U.S. patent application number 10/768694 was filed with the patent office on 2004-08-12 for method for maintenance and propagation of germline stem cells using members of the tgf-beta family of growth factors.
This patent application is currently assigned to Carnegie Institution of Washington. Invention is credited to Spradling, Allan C., Xie, Ting.
Application Number | 20040157324 10/768694 |
Document ID | / |
Family ID | 22242223 |
Filed Date | 2004-08-12 |
United States Patent
Application |
20040157324 |
Kind Code |
A1 |
Spradling, Allan C. ; et
al. |
August 12, 2004 |
Method for maintenance and propagation of germline stem cells using
members of the TGF-beta family of growth factors
Abstract
The TGF-.beta. family of growth factors, particularly the bone
morphogenetic protein (BMP)-2/4 homolog decapentaplegic (dpp), are
specifically required to maintain germline stem cells and promote
their division. Overexpression of dpp blocks germline stem cell
differentiation. Mutations in dpp or its receptor saxophone
accelerate stem cell loss and retard stem cell division. dpp
signaling is directly received by germline stem cells, and thus dpp
signaling helps define a niche that controls germline stem cell
proliferation.
Inventors: |
Spradling, Allan C.;
(Baltimore, MD) ; Xie, Ting; (Baltimore,
MD) |
Correspondence
Address: |
MORGAN LEWIS & BOCKIUS LLP
1111 PENNSYLVANIA AVENUE NW
WASHINGTON
DC
20004
US
|
Assignee: |
Carnegie Institution of
Washington
|
Family ID: |
22242223 |
Appl. No.: |
10/768694 |
Filed: |
February 2, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10768694 |
Feb 2, 2004 |
|
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09358937 |
Jul 23, 1999 |
|
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60094008 |
Jul 24, 1998 |
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Current U.S.
Class: |
435/348 |
Current CPC
Class: |
C12N 2501/155 20130101;
C12N 2510/00 20130101; C12N 5/0601 20130101; C12N 2501/41
20130101 |
Class at
Publication: |
435/348 |
International
Class: |
C12N 005/06 |
Claims
We claim:
1. A method for maintaining germline stem cells of Drosophila
comprising: (a) providing a population comprised of said germline
stem cells, and (b) stimulating signal transduction by a bone
morphogenetic protein (BMP) signaling pathway in at least one cell
of said population; wherein said stimulation maintains more
germline stem cells in said population as compared to a population
which has not had signal transduction of said BMP signaling pathway
stimulated.
2. A method according to claim 1, wherein said population is
maintained in vivo and said Drosophila has been genetically
engineered to stimulate said signal transduction.
3. A method according to claim 1, wherein said population in
maintained in vitro.
4. A method according to claim 1, wherein said germline stem cells
are from ovary.
5. A method according to claim 1, wherein said germline stem cells
are from testis.
6. A method according to claim 1 further comprising obtaining said
germline stem cells from an embryo.
7. A method according to claim 1, wherein said BMP signaling
pathway is stimulated by providing at least 10% more
Decapentaplegic (Dpp) activity to said population than is present
in wildtype.
8. A method according to claim 1, wherein said BMP signaling
pathway is stimulated by at least mutating a dpp gene to a
gain-of-function phenotype.
9. A method according to claim 1, wherein said BMP signaling
pathway is stimulated by at least providing a BMP to said
population.
10. A method according to claim 9, wherein said BMP is selected
from the group consisting of Decapentaplegic (Dpp) protein, BMP-2,
and BMP-4.
11. A method according to claim 1, wherein said BMP signaling
pathway is stimulated by at least mutating a type I or type II
decapentaplegic (dpp) receptor to a gain-of-function phenotype.
12. A method according to claim 1, wherein said BMP signaling
pathway is stimulated through at least one serine/threonine kinase
receptor which specifically recognizes a BMP.
13. A method according to claim 12, wherein said BMP receptor is
selected from the group consisting of Saxophone (Sax), Thick veins
(Tkv), and Punt (Put).
14. A method according to claim 1, wherein said BMP signaling
pathway is stimulated by altering activity of at least one signal
transducer for receptor binding to a BMP.
15. A method according to claim 14, wherein said signal transducer
is selected from the group consisting of Mothers against dpp (Mad),
Medea (Med), Daughters against dpp (Dad), Schnurri (Shn), and
Brinker (Brk).
16. A method according to claim 1, wherein said BMP signaling
pathway is stimulated by increasing expression of BMP in a cell of
said population.
17. A method according to claim 16, wherein BMP expression is
increased by hedgehog (hh)-activated transcription or wingless
(wg)-activated transcription, and BMP signaling is increased in at
least some of the germline stem cells.
18. A method according to claim 1, wherein said population is
further comprised of at least one somatic cell selected from the
group consisting of terminal filament cell, cap cell, inner sheath
cell, hub cell, and cyst progenitor cell.
19. A method according to claim 1, wherein at least one germline
stem cell is cultured in vitro in contact with feeder cells
expressing a bone morphogenetic protein (BMP).
20. A method according to claim 1, wherein at least one germline
stem cell is cultured in vitro in contact with at least some
somatic niche cells.
21. A method according to claim 1, wherein signal transduction
through said BMP signaling pathway is stimulated by in vitro
culturing said germline stem cells with a feeder layer of somatic
cells which stimulate BMP signaling.
22. A method according to claim 1, wherein signal transduction
through said BMP signaling pathway is stimulated by in vitro
culturing said germline stem cells in a culture medium which
stimulates BMP signaling.
23. A method according to claim 1 further comprising maintaining at
least one of said germline stem cells in a pluripotent state.
24. A method according to claim 1 further comprising maintaining at
least one of said germline stem cells in a totipotent state.
25. A method according to claim 1 further comprising transferring
at least one of said stimulated germline stem cells into a host
Drosophila.
26. A method according to claim 25, wherein at least one of said
transferred germline stem cells contributes to two or more
differentiated cell lineages of said host Drosophila.
27. A method according to claim 25, wherein at least one of said
transferred germline stem cells contributes to a germline cell
lineage of said host Drosophila.
28. A method according to claim 1 further comprising mutating at
least one gene of said germline stem cell's genome.
29. A method according to claim 1 further comprising introducing
one or more polynucleotides into said germline stem cell's
genome.
30. A method according to claim 1 further comprising integrating a
polynucleotide by homologous recombination at a targeted genetic
locus of said germline stem cell.
31. A method according to claim 1 further comprising targeting at
least one gene of said germline stem cell for homologous
recombination, selecting at least one germline stem cell which has
undergone homologous recombination of said gene, and transferring
said homologously recombinined germline stem cells into another
Drosophila such that said targeted gene is genetically transmitted
through said another Drosophila's germline.
32. A cell population made by a method according to claim 1,
wherein there are at least ten germline stem cells in said
population for each germline stem cell present prior to stimulation
of BMP signaling.
33. A method for maintaining Drosophila stem cells comprising: (a)
providing a population comprised of said stem cells, and (b)
stimulating decapentaplegic (dpp) signaling such that more stem
cells of said population are maintained as at least viable or
undifferentiated as compared to a population of stem cells which
has not been stimulated.
34. A method of reducing or eliminating stem cells or tumor cells
of an organism comprising: repressing signal transduction by a bone
morphogenetic protein (BMP) receptor pathway such that said stem
cells or tumor cells are reduced or eliminated.
35. A method of increasing abundance of stem cells of an organism
comprising: stimulating signal transduction by a bone morphogenetic
protein (BMP) receptor pathway such that abundance of at least some
stem cells is increased.
36. A method of increasing lifetime of stern cells of an organism
comprising: stimulating signal transduction by a bone morphogenetic
protein (BMP) receptor pathway such that said lifetime of at least
some stem cells is increased.
Description
RELATED APPLICATIONS
[0001] This application claims priority from provisional U.S.
Appln. No. 60/094,008, filed Jul. 24, 1998.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] This invention relates to members of the transforming growth
factor-.beta. family and their regulation of cell division, cell
survival, and the specification of cell fates. Particularly, the
invention relates to the bone morphogenetic protein (BMP)-2/4
homolog deca-pentaplegic (dpp) and its role in the maintenance of
stem cells. For example, a dpp-based method for maintenance and
controlling the division of germline stem cells, and a dpp-based
method for defining a niche that controls germline stem cell
proliferation are disclosed. Additionally, the invention provides a
model of ovarian tumor development. The invention further relates
to a dpp-based method for propagating stem cells in an
undifferentiated state in vivo or by culturing in vitro.
[0004] 2. Description of Related Art
[0005] In many adult tissues that undergo continuous cell turnover,
a population of stem cells is responsible for replacing lost cells.
Because of their pivotal role in controlling growth and neoplasia,
the mechanisms regulating stem cell function are of great interest
(reviewed by Potter and Loeffler, 1990; Doe and Spana, 1995; Lin,
1997; Morrison et al., 1997). Two mechanisms have been proposed to
maintain stem cell divisions and regulate the differentiation of
stem cell daughters: intrinsic factors and extracellular signals.
Asymmetrically localized intrinsic factors help specify the fates
of neuroblast daughters in Drosophila embryos (Doe and Spana,
1995). Extracellular signals from surrounding cells mediated by
cell surface-associated ligands and diffusible factors are
frequently involved (Potter and Loeffler, 1990; Morrison et al.,
1997). The identification of several of these factors has made it
possible to culture some types of stem cell in vitro.
[0006] The Drosophila ovary presents an excellent system for
studying two distinct groups of stem cells that remain active
during much of adult life (reviewed by Spradling et al., 1997). The
adult ovary contains 14-16 ovaribles each with a germarium at the
tip, within which the germline and somatic stem cells are located.
