
mobility, apparently due to additional mass from N-linked
glycosylation at the single consensus site (FIG. 10). Treatment
with Endo F converts the mobility of insect-produced noggin to that
of the bacterially produced protein (data not shown).
[0227] In the absence of reducing agents, noggin produced either in
E. coli or in baculovirus behaves as a disulfide-linked oligomeric
protein (FIG. 10). However, by gel filtration analysis and mass
spectroscopy noggin is primarily a dimeric protein (data not
shown).
[0228] Circular dichroism studies show that recombinant noggin
refolded and purified from E. coli as well as noggin purified from
insect cells have very similar conformations (FIG. 11). Secondary
structure determined by this method indicates that noggin consists
of 48% alpha-helix, 0% beta-structure, and 52% random coil.
[0229] Biological Activity of Human Noggin Produced in E. coli and
in Baculovirus
[0230] Biological activity of human noggin produced in E. coli or
in baculovirus was determined by assay of muscle actin expression
in the ventral marginal zone assay, as described supra. Results
shown in FIG. 12 indicate a positive dose response for induction of
muscle actin mRNA in VMZ exposed to either bacterially produced
human noggin, or baculovirus produced human noggin.
EXAMPLE 8
[0231] Production and Characterization of Rat Monoclonal Antibody
RP57-16 Reactive with Human Noggin.
[0232] Materials and Methods
[0233] Production of Antibody
[0234] RP57-16 rat monoclonal antibody reactive with recombinant
human and Xenopus noggin was produced by the immunization of a
female Lewis rat with four 35 .mu.g injections of purified
recombinant human noggin (produced in E. coli) over a two month
period. For the initial immunization, the protein was injected in
the rear foot pad in Freund's complete adjuvant. Subsequent
injections were given in the same foot pad in Freund's incomplete
adjuvant. The rat was euthanized 3 days after the fourth
injection.
[0235] Lymph node cells from the immunized rat were mixed with
SP2/0-E.O. mouse myeloma cells at a ratio of 2:1. After
centrifugation, the cell mixture was resuspended in 0.25 ml of 42%
(w/v) PEG 3350 (Baker) in phosphate-buffer-saline with 10% (v/v)
dimethylsulfoxide (Sigma) for a total of 3 minutes in a 37.degree.
C. water bath. Cells were plated at a density of 5.times.10.sup.4
lymphocytes per well in 96-well plates (Falcon 3072) in DMEM/F-12
(Mediatech, Inc.) containing 10% FBS (supplemented with
streptomycin, penicillin, pyruvate, and glutamine) and HMGT
(1.6.times.10-3 M thymidine, 4.0.times.10-4 methotrexate,
1.3.times.10-3 sodium bicarbonate and 1.0.times.10-2 hypoxanthine).
After 10 days in culture, supernatants were harvested and assayed
for antibody activity against recombinant human noggin by indirect
ELISA. Supernatant from COS-M5 cells transfected with the plasmid
containing the human noggin gene was air dried overnight in Probind
96-well assay plates (Falcon 3915). Non-specific binding was
eliminated by 2 hour incubation at ambient temperature with PBS/1%
BSA (Sigma). Plates were washed 2 times with PBS/0.02%Tween 20.
Culture supernants were then added and incubated at ambient
temperature for 1 hour. Plates were washed 4 times with PBS/0.02%
Tween 20. Secondary antibody, Goat anti-Rat igG (H+L) alkaline
phosphatase conjugate(Caltag) diluted 1:2000 in PBS/1% BSA was
added to each well and the plates incubated at ambient temperature
for 1 hour. Plates were again washed 4 times with PBS/0.02% Tween
20. Antibody binding was visualized by 1 hour incubation at ambient
temperature in the dark with pNPP (p-nitrophenyl phosphate, Sigma)
1 mg/mI in diethanolamine buffer, pH 9.8. The reaction was stopped
by the addition of an equal volume of 100 mM EDTA. Absorbance was
read at 405 nm on a Thermomax Microplate Reader (Molecular
Devices). A reaction was considered positive if the absorbance was
2 times that of the negative control (diluent alone followed by
secondary antibody and substrate). Positive clones were expanded
and culture supernatant containing monoclonal antibody was
collected for specificity analysis.
