Institut Jacques Monod, (UMR 7592 CNRS/Université Pierre et Marie Curie/Université Denis Diderot), Laboratoire de Génétique du Développement et Evolution, 2-4, place Jussieu, 75251 Paris Cedex 05, France
* Author for correspondence (e-mail: pret{at}ijm.jussieu.fr)
Accepted 26 November 2002
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SUMMARY |
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Key words: Oogenesis, Polar cells, Cell lineage, Apoptosis, Drosophila melanogaster
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INTRODUCTION |
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Both somatic and germline cell populations derive from stem cells present
in anterior structures of the ovary called germaria
(Lin and Spradling, 1993;
Margolis and Spradling, 1995
;
Wieschaus and Szabad, 1979
).
Each germarium is connected to a polarized string of maturing egg chambers
that constitute an autonomous structural unit termed ovariole (for reviews see
King, 1970
;
Spradling, 1993
;
Spradling et al., 1997
). The
assembly of 15-20 ovarioles forms an ovary. The germarium has been divided
into three distinct subregions according to morphological criteria. Anterior
region 1 contains 2 to 3 germline stem cells (GSCs) and their differentiating
daughter cells termed cystoblasts. Each cystoblast undergoes exactly four
mitosis with incomplete cytokinesis to produce a syncytium of 16 cystocytes
known as a germline cyst. One of these cells develops into the oocyte, while
the 15 remaining cells develop into nurse cells. Complete cysts mature through
region 2a and become enveloped individually in region 2b by inwardly migrating
somatic cells (or prefollicular cells) deriving from approximately 2 somatic
stem cells (SSCs) lying at the border between regions 2a and 2b. The
prefollicular cell population diverges soon after to give rise to
interfollicular cells organized in stalk structures, a pair of polar cells at
each extremity of the egg chamber, and follicular cells forming a polarized
epithelium around the newly formed egg chamber. Region 3 of the germarium
corresponds to a stage 1 egg chamber that will bud off upon completion of
stalk formation. The follicular cell epithelium will not remain as a uniform
layer as different follicular cell populations distinguishable by specific
gene expression patterns and morphogenetic properties are defined along the
anteroposterior axis. In particular, the terminal anterior follicular cells
become subdivided into 3 distinct follicular cell types at mid-oogenesis
[stage 9 according to Spradling
(Spradling, 1993
)]: (1) border
cells that delaminate from the epithelium and migrate between the nurse cells
to reach the oocyte, (2) stretched or squamous cells that spread to cover the
nurse cells and (3) centripetal cells that migrate between the oocyte and the
nurse cells (Spradling,
1993
).
Although polarization of the follicular epithelium along the AP axis is not
detected before stage 6, the positioning of this axis can be visualized in egg
chambers as early as stage 2, by the presence of terminal polar cells located
at each pole of the egg chamber. Polar cells are pairs of particular
follicular cells that express specific markers and differ from neighboring
follicular epithelial cells in that they have a round morphology and lack
apicobasal polarity (Ruohola et al.,
1991; Spradling,
1993
). They have been shown to assume various functions during
oogenesis, including individualization of germline cysts and control of stalk
formation in late germarial regions
(Grammont and Irvine, 2001
;
Lopez-Schier and St Johnston,
2001
), induction of planar epithelial polarity starting at stage 5
(Frydman and Spradling, 2001
)
and recruitment of border cells at stage 9
(Liu and Montell, 1999
;
Silver and Montell, 2001
).
Polar cell specification has been shown to require critical levels of both
Delta/Notch and Jak/Stat signaling pathways. Indeed, removal of either Notch
or Su(H) function in somatic cells leads to a complete loss of polar cells
(Lopez-Schier and St Johnston,
2001
). In addition, overexpression of Unpaired (Upd), a
Drosophila Jak pathway ligand, frequently results in the
disappearance of polar cell marker-expressing cells
(McGregor et al., 2002
). Once
specified, polar cells are in turn assumed to achieve their different
functions by producing specific signals, in particular early Delta1/Notch
signal to specify stalk cells
(Lopez-Schier and St Johnston,
2001
), a presently unknown signal to reorganize planar actin
filament bundles during mid-oogenesis
(Frydman and Spradling, 2001
),
and Upd-mediated Jak/Stat-activating signal to recruit border cells and
pattern anterior follicular cells (Beccari
et al., 2002
; Silver and
Montell, 2001
).