Two or three germline stem cells, located at the anterior tip of
the germarium, divide asymmetrically to generate all germline cells
in the ovariole (Wieschaus and Szabad, 1979; reviewed by Lin,
1997). Stem cell daughters known as cystoblasts undergo four rounds
of synchronous division to produce groups of two, four, eight, and
eventually 16 interconnected cystocytes, the precursors of ovarian
follicles (reviewed by de Cuevas et al., 1997). Two somatic stem
cells residing in the middle of the germarium give rise to all the
somatic follicle cells (Margolis and Spradling, 1995); their
equivalent in the testis are cyst progenitor cells. Three types of
mitotically quiescent somatic cells are located in the vicinity of
the stem cells: terminal filament and cap cells contact the
germline stem cells, while inner sheath cells lie more posteriorly
and contact both stem cell types.
[0007] Germline stem cell division is known to involve intrinsic
mechanisms. This division and subsequent cystocyte divisions are
physically unequal due to the segregation of fusomes rich in
membrane skeleton proteins such as .alpha.-spectrin and an adducin
homolog encoded by hu-li tai shao (hts) (reviewed by de Cuevas et
al., 1997). The round fusome (or "spectrosome") characteristic of
stem cells changes shape as cyst development proceeds, allowing
cysts at different stages to be identified. The bag of marbles
(bam) gene is highly expressed only in the stem cell daughter
(McKearin and Spradling, 1990). The loss of Bam protein in
cystoblasts prevents their differentiation, causing germline tumors
to form (a "tumor" in Drosophila is a large clump of proliferating
cells, the term does not imply these cells are cancerous). The
genes pumilio (pum) and nanos (nos), encoding translational
regulators, also play critical roles in the formation and
maintenance of germline stem cells (Lin and Spradling, 1997; Forbes
and Lehmann, 1998).
[0008] Less is known about the intercellular signals that control
stem cell proliferation. Two important signaling molecules,
Hedgehog (Hh) and Wingless (Wg) (reviewed by Perrimon, 1995;
Cadigan and Nusse, 1997), are expressed in terminal filament and
cap cells (Forbes et al., 1996a and 1996b). Hh signaling is
critical for proliferation and differentiation of follicle cells,
but it remained to be determined at the time the present invention
was made whether somatic stem cells or their daughters are
regulated (Forbes et al., 1996a and 1996b). The role of these
signals in the germ line was even less clear because ectopic
expression of hh did not appear to interfere with the function of
germline stem cells (Forbes et al., 1996a).
[0009] Members of the transforming growth factor-.beta.
(TGF-.beta.) family, including TGF-.beta.s, activins, and the bone
morphogenetic proteins (BMPs), elicit a broad range of cellular
responses including the regulation of cell division, survival, and
specification of cell fates (reviewed by Massague et al., 1996;
Hogan, 1996a). TGF-.beta.s were previously identified as repressing
the proliferation of stem cells as assayed by either in vitro
cultures or in vivo ectopic expression (Potter and Leoffler, 1990;
Morrison et al., 1997). Inactivation of BMP-4 and its receptor BMPR
in mice resulted in embryonic lethality for homozygous mutants.
(Winnier et al., 1995; Mishina et al., 1995), but no effect on stem
cells was noted.
[0010] Similarly dpp, encoding a vertebrate BMP-2/4 homolog in
Drosophila, functions as a local signal as well as a long-distance
morphogen to pattern the early embryo and adult appendages by
regulating cell proliferation and cell fate determination (Padgett
et al., 1987; reviewed by Lawrence and Struhl, 1996). dpp is
expressed in an anterior subset of follicle cells, and is required
for establishing egg shape and polarity during late stages of
oogenesis (Twombly et al., 1996). But an effect of dpp on
maintaining and propagating stem cells, instead of causing their
differentiation, has not been previously shown.
[0011] Major participants in the dpp signaling pathway have been
identified: saxophone (sax) and thick veins (tkv) encode type I
serine/threonine kinase transmembrane receptors, whereas punt
encodes a type II serine/threonine kinase transmembrane receptor
(Brummel et al., 1994; Nellen et al., 1994; Penton et al., 1994;
Xie et al., 1994; Ruberte et al., 1995; Letsou et al., 1995).
mothers against dpp (mad), Medea (Med), and Daughters against dpp
(Dad) encode a family of conserved TGF-.beta. transducers (Sekelsky
et al., 1995; Tsuneizumi et al., 1997; Hudson et al., 1998;
Wisotzkey et al., 1998; Das et al., 1998; Inoue et al., 1998),
collectively known as Smads. Smads are proteins which transduce
signals on behalf of TGF-.beta. family members, or inhibit
TGF-.beta. signal transduction. A paradigm for TGF-.beta. signal
transduction has been developed from several experimental systems
(Heldin et al., 1997). In Drosophila, Dpp binds both type I and II
receptors to allow the constitutively active Punt kinase to
phosphorylate and activate type I kinases, which phosphorylate Mad.
The phosphorylated Mad brings Med into the nucleus as a
transcriptional activator to stimulate dpp target gene
expression.
[0012] Enhancing Dpp or other BMP-like signaling activities can be
achieved by reducing the presence of Dad-like proteins, such as
human Smad6 and Smad7. Vertebrate Smad6 and Smad7 interact with
type I receptors, and are known to inhibit both TGF-.beta. and BMP
signaling in cultured cells and frog embryos. Thus, disinhibition
of TGF-.beta. family members by inhibiting certain Smads promotes
BMP-like signaling cascades. Additionally, Dpp or other BMP-like
signaling activities may be increased by enhancing the function of
Dpp or BMP receptors, such as Sax, Tkv, and Punt in Drosophila, and
BMP receptors BMPR-II, ActR-II, Act-IIB, BMPR-IA, and ActR-I in
humans. Other downstream positive regulators of Dpp or BMP
signaling include Mad, Med, Dad, and Schnurri proteins in
Drosophila, and Smad1, Smad4 and Smad5 in humans. See review by
Padgett (1999).
[0013] Therefore, to address the prior art's failure to identify
and characterize factors involved in germline stem cell maintenance
and propagation, we now disclose that a member of the TGF-.beta.
family of growth factors and its signaling pathway unexpectedly
provide this essential function.
SUMMARY OF THE INVENTION
[0014] It is an object of the invention to maintain and/or
propagate stem cells by stimulating signaling through a bone
morphogenetic protein (BMP) signaling pathway. In this manner a
population of stem cells can be maintained in vivo or in vitro,
and/or expanded.
[0015] Methods for maintaining germline and somatic stem cells of
an organism are provided by stimulating a bone morphogenetic
protein (BMP) signaling pathway.
[0016] The signal transduction pathway associated with a BMP
specifically binding to a receptor may involve phosphorylation of
serine/threonine residues (e.g., kinases, phosphatases) and a
cascade of components of the pathway (i.e., signal transducers such
as, for example, transcription factors) which communicate that
signal. For example, a signal may be communicated from BMP binding
at the cell surface to the nucleus where gene expression of
downstream targets are either activated or inhibited. Thus, BMP
signaling may be modulated at one or more steps in this pathway, or
by affecting upstream regulators or downstream targets of this
signaling pathway. Modulation (i.e., stimulation or repression) of
BMP signaling may be accomplished directly on the stem cell or
indirectly through other cells in a mixed cell population (e.g.,
feeder layer).
[0017] Properties of the stem cell may be maintained by stimulating
BMP signaling. Furthermore, stem cells may be increased in
abundance and/or increased in lifetime by such stimulation.
Conversely, stem cells or tumor cells in a population may be
reduced in total number or concentration, or even eliminated at the
limit of detection, by repressing BMP signaling.
[0018] Stem cells may also be propagated and isolated according to
the invention.
[0019] Our invention addresses the problem of restricted access to
and limited numbers of stem cells. The ability to maintain and to
propagate stem cells facilitates genetic manipulation and the
characterization of these rare cells.
DESCRIPTION OF SPECIFIC EMBODIMENTS
[0020] The present invention provides a method for maintaining and
controlling the division of germline stem cells in which dpp can
provide an essential role. Further, it provides a model of ovarian
tumor formation in which overexpression of dpp produces ovarian
stem cell tumors. Clonal analysis demonstrates that downstream
components (i.e., signal transducers) of the dpp signaling pathway
are required cell-autonomously in the germline stem cells for their
division and maintenance. This invention also provides a method for
control of a cellular niche by BMP signaling, in which germline
stem cells are regulated by, for example, a dpp signal that likely
derives from surrounding somatic cells.
[0021] Stem cells are thought to be regulated by positive and
negative diffusible factors, but the functions of most of these
factors have never been demonstrated in vivo. The present invention
provides a method in which Dpp directly signals to maintain
Drosophila germline stem cells and stimulate their division. The
experiments of the examples were made possible by a clonal cell
marking method that allows the function of stem cells and their
progeny to be examined directly over many cell generations. In
addition to the dpp signal, known components in the dpp signal
transduction pathway were shown to be required in these adult stem
cells. This action appears to be specific to stem cells, since germ
cells lacking dpp pathway components were still able to form
16-cell cysts. The examples demonstrate that a TGF-.beta.-like
molecule functions as a stem cell growth factor.
[0022] dpp signal transduction is required for maintaining stem
cells, on which Dpp may act in several distinct ways. Signaling
prevents germline stem cells from differentiating into cystoblasts
and gametes. The examples show that overexpressed dpp prevents stem
cell differentiation, while reduction of dpp function promotes stem
cell differentiation. An attractive candidate target of the dpp
signal transduction pathway is the Bam protein, which is normally
synthesized at much higher levels in cystoblasts than in stem cells
(McKearin and Ohlstein, 1995). The forced expression of Bam in
germline stem cells causes them to differentiate in a manner very
similar to that caused by reductions in dpp signaling (Ohlstein and
McKearin, 1997). Thus, dpp signaling may negatively regulate Bam
protein levels in germline stem cells. Two other genes, pum and
nos, are required to form and maintain germline stem cells (Lin and
Spradling, 1997; Forbes and Lehmann, 1998). In the embryo, both
proteins work together to repress the translation of target genes
such as hunchback (hb) (Baker et al., 1992; Murata and Wharton,
1995). In the ovary, dpp signaling may downregulate Bam through
effects on the Nos/Pum pathway or by an independent mechanism.
