Department of Cell Biology, BOX 3709, Duke University Medical Center, Durham, NC 27710, USA
Author for correspondence (e-mail:
h.lin{at}cellbio.duke.edu)
Accepted 14 July 2004
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SUMMARY |
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Key words: Stem cell, Germline, Primordial germ cell, Somatic signalling, Drosophila
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Introduction |
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Germline development in Drosophila provides an excellent
opportunity to study the establishment of the stem cell fate in individual
tissues. This is because the germline in Drosophila is well
characterized as a tissue lineage. Moreover, germline development typifies the
development of a stem cell-derived tissue. The Drosophila germline
originates from pole cells that form at the posterior pole of the syncytial
blastoderm. Pole cells then migrate into the abdominal region of the embryo,
where some of them coalesce with somatically derived gonadal mesodermal cells
to form two embryonic gonads (Williamson
and Lehmann, 1996). The gonadal pole cells are now generally
called primordial germ cells (PGCs). During larval development, both PGCs and
somatic gonadal cells proliferate but remain relatively undifferentiated. At
the larval-pupal transition, ovarian morphogenesis takes place, transforming
the larval gonad into the functional adult ovary
(Godt and Laski, 1995
;
King, 1970
). During this
transition, a subset of PGCs acquire the stem cell fate; they initiate
asymmetric divisions to produce differentiated daughter cells called
cystoblasts, which eventually develop into an egg chamber
(King, 1970
;
Zhu and Xie, 2003
). The
continued self-renewing division of germline stem cells (GSCs) in pupal and
adult stages leads to the growth and maintenance of the adult ovarian germline
as a large and dynamic tissue. Thus, the ovarian germline is a typical stem
cell-derived tissue, with stem cells established from a subset of their
embryonic precursors at the larval-pupal transition.
Like stem cells in mammalian tissues, GSCs in Drosophila reside in
a defined locale, called the stem cell niche, in the tissue
(Lin, 2002). In
differentiating early pupal ovaries and differentiated adult ovaries, GSCs are
located in the most anterior tip of the ovariole, the functional unit of the
ovary, in a specialized structure called the germarium. Here, GSCs are in
direct contact with somatic cap cells
(Deng and Lin, 1997
;
King, 1970
;
Lin and Spradling, 1993
;
Lin and Spradling, 1997
;
Zhu and Xie, 2003
). GSCs
divide asymmetrically with regard to cap cells, so that the daughter GSC
remains in contact with cap cells while the cystoblast becomes displaced one
cell away (Deng and Lin,
1997
). Recent studies have shown that signals from cap cells and
their neighboring terminal filament cells are essential for GSC maintenance
(Chen and McKearin, 2003
;
Cox et al., 1998
;
Cox et al., 2000
;
King and Lin, 1999
;
King et al., 2001
;
Song et al., 2004
;
Song et al., 2002
;
Xie and Spradling, 1998
;
Xie and Spradling, 2000
).
These somatic cells thus form the stem cell niche that ensures the
self-renewing ability of GSCs. The self-renewing ability of GSCs also requires
interplay between cell-autonomous genes that promote stem cell division and
those that drive differentiation (Cox et
al., 2000
; Forbes and Lehmann,
1998
; Lin and Spradling,
1997
; McKearin and Spradling,
1990
; Parisi and Lin,
1999
; Wang and Lin,
2004
).
Although much is known about mechanisms that govern GSC maintenance, how
the stem cell fate is initially determined during germline development remains
unexplored. It is known that four to seven PGCs are partitioned into each
germarium during larval-pupal transition
(King, 1970;
Parisi and Lin, 1999
).
