Howard Hughes Medical Institute, Waksman Institute and Department of
Molecular Biology and Biochemistry, Rutgers The State University of New
Jersey, Piscataway NJ 08854, USA
* Present address: INSERM UMR 384, Laboratoire de Biochimie, 28 place Henri
Dunant, 63001 Clermont-Ferrand, France
Author for correspondence (e-mail:
irvine{at}mbcl.rutgers.edu)
Accepted 6 August 2002
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SUMMARY |
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Key words: fringe, Notch, Polar cells, Oogenesis, Polarity
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INTRODUCTION |
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A Drosophila egg chamber, or follicle, is formed from a cyst of 16
inter-connected germline cells, and a similar number of somatic cells, which
surround them. One of the germline cells becomes the oocyte and adopts a
posterior localization inside the follicle, while the remainder become nurse
cells (Fig. 1)
(King, 1970;
Spradling, 1993
). Soon after
follicle formation, the somatic cells can be divided into three populations:
two pairs of polar cells at each end of the follicle; four to six
interfollicular stalk cells that separate one follicle from the next; and the
other follicular cells that surround the follicle. The polar and stalk cells
come from a common precursor, and they stop dividing in region IIb of the
germarium (Margolis and Spradling,
1995
; Tworoger et al.,
1999
). The other follicle cells continue to proliferate through
stage 6 of oogenesis, when they form a population of about 1000 cells. They
also become further subdivided into distinct groups that have specialized
roles during oogenesis, including border cells, stretched cells and
centripetal cells at the anterior of the follicle, posterior terminal cells at
the posterior of the follicle, and what we will refer to as central follicle
cells in the middle of the follicle (Fig.
1).
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The polar cells are located at the center of the distinct anterior and
posterior terminal follicle cell types
(Fig. 1), raising the
possibility that they could be involved in the process of follicle cell
patterning. Support for this possibility has begun to emerge from recent
studies on EGFR signaling (Gonzalez-Reyes
and St Johnston, 1998b), which promotes posterior follicle cell
fate; Hedgehog signaling, which is thought to exert general effects on
follicle cell proliferation and differentiation
(Forbes et al., 1996
;
Liu and Montell, 1999
;
Zhang and Kalderon, 2000
); and
JAK-STAT signaling, which influences border cell fate
(Beccari et al., 2002
;
Silver and Montell, 2001
).
Although some results from these studies suggest a role for polar cells in
patterning surrounding follicle cells, a clear understanding of polar cell
function is still lacking, as none of these studies involved a direct
manipulation of polar cell fate.
Recent studies have revealed that the polar cells are specified within the
polar-stalk lineage by the activation of the Notch signaling pathway
(Grammont and Irvine, 2001;
Lopez-Schier and St Johnston,
2001
). Notch is a transmembrane receptor protein that is involved
in a wide range of cell fate decisions during animal development (reviewed by
Artavanis-Tsakonas et al.,
1999
). Activation of Notch in the polar cells is dependent upon
the Notch pathway modulator fringe (fng), which encodes a
glycosyltransferase that can modify carbohydrates on EGF repeats of Notch
(Bruckner et al., 2000
;
Moloney et al., 2000
). In the
absence of fng or Notch function, polar cells do not form,
and the requirement for these genes in polar cell fate is strictly cell
autonomous. In most cases, the absence of polar cells during follicle
formation leads to compound follicles, in which multiple germline cysts are
enclosed within a single follicle cell epithelium. Even though the polar cells
cannot be distinguished by specific molecular markers until stage 3, these
observations established a function for polar cells much earlier, in region
IIb of the germarium. Although polar cells are required at this stage for the
separation of germline cysts into distinct follicles, in rare cases a follicle
lacking anterior or posterior polar cells can form and enclose a single
germline cyst.
The realization that fng is autonomously required for polar cell
fate and the existence of rare late stage follicles without polar cells in
fng genetic mosaics has presented for the first time the possibility
of determining the role of polar cells in follicle patterning. We report that
polar cells are required for the specification of both anterior and posterior
terminal follicle cell fates. Polar cells are also sufficient to induce at
least the immediately adjacent anterior cell fate, the border cells. Our
results indicate that the polar cells function as an organizer of terminal
follicle cell patterning. At the posterior, the terminal cells localize the
oocyte, and then participate in a reciprocal signaling process with the oocyte
that re-organizes the oocyte cytoskeleton and thereby establishes the
anteroposterior and dorsoventral axes of the oocyte
(Gonzalez-Reyes et al., 1995;
Roth et al., 1995
). Through
their influence on the posterior terminal cells, the polar cells are thus
ultimately responsible for the establishment of asymmetry during
Drosophila development.