Despite the crucial role played by polar cells, little is known about their
origin. Previous analysis of marked mitotic clones revealed that polar cells
stop dividing in region 2b of the germarium, long before the rest of the
follicular epithelial cells (which stop at stage 6), suggesting that they are
already determined at this stage (Margolis
and Spradling, 1995). A second clonal analysis confirmed this
result and further showed that polar cell pairs share common precursors with
cells of the adjacent interfollicular stalk, these precursors being distinct
from those of follicular cells (Tworoger
et al., 1999
). Nonetheless, these studies did not describe the
lineage relationship between polar cells of a given pair, nor the mechanisms
leading to specification of invariantly 2 polar cells at a given extremity. In
addition, in these studies, only stage 6 or older egg chambers were examined,
while earlier egg chambers are of particular interest since previous reports
indicate that polar cell markers can be expressed in more than 2 cells at the
extremities of some egg chambers that have just budded
(Forbes et al., 1996
;
Grammont and Irvine, 2001
;
Ruohola et al., 1991
). In the
present work, we have thus concentrated on early polar cell development by
re-investigating the polar cell lineage in early stage egg chambers. We show
that establishment of polar cell pairs is a several step process controlled,
in part, by apoptosis.
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MATERIALS AND METHODS |
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Egg chamber staining procedure
Immunocytochemistry was performed as previously described
(McKearin and Ohlstein, 1995).
The following antibodies were used in this study: mouse monoclonal
anti-Fasciclin III (1:30; Developmental Studies Hybridoma Bank), rabbit
polyclonal anti-ß-galactosidase (1:200; Boehringer Mannheim).
Flourescence-conjugated secondary antibodies were purchased either from
Jackson Immunoresearch Laboratories or from Molecular Probes. All were used at
a 1:200 dilution. Actin was labeled with Alexa-conjugated phalloidin
(Molecular Probes) at 0.1 µg/ml for 1 night in PBT1X. All samples were
mounted in cytifluor (Kent).
The TUNEL (TdT-mediated dUTP nick end labeling) assay was carried out using the In Situ Cell Death Detection Kit (Roche). Ovaries were dissected in PBS1X, then fixed for 20 minutes in 4% paraformaldehyde and rinsed three times in PBT (0.3% Triton X-100). Ovaries were then incubated in the TUNEL reaction mixture (including FITC-conjugated modified nucleotides and terminal deoxynucleotidyl transferase) for 1 hour at 37°C. The samples were rinsed in PBT and stained for other markers using the classical procedure cited above. TUNEL signal was visualized directly under a FITC filter.
Clonal analysis
For cell lineage analysis, females of the following genotypes were
analyzed: hsp-flp/+; A101(neu-lacZ)/Act>CD2>Gal4,
UAS-GFP and fuJB3,
hsp-flp/fuJB3;
A101(neu-lacZ)/Act>CD2>Gal4, UAS-GFP
(Neufeld et al., 1998).
Females were heat-shocked upon eclosion for 25-30 minutes at 32°C, placed
at 29°C, and dissected either 20 hours (±4 hours) or 44 hours
(±4 hours) later.
As described previously (Tworoger et
al., 1999), the
-squared test was applied to the clonal
studies to establish cell lineage independence between supernumerary
A101+ cells and A101- follicular epithelial cells.
Recovered GFP+ clones were divided into 3 classes: clones
containing only A101+ cells, clones containing only
A101- cells and clones containing both A101+ and
A101- cells. A fourth class was defined by chambers with no
GFP+ cells. Observed numbers for each category were respectively:
8/110, 55/110, 7/110 and 40/110, while expected numbers were: 10/110, 57/110,
5/110, 38/110. The expected outcome for each category was predicted based on
independent induction of clones, i.e. with a frequency of clones containing
both A101+ and A101- epithelial cells equivalent to the
product of the frequency of clones containing only A101+ cells and
the frequency of clones containing only A101- cells.
Somatic overexpression of p35 was carried out by first applying a heat-shock to hsp-flp/+; UAS-p35/+; Act>CD2>Gal4/+ or fuJB3 hsp-flp/fuJB3; UAS-p35/+; Act>CD2>Gal4, UAS-GFP/+ mid-pupae for 1 hour at 37°C and dissecting females 5-8 days after eclosion. This allowed generation of large clones encompassing all follicular cells of a given ovariole.