However, only two to three PGCs in direct contact with cap cells become GSCs
in pupal and adult ovaries (Deng and Lin,
1997
; King, 1970
;
Zhu and Xie, 2003
), and it
remains elusive how these two to three PGCs are selected to contact cap cells
and become stem cells. It is possible that all PGCs have an identical
potential to become stem cells. In this case, the stem cell fate would not be
determined until the onset of oogenesis during the larval-pupal transition,
when niche cells start to induce their adjacent PGCs to become GSCs (herein
called the late induction hypothesis). Alternatively, stem cell fate
determination could occur before the onset of oogenesis (herein called the
predetermination hypothesis). The predetermination could be caused by
interaction between a subset of PGCs with specific somatic cells in the
embryonic/larval gonads (herein called predetermination by induction), or by
the inherent heterogeneity of pole cells in developmental potential, so that
some of them are already destined to stem cell fate when they are formed
(herein called predetermination by lineage). Finally, both mechanisms may
participate in the predetermination process. Among these possibilities, both
the late induction and predetermination-by-induction hypotheses are supported
by the role of the stem cell niche in GSC maintenance
(Lin, 2002
), whereas the
predetermination-by-lineage hypothesis is consistent with the heterogeneity of
pole cells in polar granule content, mitotic rate, and splicing activity
towards P-transposase pre-mRNA (Kobayashi
et al., 1993
; Sonnenblick,
1950
; Technau and
Campos-Ortega, 1986
). Here, we report experiments indicating that
the GSC fate is specified by predetermination and not the late induction
mechanism. Furthermore, our data indicate the potential involvement of somatic
induction in the predetermination process.
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Materials and methods |
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`Single' pole cell transplantation
Pole-cell transplantation was conducted at 22°C as described
(Kobayashi et al., 1996).
Donor eggs and host eggs were collected at 50-minute intervals, and then
allowed to develop to 150-200 minutes after egg laying. Pole cells were
isolated from donor embryos at the cellular blastoderm stage and injected into
the posterior pole of same stage host embryos. Only one to three donor pole
cells (average 2.2 pole cells) from a single embryo were injected to a host
embryo. This number of transplanted pole cells ensures that each host receives
a single donor pole cell in its gonads at a high frequency. The injected
embryos were kept at 18°C until embryonic gonads were formed (stage
14-15). Each of these stage 14-15 embryos were transferred into a small drop
of silicone oil on the microscope slide, covered with a coverslip, and then
examined under the fluorescent microscope to locate the donor pole cell in the
host embryonic gonad. After examination, the host embryos were kept at
18°C in a moist chamber until they hatched into larvae. The hatched larvae
were collected and divided into different groups according to the number and
location of labeled pole cells in the gonads at stage 14-15. Each group of
larvae was transferred to standard Drosophila medium in individual
35-mm culture dishes (Becton Dickinson Labware, Franklin Lakes, NJ) and
incubated at 25°C until pupation. The culture dishes were supplemented
with TM6-bearing larvae so that total number of the larvae was 15-17 per dish,
as too few larvae in a dish causes low viability. The pupae were transferred
into vials with fresh Drosphila medium and raised at 25°C. The
TM6-bearing larvae and the adults were recognized by their Tubby
phenotype and the wild-type eye color, respectively, and were eliminated.
Ovaries and testes were dissected from each host animal at the late third
instar larval/prepupal stage, or at the adult stage (within 24 hours after
eclosion), and then fixed and stained separately, as described below.
Dissection of larval and adult ovaries and testes
The dissection and fixation of larval and adult ovaries and testes was
performed as previously described for the adult ovary
(Lin et al., 1994). After the
3-minute fixation and several washes, the adult samples were stained with 1
µg/ml 4',6-diamidino-2-phenyindole (DAPI) in 1xPBS for 10
minutes, then mounted in 50% glycerol in 1xPBS and 2% anti-quenching
agent DABCO, and examined by fluorescent microscopy to identify GFP-labeled
cells. For larval gonads, the GFP signal was weak, so the GFP expression was
detected by indirect immunofluorescence microscopy (see below).
Histochemical and immunological staining
Embryos were fixed and double-stained with X-gal and the rabbit anti-Vasa
antiserum (1:200) (Hay et al.,
1990), as previously described
(Asaoka-Taguchi et al., 1999
).
