1 Developmental Genetics Program, Skirball Institute at NYU School of Medicine
and Howard Hughes Medical Institute, 540 First Avenue, New York, NY 10016,
USA
2 Department of Developmental Biology, Stanford University School of Medicine,
Stanford, CA 94305-5329, USA
3 Department of Gynecology and Obstetrics, Stanford University School of
Medicine, Stanford, CA 94305-5329, USA
4 Department of Genetics, Stanford University School of Medicine, Stanford, CA
94305-5329, USA
Author for correspondence (e-mail:
lehmann{at}saturn.med.nyu.edu)
Accepted 10 September 2003
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SUMMARY |
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Key words: zpg, Drosophila, Stem cell, Germ line, Gap junction
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Introduction |
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The Drosophila ovary is composed of 16-18 units called ovarioles,
containing a germarium at the anterior and a chain of developing egg chambers
that extend posteriorly. GSCs reside in the anterior of the germarium and
closely abut the somatic terminal filament, cap and inner-sheath cells
(Fig. 1A), which comprise the
GSC niche (Lin, 1998;
Spradling, 1993
;
Xie and Spradling, 2000a
).
Several extrinsic factors, Decapentaplegic (Dpp), Piwi, Fs(1)Yb and Hedgehog,
control GSC or somatic stem cell maintenance and are expressed in different
subsets of niche cells (Cox et al.,
1998
; Cox et al.,
2000
; Forbes et al.,
1996
; King and Lin,
1999
; King et al.,
2001
; Xie and Spradling,
1998
; Xie and Spradling,
2000b
). Not much is known about how external signaling from the
niche preserves GSC fate. One protein that is likely to play a role in GSC
maintenance is Pumilio, a repressor of translation, which is required within
GSCs for GSC maintenance (Forbes and
Lehmann, 1998
; Lin and
Spradling, 1997
; Murata and
Wharton, 1995
; Zamore et al.,
1997
). However, the targets and partners of Pumilio within GSCs
are unknown.
|
Recently, we have shown that the gap junction protein Zero population
growth (Zpg) is required for an early step of both oogenesis and
spermatogenesis (Tazuke et al.,
2002). Gap junctions are intercellular, voltage-gated channels
that allow cells to selectively share ions, metabolites and other messenger
molecules. They have well-established roles in physiological and developmental
processes such as cardiac muscle contraction and ovarian follicle maturation
(Phelan and Starich, 2001
;
Simon and Goodenough, 1998
;
Simon et al., 1997
;
White and Paul, 1999
). Here we
show that GSCs lacking Zpg can divide, but the daughter cell destined to
differentiate dies. Our results suggest that zpg may be necessary for
the differentiation process itself, as well as for survival of differentiated
germ cells, and that zpg probably acts in parallel to bam
and bgcn. We further show that the differentiation of the GSC to a
cystoblast is gradual, and that many of the germ cells in `stem cell tumors'
caused either by strong mutations in bam or by overexpression of Dpp
may be at an intermediate state between GSCs and cystoblasts.
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Materials and methods |
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Three strong pum alleles were used in this study:
In(3R)Msc, pumET7 and pumET1,
described by Forbes and Lehmann (Forbes
and Lehmann, 1998). All allelic combinations were tested and
produced the described phenotypes. However, zpg,
pumET1/zpg, In(3R)Msc produced mostly empty
ovarioles, whereas zpg, pumET7/zpg, In(3R)Msc
produced more ovarioles containing germ line than other allelic combinations.
bgcnQS2 was described by Ohlstein et al.
(Ohlstein et al., 2000
) and
bam86 by McKearin and Ohlstein
(McKearin and Ohlstein, 1995
).
The hs-bam transgene on the X chromosome was described by Ohlstein
and McKearin (Ohlstein and McKearin,
1997
). hs-dpp flies were generated by Ron Blackman. The
JH1.1 strain carries eight copies of the hs-dpp transgene located at
16F-17A, 2B, 77, 96D-97C.
Antibodies
Rabbit anti-Vasa antibody was used at a dilution of 1:5000. Chicken
anti-Vasa was a generous gift from Dr K. Howard, and was used at 1:5000.
Rabbit anti-Zpg was used at 1:5000 and is described by Tazuke et al.