However, genes throughout the dpp pathway are required, including
two nuclear transcription factors, suggesting that the action of
the pathway is on transcription of target genes. Also see reviews
by Attisano and Wrana (1998), Kawabata et al. (1998), and Padgett
et al. (1998).
[0023] dpp may also function to maintain a specialized association
between the stem cells and basal terminal filament cells. Such an
association has been postulated to hold the stem cells at the
anterior of the germarium, while daughter germline cells all move
posteriorly and eventually leave the germarium. The results
presented herein indicate that the stem cell loss is due to
differentiation. Possibly, dpp signaling via its receptor regulates
the expression of adhesion molecules that reside on the cell
surface or of cytoplasmic proteins that indirectly promote stem
cell adhesion.
[0024] dpp signaling also may act to stimulate stem cell division.
dpp signaling stimulates cell proliferation at several points
during Drosophila development. In the wing imaginal disc, it is
essential for cell proliferation and/or survival (Burke and Baster,
1996), whereas it promotes the G2-M transition in the morphogenetic
furrow of the developing eye disc (Penton et al., 1997). Consistent
with such a requirement, mad mutants have greatly reduced imaginal
discs, shortened gastric caeca, and small brains (Sekelsky et al.,
1995). The requirement for dpp signaling disclosed herein suggests
that adult stem cells use strategies similar to those of embryonic
and larval somatic cells to regulate proliferation. For example,
dpp stimulates the rate of cell division for stem cells.
[0025] During aging, the number and activity of stem cells are
thought to be reduced. The examples indicate that the level of dpp
signaling controls the life span and division rate of germline stem
cells. Reduced dpp signaling caused premature stem cell loss.
Perhaps more surprising is the observation that putative increases
in signaling, caused by removal of Dad protein activity from stem
cells, permitted stem cells to be maintained longer. This finding
suggests that dpp signaling not only is necessary, but may
sometimes be rate limiting for stem cell maintenance. The
illustrative examples demonstrate for the first time a method in
which stem cell life span has been extended in an intact
organism.
[0026] These results suggest that it may be possible to extend the
life span of stem cells, a process that could be of therapeutic
significance. For example, drugs that upregulate BMP signaling to
stem cells may enhance fertility in humans and animals, such as
male fertility in patients with reduced numbers of germline stem
cells (basal cells). Such drugs may ameliorate hematologic
conditions caused by reduced stem cell functioning, for example
aplastic anemias, agammaglobulinemia, and related conditions. Drugs
enhancing BMP signaling may enhance wound healing. Aging-related
pathologies caused by loss of stem cells, such as hair loss, loss
of muscle mass, reduction of blood cell numbers, and the aging of
the skin and other stem cell-dependent tissues could be treated by
increasing BMP signal transduction. Compounds enhancing BMP
signaling may increase the average lifespan of an organism.
[0027] One method for the enhancement of dpp signal transduction
may be facilitated by removal of the dpp inhibitor Dad or other
Dad-like inhibitory protein activity (inhibitory Smad activity)
from the germline stem cells. Dad is induced by dpp signaling, but
then acts to dowregulate the very pathway that activated its
production. This method could also be practiced with other negative
regulators of the dpp signaling pathway and, in particular,
inhibitory Smads. In contrast, brinker (brk) is a target gene
repressed by dpp signaling and, because it is itself a
transcriptional repressor, the net effect of repressing expression
of the Brk repressor is to upregulate Brk-regulated target genes
(Minami et al., 1999; Campbell et al., 1999; Jazwinska et al.,
1999). This results in the increased production of Brk-regulated
target genes following dpp signaling. Hence, BMP signaling can be
stimulated or repressed by appropriate manipulation of Smads or
target genes which are regulated by BMP signaling (i.e., increasing
or decreasing their effects as appropriate to achieve stimulation
or repression of BMP signaling). The roles of Dad and Brk, like the
rest of the pathway, appear to be conserved in mammals.
[0028] Drugs that inhibit BMP signaling to stem cells may be useful
chemotherapeutic agents. For example, drugs inhibiting BMP
signaling pathways may be useful therapies against teratocarcinoma
by causing stem cell differentiation. As another example, drugs
which inhibit BMP signaling may be successful treatments against
ovarian germline tumors dependent upon BMP signaling for continued
growth.
[0029] Increased or decreased BMP signaling to stem cells might
allow populations of stem cells to expand prior to bone marrow
transplant, thereby increasing the chances of successful
transplantation and reducing the amount of donor marrow required.
Further, control of BMP signaling pathways may permit stem cells
other than those in bone marrow to be removed from a patient,
expanded in vitro, and subsequently reintroduced in to the patient
to repair tissues damaged by injury or disease, such as Parkinson's
disease.
[0030] Bone marrow from patients with hematologic tumors, such as
lymphoma and leukemia, could be tested for BMP sensitivity.
Positive test results for BMP sensitivity would allow steps to be
taken to avoid potential side effects of anti-BMP treatment in
vivo. For example, marrow removed from the patient could be
cleansed of tumors cells by inhibiting BMP signaling, thereby
inducing differentiation of tumor cells and reducing the tumor
burden. The cleansed marrow would subsequently be returned to the
patient in an autologous bone marrow transplant. Such
differentiation therapy could also be used for solid tumors like
sarcoma, carcinoma, and neuroglioma to reduce tumor burden. Therapy
may be use alone or in association with other treatments such as,
for example, chemotherapy, hyperthermia, or radiation which
preferentially kills rapidly dividing cells and surgical resection
of tumor.
[0031] Upregulation of BMP signaling to stem cells may permit the
growth of germline stem cells in culture, useful in, for example,
generating transgenic animals. Such techniques are especially
useful in organisms which have not traditionally been used as
genetic models of development and disease.
[0032] The ability to expand stem cell niches by overexpression of
TGF-.beta. members, such as dpp may allow rare human stem cells, or
alternatively rare stem cells of any species, to be purified and
propagated following transfer into living Drosophila, which have
been genetically engineered to serve as hosts.
[0033] Beside biomedical research and treatment, other uses for the
present invention include agriculture and wildlife conservation.
Stem cells could be provided in or obtained from humans, primates
(e.g., bonobo, chimpanzee, gorilla, macaque, orangutan), companion
animals (e.g., dog, cat), and farm/laboratory animals (e.g.,
cattle, donkey, goat, horse, pig, sheep; amphibians such as frog,
salamander, toad; birds such as chicken, duck, turkey, fishes such
as carp, catfish, medaka, salmon, tilapia, tuna, zebrafish;
lagomorphs such as hares, rabbits; rodents such as mice, rats).
[0034] Stem cells could be maintained and/or maintained in an
appropriate niche or in culture, and used as a source of nuclei for
cloning progeny organisms via nuclear transfer or a source of cells
for propagation of mosaic organisms via embryo aggregation. Thus,
Dpp or related BMPs provide a means for growing stem cells in vitro
or in vivo for cloning animals.
[0035] BMP signaling is unlikely to be confined to one type of BMP
and only type of BMP receptor because of the ability of
evolutionarily diverged components of the BMP signal transduction
pathway or different types of BMPs, BMP receptors, and SMADs to be
functional equivalents of each other. For example, there appears to
be crosstalk between Dpp/Tkv signaling and Gbb/Sax signaling
(Haerry et al., 1998) and one signal transducer acts in different
signaling pathways (Lagna et al., 1996). For example, a mixture of
BMPs could be added to defined culture medium or be present in
conditioned culture medium such that Dpp and Gbb would synergize in
initiating BMP signaling through more than one different types of
BMP receptor. As another example, one type of signal transducer
could stimulate signaling through more than one different types of
BMP receptor.
[0036] To stimulate BMP signaling, a positive signal transducer
could be increased in expression (e.g., more transcripts and/or
translated products) or mutated to a gain-of-function phenotype to
increase activity of that signal transducer, while a negative
signal transducer could be decreased in expression (e.g., fewer
transcripts and/or translated products) or mutated to a
loss-of-function phenotype to decrease activity of that signal
transducer. Alternatively, a downstream target gene of BMP
signaling could be directly activated or inhibited without BMP
binding to its receptor by genetic engineering using a
transactivator like GAL4 binding its UAS or ecdysone receptor
binding upstream of the target gene. Similar techniques in mice
involve induction with tetracycline or FK506.
[0037] Another method would be to increase endogenous BMP activity
in the cells or to increase exogenous BMP activity outside the
cells, especially if ligand is the limiting component in BMP
signaling. For example, BMP expression may be increased in a stem
cell and stimulate BMP signaling through an autocrine mechanism.
Alternatively, BMP expression may be increased in a non-stem cell
or a feeder cell, and then BMP activity could be secreted and taken
up by the stem cell or brought into contact with the surface of the
stem cell. BMP could also be added to the extracellular space or
culture medium. BMP activity may be increased to stimulate BMP
signaling by at least about 10%, 50%, 100%, or 200% as compared to
the amount normally present in the animal or the culture.
[0038] Properties of the stem cell which may be maintained include
the following: pluripotency, totipotency, committing to one or more
differentiating cell lineages, giving rise to multiple different
types of progenitors and/or differentiated cells, contributing to
the germline, and combinations thereof. Thus, the growth and/or
survival of stem cells may be maintained without commitment to a
program of differentiation, while retaining the capacity to
differentiate normally under appropriate conditions following
reduction or elimination of BMP signaling. More simply, stem cells
in a population may be expanded in total number or concentration
relative to non-stem cells (i.e., an increase in abundance),
extended in the time between a stem cell's birth and its death or
apoptosis (i.e., an increase in lifetime), or combinations thereof.
Conversely, stem cells or tumor cells in a population may be
reduced in total number or concentration, or even eliminated at the
limit of detection, by repressing BMP signaling.