X-gal staining of ovaries and testes was performed as described previously
(Lin et al., 1994
). Antibody
staining of larval/prepupal ovaries and testes were performed as described
previously (King and Lin,
1999
). The following antibodies were used: rabbit anti-GFP
antibody (1:1000, Molecular Probes), mouse anti-1B1 antibody (1:100;
Developmental Studies Hybridoma Bank), guinea pig anti-ß-galactosidase
antibody (1:1000, a gift from T. Isshiki), rat anti-DE-cadherin antibody DCAD2
(1:200, a gift from T. Uemura), rabbit anti-Vasa antibody (1:200, a gift from
Y. Jan) and rat anti-Vasa antibody (1:2000, a gift from A. Nakamura). For
confocal analysis, secondary antibodies conjugated with different fluorophores
anti-rabbit Alexafluor-488 (1:200), anti-mouse Cy3 (1:500), anti-mouse
Cy5 (1:500), anti-guinea pig Cy3 (1:500), anti-rat Cy3 (1:500), anti-rabbit
FITC (1:500) and anti-rat FITC (1:500) were used. All the secondary
antibodies were obtained from Jackson Immunoresearch Laboratories, except for
Alexafluor-488-conjugated anti-rabbit secondary antibody (Molecular Probes).
Confocal images were collected using a Zeiss LSM510-META confocal
microscope.
For ovary staining with anti-DE-cadherin antibody, ovaries were dissected
in 1xEBR (Lin et al.,
1994) and fixed in 3.7% formaldehyde in PCM (100 mM PIPES pH 6.9,
1 mM CaCl2, 2 mM MgSO4) for 15 minutes. The staining
procedures and image collection were the same as described above, except that
a PBT solution containing 0.4% of Triton X-100 was used instead of the
standard PBT solution.
Germline clone analysis
The shotgun (shg) germline clone was generated using the
FLP-DFS technique, as described previously
(Chou and Perrimon, 1996).
w hs-FLP /Y; FRT arm-lacZ males were mated with
FRT42DshgR69/CyO virgin females. Egg
collection was performed in the culture tubes for 12 hours. The culture tubes
containing the first instar larvae (34-46 hours after egg laying) were heat
shocked for 1 hour in a 37°C water bath to induce mitotic recombination.
The ovaries of the heat-shocked larvae were dissected at late third instar
larval stage, and processed for immunostaining and confocal microscope
analysis. shg mutant clones and siblings were identified as
lacZ-negative and lacZ-overexpressed PGCs, respectively.
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Results |
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Anterior somatic cells in the embryonic gonad are likely to be the precursors of GSC niche cells in the ovaries and testes
In adult ovaries and testes, anterior somatic cells constitute a stem cell
niche that plays a crucial role in maintaining GSCs
(Lin, 2002). In ovaries, niche
cells include terminal filament and cap cells, and possibly inner germarial
sheath cells (Fig. 1C). These
cell types form in the anterior region of ovaries at the larval-pupal
transition stage (Godt and Laski,
1995
; King, 1970
).
In testes, the niche is composed of hub cells, somatic cyst progenitor cells,
and, possibly, early somatic cyst cells
(Fig. 1D). Testicular niche
morphogenesis begins near the end of embryogenesis
(Cooper, 1950
). To determine
whether the 3914 enhancer activity correlates with the signaling
ability of somatic cells, we examined 3914 expression in larval and
adult ovaries, as well as in the adult testis.
In late third instar larval ovaries, the 3914 enhancer trap is expressed in the anterior somatic region, where forming terminal filament and cap cells reside (Fig. 1B). Particularly, strong expression was detected in developing terminal filament and cap cells. However, expression was never detected in the medial region that contains PGCs and somatic interstitial cells, or in the posterior region that includes the precursors of basal stalk cells.
In the adult ovary, 3914 is expressed strongly in cap cells, the central component of the niche, with weaker expression in terminal filament and inner germarial sheath cells (Fig. 1C). The expression pattern of 3914 in embryonic gonads, larval and adult ovaries suggests that the anterior somatic cells in the embryonic gonad are likely to be the precursors of stem cell niche cells in the larval and adult ovaries, and that they may also have a signaling function.