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MATERIALS AND METHODS |
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Follicle staining
Histochemical and immunofluorescent staining of follicles was carried out
as described previously (Grammont and
Irvine, 2001). For Fig.
6G-J, X-gal and peroxidase staining were employed and the
fng clones are unmarked because the fixation protocol required to
detect cytoskeletal ß-galactosidase in the germline and MYC staining in
the soma by immunofluorescence was not consistent enough to reliably determine
the orientation of the cytoskeleton in those rare follicles with ectopic polar
cells. We used the same method to analyze posterior follicle fate
(Fig. 6E,F).
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Follicles were staged according to King
(King, 1970) and Spradling
(Spradling, 1993
). Because
fng mosaic follicles can be abnormally shaped, and lack border,
stretched and centripetal cells, we relied upon the following criteria: the
size of the follicle, the ratio of volume of the oocyte to the volume of the
nurse cells, the migration of the central follicle cells towards the oocyte,
and the stage of neighboring follicles within the same ovariole.
RESULTS
The primary strategy we pursued to investigate the role of the polar cells
in terminal follicle cell patterning was to genetically ablate the polar cells
by mutation of fng, and then to use molecular and morphological
criteria to assay the fates of the surrounding cells in these follicles.
Somatic follicle cells derive from two stem cells that are located in the
germarium (Margolis and Spradling,
1995). Follicles genetically mosaic for fng were created
by inducing mitotic recombination in animals heterozygous for a fng
null allele, and marked by the absence of expression of a synthetic Myc-tagged
protein in fng mutant cells. When this recombination event occurs in
one of the two follicle stem cells, many of the follicles that develop in the
ovariole will be a mosaic of wild-type cells derived from one stem cell and
fng mutant cells derived from the other stem cell. When the cells
that would ordinarily have formed polar cells are mutant for fng, no
polar cells form (Grammont and Irvine,
2001
). The absence of polar cells in these follicles can be
monitored by staining with antibodies that recognize the FasIII protein, which
is expressed specifically in the polar cells from stage 4 through the end of
oogenesis (Ruohola et al.,
1991
). Both Notch and fng are autonomously
required within polar cells, but we chose to focus our analysis on
fng because fng is only required in the polar cells during
follicle formation (Grammont and Irvine,
2001
). Even in the case of fng, the majority of mosaic
follicles lacking polar cells either form compound egg chambers or fail to
develop to stages late enough for the patterning of terminal follicle cells to
be assessed, and several thousand follicles had to be examined in order to
identify the examples described below.
Anterior polar cells are required for border cell formation
The border cells are a cluster of six to ten cells that are specified at
the extreme anterior of the follicle. They include the two anterior polar
cells, plus an additional four to eight adjacent terminal cells, the outer
border cells. The border cells leave the follicle epithelium at the beginning
of stage 9 of oogenesis, and then migrate between the nurse cells to the
oocyte. They can be recognized by their unique morphology and behavior during
stages 9 and 10 of oogenesis, when the small diploid border cells can be found
migrating through the large polyploid nurse cells
(Fig. 1B), and by the
expression of the slow border cells (slbo) gene, which
starts at stage 8 of oogenesis in the border cells during oogenesis, and is
required for their migration (Montell et
al., 1992; Rorth et al.,
2000
). We have thus also used a lacZ enhancer trap
insertion in slbo, slbo-lacZ, to aid in the identification of border
cells.
Altogether, we identified nine fng mosaic follicles at early stage 9 to late stage 10B of oogenesis that lacked anterior polar cells. All nine of these follicles lacked detectable border cells, as visualized by DNA staining (Fig. 2A). In addition, five of the nine carried the slbo-lacZ enhancer trap, which was not detectably expressed in any of these mosaic follicles (Fig. 2A). These observations indicate that fng is required for border cell fate, but because both border cells and polar cells are mutant for fng in these mosaics, they do not strictly localize the requirement for fng. Importantly, however, we also identified ten counter examples in which all of the most anterior terminal cells, including the outer border cells, were fng-, except for the two polar cells, which were fng+. In all cases, slbo-lacZ was expressed normally, the number of border cells appeared normal, and they migrated away from the anterior of the follicle (Fig. 2B). Together, these observations demonstrate that fng is not required within the outer border cells themselves, and instead that polar cells are essential for the specification of border cell fate.