[0039] Stem cells made according to the present invention may be
totipotent or pluripotent, male or female, germline or somatic,
dividing or quiescent, vertebrate or invertebrate, present in situ
or isolated, partially or substantially purified of differentiate
cells, and combinations thereof. Proliferating stem cells are
diploid, entering meiosis and the later stages of gametogenesis is
part of the program of differentiation for male or female germline
stem cells that is prevented by the present invention. Stem cells
may be present in or obtained from testis, ovary, especially apical
tips of Drosophila testes and/or ovarioles, or other adult or
embryonic tissues. By differentiating, stem cells may differentiate
into cells of the hematopoietic, immune, or nervous systems or the
like. Preferably, stem cells maintained and/or propagated by the
present invention retain the potential to later differentiate and
thereby contribute to oogenesis or spermatogenesis, all three germ
layers (i.e., endoderm, mesoderm, ectoderm), multiple
differentiated cell lineages, and combinations thereof.
[0040] Somatic cells include terminal filament cells, cap cells,
and inner sheath cells from the ovary and hub cells from the
testis. Preferably, the present invention reduces the proportion of
somatic cells in a population relative to germline cells during
maintenance and/or propagation. A niche defined by surrounding
somatic cells or a feeder layer comprised of somatic cells may
provide cell contact and other extracellular signals to maintain
and/or propagate germline cells. A feeder layer may be provided
that provides certain essential extracellular signals by, for
example, genetically manipulating cultured cells to express and
secrete a BMP which then binds to its receptor on the stem
cells.
[0041] Cell populations may be derived from the germline or somatic
(or mixed), male or female, dividing or quiescent, vertebrate or
invertebrate, present in situ or isolated, partially or
substantially purified, and combinations thereof. Preferably, cell
populations include cells expressing one or more BMPs; more
preferably, BMP is secreted by non-stem cells and binds to
receptors of stem cells to stimulate BMP signaling. Thus, stem
cells of the present invention contain receptors for BMP,
especially Dpp or a homolog, or are at least responsive to BMP
signaling.
[0042] Besides mammals, amphibians, birds, and fishes, other
organisms may be used in the present invention such as
invertebrates like worms (e.g., Helminthes, Nematodes) and insects
(e.g., Anopheles, Drosophila). In particular, comparison of
components of the BMP signaling pathway, upstream regulators, and
downstream targets show them to be highly conserved (Bitgood and
McMahon, 1995; Padgett et al., 1998). Thus, the present invention
should not be limited in its usefulness to Drosophila melanogaster.
Other species which show conservation of dpp (Newfeld et al., 1997)
and are likely to be useful are D. simulans, D. pseudoobscura, and
D. virilis. For metazoan species in which there has been a diligent
search, a dpp-like gene has been identified.
[0043] Mammalian homologs of dpp, glass bottom boat (gbb), and
screw (scw) have been identified as BMP-2/4, BMP-5/8, and BMP-6,
respectively (Hoffmann, 1997; Raftery and Sutherland, 1999; Wharton
et al., 1999). A mammalian serine/threonine kinase receptor has
been identified that specifically binds BMP-2 and BMP-4 (Yamaji et
al., 1994). Other related members of the TGF-.beta. family, their
receptors, or other components of their signaling pathways, might
also be used in the present invention. See also U.S. Pat. Nos.
5,011,691, 5,013,649, 5166,058, 5,168,050, 5,216,126, 5,324,819,
5,354,557, 5,635,372, 5,639,638, 5,650,276, and 5,854,207.
[0044] Furthermore, mutational analysis and determination of
structure-function relationships have identified conserved residues
and essential residues for Dpp signaling (Wharton et al., 1996).
Bacterially expressed Dpp can be refolded, then biochemically and
biophysically characterized (Groppe et al., 1998). Homologs of a
member of the BMP family, their receptors, and other components of
the signaling pathway can be identified by a high level of
structural conservation when amino acid sequences are compared,
and/or functional conservation when homologs rescue mutant
phenotypes or otherwise replace BMP activity.
[0045] Cell types have been identified by markers and are well
characterized by genetic mutants and developmental studies. Stem
cells may be provided in situ as part of an intact organism or they
may be cultured in vitro. Germline stem cells and surrounding cells
may be from an adult (e.g., ovary, testis) or an embryo. For in
vitro culturing, cells may be obtained directly from an organism
(i.e., primary culture) but it would be convenient to passage them
through several cultures (e.g., at least five, ten, or twenty
times) to expand their number (e.g., at least two, ten, or 100
times more than the original number).
[0046] Stem cells may be isolated from a donor organism with or
without increasing cell number by stimulating BMP signaling;
manipulated during transient in vitro culturing under conditions
for maintenance and/or propagation by treating with one or more
chemicals, introducing genetic material, fusing with another cell,
mutating one or more genes, selecting a desired genotype or
phenotype, or combinations thereof; and transplanting stem cells
back into a host which is identical to the donor (i.e., autologous
transplantation), similar to the donor but different (i.e.,
allogeneic transplantation), or is totally different from the donor
(i.e., xenogeneic transplantation). In vitro culture conditions,
genetic engineering of Drosophila by transfection and site-specific
recombination, and cell or nuclear transplantation are known in the
art.
[0047] For Drosphila, there are only about 10 germline stem cells
per testis and about 32-48 germline stem cells per ovary (i.e.,
there are about 16 ovarioles per ovary and about two or three
germline stem cells per ovary). The present invention provides
greatly increased numbers of stem cells to be produced in vivo in
an adult or embryo, and then cultured in vitro. In vitro culture of
cells may be carried out by initially generating flies with a large
number of germline stem cells in each ovariole. Then ovaries may be
removed surgically into sterile culture medium and the germ cells
released (they do not adhere and, thus, do not need to be
disaggregated). Alternatively, disaggregated embryos may also be
used as a source of germline stem cells. Although the number of
germ cells per embryo is similar to the number per ovary and
testis, it is possible to start with 100,000 embryos but only a few
hundred gonads can be easily obtained. Schneider (1972) shows
derivation of a cell line from Drosophila.
[0048] Drosophila cells may be plated into small wells containing
feeder layers of cells expressing Dpp (e.g., Panganiban et al.,
1990) or Hh (e.g., Lee et al., 1994), or culture media prepared by
conditioning the media with cells secreting soluble factors or
simply adding a recombinantly produced soluble factor (e.g., Dpp
produced according to Groppe et al., 1998). In vitro culture media
for growing Drosophila cells are commercially available such as,
for example, Schneider's Drosophila medium. Drosophila cells can
also be adapted and grown in mammalian tissue culture media
(Spradling et al., 1975; Lengyel et al., 1975). Drosophila cells
can be transfected like mammalian cells (Burke et al., 1984).
Constructs and strategies for homologous recombination in somatic,
embryonic stem (ES), and embryonic (EG) cells could be adapted for
use with in vitro cultured Drosophila cells (Capecchi, 1989; Koller
and Smithies, 1992). Cultured cells or their nuclei may then be
transferred into Drosophila (Okada et al., 1974; Van Deusen,
1977).
[0049] Previous attempts at culturing germline stem cells utilized
the 40 germline cells present in each embryo at a certain stage of
development. But no dpp was provided, and these cells
differentiated in culture (Allis et al., 1979). Inducing BMP
expression in cells of such cultures or adding exogenous BMP to
them would be a simple way of maintaining and/or propagating
germline stem cells in vitro.
[0050] A BMP may also be used in replacement of, or combination
with, known stem growth factors such as, for example, fibroblast
growth factor (FGF), leukemia inhibitory factor (LIF), and steel
factor (SF). Thus, BMP activity as observed herein might also be
demonstrated using the techniques taught in U.S. Pat. Nos.
5,453,357 and 5,690,926.
[0051] Ex vivo culturing of stem cells with stimulation of BMP
signaling only performed outside the body is preferred to avoid
systemic effects of BMP signaling on the organism.
[0052] Vascular or organ engineering may be accomplished with stem
cells that differentiate into endothelium or parenchyma,
respectively, with or without an implantable support (e.g., stent,
hollow fiber or particle) on which stem cells have been coated or
impregnated. If not autologously transplanted and in an organism
with an immune system recognizing histoincompatibility,
transplantation of allogeneic or xenogeneic tissue may require
immunosuppression of the host (e.g., cyclosporine A or FK506
treatment). Differentiation of stem cells into tissue with the
activity and/or structure of adrenal gland, bone marrow, brain,
liver, ovary or testis, pancreas, peripheral neurons or glia, red
or white blood cells, skeletal or smooth muscle, skin, thyroid
gland, or combinations thereof is preferred.
[0053] One or more genes of the stem cell may be activated or
inhibited by chemical or environmental induction, antisense,
ribozyme, chimeric repair vector, RNAi, or random/sequence-specific
insertion. Ectopic expression of a gene may be controlled in a
particular spatial or temporal manner, mimic pathologic or disease
states, or create phenocopies of mutations in the endogenous gene.
Homologous recombination is preferred to achieve gene knockout or
replacement (see, e.g., U.S. Pat. Nos. 5,569,824, 5,602,307,
5,614,396, 5,683,906, and 5,830,682). For example, stem cells may
be transfected with a polynucleotide, the polynucleotide or a
portion thereof integrates into the genome of transfected stem
cells at a random site or in a sequence-specific manner, homologous
recombinants at a genetic loci of interest are selected, and the
selected stem cells are transplanted into a host organism. Physical
introduction of polynucleotides (e.g., biolistics, electroporation,
microinjection) is preferred. Alternatively, insertion of P
elements may be genetically engineered in vivo or in vitro in a
stem cell maintained and/or propagated according to the present
invention to disrupt genes (cf. Zhang and Spradling, 1994;
Spradling et al., 1995).