In the adult testis, 3914 was also expressed in the niche cells,
with strong expression in hub cells, the central component of the niche, and
weaker expression in somatic cyst progenitor cells and their daughter cells,
the somatic cyst cells (Fig.
1D). This corroborates a previous enhancer-trap study that
suggests that the hub precursors are a subset of cells located at the anterior
tip of the embryonic gonad (Gönczy et
al., 1992). Moreover, the broader expression pattern of
3914, covering the entire anterior half of the embryonic gonad,
suggests that these anterior somatic cells not only give rise to hub cells,
but also to somatic cyst progenitor cells and their daughter cyst cells that
form the niche in the apical region of the testis.
Anterior and posterior pole cells in the embryonic gonad give rise to GSCs and cystoblasts, respectively
Our analysis of 3914 expression raises the possibility that
anterior somatic cells of the embryonic gonad are involved in determining the
GSC fate, and that this fate could be determined as early as in the early
embryonic gonad. To test this possibility, we investigated whether only those
pole cells in contact with the anterior somatic cells in the embryonic gonad
give rise to GSCs in the adult ovary. To this end, we traced the position and
fate of single pole cells from early embryonic gonadal stage to the adult
stage using a vasa-EGFP gene that is specifically and continuously
expressed in the germline (Sano et al.,
2002). The following strategy was used for the lineage tracing
(see Materials and methods). A single EGFP-marked wild-type pole cell was
transplanted into the pole cell region of a wild-type recipient embryo at the
cellular blastoderm stage. The recipient embryos then continued to develop to
stage 14-15, at which time the position of the GFP-marked pole cells in the
newly formed embryonic gonad was examined and recorded. The embryo was then
allowed to develop to adulthood. Its ovaries or testes were then dissected and
analyzed to determine the fate of the EGFP-marked pole cell.
We transplanted EGFP-marked pole cells into 1425 cellular blastoderm embryos, 694 of which incorporated a marked pole cell in their gonads following gonadal formation (stages 14-15; Fig. 2A-D). Eventually, 45 of such marked embryos developed into pupal-adult females. Of these, 19 females were developed from embryos with the marked pole cell located in the anterior half of the embryonic gonad (anterior pole cells; Fig. 2A,B). Seven such females still retained EGFP-marked germ cells in the ovary. Twenty-four females developed from embryos with a marked pole cell in the posterior half region of the embryonic gonad (posterior pole cell; Fig. 2C,D). Ten such females still retained EGFP-marked germ cells in the ovary. Thus, the survival rates for the transplanted anterior and posterior pole cells are very similar (37% versus 42%, P>0.1). Finally, two females developed from embryos with a marked pole cell on the exact midline of the embryonic gonad were excluded from this analysis.
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PGCs derived from a single pole cell are not anchored to each other or to specific somatic cells during proliferation
How can an anterior pole cell in the embryonic gonad give rise to GSCs?
Three recent findings suggest that anterior pole cells are anchored to
anterior somatic cells in the embryonic gonad and that this anchorage is
maintained during larval development so that a pole cell gives rise to a clone
of PGCs that are partitioned only into a few adjacent germaria during the
larval-pupal transition. First, E-cadherin is required in both pole cells and
somatic gonadal precursor cells for the formation of embryonic gonad
(Jenkins et al., 2003).
Second, in the early pupal ovary, a PGC in contact with newly formed cap cells
tends to divide along the plane of cap cells, generating two to three GSCs
that all contact cap cells within a germarium [`clonal expansion' theory
(Zhu and Xie, 2003
)]. Third,
in the adult ovary, E-cadherin is essential for anchoring GSCs to cap cells to
maintain their stem cell fate (Song et
al., 2002
).