(Tazuke et al., 2002). Rat
anti-BamC was a gift of Dr D. McKearin and was used at 1:500. Rabbit anti
p-Mad (antiphosphorylated SMAD1, PS1)
(Persson et al., 1998
) was a
gift of P. ten-Dijke and was used at 1:1000. Rabbit anti-phospho-Histone H3
was from Upstate Biotechnology and was used according to the manufacturer's
protocol. 1B1 monoclonal supernatant
(Zaccai and Lipshitz, 1996
)
was from the Developmental Studies Hybridoma Bank and was used at 1:25. In all
cases, secondary antibodies, coupled to FITC, Cy3 or Cy5, were purchased from
Jackson ImmunoResearch Laboratories and were used at 1:500.
Heat shock
Newly eclosed flies were heat-shocked twice (hs-bam) or three
times (hs-dpp) a day at 37°C, for 1 hour, for four days. Flies
were dissected the following day and stained with the appropriate
antibodies.
Immunofluorescence
Fixation and immunostaining of ovaries were according to standard
protocols. Imaging was performed on a Leica DM RBE confocal microscope, using
the Leica TCS NT program.
Stem cell division counts
Newly eclosed flies were dissected and double-stained with chicken
anti-Vasa and rabbit anti-phospho-Histone H3 antibodies. Ovarioles were
observed under a fluorescent microscope to score for cells that had
anti-phospho-Histone staining. Only the cells at the anterior tip of the
ovariole were defined as `GSCs'. The number of cells that double stained for
Vasa and phospho-Histone H3 was divided by the total number of ovarioles
scored for each genotype.
Clonal analysis
One- to 3-day-old flies of the genotype
hs-flp122; FRT82, armadillo-LacZ /
FRT82 were heat shocked once for 15 minutes, at 37°C. Under these
mild conditions, only 30-50% of the ovarioles, depending on the experiment,
were marked, and marked ovarioles mostly had only one marked GSC. Flies were
dissected 2-3 days after heat shock and stained with anti-Vasa and
anti-ß-Gal. Cells that carry no copies, one copy or two copies of the
transgene can be distinguished by the intensity of anti-ß-Gal
staining.
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Results |
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In some zpg ovarioles we observed clusters of 2-3 cells that
stained positive with anti-BamC antibodies. We assume that these may be the
developing cysts that give rise to the rare egg chambers observed in these
mutants (Tazuke et al., 2002).
As in wild type, the rare developing zpg cysts were interconnected by
ring canals. However, the fusome, which normally spreads through the dividing
cyst, was either aberrant or absent. The rare egg chambers were almost always
composed of less than 16 germ line cells, and DAPI staining indicated
degeneration of the chamber (data not shown). As the fusome controls the
divisions of the cyst (de Cuevas et al.,
1996
; Lilly et al.,
2000
; Lin et al.,
1994
), the abnormal number of germ cells in the egg chamber is
likely to be the result of a severed fusome in the mutants.
Using an antibody raised against the cytoplasmic tail of Zpg
(Tazuke et al., 2002), we
observed staining in germ cells of every stage of development from GSCs to
budding cysts of wild-type germaria. The staining appeared to be at the
membrane of the cells and was somewhat stronger in region 2
(Fig. 1E). The presence of Zpg
staining in GSCs and dividing cysts correlates with the early defect in germ
cell differentiation in zpg mutants and suggests that Zpg acts in the
germ line.
zpg is required for survival of differentiating early germ
cells
The low number of cystoblasts in zpg ovarioles could be the result
of defects in GSC division or in survival of the GSC daughter cell destined to
differentiate. To distinguish between these two possibilities, we stained
zpg or wild-type germ cells for the mitotic marker phospho-Histone H3
(Fig. 2A,B). The percentage of
marked zpg cells at the stem cell position per ovariole was not
statistically different from that of wild-type GSCs
(Table 1). Similarly, the
number of wild-type GSCs per ovariole in S phase (detected by BrdU labeling)
was comparable to that of zpg cells (data not shown). Taken together,
these data show that zpg and wild-type cells spend a similar
proportion of time in M and S phase. Although we cannot rule out the
possibility that zpg cells have an overall slower cell cycle, as
compared to wild-type GSCs, our data show that zpg cells divide. The
lack of differentiating cells in zpg ovarioles may therefore indicate
that zpg germ cells die when they commence differentiation.