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Anterior polar cells are required for stretched cell formation
The stretched cells comprise a population of about 40 to 50 cells that
undergo a transition from a columnar to a squamous epithelium during stage 9
of oogenesis (Fig. 1). This
morphological transition allows them to expand and cover the nurse cells when
the central follicle cells migrate towards and over the oocyte. The stretched
cells can be recognized morphologically by the middle of stage 9. At this
time, they also begin to express an enhancer trap insertion in an unknown
gene, MA33.
We identified six fng mosaic follicles from late stage 9 to late stage 10 of oogenesis without anterior polar cells. All six lacked detectable stretched cells as visualized by DNA staining (Fig. 3A). In addition, four out of the six also carried the MA33 enhancer trap line, but it was not detectably expressed in any of them (Fig. 3A). Interestingly, despite the absence of the stretched cells, the anterior follicular cells continue to migrate over the oocyte, such that many nurse cells are no longer covered by a follicular epithelium (Fig. 3A, Fig. 4A). These data demonstrate that fng is required for stretched cell determination. The requirement for fng was localized to the polar cells by the identification of 12 counter examples in which most anterior cells were fng- except the two anterior polar cells, which were fng+. In all 12 cases, MA33 was expressed and the stretched cells appeared morphologically normal (Fig. 3B). Thus, the polar cells are required for the specification of stretched cell fate.
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Anterior polar cells are required for centripetal cell formation
The centripetal cells consist of 30 to 40 follicle cells that migrate
during stage 10B of oogenesis in between the nurse cells and the anterior of
the oocyte. They are morphologically recognizable from stage 10B of oogenesis,
and can also be distinguished at this time by their expression of an enhancer
trap insertion in an unknown gene, BB127, and by expression of
slbo.
We identified three late stage 10B fng mosaic follicles without anterior polar cells. All three lacked centripetal cells, as visualized by DNA staining (Fig. 4A). In addition, one carried the slbo-lacZ enhancer trap, but it was not detectably expressed. Thus, fng is required for centripetal cell formation. The anterior of the oocyte is abnormally shaped in these follicles, which we attribute to the lack of centripetal cells (Fig. 4A). The requirement for fng was localized to the polar cells by the identification of seven counter examples in which all centripetal cells were fng- and the two anterior polar cells were fng+. In all cases, centripetal cells in the process of migrating between the nurse cells and the oocyte could be identified (Fig. 4B). Three of the follicles were from animals that carried the BB127 enhancer trap, and one was from an animal that carried slbo-lacZ. In all four cases, these markers of centripetal cell fate were expressed normally (Fig. 4B). Thus, the polar cells are required for the correct specification of centripetal cells.
Extra Polar cells are sufficient to induce additional border
cells
The observations described above demonstrate that anterior polar cells are
required for the specification of other anterior terminal cell fates. In order
to investigate whether polar cells are sufficient to induce these fates, we
assayed the consequences of increasing the number of polar cells by inducing
the production of clones of cells expressing a constitutively-activated form
of the Notch receptor. Expression of activated-Notch can induce additional
polar cells (Grammont and Irvine,
2001). However, only cells at the extreme termini of the follicle
(presumably cells of the polar/stalk lineage) are competent to respond to
Notch activation by becoming polar cells. Thus, expression of activated-Notch
can increase the number of polar cells at their normal location at the end of
the follicle, but can not induce polar cell formation at ectopic
locations.