[0054] TGF-.beta. signaling has been shown to limit the growth of
germline cysts during Drosophila spermatogenesis (Matunis et al.,
1997). When punt or shn function is removed in clones of somatic
cells that surround germ cells, cysts continue dividing after four
rounds of mitosis (Matunis et al., 1997). However, these
investigators did not address whether this pathway functions in
male germline stem cells. In the embryo and imaginal discs, punt
and shn can function downstream of dpp (Ruberte et al., 1995;
Letsou et al., 1995; Arora et al., 1995; Grieder et al., 1995), but
it was not known whether dpp or another TGF-.beta. family member is
utilized to send the signal. Clonal analysis of mutants in dpp
downstream components in male germline stem cells, like those
reported here in the ovary, could show whether Dpp and/or other
TGF-.beta.-like molecules are required for their division and
maintenance in the testis.
[0055] In mouse, the BMP family members BMP-2 and -4 are most
closely related to dpp, with greater than 75% identity, and can
function to rescue dpp mutants in embryos (Padget et al., 1993).
Recently, both genes have been inactivated by homologous
recombination, but the homozygous embryos die too early to assess
possible functions in the gonads (Winnier et al., 1995; Zhang and
Bradley, 1996; reviewed by Hogan, 1996b). Consistent with our
findings, Lawson et al. (1999) report that BMP-4 affects the number
of primordial germ cells; moreover, BMP4 was needed in somatic
tissue, and presumably stimulated BMP signal transduction in
germline cells, although this was not shown directly. The roles
during spermatogenesis of two other BMP family members, BMP-8A and
BMP-8B, have been tested (Zhao et al., 1996; 1998). BMP-8B is
required for the resumption of male germline cell proliferation in
early puberty, and for germline cell survival in the adult, whereas
BMP-8A plays a role in the maintenance of adult
spermatogenesis.
[0056] The "niche" hypothesis postulates that stem cells reside in
optimal microenvironments or "niches" (Schofield, 1978). When a
stem cell divides, only one daughter can remain in the niche while
the other becomes committed to differentiate. A stem cell within
the niche would have a high probability of self-renewal, but a low
probability of entry into the differentiation pathway. This model
is consistent with the observations that stem cells require the
addition of growth factors for proliferation and differentiation in
many in vitro culture systems (Potter and Loeffler, 1990; Morrison
et al., 1997). The molecular nature of the microenvironment within
a niche has yet to be defined in any system, although the
Drosophila germarium appears to contain such a niche. Anteriorly,
the stem cells abut terminal filament and cap cells, which both
express hh, while only the latter express armadillo (arm) and wg
(Forbes et al., 1996a; 1996b). Stem cell daughters lie more to the
posterior, and probably directly contact inner germarial sheath
cells, which express patched (ptc) and hh (Forbes et al., 1996b).
This asymmetry in structure and signals may allow germline stem
cells to receive different levels of signals from their daughters.
Consistent with the existence of a niche, two wildtype stem cells
in germaria that recently lost a marked mutant stem cell were
occasionally observed, suggesting that a vacated niche could be
reoccupied.
[0057] The existence of the germline stem cell niche is also
consistent with stem cell proliferation when local dpp is
overexpressed. Under these conditions, the size of the niche may be
substantially enlarged. Conversely, reduction of dpp function may
weaken the ability of the niche to maintain germline stem cells,
leading to accelerated losses. These results suggest that dpp is an
essential niche signal. However, dpp likely interacts with other
signals from surrounding somatic cells to make a functional niche
for germline stem cells. Nonetheless, the identification of dpp as
a key niche signal should greatly facilitate efforts to culture
Drosophila germline stem cells in vitro.
[0058] Technical limitations have previously prevented
identification of the source of the dpp signal that is received by
germline stem cells. Ideally, analysis of clones of a null dpp
allele would reveal which cells produce the signal. However, the
somatic cells adjacent to the stem cells cease division early in
ovary development and make induction of specific small clones
difficult. The pattern of dpp expression in the germarium should
also provide some insight into the origins of the signal. However,
the only available dpp-lacZ fusion line and whole mount in situ
experiments failed to detect expression in the germarium, although
follicle cell expression in late stage egg chambers was observed.
We now show that somatic cells in the niche express dpp. In many
systems, low levels of dpp expression are known to be sufficient
for biological effects so it may be sufficient to provide only low
levels of BMP in the present invention.
[0059] In the Drosophila leg, antenna and genital discs, dpp and wg
are induced in the anterior compartment by hh, and the mutual
repression of dpp and wg restricts them to their appropriate
domains (Brook and Cohen, 1996; Jiang and Struhl, 1996; Chen and
Baker, 1996). In vertebrate limb development, sonic hedgehog (shh)
can induce the expression of BMP-2 (Johnson and Tabin, 1995). The
somatic terminal filament, cap, and inner sheath cells express hh
and lie adjacent to the germline stem cells (Forbes et al., 1996a,
1996b). wg and dpp expression may be induced by hh, and signal to
germline stem cells for their proliferation and maintenance. The
data indicate that these and possibly additional signals from the
anterior somatic cells define a niche for germline stem cells at
the tip of germarium. Thus, agents which modify hedgehog signaling
may be used to alter local BMP signaling, thereby regulating stem
cell maintenance and/or propagation.
EXAMPLES
Example 1
Ectopic Dpp Expression Induces Germ Cell Tumors
[0060] To assess whether Dpp can regulate germline stem cells in
the Drosophila adult ovary, Dpp was ectopically expressed in the
germarium using hsp70-GAL4 (hs-GAL4) and UAS-dpp (Brand and
Perrimon, 1993). To distinguish different cell types in the
germarium, we used anti-Hts and anti-Vase antibodies to visualize
somatic and germline cells, respectively. The anti-Hts antibody
also recognizes spectrosomes and fusomes in the germline cells of
the germarium (see de Cuevas et al., 1997).
[0061] Only germline stem cells and cystoblasts have a big round
spectrosome, while cysts have a characteristic branched fusome. In
the wildtype germarium, two germline stem cells are more anteriorly
located than cystoblasts. Developing cysts in germarial regions 1
and 2a (i.e., the anterior half), which are more posterior than
germline stem cells and cystoblasts, are connected by fusomes. In
the germarial regions 2b and 3, both lens-shaped and round cysts
span across the germarium, and become surrounded by somatic
follicle cells. Fusome structures begin degeneration in these older
cysts.
[0062] The germaria from hs-GAL4 females subjected to heat shock,
and those from females carrying hs-GAL4 and UAS-dpp in the absence
of a heat shock, were indistinguishable from wildtype. In these
heat shock-treated germaria, large single germline cells filling
the corresponding wildtype germarial regions 1 and 2a contained
spectrosomes but showed no evidence of cyst formation. In the
corresponding wildtype regions 2b and 3, both lens-shaped and round
cysts were observed that probably derived from differentiated
cystoblasts or cysts that had formed before the initial heat
shock.
[0063] Consistent with this interpretation, after 4-5 days of heat
shock, all germ line cells in the corresponding regions 1 and 2
were single cells containing spectrosomes and developing cysts
containing branched fusomes were rarely detected. Only somatic
follicle cells were detected, there were no germline cells. This
phenotype is very similar to that of bam and benign gonial cell
neoplasm (bgcn) mutants (McKearin and Spradling, 1990; Gateff and
Mechler, 1989).
[0064] Here, instead of the wildtype number of two or three
germline stem cells per ovariole, dozens were present in a single
ovariole. Moreover, the number present was 2-3 times greater after
4-5 days than after 3 days. Because there are 16 ovarioles per
ovary and two ovaries per female fly, all of the above numbers
should be multiplied by 32 to calculate the number of female
germline stem cells per fly. The germline stem cells proliferate
following induction of dpp to form a large mass of normal
appearing, normal functioning germline stem cells. The
proliferating cells were shown to be germline stem cells based on
(1) general size and appearance, (2) fusome morphology, (3)
expression of the germ cell-specific gene vasa, (4) absence of
expression of cytoplasmic Bam (i.e., a sensitive indicator that
germline stem cells have differentiated into cystoblasts), and (5)
ability to differentiate along the normal pathway for germline
cells following removal of dpp.
Example 2
Dpp-Induced Tumor Cells Resemble Germline Stem Cells
[0065] Cystoblasts and early mitotic cysts can be distinguished
from stem cells because the former express cytoplasmic Bam protein
from the cystoblast stage to the end of the 8-cell stage cyst
stage. Immunofluorescent staining of the wildtype germarium with
anti-BamC and anti-.alpha.-spectrin antibodies document that
cystoblasts and developing cysts, but not germline stem cells,
express cytoplasmic Bam protein. Immunofluorescent staining of
dpp-induced germaria with anti-BamC and anti-.alpha.-spectrin
revealed that amplified single germline cells failed to express the
cytoplasmic Bam protein. In dpp-induced germaria, a few, rare
BamC-positive cells were observed that appeared to be growing
cysts. These data show that the large number of single germline
cells induced by dpp overexpression resemble stem cells rather than
differentiated cystoblasts.
[0066] To determine that this absence of BamC-staining was not due
to growth arrest of the accumulated single germline cells,
dpp-induced germaria were stained with anti-BrdU and
anti-.alpha.-spectrin antibodies following incorporation of the
nucleotide analog BrdU for one hour. Mitotically active germline
cells in their S-phase of the cell cycle can incorporate BrdU. In
the dpp-induced germaria, some single germline cells incorporated
BrdU, indicating that these single germline cells have not
undergone growth arrest.
[0067] These results show that the tumor cells induced by dpp
overexpression continue to divide, and resemble stem cells in their
fusome morphology and absence of Bam protein. They represent an
increased number of germline stem cells.
[0068] To determine if these dpp-induced stem cells retain the
capacity to differentiate, their behavior was examined.
hs-GAL4/UAS-dpp Drosophila were induced by four days of heat
shock-treatment, and then returned to room temperature for 2 or 4
days prior to staining with anti-Hts and anti-Vase antibodies.