To examine whether PGCs derived from an anterior pole cell adhere to each
other and to anterior somatic cells during their proliferation, we first
looked at the distribution of daughter cells of a single, labeled anterior
pole cell in larval/prepupal ovaries. We transplanted EGFP-labeled pole cells
into 520 blastoderm embryos, recorded the position of the single, labeled pole
cell in the newly formed gonad of the recipient embryos (stage 14-15), allowed
the embryo to develop to the late third instar larval or prepupal stage, and
then isolated ovaries for immunofluorescence analysis (see Materials and
methods). Such ovaries were stained with anti-GFP antibody to identify PGCs
derived from the marked pole cell, and with anti-1B1 antibody, both to outline
the developing terminal filament and cap cells and to label spectrosomes
(Deng and Lin, 1997).
To our surprise, only 14% of ovaries contained a clone of labeled germ cells in contact with one another (n=14). By contrast, in 72% of ovaries, labeled cells were dispersed widely along both the anteroposterior and mediolateral axes, with some PGCs even passing across the midline into the posterior region (17% of ovaries, Fig. 4C, Fig. 5A). In particular, in 43% of ovaries, all labeled daughter cells were highly dispersed, whereas a small number of labeled cells were associated in another 29% of ovaries. Even in these 29% of ovaries, most PGCs were dispersed several cells away from each other. The remaining 14% of ovaries had only one labeled daughter cell. These results indicate that PGCs derived from an anterior pole cell do not adhere to each other or to anterior somatic cells during their proliferation. Instead, they become dispersed and intermixed with other pole cell progeny during larval development. Furthermore, as there was no difference in the extent of dispersion between daughter cells derived from anterior versus posterior pole cells, it can be surmised that PGCs derived from a single pole cell are dispersed during proliferation irrespective of the original position of the pole cell in the embryonic gonad.
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A similar frequency of dispersion was observed for PGCs derived from posterior pole cells, as inferred from the labeling pattern in the adult ovary. A single, labeled posterior pole cell produced 2-35 differentiated daughter cells (average 16.4) that were distributed into 1-13 ovarioles (average 7.4), which represents, on average, 31% of ovarioles in an ovary [n(ovariole)=236; n(ovary)=10]. In 45% of these ovarioles, only one germline cyst or egg chamber was labeled (n=74). These results indicate that PGCs derived from a single posterior pole cell are also dispersed during ovarian morphogenesis.
PGCs derived from anterior pole cells preferentially enter nascent stem cell niches
As only anterior pole cells give rise to GSCs, we would expect their
daughter PGCs to be much more accessible to nascent stem cell niches in
larval/prepupal ovaries, even though they are not anchored to anterior somatic
cells in the gonad. To test this hypothesis, we determined whether only PGCs
derived from anterior pole cells would contact developing cap cells in late
third instar larval and prepupal ovaries. As expected, 90% of PGCs derived
from anterior pole cells were in contact with developing cap cells
[Fig. 4A,A';
n(labelled PGC)=19; n(ovary)=6]. Only 10% of the PGCs were
not in contact with developing cap cells. This frequency corresponds well with
the observation that 14% of anterior pole cells produce only differentiating
germline cyst or egg chambers in the adult ovaries (see above). By contrast,
92% of PGCs derived from posterior pole cells were in not contact with
developing cap cells in the larval/prepupal ovaries
[Fig. 4B,B'; n(labelled PGC)=38; n(ovary)=8]. The difference between the
frequencies of anterior and posterior pole cells in giving rise to cap
cell-contacting PGCs (90% versus 8%, respectively) is highly significant
(P<0.0001). These results show that PGCs derived from anterior
pole cells have a strong tendency to become stem cells because they
preferentially enter the nascent stem cell niche in the larval/pupal
transitional ovaries.