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RESULTS |
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This progressive definition of polar-like cells is not specific to the A101 enhancer-trap line since we obtained similar results using other specific polar cell fate markers such as PZ80/unpaired-lacZ (Fig. 1C), fringe-lacZ and Fasciclin III (data not shown).
Thus, restriction in the number of polar cell marker-expressing cells seems to be a progressive process that is not completed before stage 5 of oogenesis.
Each group of A101+ cells originates from several polar
cell precursors
Given that groups of 3 to 5 polar cell marker-expressing cells were
observed in early stage egg chambers, we aimed at characterizing the lineage
relationship between these cells, in particular to determine whether they
actually originate from the polar cell lineage. Previous cell lineage studies
did not address these groups of A101+ cells, which are
characterized here for the first time
(Margolis and Spradling, 1995;
Tworoger et al., 1999
). In
addition, though the most recent of these studies examined the lineage
relationship between mature polar cells and stalk cells, or between stalk
cells and neighboring follicular cells, the lineage relationship between cells
within a given polar cell pair was not clearly described.
We therefore undertook a cell lineage analysis by generating dominantly marked clones using the flip-out/Gal4 system and a UAS-GFP reporter transgene (see Materials and Methods). Flipase expression was induced by mild heat-shock treatment of adult females in order to minimize the frequency of recombination events. Examination of stage 3 egg chambers 20 hours (±4 hours) after this treatment, or of stage 6 egg chambers 44 hours (±4 hours) after, allowed us to visualize small transient GFP+ clones whose progenitors were in region 2b at the time of flipase-induced GFP expression, and therefore to avoid observation of large somatic stem cell- or early prefollicular cell-derived clones.
We first analyzed the distribution of GFP+ cells within clones in which a recombination event occurred specifically within the population of precursors common to stalk cells and polar cells (i.e. clones containing both GFP+ stalk and A101+ cells). Examination of stage 3 egg chambers containing groups of 3 to 5 A101+ cells revealed that among 24 clones recovered, no GFP uniformly-marked groups of A101+ cells were found (Fig. 2A,B). This suggests that groups of 3 to 5 A101+ cells found in early stage egg chambers have a polyclonal origin. In addition, clones contained either 1 or 2 GFP+ stalk cells, coupled with either 1 or 2 GFP+/A101+ cells (Table 2). Whether 3, 4 or 5 A101+ cells were present within these clusters, no more than 2 GFP+/A101+ cells were ever observed. Since clones containing up to eight follicular cells could be found under these conditions (data not shown), this shows that A101+ cells and their direct precursors stop dividing earlier than follicular cells.
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Interestingly, as described for stalk cells
(Tworoger et al., 1999), no
clustering of sister cells was observed. Indeed, GFP+ cells
separated by unmarked (GFP-) cells could be found (data not shown).
In addition, GFP+ cells were found either in the middle
(Fig. 2A), or at the border
(Fig. 2B) of groups of 3-5
A101+ cells.
Since we show that early groups of 3 to 4 A101+ cells derive
from several precursors, it is possible that some of these cells do not derive
exclusively from the polar cell lineage, but could be recruited from the
follicular cell lineage, their proximity to the poles being responsible for
the induction of polar cell markers. In order to address this point, we
compared the relative proportions of egg chambers with GFP+ clones
containing only A101+ cells, only A101- epithelial
follicle cells, or both A101+ and A101- cells. If some
of the A101+ cells belong to the follicular cell lineage, we would
expect a high frequency of egg chambers with GFP+ groups containing
both A101+ cells and A101- follicular epithelial cells
in a given egg chamber extremity. In contrast, we found a low frequency of
this type of clone-containing egg chamber (6.36%, n=110),
statistically consistent with the frequency expected as a result of
independent flip-out events (=0.71, see Materials and Methods). All
precursors of A101+ cells have thus already diverged from the
follicular cell lineage in germarial region 2b.
Given that early groups of A101+ cells derive from several polar cell precursors, we wondered how the final pairs of polar cells are selected, and whether the mature polar cells always derive from the same direct precursor. We therefore examined GFP+ clones containing both stalk and polar cells, specifically in stage 3 and stage 6 egg chambers with only 2 polar cells at a given extremity. Among 10 clones recovered, clones containing 1 polar cell/1 stalk cell (6/10); 1 polar cell/2 stalk cells (1/10); 2 polar cells/1 stalk cell (2/10), or 2 polar cells/2 stalk cells (1/10) were found. The fact that polar cell pairs containing either 2 GFP+ cells (Fig. 2C), or 1 GFP+ and 1 GFP- cell (Fig. 2D) were observed suggests that polar cells of a given pair do not exclusively derive from the same precursor, and therefore that selection of mature polar cells does not result from a strictly lineage-based mechanism.