Germline cysts were observed starting to form two days after the
temperature down-shift and always formed initially in the most
posterior region of the tumor. Many 16-cell cysts were seen 4 days
after the shift back to room temperature. Based on their location
and number, these cysts must derive from dpp-induced germline stem
cells, rather than from stem cell divisions that occur after the
downshift. But not all the dpp-induced germline stem cells were
able to form complete cysts, because some ovarioles contained cysts
with one, two, four, or eight cells in region 3.
Example 3
Overexpressed dpp Acts Directly on Germline Stem Cells
[0069] Two different models could explain the Dpp effect on
germline stem cells: direct signaling to the germline stem cells
and relay signaling. The relay signaling model predicts that
ectopic Dpp turns on a secondary signal in the somatic cells
surrounding germline stem cells.
[0070] To directly test the relay model, the hs-GAL4/UAS system was
used to activate Dpp type I receptors. The hs-GAL4/UAS system can
express a target gene at high levels in somatic cells of the adult
ovary, but not in germline cells (Manseau et al., 1997). Both
activated tkv (tkv*) (Nellen et al., 1996) and activated sax (sax*)
(Des et al., 1998) have been shown to mimic dpp signaling pathway
activation in many developmental processes.
[0071] Overexpression of activated Dpp type I receptors in the
somatic cells of the germarium does not mimic the effect of ectopic
dpp expression. Flies of the following genotypes were subjected to
heat shock-treatment for three days, and germaria were subsequently
labeled with anti-Vase and anti-Hts antibodies: hsGAL41UAS-sax*,
hsGAL4/UAS-tkv*, and UAS-sax*/+;hsGAL4/UAS-tkv*. Two independent
lines containing the UAS-tkv* and UAS-sax* insertions at different
chromosomal sites were tested. When activated sax* or tkv*, or
both, were overexpressed in the somatic cells of the germarium
using hs-GAL4, the same driver for dpp overexpression, no germline
stem cell proliferation was observed. But egg chamber budding was
frequently affected in region 3 cysts in the hsGAL4/UAS-tkv*, and
UAS-sax*/+;hsGAL4/UAS-tkv* lines, suggesting that somatic follicle
cell function was defective at a later stage.
[0072] These results suggest that relay signaling, regardless of
its mechanism, is by itself not sufficient to inhibit germline stem
cell differentiation. Since overexpressed Dpp does not appear to
act by a relay signal, it likely acts directly on germline cells
via functional Dpp receptors to inhibit cystoblast
differentiation.
Example 4
Dpp and Sax are Required for Germline Stem Cell Division and
Maintenance
[0073] To directly test the role of dpp, we examined mutations that
reduce its function and that of the Dpp receptor sax. Dpp signaling
is essential at many points during Drosophila development. Several
temperature-sensitive allelic combinations of dpp mutants,
including dpp.sup.e90/dpp.sup.hr56 and dpp.sup.hr4/dpp.sup.hr56,
can develop into adults at 18.degree. C. (Wharton et al., 1996).
These heteroallelic combinations allowed us to examine the mutant
phenotypes of dpp in the germarium after the shift to 28.degree. C.
Forty to 50% of germaria from these genotypes examined one week
after the temperature shift were significantly smaller than
heterozygotes, and more severe reductions were seen in older
females maintained at the higher temperature. To determine if stem
cells were being lost, ovaries from the mutant females were stained
with anti-Hts and anti-Vase antibodies and the number of stem cells
in each ovariole were directly counted (Table 1). There was a
dramatic reduction in germline stem cell number in both tested
genotypes over a two week period. The stronger of the two,
dpp.sup.hr4/dpp.sup.hr56- , almost completely eliminated stem cells
within two weeks. This combination produces many fewer adult flies
and is known to disrupt embryonic development more severely than
dpp.sup.e90/dpp.sup.hr56 (Wharton et al., 1996).
[0074] If the mutations act specifically on germline stem cells,
cystoblasts and cysts should continue to divide and develop. To
examine this, the morphology of fusomes in the mutant ovarioles
were analyzed. Ovarioles from the Dpp receptor mutant sax.sup.P,
which has a weaker effect on stem cell number were also studied.
The timing of stem cell loss is expected to vary among individual
germaria, because stem cell loss is a random process (Margolis and
Spradling, 1995).
[0075] Control germaria from one week-old sax.sup.P/+ and
dpp.sup.e90/CyOP23 females were double labeled with anti-Vase and
anti-Hts antibodies. In both cases, germaria from one week-old
females heterozygous for the dpp or sax alleles generally contained
two stem cells at the anterior. The mutant germania were also
double labeled with anti-Vase and anti-Hts antibodies.
[0076] Mutant sax.sup.P/sax.sup.P germaria from one week-old
females were smaller than wildtype. In one case, two stem cells
were observed but the number of cysts was reduced. In another case,
one stem cell remained and regions 1 and 2a were much reduced as
indicated by the start of region 2b. This indicates that stem cells
were being lost and their division slowed.
[0077] Many germaria in two week-old mutant sax.sup.P/sax.sup.P
females had lost both stem cells and no mitotic cysts were present,
although cysts and egg chambers at later developmental stages
remained (e.g., the most anterior cyst corresponding to region
2b).
[0078] Mutant dPP.sup.e90/dPP.sup.hr56 females showed a more rapid
loss of stem cells at 28.degree. C. Such germaria frequently
contained one or zero stem cells after one week. In one case, only
one stem cell and no mitotic cysts were found; the most anterior
cyst contained 16 cells. In another case, no stem cells were
present; an 8-cell cyst and a 16-cell cyst lay at the anterior.
After two weeks, most ovarioles lacked stem cells entirely, but
some still contained 16-cell cysts or older follicles.
[0079] Because normal cystocyte development continued throughout
the germarium, the effects of these mutations appear to be limited
largely to stem cell division and maintenance. Some abnormalities
in a later process, egg chamber budding, were observed. Stem cell
loss might be caused by either cell death or differentiation.
Apoptotic cells were not observed in the most anterior region of
these germaria where germline stem cells are located based on DAPI
staining.
[0080] These results indicate that a reduction in the level of dpp
signaling promotes the differentiation of germline stem cells into
cysts, and thus causes stem cell loss. Consistent with previous
studies (Twombly et al., 1996), we observed some partially
ventralized eggs with anterior defects in these dpp mutants and the
sax.sup.P mutant.
Example 5
Put, Tkv, Mad, Med, and Dad are Required Cell-Autonomously for
Germline Stem Cell Maintenance.
[0081] To demonstrate definitively that dpp signaling was received
by the germ line, studies were conducted to assess whether
components of the signal transduction pathway are autonomously
required in these cells. Flp-induced mitotic recombination was
employed to generate marked clones homozygous for loss-of-function
mutations in the germline stem cells of adult ovaries (see
Experimental Procedures). Genes downstream of dpp in the signal
transduction pathway are required in the germline stem cells for
their division and maintenance. Germaria lacking or bearing stem
cell clones of the indicated genotypes were generated, and then
labeled with anti-lacZ and anti-Hts antibodies. Marked stem cells
and their progeny cysts were indicated by the absence of lacZ
protein.
[0082] Clones were marked using armadillo-lacZ, which is strongly
expressed in all cells within the germarium when wildtype flies are
not subjected to heat shock. Stem cell clones can be recognized
because only stem cells persist in the germarium more than 5 days
after a mitotic recombination event (Margolis and Spradling, 1995).
As recombination events can take place only in mitotically active
adult cells, this method will not produce mutant clones in the
terminally-differentiated terminal filament, cap cells, and inner
sheath cells. Consequently, this approach excludes potential
complications due to mutant clones in these surrounding somatic
cells, allowing the autonomous function of genes to be tested in
germline stem cells. This method has three major additional
advantages. Firstly, the persistent mutant clones can be studied
over a long period of time allowing germline stem cell maintenance
to be quantified. Secondly, the existence of both a mutant and a
wildtype stem cell side-by-side in the same germarium provides a
control for the effects of gene removal by direct comparison. Thus,
the relative division rates of these two stem cells can be
determined simply by counting the number of mutant and wildtype
cysts in germania with one mutant and one wildtype stem cell.
Finally, germline stem cell-specific effects of the mutations can
be assessed by looking at the developmental status of marked
cystoblasts, cysts, and egg chambers.
[0083] Germline stem cell clones of punt-, tkv-, mad-, Med-, and
Dad- were generated by subjecting females of the appropriate
genotype to heat shock and examining their ovaries beginning one
week later. Stem cells in the Drosophila ovary have a finite life
span with a half-life of about 4.6 weeks (Margolis and Spradling,
1995; Table 2). In contrast to wildtype clones, stem cells mutant
for each of the tested genes (except Dad) were lost more rapidly
(Table 2). For example, after one week, the punt.sup.135 mutant
germline stem cell was either still present or had only recently
been lost, as indicated by the presence of relatively young mutant
cysts. However, after two weeks, the punt.sup.135 mutant germline
stem cell had usually been lost and only a few advanced mutant
cysts remained.
[0084] mad.sup.12 mutant stem cells were lost even more rapidly.
After one week, the mad.sup.12 mutant germline stem cell sometimes
remained, but did not proliferate well as indicated by the lack of
progeny cysts. More frequently, the germline stem cell was already
lost and a more developed cyst (or cysts) was observed. After two
weeks, mad.sup.12 mutant germline stem cells rarely remained so
there were no mutant cysts, but older mutant egg chambers were
present. Surprisingly, two wildtype germline stem cells were
occasionally observed after the mutant stem cell was lost. These
results indicate that the dpp signal directly acts on germline stem
cells to regulate their maintenance. However, no effects were
observed on the formation of 16-cell cysts or the subsequent
development of germline cells.