Sixteen examples of stage 9 or 10 follicles with additional polar cells, as assayed by expression of FasIII or neuralized-lacZA101, were identified and characterized. All sixteen were associated with an increase in the number of outer border cells. In eleven of these cases, all cells in the follicle expressed activated-Notch (Fig. 5A,C), yet aside from the formation of extra polar and border cells, no morphological defects were observed. In the other five examples, activated-Notch was only expressed in two or three cells (Fig. 5B). These cells were polar cells and were surrounded by border cells. Thus, the formation of additional border cells does not require the expression of the activated Notch in any follicular cells besides the polar cells. In three of the examples, the wild-type polar cells could be clearly identified, as they did not express activated-Notch. The presence of extra polar cells thus does not inhibit the formation of the normal polar cells, nor does it inhibit their function as these wild-type polar cells were surrounded by outer border cells and they migrated normally (Fig. 5B). In all cases, the additional polar cells are also surrounded by outer border cells, and they migrate normally through the nurse cells. In six examples, all of the border cells were together in one large cluster (Fig. 5C), while in ten examples the border cells were split into up to four independently migrating groups (Fig. 5A,B). These additional clusters of migrating border cells always include at least one polar cell. In eight of these cases, the migrating border cells included only a single polar cell, demonstrating that one polar cell is sufficient for the formation and the migration of a border cell cluster (Fig. 5A,B).
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Notably, the number of outer border cells in each migrating cluster correlates with the number of polar cells. When only a single polar cell was detected at the center of a cluster, an average of three outer border cells was observed (Fig. 5A,B). When two polar cells are observed, they were surrounded by approximately six outer border cells, as in wild-type follicles (12 examples, Fig. 5A,B). In the six examples where four polar cells were identified at the center of a cluster of border cells, they were surrounded by approximately twelve outer border cells (Fig. 5C). Thus, the number of outer border cells that are specified and migrate with each cluster is proportional to the number of polar cells.
Posterior polar cells control posterior localization of the
oocyte
Posterior terminal cells have two crucial functions during oogenesis: to
localize the oocyte to the posterior of the follicle at stage 1, and to
establish the developmental axes of the oocyte at stage 6 (see below). The
posterior localization of the oocyte is achieved through differential
adhesion: the oocyte expresses higher levels of E-cadherin than do other
germline cells, and the posterior follicular cells express higher levels of
E-cadherin than do central follicular cells
(Godt and Tepass, 1998;
Gonzalez-Reyes and St Johnston,
1998a
). Homophilic adhesion mediated by E-cadherin then maintains
the oocyte at the posterior of the follicle. Mosaic analysis has demonstrated
that the requirement for E-cadherin maps to posterior terminal cells, but not
specifically to the polar cells. To examine the role of the polar cells in
oocyte localization, we searched for fng mosaic follicles that lacked
posterior polar cells.
Four fng mosaic follicles without posterior polar cells were
identified, and in all cases the oocyte was abnormally localized to the
anterior of the follicle, in contact with the anterior polar cells
(Fig. 6A). The anterior
terminal cells also express elevated levels of E-cadherin, and posterior
clones mutant for E-cadherin similarly result in mislocalization of the oocyte
to the anterior. Importantly, fng mosaics in which only the two polar
cells are fng+, and all other posterior cells are mutant,
form wild-type follicles with a posteriorly located oocyte (four examples), as
described previously (Grammont and Irvine,
2001). Together, these observations localize the requirement for
fng in oocyte positioning to the polar cells, and demonstrate that
posterior follicle cells are unable to localize the oocyte in the absence of
polar cells.
To determine whether a polar cell-dependent terminal cell fate is required for the initial asymmetric localization of the oocyte within the follicle, or only for its maintenance, we examined fng mosaics at stage 1, when the oocyte first becomes localized to the posterior. As no markers are available for polar cells at this stage, we looked for stage 1 follicles surrounded entirely by fng- cells, which can not form polar cells. We identified ten fng- stage 1 follicles, and the oocyte was mislocalized in all ten (Fig. 6D, compare with 6C). Thus, the dependence of oocyte positioning on fng begins during follicle formation and not later, during follicle maturation. Because fng acts cell-autonomously to specify polar cell fate, and is later not required in any other follicle cells for oocyte localization, these observations imply that the polar cells have an essential role during early oogenesis in oocyte localization, well before they become recognizable through their expression of specific molecular markers.
In 33 cases, fng mosaic follicles were recovered that lacked
posterior polar cells, but instead had two polar cells along the lateral side
of the follicle (Fig.