|
|
zpg is necessary for differentiation of pum mutant
GSCs
To further explore the role of Zpg in early germ cell differentiation and
survival, we recombined zpg alleles with mutant alleles of the gene
pumilio (pum) (Forbes
and Lehmann, 1998; Lin and
Spradling, 1997
). pum mutant ovaries exhibit a compound
phenotype. Many ovarioles lack germ line completely
(Fig. 3B,D), a defect that may
be attributed to preoogenic defects
(Asaoka-Taguchi et al., 1999
;
Forbes and Lehmann, 1998
;
Lin and Spradling, 1997
;
Parisi and Lin, 1999
).
Ovarioles with germ line have a germ line-depleted germarium connected to a
few defective egg chambers (Fig.
3A). This phenotype suggests that Pum has roles in GSC maintenance
(Forbes and Lehmann, 1998
;
Lin and Spradling, 1997
). In
ovaries from zpg, pum double-mutant females, many ovarioles were
empty (see Materials and methods). This is consistent with the embryonic and
larval requirement for pum (Fig.
3D). Ovarioles occupied by germ line exhibited a phenotype more
similar to zpg than to pum mutants: few germ cells at the
tip of the ovariole (Fig.
3C,D). Thus Zpg function is required for the differentiation of
pum mutant germ cells. The apparent difference between the zpg,
pum and hs-Bam; zpg phenotypes may reflect the different roles
of Pum and Bam in GSC differentiation. Pum, as a translational repressor may
permit GSC maintenance by repressing differentiation, which requires Zpg. By
contrast, Bam may have a more instructive role in GSC differentiation, such
that its overexpression can overrule GSC-maintenance cues emanating from the
niche, independent of zpg
(Ohlstein and McKearin, 1997
)
(this study).
|
bam and bgcn `stem cell' tumors do not develop in
the absence of zpg
Our analysis suggests that Zpg wild-type function is required for the
differentiation of GSCs. At least two other genes, bam and
bgcn, are required for early germ cell differentiation
(Gonczy et al., 1997;
Lavoie et al., 1999
;
McKearin and Ohlstein, 1995
;
Ohlstein et al., 2000
).
However, the phenotype of bam and bgcn mutant ovaries is
strikingly different from that of zpg mutants. Ovaries mutant for
bam or bgcn are filled with many undifferentiated single
germ cells harboring a spherical spectrosome, which have been described as GSC
tumors (Fig. 4A)
(McKearin and Ohlstein, 1995
;
McKearin and Spradling, 1990
;
Ohlstein et al., 2000
). By
contrast, ovaries from zpg flies have only a few germ cells at the
tip of the ovariole (Fig. 4B).
To determine the functional relationship between these genes, we made flies
double mutant for zpg with either bam or bgcn.
Ovaries from newly eclosed females were stained to visualize the germ line and
the spectrosomes. In the double mutant lacking both zpg and
bam, only a few germ cells were detected at the ovariole tip
(Fig. 4C). However, most
double-mutant ovarioles had somewhat more germ cells than zpg
ovarioles (average for zpg, bam=6.4, s.d.=3.2, n=110;
average for zpg=3.8, s.d.=1.9, n=464). Similar results were
obtained with double mutants of zpg and bgcn
(Fig. 4D).
|
Dpp cannot support early germ cell over-proliferation in the absence
of zpg
To investigate further a possible role for Zpg-mediated intercellular
communication in the development of germ cell tumors, we analyzed the genetic
interaction between dpp and zpg. It had been proposed that
an increased Dpp signal induced over-proliferation of GSC-like cells
(Xie and Spradling, 1998;
Xie and Spradling, 2000b
). We
therefore reasoned that an increased Dpp signal could induce zpg
cells to over-proliferate. To test this, we crossed flies carrying several
insertions of a heat-shock dpp transgene (hs-dpp) into a
zpg background. Flies were heat shocked, and then dissected and
stained to reveal the germ line and spectrosomes. Control animals heterozygous
for zpg, carrying a subset of the hs-dpp transgenes, had
more single germ cells with spherical spectrosomes than did wild type
(Fig. 5A). Homozygous
zpg animals, which carried at least the same number of
hs-dpp transgenes as the control, did not show an increase in germ
cell number (Fig. 5B). To test
whether zpg cells could respond to a Dpp signal, we stained ovaries
of zpg animals with antibodies against phosphorylated Mad (p-Mad)
(Persson et al., 1998
). Mad is
a component of the Dpp signal transduction pathway and is phosphorylated upon
activation of the pathway (Massague,
1998
). In wild type, p-Mad staining could be detected in GSCs,
cystoblasts and dividing cysts in region 1 of the germarium. The highest level
of staining was observed in early germ cells; staining then gradually declined
towards the posterior (Fig.