DE-cadherin is not required for PGCs to enter the nascent stem cell niche
Then, how do PGCs derived from anterior pole cells preferentially enter the
nascent stem cell niche? It may be due to one of the following four
mechanisms, or a particular combination of them. (1) Anterior pole cells and
their progeny PGCs are anchored to the anterior somatic gonadal cells and
their progeny via static cell-cell adhesion, such as adherens or desmosomes
junctions (static anchorage mechanism). (2) PGC divisions occur more
frequently along the niche cell/PGC interface, owing to somatic induction or
mechanical constraints of the ovary (differential mitotic orientation
mechanism). (3) PGCs derived from an anterior pole cell divide at a slower
rate so that their progeny is more confined to the anterior region
(differential mitotic rate mechanism). (4) The dispersed PGCs derived from the
anterior pole cells actively home back to the niche cell/PGC interface in
response to somatic induction (active homing mechanism).
The static anchorage mechanism is unlikely to play a significant role,
because, as described above, the progeny of a single pole cell are widely
dispersed in the larval gonad both laterally and anteroposteriorly,
irrespective of the position of the original pole cell (see above;
Fig. 4C,
Fig. 5A). Thus, even though
cell adhesion molecules such as E-cadherin are required for the association of
pole cells and somatic cells in the embryonic gonad, and for the later
anchoring of GSCs to cap cells in adult ovaries
(Jenkins et al., 2003;
Van Doren et al., 2003
;
Song et al., 2002
), such
adhesion may not function during PGC proliferation in the larval gonad. In
support of this reasoning, DE-cadherin and ß-catenin only start to
accumulate at the interface between nascent cap cells and future GSCs at the
late third instar larval stage (Song et
al., 2002
).
To determine whether DE-cadherin is required for PGCs even to enter the nascent stem cell niches, we used a FLP-mediated mitotic recombination technique to induce the production of PGCs that are null for the DE-cadherin gene shotgun (shg) in the shg/+ first instar larvae (see Materials and methods). The shg/shg PGCs and their +/+ sibling PGCs are marked by zero and two copies of the lacZ gene, respectively (Fig. 5). We then compared the locations of the marked DE-cadherin-deficient PGCs and their wild-type sibling PGCs in late third instar larval ovaries. The marked DE-caderin-deficient PGCs and their wild-type sibling PGCs show similar distributions in the anterior and posterior regions of the ovary, with the wild-type PGCs actually distributed somewhat more in the posterior region (Fig. 5E). In particular, 16.8±4.6% of DE-cadherin-deficient PGCs were in contact with cap cells, as compared with 6.1±3.2% of their siblings (P=0.066, t test, Fig. 5E). These data indicate that DE-cadherin is not required for PGCs to enter the nascent niches, and that it might constrain PGCs from actively reaching cap cells.
The preferential niche occupancy of PGCs derived from anterior pole cells is not due to differential orientation or rate of divisions
We next examined whether PGCs derived from anterior pole cells
preferentially enter the nascent stem cell niche as a result of differential
mitotic orientation or rate. In third instar larval ovaries, the divisional
orientation of PGCs can be easily observed at the prolonged telophase/G1
phase, when the dividing GSC pair displays dumbbell morphology with an
elongated spectrosome located in the neck region
(Fig. 6A-D). Moreover, the
frequency of the prolonged telophase/G1 phase GSC pairs reflects the frequency
of PGC divisions. The mitotic orientation of cap cell-contacting PGCs displays
a random distribution that is very similar to anterior PGCs that are not in
contact with cap cells and posterior PGCs
(Fig. 6). These results show
that the strongly preferential entry of PGCs derived from anterior pole cells
into the nascent stem cell niche is not due to the differential orientation
mechanism.
|
Both anterior and posterior pole cells can give rise to GSCs in the testis
We also determined whether the anterior pole cells preferentially give rise
to GSCs in the testis, by using the same experimental approach described above
(also see Materials and methods). In male gonads, GSCs are established by the
first instar larval stage (Cooper,
1950). By the third instar larval stage, the testis contains 16-18
GSCs, all of which are anchored around hub cells
(Hardy et al., 1979
;
Lin, 2002
). In the adult
testis, the number of GSCs is reduced to five to nine
(Hardy et al., 1979
). Similar
to in the female gonad, anterior pole cells in the male embryonic gonad gave
rise to GSCs with a 100% frequency in larval-adult testes (n=5). In
these testes, labeled germ cells included one to five GSCs, and many
differentiated germ cells ranging from gonialblasts to sperm
(Fig. 7A-C). However, unlike in
female gonads, labeled GSCs were also generated from posterior pole cells.