6B,F,H,J). Although we do not understand how these abnormally
constructed follicles arise, they presented an opportunity to investigate the
ability of ectopic polar cells to control the position of the oocyte. In all
cases, the oocyte was in contact with these mislocalized polar cells rather
than at the posterior or anterior termini of the follicle. As prior studies
have ruled out any role for the oocyte in the induction of polar cell fate
(Oh and Steward, 2001) and for
the polar cells in the specification of the oocyte
(Grammont and Irvine, 2001
;
Lopez-Schier and St Johnston,
2001
), we conclude from the co-localization of the oocyte and the
polar cells that the polar cells are not only necessary but also sufficient to
direct the localization of the oocyte within the follicle.
We note also that when these follicles lack normal anterior as well as
posterior polar cells, they tend to be round, or even elongated perpendicular
to the axis of the ovariole (Fig.
6J). This phenotype is reminiscent of that of Leucocyte
antigen related (Lar) mutations
(Frydman and Spradling, 2001).
Lar encodes for a receptor-like tyrosine phosphatase, which is
required for epithelial planar polarity during oogenesis. Our observations are
thus consistent with a hypothesized role for the polar cells in a
Lar-dependent reorganization of actin filaments that influences
follicle elongation (Frydman and
Spradling, 2001
).
The posterior polar cells control responsiveness to the EGFR signal
that establishes the anteroposterior and dorsoventral axes of the oocyte
Posterior terminal cells also have a second crucial function during
oogenesis, in establishing the anteroposterior and dorsoventral axes of the
egg through reciprocal signaling with the oocyte. Prior to stage 6, a
microtubule organizing center (MTOC) is located at the posterior of the
oocyte, resulting in a network of microtubules with their minus ends at the
posterior of the oocyte and their plus ends at the anterior of the follicle
(Theurkauf et al., 1992). At
stage 6, the oocyte signals to the follicle cells through the EGFR ligand
Gurken (Gonzalez-Reyes et al.,
1995
; Roth et al.,
1995
). This signal represses anterior terminal follicle fate and
establishes posterior terminal follicle fate
(Gonzalez-Reyes and St Johnston,
1998b
). The posterior follicular cells then send back an unknown
signal to the oocyte that inactivates the existing MTOC
(Deng and Ruohola-Baker, 2000
;
Gonzalez-Reyes et al., 1995
;
MacDougall et al., 2001
;
Roth et al., 1995
). In
parallel, a new microtubule network is established with the minus ends of the
microtubules at the anterior of the oocyte and the plus ends at the posterior
(Theurkauf et al., 1992
). This
new microtubule network is essential for the correct localization of the
anterior, posterior and dorsal determinants within the oocyte, and
consequently for the later establishment of embryo polarity
(Gonzalez-Reyes et al., 1995
;
Neuman-Silberberg and Schupbach,
1993
; Roth et al.,
1995
). The posterior terminal cells also express specific genes or
markers of posterior identity, such as the pointed gene
(Fig. 6E)
(Gonzalez-Reyes and St Johnston,
1998b
).
Prior studies have established that only posterior terminal cells, and not
central follicle cells, are competent to express these genes and to signal
back and inactivate the first MTOC in response to the Gurken signal
(Gonzalez-Reyes et al., 1997;
Gonzalez-Reyes and St Johnston,
1998b
). These studies also show that this competence does not
depend on signaling from the oocyte
(Gonzalez-Reyes and St Johnston,
1998b
). Although this competence maps to posterior terminal cells
and not specifically to polar cells, we hypothesized that the distinct
behavior of the posterior terminal cells could nonetheless be established by
signaling from the polar cells. It is not possible to examine the fate of
posterior cells or the microtubule network in oocytes without posterior polar
cells, as in such mosaics the oocyte simply relocalizes to the anterior. Thus,
to address the question of whether the polar cells can induce a terminal fate
in neighboring follicular cells, we analyzed fng mosaic follicles
that lacked posterior polar cells and instead possessed two lateral polar
cells. As described above, the oocyte localizes to the side of the follicle in
these cases.