Altogether, our results indicate that early groups of A101+ cells (that we name pre-polar cells) derive from at least 2 different precursor cells, and that these precursors belong specifically to the polar cell lineage. In addition, our results also imply that a stochastic selection of 2 polar cells takes place from among the pre-polar cells produced in the germarium.
Supernumerary pre-polar cells are eliminated in an
apoptosis-dependent process
As described above, each pair of polar cells seems to be selected from a
group of several pre-polar cells. The next question addressed was how
supernumerary pre-polar cells are eliminated. We considered 2 possibilities:
first supernumerary pre-polar cells could loose their specific properties
(including marker expression and lack of cell polarity), thus becoming
indistinguishable from neighboring follicular cells, second, supernumerary
pre-polar cells could be physically eliminated. The second possibility was
tested by looking for apoptosis events using the TUNEL assay. Analysis of
ovaries from A101/TM6 females revealed that, from early stage 2 to early stage
5 egg chambers, some groups of 3 or more A101+ cells contain 1
TUNEL-positive cell (Fig. 3A,a2
arrow). This cell could be located at any position within the group (data not
shown). In addition, it was typically smaller and rounder than other
A101+ neighboring cells (Fig.
3a3, arrow) and also contained fragmented nuclei visible after
DAPI staining (Fig. 3a1, arrow). Detection of apoptosis events affecting pre-polar cells earlier was
largely hindered by the lack of an early polar cell marker. However, using
anti-Fasciclin III (Fas III) antibodies, we stained region 2b/3 prefollicular
cells and found Fas III+ cells also positively stained with the
TUNEL reagent (Fig. 3B',
arrowheads). Such cells could thus also correspond to pre-polar cells in the
process of being eliminated. Altogether, these results suggest that the
restriction in the number of polar cells occurs via apoptosis-induced cell
death.
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Given these results, blocking the endogenous apoptosis program would be
expected to prevent elimination of pre-polar cells and thereby to produce
supernumerary polar cells. We thus overexpressed the bacculoviral p35 caspase
inhibitor protein in somatic cells of the ovary using the flip-out/Gal4 system
(Hay et al., 1994). Induction
of large somatic clones covering entire ovarioles resulted in a significant
increase in the number of cells strongly expressing Fas III at the poles of
p35-overexpressing egg chambers compared to control egg chambers (3.47 versus
2.13 respectively, n=178, P<0.001). Groups of 3, 4, 5 and
even 6 Fas III+ cells were found at the poles of stage 5 and later
stage clonal egg chambers (Fig.
3D,D', compare with Fig. 3C,C', and data not shown).
Furthermore, restriction of p35 overexpression to the polar cell lineage using
a neuralized-Gal4 transgene
(Bellaiche et al., 2001
) also
produced supernumerary polar cells in advanced egg chambers
(Fig. 3E,E') and groups
of up to 6 Fas III+ cells could be found (data not shown).
Interestingly, in the clusters of polar cells produced under these conditions,
consistently, 2 cells do not express a reaper-lacZ construct while
the supernumerary cells do (Fig.
3F,F'). The reaper gene encodes a pro-apoptotic
protein that has been shown to function upstream of caspases to induce cell
death (White et al., 1996
). In
addition, expression of this gene is confined to cells destined to die. In
wild-type ovarioles, however, we did not detect expression of
reaper-lacZ in the germarium or in early groups of pre-polar cells,
possibly because of transient expression of reaper and rapid
elimination of apoptotic cells. In contrast, in ovarioles overexpressing p35
within the polar cell lineage, the terminal block in the cell death program,
downstream of reaper induction, probably leads to the accumulation of
a high level of ß-galactosidase (Fig.
3F,F'). Such a phenomenon has already been described in
midglial cells of the embryonic nerve cord where reaper expression is
difficult to detect by in situ hybridization in wild-type embryos, but easily
detectable after p35 overexpression (Zhou
et al., 1997
).
Altogether, these results strongly suggest that apoptosis-mediated cell loss occurs within the polar cell lineage in wildtype ovarioles to allow selection of 2 mature polar cells from a pool of pre-polar cells.