5C). p-Mad was also detected in the single cells that accumulate
following Dpp overexpression (Fig.
5D). Similarly, p-Mad staining was detected in zpg germ
cells (Fig. 5E,F), suggesting
that the mutant cells are able to receive the Dpp signal. There may be several
explanations for the failure of zpg cells to proliferate or survive
in response to a Dpp signal. One is that the Dpp pathway is blocked downstream
of Mad in zpg cells. Second, zpg cells may not be able to
survive when unattached to the tip of the ovary. Third, proliferating cells in
hs-dpp flies, which move away from the niche, are in a more
differentiated state than the cells in the niche, and therefore die in a
zpg background (see Discussion).
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Discussion |
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Zpg is a gap junction protein necessary for GSC
differentiation
zpg encodes innexin 4 - one of eight innexins in
Drosophila (FlyBase) (Phelan and
Starich, 2001; Stebbings et
al., 2002
; Tazuke et al.,
2002
). Innexins are the functional homologues of the vertebrate
connexins, or gap junction proteins
(Phelan and Starich, 2001
). In
mammalian oogenesis, gap junctions have been implicated in cell-cell
signaling. Early luteinization of granulosa cells is observed either when the
oocyte is physically removed from immature wild-type follicles
(el-Fouly et al., 1970
), or in
mice lacking connexin 37 (Simon et al.,
1997
), suggesting a gap junction-mediated signaling mechanism
between the oocyte and granulosa cells.
Here we show that the Drosophila gap junction protein Zpg is
required for the survival of the germ line stem cell daughter as it moves away
from the niche and begins to differentiate. From its expression pattern, and
the specificity of its function, it is clear that Zpg acts in a germ line
autonomous way. However, we do not yet know whether Zpg facilitates
communication between germ cells, or between germ line and soma. The germarium
is a compact structure where early germ cells contact each other, the somatic
cap cells contact GSCs, and inner-sheath cells contact GSCs and their
daughters (King, 1970;
Schulz et al., 2002
). Further
study is needed to clarify which cells communicate with germ cells through
Zpg-gap junctions. Likewise, the nature of the requirement for zpg in
GSC differentiation is still uncertain. Gap junctions may be used to supply
the GSC daughter cell with nutrients, or with a survival factor required for
its subsequent growth. Alternatively, gap junctions may be used to deposit a
factor that is required for the differentiation process itself, rather than
for survival, while an accessory mechanism eliminates cells that begin
differentiating without that factor. The major argument in favor of a role for
Zpg in differentiation at the stem cell stage comes from our phenotypic
analysis of zpg, pum double mutants in which, unlike in pum
single mutants, GSCs do not differentiate. Although Pum-mediated repression is
removed in the double mutants, GSCs cannot differentiate as they may lack a
differentiation signal provided by Zpg. It is harder to imagine how
nutritional deficiency per se could block differentiation of the double-mutant
cells because single-mutant zpg cells do begin to differentiate (and
then die).
The nature of GSC differentiation
Recent studies suggest that the niche promotes stem cell fate through Dpp
signaling (Xie and Spradling,
1998; Xie and Spradling,
2000b
; Zhu and Xie,
2003
). This may be achieved through repression of bam
(Chen and McKearin, 2003
). GSCs
are also tightly tethered to the niche via adherens junctions
(Song et al., 2002
). Other,
as-yet unidentified mechanisms may be used by the niche to protect GSCs.
Once germ cells leave the niche they activate the differentiation pathway.
We propose that differentiation of GCSs to cystoblasts is not direct but
proceeds via an intermediate state (Fig.