In 7/7 fng follicles with lateral polar cells that carried an
enhancer trap in the pointed gene,
pnt-lacZS99812, ß-galactosidase staining was observed
in follicle cells surrounding the ectopic polar cells
(Fig. 6F). Thus, ectopic polar
cells induce a competence in neighboring cells to respond to the Gurken signal
by expressing a posterior follicle marker. If these cells are fully functional
posterior cells, they should also be able to signal back to the oocyte to
inactivate the first MTOC. To examine the polarity of the microtubule
cytoskeleton, we took advantage of previously established nod:lacZ
and kin:lacZ reporter constructs
(Clark et al., 1997). These
constructs fuse the minus end directed motor Nod or the plus-end-directed
motor kinesin to ß-galactosidase, and consequently they serve as
reporters of the minus and plus ends of microtubules
(Fig. 6G,I). In seven out of
seven fng follicles with lateral polar cells that carried the
kin:lacZ marker, ß-galactosidase staining was observed where the
oocyte contacts follicle, near the polar cells
(Fig. 6H). In seven out of
seven fng follicles with lateral polar cells that carried the
nod:lacZ marker, ß-galactosidase staining was observed where the
oocyte contacts the nurse cells, far from the polar cells
(Fig. 6J). Thus, in these
lateral oocytes the microtubule cytoskeleton is oriented perpendicular to the
normal AP axis of the follicle, but is nonetheless correctly established with
respect to the polar cells. We conclude from this that the follicle cells
surrounding these lateral polar cells have been instructed by the polar cells
to adopt a terminal follicular fate that renders them competent to adopt a
posterior fate in response to Gurken signaling from the oocyte.
upd is not required for terminal follicle cell fates
The results of the experiments described above indicate that that polar
cells signal to neighboring anterior and posterior terminal cells to influence
their fate. The upd gene encodes a ligand for the JAK-STAT pathway
that is expressed specifically in the polar cells and is required for normal
follicle formation (Baksa et al.,
2002; Harrison et al.,
1998
; McGregor et al.,
2002
; Sefton et al.,
2000
). In order to investigate whether upd could account
for some or all of the signaling capabilities of the polar cells, we initiated
experiments to analyze the consequences of removal of upd on polar
cell-dependent terminal fates. Recently, two other laboratories have published
the results of investigations into the role of the JAK-STAT pathway in
follicle patterning (Beccari et al.,
2002
; Silver and Montell,
2001
). These studies demonstrated that upd is required
for the normal recruitment and migration of the border cells, but did not
directly address the potential role of upd in other terminal fates.
Thus, we present here briefly the results of our own analysis of follicles
that are mosaic for a null allele of upd.
We examined eight stage 9-10A mosaic follicles with
upd- anterior polar cells. As judged by DNA staining,
these follicles contain fewer than normal border cells, and these border cells
fail to migrate (data not shown) (Silver
and Montell, 2001). Although this indicates that upd
influences the outer border cells, this phenotype is significantly milder than
the complete absence of border cells that is observed upon genetic ablation of
the anterior polar cells. Examination of DNA staining in stage 9 to 10
follicles with upd- anterior polar cells revealed the
presence of morphologically normal stretched cells (eight examples,
Fig. 7A), while examination of
DNA staining in stage 10B follicles with upd- anterior
polar cells revealed the presence of morphologically normal centripetal cells
migrating at the interface between the nurse cells and the oocyte (six
examples, Fig. 7A). Thus,
upd is not essential for the differentiation of stretched or
centripetal cells.
|
We also identified 17 mosaic follicles with upd- posterior polar cells. In all cases, DNA staining and Nomarsky optics revealed the presence of a normally localized oocyte at the posterior part of the follicle (Fig. 7B). As a marker of the establishment of anteroposterior polarity, we determined the position of the oocyte nucleus in stage 9 or 10 follicles. In all cases (11 examples, Fig. 7B), the oocyte nucleus was correctly positioned in the anterior part of the ooplasm. Thus, upd is also not essential for the specification of surrounding terminal cells at the posterior of the follicle. The observation that upd mutant polar cells are able to assume most of the functions of polar cells in follicle patterning further implies that upd is not required for polar cell fate.
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DISCUSSION |
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At the anterior of the follicle, each of the three distinct cell types that
surround the polar cells, the border cells, the stretched cells, and the
centripetal cells, fail to form in the absence of polar cells. Instead, these
cells appear to adopt the fate of central follicle cells, which is to migrate
over the nurse cells towards and around the oocyte, leaving the anterior germ
cells uncovered. The conclusion that the polar cells serve as organizers of
follicle patterning is also supported by the ability of additional or
mispositioned polar cells to redirect the fates of neighboring cells. When the
number of anterior polar cells is increased by expression of an activated form
of the Notch receptor, the number of border cells is increased, and this
increase occurs in proportion to the number of extra polar cells. Similarly,
activation of the HH pathway can result in both extra polar cells and a
corresponding increase in border cells
(Forbes et al., 1996;
Liu and Montell, 1999
;
Zhang and Kalderon, 2000
).