Mutations in the Hedgehog signal transducer fused result in
a delay in the polar cell differentiation program and allow visualization of
putative prepolar cells
The fused (fu) gene encodes a serine/threonine kinase
identified as a positive effector of the Hedgehog signal transduction pathway
in Drosophila embryo and imaginal disc development
(Alves et al., 1998;
Ingham, 1993
;
Limbourg-Bouchon et al., 1991
;
Sanchez-Herrero et al., 1996
).
In the ovary, Hedgehog signal transduction has been shown to control somatic
stem cell (SSC) proliferation. Indeed, SSC self-renewing properties are not
maintained in the absence of Hh signaling, whereas excessive Hh signaling
produces supernumerary stem cells and leads to the accumulation of poorly
differentiated somatic cells between egg chambers
(Forbes et al., 1996
;
Zhang and Kalderon, 2000
;
Zhang and Kalderon, 2001
). Our
previous analysis of fu mutations had indicated that fu
function is not involved in this process. Rather, we showed that
fu-dependent Hedgehog signal transduction is necessary for somatic
prefollicular cell differentiation and morphogenesis
(Besse et al., 2002
). In
particular, fu function seems to be required for correct timing of
the polar cell differentiation program. Indeed, fu mutant females
exhibit a global shift in the dynamics of A101 staining, as visualized after
anti-ß-galactosidase staining of
fuJB3/fuJB3; A101/+ females
(Fig. 4A', compare with
Fig. 1A). First, the appearance
of A101 staining is delayed, as 28% (10/36) of stage 2 fu egg
chambers do not exhibit any marked anterior cells compared to 19% (10/52) in
heterozygous sisters (Fig. 4C).
Second, restriction of A101 staining to 2 polar cells is also delayed, as 60%
(26/43) of stage 3 and 19% (7/36) of stage 4 fu egg chambers
contained 3 or more stained anterior cells compared to 33% (17/52) and 4%
(2/48), respectively, for fu+ egg chambers
(Fig. 4C). Strikingly, 100% of
stage 5 fu mutant egg chambers exhibit only 2 A101+ cells
(Fig. 4C), indicating that
restriction in the number of polar cells does eventually occur as in wild-type
ovarioles. Altogether, these results suggest that fu mutations lead
to a delay in the polar cell differentiation program.
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Close examination of fu ovarioles revealed that a higher
proportion of groups containing 4 A101+ cells (17/156), 5
A101+ cells (8/156), and even 6 A101+ cells (1/156) can
be found in stage 2 as well as stage 3 fu mutant egg chambers
compared to the wild-type situation (Fig.
4B', inset, C). We reasoned that the presence of such groups
of cells could result either from abnormally slow apoptosis-dependent
elimination of pre-polar cells, or from an overproduction of pre-polar cells
or their precursors. We could not directly test the first hypothesis because
the relatively low number of TUNEL-positive cells found in both wild-type and
fused females (possibly due to rapid elimination of apoptotic cells)
made it impossible to compare quantitatively the dynamics of polar cell
apoptotic cell death between these two contexts. We therefore tested the
second hypothesis and looked for defects in polar and pre-polar cell
proliferation, or in the number of polar cell precursor cells. First, polar
cell proliferative properties do not seem to be altered in the vitellarium of
fu ovarioles since (1) no increase in the size of A101+
terminal clusters is observed with increasing age of fu egg chambers
from stage 2 to 5 (Fig. 4C),
and (2) no prolongation beyond stage 6 of somatic cell mitotic activity is
observed in fu ovarioles (data not shown). Second, using the
dominantly marked clone approach described above, we showed that early
clusters of 4-6 A101+ cells found in fu females never
contain more than 2 GFP+ cells (n=35, data not shown), and
therefore that they do not result from extra divisions of precursor cells
within the germarium. Third, we reasoned that preventing apoptosis in the
polar cell lineage in a fu mutant context should give us an
indication about the number of polar cell precursors present in these flies.
If the number of such precursors is greater in fu females than in
wild-type females, then blocking apoptosis should result in a greater number
of "rescued" cells in polar cell clusters than in a wild-type
context (that is more than 6 cells). Therefore, we used the flp-out/Gal4
system to generate large somatic clones of p35 overexpressing cells in a
fu mutant context. The number of Fas III+ cells found in
terminal clusters of fuJB3
hsp-flp/fuJB3; UAS-p35/+; Act>CD2>Gal4 UAS-GFP/+
females was scored and compared to that of control fuJB3
hsp-flp/fuJB3; Sp/+; Act>CD2>Gal4 UAS-GFP/+ sisters.