Fifty percent of the examined pole cells gave rise to GSCs, while the
remaining 50% differentiated directly into gonialblasts
(Fig. 7D-F; n=6).
These results are consistent with the observation that
3914-expressing somatic cells exist in the posterior region of late
male embryonic gonad (see above). These observations indicate that the
GSC-inducing somatic cells may be present in the posterior region of the male
embryonic gonad (see Discussion).
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Discussion |
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The GSC fate is predetermined prior to GSC establishment
The process of cell fate determination is known to precede the
establishment of the determined cell type in a few studied systems. For
example, the initial cue for the determination of the anteroposterior axis of
the Drosophila embryo lies in its remote ancestor, the 16-cell
germline cyst that resides in the germarium of the ovary of the mother
(Manseau and Schüpbach,
1989). The posterior location of the oocyte in the cyst determines
the anteroposterior axis of the future embryo. As another example, the initial
determining cue for the oocyte fate within the 16-cell germline cyst appears
to be already present in its four-generation-removed precursor cell, the
cystoblast (Lin and Spradling,
1995
). The asymmetric retention of the spectrosomal material in
the daughter `cystoblast' during the subsequent four rounds of division
determines its fate as an oocyte. Here, we have shown that the GSC fate is
predetermined prior to oogenesis, with the predetermination apparently
occurring as a continuous process (see below), starting at least as early as
in the newly formed embryonic gonad; the location of a subset of pole cells in
the anterior half of the gonad provides the first positional cue that
predetermines the strong preference of their progeny for entering nascent stem
cell niches in the late third instar larval ovaries, and eventually their fate
as GSCs.
Embryonic gonad exerts somatic induction in GSC fate predetermination
Then, how does this positional cue determine the fate of future GSCs? Our
study suggests that, at least, a somatic induction mechanism is involved in
the process. Even if a subset of pole cells is destined to become GSCs upon
their formation as a result of the heterogeneous lineage mechanism, specific
interactions must occur between these `privileged' pole cells and anterior
somatic cells. Otherwise, such pole cells could locate to any region of the
gonad. This signaling function of the anterior somatic cells in the embryonic
gonad is consistent with their being the likely precursor cells of the future
niche cells.
The signaling from the anterior somatic cells to anterior pole cells must
be via direct cell-cell contact, because every somatic cell in the embryonic
gonads extends its cellular processes to envelop individual pole cells
(Jenkins et al., 2003;
Van Doren et al., 2003
).
Therefore somatic signalling via direct cell-cell contact would ensure that
anterior pole cells receive the signal, while preventing posterior pole cells
from receiving the signal.
Somatic induction may exist in the larval gonad for PGCs to enter the nascent niche by an active homing mechanism
Even though somatic induction in the embryonic gonad, either alone or
together with a heterogeneous lineage mechanism, is likely to be responsible
for initiating the GSC predetermination process, this embryonic mechanism
alone is not sufficient for GSC fate determination. This is because different
PGCs derived from the same pole cells can adapt different fates. We found that
approximately 17% of single anterior pole cells gave rise to both PGCs that
are in contact with cap cells in the late third instar larval ovary and PGCs
that are distant to cap cells (Fig.
4C). Correspondingly, approximately 14% of anterior pole cells
gave rise to both GSCs and cystoblasts in the adult ovary (data not shown).
These observations suggest that predetermination in the embryonic gonads is
somewhat flexible. Only those PGCs in contact with nascent niche cells in the
late third instar larval ovary appear to become GSCs.