6). We have shown that most wild-type cells, which by their
position within the germarium were judged to be cystoblasts, are not stained
with BamC antibody. Our finding concurs with Ohlstein and McKearin, who
observed cytoplasmic Bam just before the cystoblasts divide to form a two-cell
cyst (Ohlstein and McKearin,
1997), and proposed the existence of an
intermediate/pre-cystoblast state between GSCs and cystoblasts. In other stem
cell systems, the intermediate cell population has a biological function,
namely increasing the progeny of a single stem cell division. Our results
indicate that in Drosophila females, cells at the intermediate state
do not constitute a transit-amplifying cell population. However, the
`pre-cystoblast state' may have a different biological significance. A vacant
niche can be filled by a neighboring GSC that divides `sideways' instead of
along the anteroposterior axis (Xie and
Spradling, 2000b
). An alternative for filling a vacant GSC
position might be for a cell at the intermediate state to reoccupy the
niche.
The suggested model, adding a transitory state between the stem cell and
the cystoblast, raises an interesting question. Is the differentiation of a
GSC to a cystoblast a continuous process or a discrete one? It is notable that
none of the markers we currently use is specific to the stem cell, the
cystoblast or the intermediate. Bam is present (in its fusomal form) already
in the stem cell, and BamC gradually accumulates in the cystoblast
(Ohlstein and McKearin, 1997)
(this study). Pumilio protein and phosphorylated Mad are also detected from
GSCs to cystoblasts and early cysts
(Forbes and Lehmann, 1998
)
(this work). In other stem cell systems, including mammalian hematopoiesis,
many intermediate cell types are known, and can be isolated by specific marker
combinations (reviewed by Melchers and
Rolink, 2000
). Due to the relative lack of markers, the isolation
of these `cell types' from Drosophila ovaries is currently
impossible. The overlap of expression patterns of the proteins that are known
to have a role in stem cell maintenance and differentiation may indicate that
differentiation is gradual, and possibly reversible.
The niche promotes germ line stem cell fate
It has been suggested that in bam or bgcn mutant ovaries,
or when Dpp is overexpressed, the germaria are filled with GSC tumor cells
(Gonczy et al., 1997;
Lavoie et al., 1999
;
McKearin and Ohlstein, 1995
;
Ohlstein et al., 2000
;
Xie and Spradling, 1998
;
Xie and Spradling, 2000b
). Our
findings of an intermediate cell population in wild type raises the
possibility that GSC tumor cells share some properties with precystoblasts.
Both these populations are single, do not stain for BamC, but exist outside of
the niche. Some support to the analogy between pre-cystoblasts and `GSC
tumors' is evident in the fact that the latter do not survive past the niche
in a zpg background. Thus, the zpg double mutants allow us
to distinguish two cell populations in `GSC tumors' - cells that are inside or
outside of the niche. We suggest that when Dpp is overexpressed, or in
bam/bgcn mutants, cells outside the niche cannot fully
activate the differentiation program and are at an intermediate state between
a GSC and a cystoblast. These cells die in a zpg background, whereas
the tumor cells in contact with the niche survive. Our results thus suggest
that beyond Zpg gap junctions and Dpp signaling, there must be additional
signaling between GSCs and the niche, which helps maintain GSCs. Additional
markers are needed to determine unequivocally whether bam tumors are
similar to Dpp tumors, and whether they share properties with wild-type
precystoblasts.
Differentiation of the stem cell daughter requires gap junctions. We assume
zpg is acting in parallel to pum, dpp, bam and bgcn
because the double mutants showed incomplete epistasis of zpg over
each of these genes (Fig. 7).
Although a role for gap junction proteins has been established in mammalian
oogenesis (Ackert et al., 2001;
Carabatsos et al., 2000
;
Juneja et al., 1999
;
Simon et al., 1997
), to our
knowledge, this is the first instance where a gap junction protein is shown to
control stem cell differentiation. What passes through these gap junctions,
and which cells are connected to GSCs through Zpg channels, is still unknown.
One intriguing option is that Zpg forms part of the channels that connect GSCs
to the surrounding somatic niche cells. If so, that would suggest that the
niche in the Drosophila germarium is necessary, not only for stem
cell maintenance, but also for stem cell differentiation.
|
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ACKNOWLEDGMENTS |
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Footnotes |
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Present address: Center for Research on Reproduction and Women's Health,
University of Pennsylvania School of Medicine, 1355 BRB II/III, 421 Curie
Boulevard, Philadelphia, PA 19104-6142, USA
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