[0085] Unlike the other tested genes, Dad is a negative regulator
of dpp signaling. The Dad gene is induced by the dpp signaling
pathway and antagonizes the function of dpp (Tsuneizumi et al.,
1997). The Dad.sup.271-68 allele is a severe allele in which the
entire C-terminal conserved domain was deleted (Tsuneizumi et al.,
1997). Strikingly, germline stem cells mutant for Dad.sup.271-68
were not lost (e.g., a mutant germline stem cell and its progeny
cysts may be present), even if both germline stem cells lacked this
gene (e.g., two mutant germline stem cells and a normal complement
of progeny cysts were present). No turnover could be detected even
after three weeks of clone induction, suggesting that increasing
dpp signaling can prolong germline stem cell lifetime.
[0086] To compare the magnitude of the effects of different
mutations on stem cells, the half-life of mutant germline stem
cells was measured (Table 2; Experimental Procedures).
punt.sup.10460 is a hypomorphic allele of the Dpp type II receptor
whereas punt.sup.135 is a strong allele (Arora et al., 1995; Letsou
et al., 1995). In punt.sup.10460 clones, germline stem cell
half-life was reduced from about 4.6 to 0.90 weeks, whereas the
stronger punt.sup.135 allele reduced germline stem cell half-life
to about 0.41 weeks. tkv.sup.8 is a strong allele of the type I
receptor (Brummel et al., 1994; Nellen et al., 1994; Penton et al.,
1994). tkv.sup.8 stem cell clones reduced germline stem cell
half-life to about 0.69 weeks. Clones of two alleles of the
downstream signal transducer, mad.sup.9 and mad.sup.12, reduced
germline stem cell half-life to 2.5 weeks and 0.25 weeks,
respectively. Consistent with this observation, mad is a much
stronger allele than mad.sup.9 (Sekelsky et al., 1995). Med.sup.26
is a strong allele of another down-stream transducer (Des et al.,
1998). Med.sup.26 germline stem cells turned over with a half-life
of about 0.38 weeks.
Example 6
Punt, Tkv, Mad, Med Are Required Cell-Autonomously to Stimulate
Germline Stem Cell Division.
[0087] To further define the role of the dpp pathway in the
regulation of germline stem cell division, the number of mutant and
wildtype cysts in germaria carrying one mutant and one wildtype
germline stem cell were compared. Since each cyst represents one
germline stem cell division, counting the number of wildtype and
mutant cysts allowed the measure of relative germline stem cell
division rates. All germaria that still retained a mutant germline
stem cell from all three time points were counted and compared to
the number of wildtype cysts. In controls containing a marked but
genetically wildtype germline stem cell, approximately 50% of cysts
were marked, indicating that two germline stem cells are present in
one week-old adult germaria and divide at similar rates (see Table
2).
[0088] As expected based on previous experiments punt-, tkv-, mad-,
and Med- mutant germline stem cells all divided more slowly than
wildtype (see Table 2). While the relative division rate of marked
wildtype germline stem cells was about 0.93, the rates in the
tested genotypes ranged from about 0.21 to 0.60. These reductions
mostly correlated with the known strength of these alleles, and
with their effects on germline stem cell maintenance. However, both
punt.sup.10460 and punt.sup.135 mutant germline stem cells
proliferated about three-fold slower than the wildtype, despite the
fact that they differ in strength. Differences between the effects
of these mutants on maintenance and division may reflect branch
points in the pathway, and may suggest that at least one additional
type II receptor also mediates germline stem cell behavior.
Interestingly, Dad.sup.271-68 mutant germline stem cells, which
were more stable than wildtype, divided at a similar or slightly
slower rate than wildtype ones. These results demonstrate that
components of the dpp signaling pathway are required autonomously
for the proliferation of germline stem cells.
[0089] As shown previously, cysts produced in the presence of
overexpressed dpp driven by hs-GAL4 always contained 16 cells. To
verify that dpp signaling is not involved in regulating the
cystoblast and cystocyte divisions, the number of germline cells in
individual cysts mutant for punt.sup.10460, mad.sup.9, mad.sup.12,
Med.sup.26, and Dad.sup.271-68 were counted. In every case, these
cysts contained 16 cells, including a single oocyte. Therefore, the
dpp signaling pathway specifically acts on stem cells within the
germ line.
Example 7
dpp is Expressed in Differentiated Somatic Cells Surrounding
Germline Stem Cells.
[0090] To directly localize the source of the Dpp signal, a whole
mount mRNA in situ hybridization was performed to visualize
expression of the dpp gene on two day-old wildtype females which
were dissected and fixed. A standard protocol was used (Yue and
Spradling, 1992) except that protease digestion was performed at 50
gm/ml for 5 min. No staining was observed using the dpp sense RNA
probe as a control. Under the same conditions, the dpp anti-sense
probe detected the dpp mRNA in the inner sheath cells and cap cells
adjacent to germline stem cells, and in the posterior somatic
follicle cells, but not in germline and terminal filament cells.
These expression data further support our finding that surrounding
differentiated somatic cells constitute a niche for germline stem
cells.
Example 8
A Lost Germline Stem Cell can be Replaced by the Daughter of the
Other Stem Cell in the Same Germarium.
[0091] To provide further evidence that the stem cells in the
ovariole reside within a niche, we showed that lost germline stem
cells can be replaced and function as germline stem cells by cells
that would otherwise differentiate. In one week-old germaria in
which one stem cell is marked, the marked cell contributes almost
50% of cysts, suggesting there are an average of two germline stem
cells per germarium. Since we have shown that wildtype germline
stem cells turn over with a half-life of 4.6 weeks, ovarioles
containing only one or zero germline stem cells would arise at a
predictable rate unless they are replaced. For example, after 4.6
weeks, 25%, 50% and 25% of the germaria are expected to have two,
one, and zero germline stem cells. In contrast, we observed that
more than 71% of five week-old germaria still contain two germline
stem cells, 20% contained one germline stem cell, and 9% contained
none. These results were unexpected and demonstrate that following
loss, germline stem cells are replaced 62% of the time over this
time period.
[0092] To determine how replacement occurs, we identified ovarioles
where a marked stem cell had just been lost and was in the process
of being replaced Such ovarioles contain a marked cystoblast and
marked developing cysts, but no marked stem cell. We observed an
unusual division of the remaining stem cell in a plane
perpendicular to the axis of the ovariole. Such a division would
place the stem cell daughter in the same location as the recently
lost stem cell. Normally, a germline stem cell divides along the
anterior-posterior axis, and the posterior daughter differentiates
into a cystoblast. These findings indicate that the fate of the
stem cell daughter is determined by the environment. This
environment within the niche maintains the stem cell fate, while
the environment more posteriorly in the ovariole promotes
differentiation as a cystoblast.
Example 9
Dpp is Required for Maintenance of Male Germline Stem Cells
[0093] Temperature sensitive dpp mutant genotypes were generated by
crossing dpp.sup.hr56/CyO with dpp.sup.hr4 and dpp.sup.e90/CyO.
Temperature-sensitive punt mutant males were generated by crossing
punt.sup.10460/TM3 with punt.sup.135/TM3 Sb. The
dPP.sup.hr56/dPP.sup.e90- , dpp.sup.hr56/dpp.sup.hr4 and
punt.sup.104601/punt.sup.135 adult males were raised at 28.degree.
C. (i.e., the restrictive temperature) for one week. Heterozygotes
controls were also examined at 28.degree. C.; testes were dissected
out and stained with rabbit anti-Vasa and mouse anti-Hts
antibodies. Cy3-conjungated goat anti-rabbit and FITC-conjungated
goat anti-mouse secondary antibodies were used to visualize the
Vasa protein (red) and Hts protein (green) with a Leica TCS-NT
confocal microscope. The germline stem cells are located at the tip
of testes, and can be recognized by their expression of Vasa
protein (red) and also contain round fusomes (yellow), and by their
association with hub cells (green). The differentiated germline
cells lie more distant from the tip, and also contain either round
fusomes or branched fusomes.
[0094] To directly show that dpp regulates male germline stem cells
in Drosophila testes, the number of stem cells was examined in
dpp.sup.hr56/dpp.sup.e90, dpp.sup.hr56/dpp.sup.hr4 and
punt.sup.10460/punt.sup.135 mutant testes under restrictive
conditions, and compared to heterozygote control testes. In a
heterozygous testis (control), there were between seven and nine
germline stem cells located adjacent to somatic hub cells and
contain round fusomes like their counterparts in the ovary; these
testes were full of developing germline cysts and primary
spermatids. After one week at the restrictive temperature, there
were still over seven germline stem cells. These values are
indistinguishable from the typical wildtype testis. In contrast,
one week-old dpp.sup.hr56/dpp.sup.e90, dpp.sup.hr56/dpp.sup.hr4 and
punt.sup.10460/punt.sup.135 mutant testes from males that had been
raised at the restrictive temperature contained a reduced number of
germline stem cells, ranging in number from 2-7 per testis. As a
consequence of this loss, these testes were also significantly
smaller than the controls and contained fewer developing germline
cysts and primary spermatids. Because a testis starts with a much
larger number of germline stem cells than an ovariole, complete
loss would not be expected within one week even if they require dpp
and punt to the same degree as female germline stem cells. These
results demonstrate that dpp and punt are required for maintaining
male germline stem cells.
Example 10
Shn is Required for Germline Stem Cell Maintenance
[0095] schnurri (shn) encodes a zinc-finger protein homologous to
human MPB1. It is required for dpp signaling in the Drosophila
embryo (Arora et al., 1995; Greider et al., 1995). Mutant shn
germline stem cell clones were generated as described above. FRT42D
arm-lacZ/FRT42D arm-lacZ virgin females were crossed to FRT42D
shn/CyO and FRT421D+/FRT42D+ (control) males, respectively. Two
day-old adult non-CyO females carrying an arm-lacZ transgene in
trans to the shn mutant-bearing chromosomes were heat shocked twice
at 37.degree. C. for 60 min each separated by eight hours. Germline
stem cells were examined in the same manner as described above.