Although an increase in the average size of the terminal Fas III+
cell cluster was observed after p35 overexpression (a mean number of 3.64
cells per cluster was observed in egg chambers from stage 3 to 7 compared to
2.45 in control females, n426, P<0.001), only groups
containing 2 to 6 cells were recovered. This indicates that fused
females contain the same number of polar cell precursors as wild-type
females.
Therefore, we interpret the supernumerary polar cells in both wild-type and fused mutant contexts to represent pre-polar cells, and propose that slower apoptosis-mediated reduction in the number of these cells in a fused context allows easier visualization of these cells. Thus, fused mutations, by delaying the somatic cell differentiation program, confirm the existence of pre-polar cell clusters and allow detection of up to 6 pre-polar cells. However, restriction in the final number of polar cells is achieved by stage 5 (Fig. 4C) and is probably also mediated by apoptosis since TUNEL-positive A101+ cells are found in fused females (data not shown).
Restriction in the number of polar cells is required for late egg
chamber development
Restriction in the number of polar cells, though progressive, seems to be
tightly controlled, as 100% of wild-type stage 5 egg chambers contain exactly
2 pairs of polar cells (Table
1). In order to determine the significance of this process, we
looked for possible defects associated with the production of extra polar
cells upon p35 overexpression. No obvious defects in early development of egg
chambers, for example in individualization of germline cysts or formation of
stalk structures, were observed (data not shown). Given that polar cells are
assumed to produce a signal required for planar polarization of follicular
cell actin filament bundles (Frydman and
Spradling, 2001), we then examined actin organization in somatic
epithelial cells of p35-overexpressing ovarioles. In wild-type ovarioles,
actin filament bundles are present basally and orient themselves
perpendicularly to the AP axis (Fig.
5A, insets). Polarization has been shown to arise gradually,
beginning at stage 5, and proceeding from the poles toward the center
(Frydman and Spradling, 2001
).
No perturbation, in either establishment or maintenance of actin filament
orientation was detected in egg chambers containing extra polar cells
(Fig. 5B and data not shown).
However, two aspects of anterior somatic cell morphogenesis seem to be
compromised in such ovarioles, whether p35 overexpression is ubiquitous or
restricted to the polar cell lineage. Normally, starting at stage 9,
anterior-most somatic follicular cells (or squamous cells) begin to stretch
over nurse cells, which results in both the elongation of these cells
(Fig. 5C') and thinning
of the epithelium (Fig.
5C'', inset) as visualized after phalloidin staining of actin
in these cells. Other consequences of these morphogenetic changes are a
reduction in the density of nuclei at the anterior pole and the development of
a pointed shape at the anterior end of the egg chamber, as observed upon DAPI
and phalloidin staining (Fig.
5C,C'). In p35-overexpressing ovarioles, anterior somatic
cell stretching is perturbed as about 80% (35/44) of stage 9 egg chambers with
multiple polar cells exhibited round anterior follicular cells
(Fig. 5D'), a constant
thickness of epithelial cells along the AP axis
(Fig. 5D'', insert) and a
homogeneous distribution of anterior follicular cell nuclei
(Fig. 5D). Nonetheless, stage
10 egg chambers with extra polar cells were produced, but 91% (21/23) of these
presented defects in delamination and migration of border cells. In wild-type
ovarioles, groups of 6-8 border cells detach from the anterior epithelium and
migrate through the nurse cells during stage 9, and reach the oocyte at stage
10. Anterior polar cells, though not by themselves migratory, are dragged
along by outer border cells and therefore can be used to mark the migratory
pathway (Fig. 5E,E', insets). In stage 10 egg chambers with extra polar cells, clusters of border
cells were either still stuck at the anterior pole
(Fig. 5F,F', insets) or
only partway between the anterior pole and the oocyte (data not shown). In
addition, these clusters were often observed to contain supernumerary border
cells, as a mean of 11 border cells was found in extra polar cell-containing
stage 10 egg chambers (compared to 7.2 in control females, n
26).
Interestingly, clusters of 5 or 6 polar cells recruited a higher number of
border cells than those containing only 3 or 4 (data not shown).