[0236] RP57-16 was cloned in soft agar. Cloned hybrid cells were
expanded in DMEM/F-12 (Mediatech, Inc.) containing 10% FBS
(supplemented with streptomycin, penicillin, pyruvate, and
glutamine). Supernatant containing antibody was aliquoted and
stored at -70.degree. C. until use.
[0237] Specificity Analysis
[0238] ELISA
[0239] 100 ng of purified recombinant human noggin, Xenopus noggin,
BDNF, NT-3, and NT4 protein was individually passively adsorbed to
Probind 96-well assay plates by overnight incubation at 4.degree.
C. in 50 mM bicarbonate buffer, pH 9.6. BDNF, NT-3 and NT-4 were
used to assess non-specific binding of rat monoclonal antibody
RP57-16. Supernatants from COS-M5 cells transfected with either the
plasmid containing the human noggin gene or the plasmid containing
the flg C-terminal tagged Xenopus noggin gene were air dried to
Probind 96-well plates overnight. Non-specific binding was
eliminated by 2 hour incubation at ambient temperature with PBS/1%
BSA (Sigma). Plates were washed 2 times with PBS/0.02% Tween 20.
Undiluted RP57-16 was added and incubated at ambient temperature
for 1 hour. Plates were washed 4 times with PBS/0.02% Tween 20.
Secondary antibody, Goat anti-Rat IgG (H+L) alkaline phosphatase
conjugate(Caltag) diluted 1:2000 in PBS/1% BSA was added to each
well and the plates incubated at ambient temperature for 1 hour.
Plates were again washed 4 times with PBS/0.02% Tween 20. Antibody
binding was visualized by 1 hour incubation at ambient temperature
in the dark with pNPP (p-nitrophenyl phosphate, Sigma) 1 mg/ml in
diethanolamine buffer, pH 9.8. The reaction was stopped by the
addition of an equal volume of 100 mM EDTA. Absorbance was read at
405 nm on a Thermomax Microplate Reader (Molecular Devices). A
reaction was considered positive if the absorbance was 2 times that
of the negative control (diluent alone followed by secondary
antibody and substrate).
[0240] Electrophoresis and Western Blotting
[0241] Rat monoclonal antibody RP57-16 was also analyzed by Western
blotting. 50 ng of recombinant human noggin, non-reduced and
reduced, were electrophoresed on 12.5% SDS-polyacrylamide gels and
electroblotted on nitrocellulose membranes. Membranes were blocked
with PBS/1% Casein/0.1% Tween 20, and then incubated for 2 hours
with undiluted RP57-16 culture supernatant. Following 4 washes in
PBS/0.02% Tween 20, the membranes were incubated with a 1:5000
dilution of Goat anti-Rat IgG (H+L) horseradish peroxidase
conjugate (Pelfreeze) in PBS/1% BSA/0.1% Tween 20. Membranes were
washed 4 times with PBS/0.02% Tween 20. Proteins were visualized
with ECL Western Blotting Reagents (Amersham) according to the
manufacturer's instructions. Membranes were then exposed to XAR 5
Scientific Imaging film (Kodak) for 5 seconds.
[0242] Results
[0243] Rat monoclonal antibody RP57-16 reacts with both recombinant
human and Xenopus noggin and with recombinant human noggin produced
in E. coli, in insect cells, and in COS-M5 cells. The antibody does
not react with the neurotrophins BDNF, NT-3 and NT-4, Western
blotting showed that the antibody detects both reduced and
non-reduced protein.
[0244] Deposit of Microorganisms
[0245] The following were deposited with the American Type Culture
Collection, 12301 Parklawn Drive, Rockville, Md. 20852 under the
terms of the Budapest Treaty:
8 ATCC Accession Date of No. Deposit phage hnog.lambda.-5 75311
9-23-92 phage hnog.lambda.-7 75309 9-23-92 phage hnog.lambda.-9
75310 9-23-92 phage hnog.lambda.-10 75308 9-23-92 hybridoma RP57-16
CRL 11446 8-25-93
[0246] It is to be understood that while the invention has been
described above in conjunction with preferred specific embodiments,
the description and examples are intended to illustrate and not
limit the scope of the invention, which is defined by the scope of
the appended claims.
Sequence CWU 0
0
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