These results demonstrate that shn is also required in germline
stem cells for their maintenance and division.
Experimental Procedures
[0096] A description of materials and methods useful for practicing
the present invention is given in the following general references:
Lindsley and Grell (Genetic Variations of Drosophila melanogaster,
Carnegie Inst. of Wash., 1968); Ashburner (Drosophila: A laboratory
handbook and A laboratory manual, Cold Spring Harbor Lab., 1989);
Lindsley and Zimm (The Genome of Drosophila melanogaser, Academic,
1992); Bate and Arias (The Development of Drosophila melanogaster,
Cold Spring Harbor Lab., 1993); and Greenspan (Fly Pushing: The
Theory and Practice of Drosophila Genetics, Cold Spring Harbor
Lab., 1997). Drosophila stocks may be obtained from the Bloomington
Stock Center at Indiana University. Information relevant to
Drosophila genetics and molecular biology, including recombinant
clones and nucleotide/amino acid sequences obtained through the
Drosophila genome project, is publicly available in the FLYBASE
relational database (see Nucl. Acids Res. 27, 85-88, 1999).
[0097] Drosophila Stocks and Genetics
[0098] The following fly stocks used in this study were described
either in the FlyBase or otherwise specified: tkv.sup.8;
punt.sup.10460 and punt.sup.135; mad.sup.9 and mad.sup.12;
Med.sup.26 (Des et al., 1998); Dad.sup.271-68; sax.sup.P;
dpp.sup.hr56 dpp.sup.hr4, dpp.sup.e90; UAS-dpp; hs-GAL4; HSFlp;
FRT40A armadillo-lacZ and HSFLP;FRT82B armadillo-lacZ (Lecuit and
Cohen, 1997); UAS-tkv* (activated) and UAS-sax* (activated) on both
chromosomes 2 and 3 (Des et al., 1998). Most stocks were cultured
at room temperature. To maximize their effects, sax.sup.P and dpp
mutants were cultured at 28.degree. C. for 1-2 weeks.
[0099] Generating Mutant Germline Stem Cell Clones and
Overexpression
[0100] Clones of mutant cells were generated by Flp-mediated
mitotic recombination as described previously (Xu and Rubin, 1993).
To generate the stocks for stem cell clonal analysis, +FRT40A/CyO,
tkv.sup.8 FRT40A/CyO, mad.sup.9 FRT40A/CyO, and mad.sup.12
FRT40A/CyO males were mated with virgin females w HSFlp1;
armadillo-lacZ FRT40A, respectively. FRT82B Med.sup.26/TM3 Sb,
FRT82B punt.sup.135/TM3 Sb, FRT82B punt.sup.10460/TM3 Sb, FRT82B
Dad.sup.271-68/TM3 Sb males were mated with virgin females w
HSFlp1; FRT82B armadillo-lacZ, respectively. Two day-old adult
non-CyO or non-Sb females carrying an armadillo-lacZ transgene in
trans to the mutant-bearing chromosome were heat shocked at
37.degree. C. for 60 min. The females were transferred to fresh
food every day at room temperature, and ovaries were removed one
week, two weeks, or three weeks after the last heat shock-treatment
and processed for antibody staining.
[0101] To construct the stocks for overexpressing dpp and activated
receptors, the hsGAL4 virgins were crossed with UAS-dpp,
UAS-tkv*/CyO, UAS-tkv*/TM3 Sb, UAS-sax*/CyO, UAS-sax*/TM3 Sb,
UAS-tkv*/CyO; UAS-sax*/TM6, UAS-sax*/CyO; UAS-tkv*/TM6 males,
respectively. The females which did not carry balancer chromosomes
were heat shocked at 37.degree. C. for 30 min each time with the
interval of 12 hr for 3-5 days.
[0102] Calculations
[0103] To determine stem cell life spans, stem cells were marked in
one to two day-old females of the appropriate genotype by a single
heat pulse. Subsequently, ovaries were dissected from some of the
females one, two, and three weeks later and stained with anti-Hts
and anti-lacZ antibodies. The percentage of germaria containing a
marked stem cell was determined by counts of 60-200 germaria at
each time point, and used to calculate the stem cell half-life.
[0104] To measure stem cell division rates, we determined the
relative number of wildtype and mutant cysts in germaria that
contained one wildtype and one mutant stem cell. A relative
division rate of 1.0 would indicate normal division. For a given
genotype, these values were similar at each time point, and the
average is presented in Table 2. Marked wildtype stem cells gave a
value of 0.93 rather than 1.0 probably due to a small fraction of
germaria that contained three rather than two germline stem
cells.
[0105] To measure stem cell loss, germaria with two, one, or no
germline stem cells, were counted from the ovaries of the one and
two week-old females. Heterozygous females carrying one copy of the
mutant gene in combination with a CyO balancer chromosome
containing a dpp transgene (Hursh et al., 1993) served as a
control. Values are expressed as the percentage of ovarioles with
the indicated stem cell compositions.
[0106] Immunohistochemistry
[0107] The following antisera at the indicated dilutions were used:
polyclonal anti-Vasa antibody (1:2000) (Liang et al., 1990);
monoclonal anti-Hts antibody IB 1 (1:5) (Zacci and Lipshitz, 1996);
poLyclonal anti-.alpha.-spectrin antibody (1:100) (Byers et al.,
1987); rat anti-Bam antibody (1:100) (McKearin and Ohlstein, 1995);
monoclonal anti-BrdU antibody (1:50) (Becton-Dickinson); polyclonal
anti-.beta.-galactosidase antibody (1:1000) (Cappel). Labeling with
BrdU was carried out for 1 hour at room temperature as described by
de Cuevas and Spradling (1998). All photomicrographs were taken
using a Leica TCS-NT confocal microscope.
1TABLE 1 Dpp is Required for Germline Stem Cell Maintenance. One
week Two weeks No One Two No One Two Genotypes GSC GSC GSC GSC GSC
GSC dpp.sup.e90/CyOP23 0.0% 4.4% 95.6% 0.5% 17.5% 82.0% (0) (5)
(108) (1) (36) (168) dpp.sup.hr4/CyOP23 0.0% 3.5% 96.5% 1.5% 23.9%
74.6% (0) (6) (165) (3) (48) (150) dpp.sup.e90/dpp.sup.hr56 16.0%
29.3% 54.7% 47.3% 39.8% 12.9% (17) (31) (58) (140) (118) (38)
dpp.sup.hr4/dpp.sup.hr56 18.1% 33.9% 48.0% 98.4% 1.6% 0.0% (22)
(41) (58) (122) (2) (0)
[0108] The percentage of ovarioles with zero, one or two germline
stem cells is given for each genotype. Actual numbers are given in
parentheses. .sup.aP23 is a dpp transgene on the CyO chromosome
(Hursh et al., 1993).
2TABLE 2 Downstream Components of the dpp Pathway are Required in
Germline Stem Cells for their Maintenance and Division. Percent of
Germaria.sup.a GSC.sup.b Relative.sup.c with a Marked GSC Half-Life
Division Strains 1 week 2 weeks 3 weeks (weeks) Rate Control 37.7
(138) 34.4 (161) 27.5 (160) 4.6 0.93 (1410) punt.sup.10460 43.2
(118) 26.4 (182) 9.5 (116) 0.90 0.36 (1126) punt.sup.135 27.4 (95)
5.1 (138) 0 (114) 0.41 0.37 (329) tkv.sup.8 38.6 (132) 16.4 (176)
6.1 (197) 0.69 0.29 (744) mad.sup.9 43.6 (g4) 29.3 (208) 25.8 (155)
2.5 0.60 (1116) mad.sup.12 17.8 (124) 0 (108) 0.7 (136) 0.25 0.21
(214) Med.sup.26 23.8 (172) 7.3 (110) 0 (122) 0.38 0.39 (512)
Dad.sup.271-68 28.0 (107) 32.6 (86) 32.3 (62) >>4.6 0.84
(770) Control 41.3 (235) 33.8 (185) 32.1 (379) 4.7 1.16 (316) shnP
38.9 (126) 23.7 (228) 16.5 (332) 2.2 0.53 (331) .sup.aNumber of
germaria with lacZ-negative germline stem cell clone/total germaria
.times. 100. The actual number of germaria counted is given in
parentheses. .sup.bCalculated as described in Experimental
Procedures. .sup.cCalculated as described in Experimental
Procedures. The number of cysts counted is given in
parentheses.
[0109] While the present invention has been described by what is
presently considered to be practical and preferred embodiments, it
is to be understood that variations in the claimed invention will
be obvious to skilled artisans without departing from the novel
aspects of the present invention and that such variations are
intended to come within the scope of the claims.
[0110] For example, components of the dpp signaling pathway are
conserved in structure (e.g., amino acid residues are identical or
chemically analogous in a high proportion of positions when
sequences are aligned) and function such that mammalian proteins
can rescue Drosophila mutant phenotypes which result from mutations
in homologous gene of the pathway. Equivalents to the Drosophila
genes and proteins identified herein, as well as mutants thereof,
would be known to skilled artisans practicing the present invention
by their similarity in amino acid sequence (e.g., members of the
TGF-.beta. family) and/or their ability to at least partially
rescue mutant phenotypes or to create phenocopies of such
phenotypes.
[0111] Thus, the extent of legal protection will be determined by
the limitations recited in the allowed claims and their
equivalents. Unless explicitly recited, other aspects of the
present invention as described in this specification do not limit
the scope of the claims. In this regard, the mechanisms of action
suggested in the specification (e.g., models for BMP signaling) are
merely possible explanations for our observations while operation
of the claimed invention is not necessarily dependent thereon.
[0112] All references, patent applications, and patents cited in
this disclosure are hereby incorporated herein by reference in
their entirety and indicate the high skill of artisans in this
field. In particular, some of the results shown above were
published by Xie and Spradling in Cell 94, 251-260 (1998) after the
filing date of priority U.S. Appln. No. 60/094,008.
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