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Thus, prevention of pre-polar cell death leads to defects in both squamous and border cell morphogenetic properties, showing that polar cells act in a non cell-autonomous fashion to define anterior follicular cell identity.
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DISCUSSION |
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In addition, Tworoger and co-workers showed that polar and stalk cells
derive from a common population of precursor cells and together form a lineage
distinct from that of main body follicular cells
(Tworoger et al., 1999). Based
on this study, the authors proposed that all polar cells and stalk cells
separating two consecutive egg chambers derive from common precursors and
therefore form a so-called `polar cells-stalk cells-polar cells' unit.
However, since neither their work, nor our work indicates a lineage
relationship between polar cells at the extremities of a given unit, we prefer
to consider half of such a unit in the following working model. As shown in
Fig. 6, we propose that mature
polar cells and stalk cells originate from a several step process. First,
polar cell and stalk cell precursors (white circles, first row) would divide
at least twice to generate groups of cells within which pre-stalk and
pre-polar cells are specified. Divergence between stalk cell (blue circles)
and polar cell (pink circles) fate could occur either through a lineage-based
process (arrow 1), or after completion of divisions (arrow 2), through
cell-cell interactions between stalk and polar cell precursors (lateral
inhibition-type mechanism) or between stalk/polar cell precursors and other
neighboring cell types. A subsequent step would then be final differentiation
of stalk cells and polar cells from groups of pre-stalk and pre-polar cells.
Although the number and fate of pre-stalk cells remains to be determined, our
results indicate that polar cell pairs are generated through
apoptosis-mediated elimination of supernumerary pre-polar cells (see
below).
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Apoptosis as a way to refine cell lineage
Selective removal of cells by programmed cell death is an evolutionarily
conserved and widely used mechanism that plays a fundamental role in pattern
formation and morphogenesis during embryonic and adult development of
multicellular organisms (Rusconi et al.,
2000; Vaux and Korsmeyer,
1999
). Apoptosis, the most well studied form of programmed cell
death, is also involved in precise elimination of either unnecessary or
deleterious supernumerary cells within specific cell lineages, as exemplified
during C. elegans hermaphrodite development during which exactly 131
somatic cells are invariantly fated to die
(Ellis and Horvitz, 1986
).
Our study of the Drosophila ovarian polar cell lineage reveals
that each pair of mature polar cells derives from a pool of precursor
pre-polar cells within which supernumerary cells are eliminated via an
apoptosis-dependent mechanism. This mechanism probably requires both caspase
activity and the `death' gene reaper since it is inhibited by ectopic
expression of the bacculoviral p35 protein and is associated with specific
induction of reaper expression
(Hay et al., 1994;
Nordstrom et al., 1996
).
However, whereas the self-death machinery appears to be evolutionary conserved
(Meier et al., 2000
;
Song and Steller, 1999
), a
wide range of distinct signaling mechanisms can be used to elicit apoptosis.
Interestingly, a stochastic cell loss phenomenon has been documented in the
Drosophila embryonic nerve cord where a subset of midline glial cells
undergo apoptosis-mediated reduction in cell number between stages 13 and 16,
after these cells have facilitated commissure tract morphogenesis
(Sonnenfeld and Jacobs, 1995
).
In this system, induction of midline expression of p35 prevents the normal
reduction of midline glial cell number from 6 to, on average, 3.2, resulting
in a subsequent disruption of the central nervous system (CNS) midline
(Zhou et al., 1997
). At least
two cell-cell signaling pathways, the EGF and Notch pathways, are thought to
mediate cell-death in the ventral midline. However, although modulation of EGF
receptor activity clearly alters the pattern of midline cell death, the role
of the Notch signal transduction pathway is less clear, in part because the
latter is also required for cell fate specification
(Lanoue and Jacobs, 1999
;
Menne and Klambt, 1994
;
Stemerdink and Jacobs, 1997
).
In the ovarian system, cellular interactions within or without the pre-polar
cell cluster may also be crucial for regulation of the selective pre-polar
cell loss. In the present study, we were not able to make any correlation
between pre-polar cell position and cell removal, at least for apoptosis
events occurring after egg chamber budding. It would be interesting
nonetheless to examine Notch signaling as a survival factor in this system.
Indeed, induction of Notch loss-of-function clones in prefollicular cells is
associated with absence of polar cells
(Lopez-Schier and St Johnston,
2001
). Conversely, egg chambers with terminal clones expressing an
activated form of Notch contain up to 6 polar cell marker-positive cells
(Grammont and Irvine, 2001
).