In contrast to the anterior terminal cells, the posterior terminal cells do not exhibit obvious differences in morphology or behavior from central follicle cells. Nor are distinct molecular markers available, because all of the known posterior-specific genes are also targets of EGFR signaling from the oocyte, which cannot occur in the absence of polar cells due to the mislocalization of the oocyte. However, fng mosaic follicles are sometimes abnormally constructed such that polar cells are formed along the sides of the follicle, rather than at the posterior. These ectopic polar cells are sufficient to confer to the neighboring cells a posterior identity, which then directs the reorganization of the oocyte cytoskeleton.
Thus, we conclude that polar cells are both necessary and sufficient to
direct the fates of surrounding cells. The polar cells exhibit the hallmarks
of an organizer because they not only influence the fates of surrounding
cells, but they establish distinct cell fates at different distances, and they
can redirect the fates of surrounding cells at ectopic locations. Although the
induction of distinct fates at different distances normally only occurs at the
anterior of the follicle, the capacity to establish these distinct fates also
exists at the posterior, but is suppressed there by EGFR signaling
(Gonzalez-Reyes and St Johnston,
1998b). The observations that distinct cell fates arise in rings
at discrete distances from the polar cells at both poles in the absence of
EGFR signaling pointed to the polar cells as a potential source of a signal
that directs terminal fate (Gonzalez-Reyes
and St Johnston, 1998b
). Our analysis confirms this hypothesis and
leads to an understanding of terminal patterning as a two-step process in
which cells at each end of the follicle first receive identical polar-cell
signals that distinguish terminal from central follicle cell fates, and then
later GRK signaling from the oocyte represses anterior fates and promotes
posterior fate (Fig. 8).
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As is the case for boundary organizers in the Drosophila wing and
eye, the polar cells are specified by Notch activation. Prior studies have
revealed that the Notch pathway is required for the differentiation of the
border and centripetal cells, and for the establishment of posterior terminal
identity. These studies were conducted with a temperature sensitive allele of
Notch, and the location of the requirements for Notch was
not mapped (Gonzalez-Reyes and St
Johnston, 1998b; Keller Larkin
et al., 1999
). Our results raise the possibility that these
previously established requirements for Notch in terminal cell
patterning can be accounted for by its requirement for polar cell
specification. However, Notch also has other roles in oogenesis, and
the differentiation of all follicle cells, including the terminal cells,
appears to be additionally influenced directly by Notch activation that is
dependent upon Delta signaling from germline cells
(Deng et al., 2001
;
Lopez-Schier and St Johnston,
2001
).
Nature of the polar cell signal
Although more complex models are possible, two basic mechanisms for
organizer activity are a morphogen or a signal relay. In the morphogen model,
a signal would be produced by polar cells and then spread to all of the cells
whose fate is polar-cell dependent. The signal would exist in a concentration
gradient, with different amounts of the signal being required to induce the
distinct border, stretched and centripetal cell fates. In the signal relay
model, the polar cells would produce a short range signal that induced border
cells, which would then produce a second signal that induced stretched cells,
which would then in turn express a third signal that induced centripetal
cells. Although these models can not be definitively distinguished until the
polar cell signal(s) are identified, several observations together suggest
that a combination of the morphogen and signal relay mechanisms are actually
employed (Fig. 8).
In support of a single long-range signal, reduction in the number of border
cells by mutation of components of the JAK-STAT pathway does not result in any
obvious reduction of the number of stretched or centripetal cells. Nor does
ablation of the border cells by expression of a toxic protein exert obvious
effects on stretched or centripetal cell fate
(Han et al., 2000). Thus, the
establishment of more distant polar-cell dependent cell fates does not require
the establishment of intervening cell fates. Conversely, however, the
determination that the specification of the centripetal cells depends in part
upon DPP signaling from the stretched cells supports the idea of a signal
relay from the stretched to the centripetal cells
(Deng and Bownes, 1997
;
Dobens et al., 2000
;
Peri and Roth, 2000
).