How, then, do PGCs derived from anterior pole cells maintain their contact
with anterior somatic cells during larval development? Our study rules out any
significant role of static anchorage, differential mitotic orientation or
differential mitotic rate in the process (see Results). One possibility is
that, even though PGCs derived from an anterior pole cell become passively
dispersed during proliferation, they tend to populate the anterior region. Our
data do not rule out this mechanism. However, these data favor the active
homing mechanism. Passively dispersed PGCs derived from an anterior pole cell
are selectively sorted out into the niche cell/PGC interface in the larval
gonad as a result of niche signaling, where they become anchored to newly
formed cap cells during the larval-pupal transition. Such somatic signaling
could start in embryonic gonads. Consistent with this, a gap junction protein,
Drosophila Innexin 4, is present in pole cells in embryonic gonads,
even though its function at this stage is not clear
(Tazuke et al., 2002).
Regardless of when the somatic induction is initiated, it probably plays an
important role in ensuring that the progeny of anterior pole cells in the
embryonic gonad will sustain their proximity to the future stem cell
niche.
Potential role of the heterogeneous lineage mechanism in GSC fate determination
This study does not directly address the role of the heterogeneous lineage
mechanism in initial GSC fate determination during embryogenesis because we
did not distinguish individual donor pole cells for their intrinsic
properties, such as polar granule content or their initial position in the
embryo. However, our study suggests that the lineage mechanism is unlikely to
play any significant role in the maintenance phase of the predetermination
process during larval development, because a labeled pole cell, either
anterior or posterior, can produce both cap cell-contacting and non-contacting
PGCs in the larval ovary, as well as both GSC and cystoblasts in the adult
ovary (see Results). These results suggest that the lineage mechanism does not
govern the fate of pole cell progeny in the larval ovary.
A model for the determination of stem cell fate in the female germline
The discussion above allows us to propose a somatic induction hypothesis
for GSC fate determination during female germline development
(Fig. 8). In this model, the
anterior positioning of pole cells in the embryonic gonad provides the initial
positional cue that predetermines the fate of future GSCs. Such
predetermination is initiated by cell-cell interactions between somatic cells
and pole cells in the anterior half of the embryonic gonad, and is maintained
by signalling from anterior somatic cells in the larval gonad. Such somatic
signaling would lead to the active homing of PGCs derived from the anterior
pole cells to maintain their contact with the signaling cells in the larval
gonad. At the late third instar larval stage, the developing terminal filament
and cap cells express DE-cadherins and other molecules to anchor the adjacent
PGCs, transforming them into GSCs. Finally, it is possible that pole cells are
heterogeneous in their potential upon their formation, which might affect
their initial choice of position in the embryonic gonad. If so, the lineage
and somatic induction mechanisms would act in sequence to determine the GSC
fate.
|
Sex incompatibility of transplantation
As we did not select donor and host embryos according to their sex for
transplantation, 50% of transplanted embryos should have received pole cells
of the opposite sex. Previous studies have shown that XY pole cells in female
gonads never enter oogenesis. They can sometimes survive to enter
spermatogenesis, producing germline cysts containing 30-1000 cells. These
cells include undifferentiated germ cells, spermatocytes and many degenerating
cells (Steinmann-Zwicky et al.,
1989). In our experiments, however, no such cyst was detected,
suggesting that the transplanted male pole cells have degenerated in female
hosts before the adult stage. This degeneration could be due to the difference
of hosts: Steinmann-Zwicky et al.
(Steinmann-Zwicky et al.,
1989
) transplanted multiple pole cells into germlineless embryos,
whereas we transplanted only a few pole cells into wild-type embryos
containing a normal complement of endogenous pole cells. Thus, XY germ cells
may be effectively out-competed by the XX poles cells in the female
gonads.
XX pole cells in male gonads can also enter an abortive spermatogeneic
pathway. However, most of them degenerate before adult stage, even when they
were transplanted into an agametic gonad
(Steinmann-Zwicky et al.,
1989). As expected, we saw no oogenic cells in the testis.
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ACKNOWLEDGMENTS |
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Footnotes |
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