Such phenotypes, interpreted as reflecting a role for Notch signaling in polar
cell specification, could also correspond to a Notch-dependent control of
apoptosis within the pre-polar cell lineage.
Considering both the embryonic and oogenesis systems, the question can be asked as to the biological significance of creating supernumerary cells to remove them afterwards. One proposed explanation for what is observed in the embryonic CNS is that a differential number of glial cells may be required depending on the developmental stage. By analogy, at least 6 pre-polar cells may be required in the ovary to assume early germarial functions, whereas only 2 polar cells may be needed during later egg chamber maturation. Another possibility is that the process of polar cell production corresponds to an evolutionarily conserved mechanism to which removal of deleterious cells would have been added later. Further studies are now needed to determine whether the pre-polar cell clusters have a functional role in early oogenesis.
Function of polar cells in the patterning of anterior follicular
somatic cells
Polar cells are involved in several important signaling processes during
oogenesis, including posterior positioning of the oocyte
(Tworoger et al., 1999;
Zhang and Kalderon, 2000
),
induction of stalk cells in the germarium
(Grammont and Irvine, 2001
;
Lopez-Schier and St Johnston,
2001
), organization of follicular cell epithelial planar polarity
during mid-oogenesis (Frydman and
Spradling, 2001
) and anteroposterior patterning of follicular
epithelial cells at stage 9 (Beccari et
al., 2002
).
Strikingly, polarization of basal actin filament bundles in the follicular
epithelium arises progressively, starting at stage 5 and proceeding from the
poles, suggesting the existence of a diffusible signal produced by polar
cells. Consistent with this, ectopic polar cells generated upon
hedgehog overexpression have the capacity to reorganize actin bundles
locally (Frydman and Spradling,
2001). However, orientation of planar actin filaments probably
does not require a precise level of polarizing signal since we have shown here
that an increase in polar cell number does not affect the establishment or
maintenance of planar polarity.
In contrast, we find that the restriction in the number of polar cells
seems to be required for correct anterior patterning of follicular epithelial
cells. Indeed, preventing the elimination of supernumerary pre-polar cells
results in morphogenetic defects affecting both stretching of anterior
squamous cells and migration of border cell clusters. The latter is also
accompanied by an increase in the number of recruited border cells. Production
of ectopic polar cells has been described to induce ectopic and poorly
migrating border cells at stage 9 (Liu and
Montell, 1999; Zhang and
Kalderon, 2000
). Yet, this phenomenon was observed upon ectopic
activation of hedgehog signal transduction (patched and
Costal 2 mutant contexts). Therefore it is not clear in these cases
whether migration defects are a direct consequence of extra border cell number
or whether they reflect an additional effect of the Hedgehog signaling pathway
on border cell migration and/or specification. In our study, we were able to
observe defective border cell migration after having prevented cell death
specifically within neuralized-expressing pre-polar and polar cells.
This suggests, first, that rescued pre-polar cells are not inert and, second,
that final polar cell number per se is critical for both border cell
recruitment and migration. Interestingly, polar cells show specific expression
of Unpaired (Upd), an extracellular ligand described to activate the conserved
JAK/Stat signaling pathway (Beccari et al.,
2002
; McGregor et al.,
2002
; Silver and Montell,
2001
). In the absence of positive effectors of this pathway, such
as Unpaired, Hopscotch or STAT92E, defects in both recruitment and migration
of border cells and sometimes also in stretching of squamous cells are
observed. In addition, ectopic expression of Upd in a subset of anterior
somatic cells is sufficient to induce expression of border cell markers in
adjacent squamous cells (Beccari et al.,
2002
; Silver and Montell,
2001
). Interestingly, high levels of Upd result in the formation
of egg chambers in which both normal and supernumerary border cells frequently
fail to migrate. Altogether, this suggests that Upd could act as a morphogen
produced by polar cells and necessary for establishing anteroposterior
patterning of the follicular epithelium. In the present study, prevention of
pre-polar cell death and subsequent generation of supernumerary polar cells
may lead to production of an excess of signaling molecules, such as Upd, and
alteration of endogenous morphogen gradients, which could explain why both
squamous cells and border cells exhibit aberrant behavior. Our results
therefore provide further evidence for a non cell-autonomous role for anterior
polar cells in patterning of the follicular epithelium.
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ACKNOWLEDGMENTS |
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