Further insight into the nature of the polar cell signal may be gleaned
from the time and distance over which it acts. Polar cell signaling must occur
at the anterior and at the posterior of the follicle prior to stage six, when
terminal cells are required to confer a distinct responsiveness to EGF-R
signaling from the oocyte (Gonzalez-Reyes
and St Johnston, 1998b). Additionally, recent observations suggest
that the polar cells behave as polarization centers with respect to the
organization of F-actin within the follicular cells, which gradually aligns
from the poles to the center of the follicle during stages 5-7
(Frydman and Spradling, 2001
).
This phenomenon suggests that a signal should exist from the polar cells even
before the end of follicle cell proliferation at stage 5.
Only one gene, upd, is known that encodes for a signaling molecule
that is expressed by polar cells (Baksa et
al., 2002; Harrison et al.,
1998
; McGregor et al.,
2002
; Sefton et al.,
2000
). Although loss of upd, or other components of the
JAK-STAT pathway, reduces the number of border cells
(Beccari et al., 2002
;
Silver and Montell, 2001
),
this contrasts markedly with the complete elimination of border cells observed
in the absence of polar cells. Moreover, loss of upd does not have
obvious effects on any of the other terminal cell fates that are polar-cell
dependent. Thus, the existence of additional signaling molecules must be
invoked to account for the organizing activity of the polar cells.
Establishment of asymmetry in Drosophila development
The anteroposterior and dorsoventral axes of Drosophila are
established during oogenesis by localized determinants. These consist of mRNAs
for bcd and nanos localized, respectively, at the anterior
and the posterior pole for the AP axis, and mRNAs for grk around the
oocyte nucleus for the DV axis
(Neuman-Silberberg and Schupbach,
1996; Riechmann and Ephrussi,
2001
) (Fig. 8). The
localization of these mRNAs is dependent upon the establishment of the correct
polarity of the microtubule cytoskeleton. Thus, prior work has made it
possible to trace the establishment of both the AP and the DV axes of
Drosophila back to the signaling process between oocyte and terminal
follicle cells that regulates the oocyte cytoskeleton
(Gonzalez-Reyes et al., 1995
;
Roth et al., 1995
;
Theurkauf et al., 1992
). This
signaling further requires that the oocyte be correctly localized to the end
of the follicle, which is dependent upon differential cadherin expression in
the germarium (Godt and Tepass,
1998
; Gonzalez-Reyes and St
Johnston, 1998a
).
Our observations now allow us to trace the origin of asymmetry back further, to the specification of the polar cells in the germarium, and their initial contact with the oocyte (Fig. 8). As the germline cysts move from region IIb to region III of the germarium, the oocyte localizes to the posterior of the cyst. This localization is dependent upon the polar cells, presumably because of their ability to upregulate the expression of E-cadherin. Although we cannot distinguish between the possibilities that the autonomous upregulation of E-cadherin effects oocyte localization, or that the non-autonomous upregulation induced by polar cell signaling also contributes to oocyte localization, in either case, oocyte localization and hence the initial AP asymmetry of the follicle, is established by the polar cells. All available evidence indicates that the anterior and posterior polar cells are equivalent, and the posterior localization of the oocyte is likely a consequence of the more advanced development of the posterior follicle cells surrounding region IIb cysts, which thus have the first opportunity to localize the oocyte (Fig. 8).
The localization of the oocyte at the posterior is then a necessary precondition for the second essential role of the polar cells in establishing oocyte polarity, which is to promote terminal follicle cell fate. Terminal follicle cell fate then confers a distinct responsiveness to EGFR signaling from the oocyte, which is manifest in their ability to signal back to the oocyte to destroy the initial posterior MTOC. Destruction of the initial MTOC in turn allows establishment of the correctly polarized microtubule cytoskeleton that is necessary for the ultimate establishment of the AP and DV axes. Notably, the dual roles of the polar cells in initiating the establishment of the axes of the oocyte thus work in concert, as the localization of the oocyte to the posterior of the follicle by the polar cells places the oocyte in the correct position to participate in the later reciprocal signaling process with terminal follicle cells.
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ACKNOWLEDGMENTS |
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