Institute of Signaling, Developmental Biology and Cancer, UMR 6543 CNRS, Parc Valrose, 06108 NICE cedex 2, France
Author for correspondence (e-mail:
noselli{at}unice.fr)
Accepted 30 August 2002
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SUMMARY |
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Key words: JAK/STAT pathway, Cytokine, Crumbs, Border cells, Follicle cells, Egg chamber, Drosophila melanogaster
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INTRODUCTION |
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In Drosophila, homologs of the mammalian JAK/STAT pathway
components have been isolated. hopscotch (hop) encodes a
Drosophila JAK homolog (Binari and
Perrimon, 1994) and marelle (mrl) codes for a
STAT factor (also known as Stat92E)
(Hou et al., 1996
;
Yan et al., 1996
). The only
known ligand for the Drosophila JAK/STAT pathway is encoded by the
unpaired (upd/os) gene
(Harrison et al., 1998
). Only
recently, Domeless (Dome; also known as Mom)
(Chen et al., 2002
) has been
identified as the first putative receptor for the JAK/STAT pathway in
Drosophila embryos (Brown et al.,
2001
). The reduced number of JAK/STAT pathway genes in
Drosophila contrasts with the multiplicity of JAK/STAT homologs found
in mammals, making Drosophila a suitable system to study JAK/STAT
signaling (Luo and Dearolf,
2001
; Mathey-Prevot and
Perrimon, 1998
; Zeidler et
al., 2000
). The Drosophila JAK/STAT pathway was
originally identified for its role in embryonic segmentation. Further work has
shown that this pathway also participates in blood cell determination and
proliferation, eye and wing development
(Luo and Dearolf, 2001
;
Zeidler et al., 2000
;
Zeidler et al., 1999
), sex
determination (Jinks et al.,
2000
; Sefton et al.,
2000
; Zeidler and Perrimon,
2000
) and stem cell differentiation
(Kiger et al., 2001
;
Tulina and Matunis, 2001
).
More recently it has been shown to play a role in oogenesis for stalk and BC
differentiation (Baksa et al.,
2002
; Beccari et al.,
2002
; McGregor et al.,
2002
; Silver and Montell,
2001
). Thus, as in mammals, the Drosophila JAK/STAT
pathway has a pleiotropic role and a common function in cell differentiation
and blood cell development.
Here we show that dome is involved in BC determination and migration. In addition, dome is required for follicle cell polarization through the control of the apical determinant Crumbs. The Dome protein is associated with apicolateral membranes in follicle cells, and becomes internalized upon exposure to Upd, its putative ligand. Our loss- and gain-of-function analyses show that dome is autoregulated in follicle cells, and that both the intracellular and extracellular domains are required for Upd transduction and BC migration. Our data show that Dome is an essential receptor molecule for Upd and JAK/STAT signaling during oogenesis.
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MATERIALS AND METHODS |
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Molecular biology and transgenic lines
Several independent P-element insertions (PL58, PL100, PG14, 12030, PG5,
PG35) (Bourbon et al., 2002)
have been identified in domeless and mapped in the 5'UTR region
of the gene between position -20 and +405 of the putative transcription start
site. A full-length cDNA (LD46805) was obtained from the Berkeley Drosophila
Genome Project (BDGP) and fully sequenced using an automated ABI DNA sequencer
(Accession Number, AY147847). The dome cDNA is 4805 bp long and
contains 478 bp of 5'UTR, 3846 bp of coding region and 480 bp of
3'UTR. It encodes a protein of 1282 amino acids, whose sequence is
identical to the one described previously
(Brown et al., 2001
;
Chen et al., 2002
). The
pUAS-dome construct was made by cloning a full-length dome
cDNA (LD46805) cut with EcoRI and XhoI and ligated into the
EcoRI and XhoI sites of the transformation vector pUAST.
pUAS-dome
EXT was constructed by cloning a
EcoRI-XhoI fragment containing the 5'UTR, and the
sequence encoding amino acids 1-27 (signal peptide) fused to amino acids
873-1281 (transmembrane and intracellular region) into the transformation
vector pUAST cut with EcoRI and XhoI.
pUAS-domeGFP was constructed by single step ligation of the following DNA fragments: (i) EcoRI-XhoI fragment containing the 5'UTR and coding region (1-1281), (ii) a XhoI-XbaI fragment encoding EGFP and (iii) the transformation vector pUAST cut with EcoRI and XbaI. This results in the insertion of GFP at the C terminus of a wild-type Dome protein. For each construct, several independent transgenic lines have been generated and tested.
Protein purification and antibody production
A glutathione S-transferase (GST) fusion protein containing Dome amino
acids 918-1113 was produced in E. coli and used to immunize female
New Zealand rabbits, according to standard protocols. The sera were collected
and tested by western blot analysis to ensure specificity (data not shown). To
purify the sera, a His-tagged-Dome (aa 918-1113) protein was produced in
E. coli. 1 mg of this protein was separated by SDS-PAGE, and blotted
onto a nitrocellulose filter. A strip containing the purified protein was used
to affinity purify the Dome antibodies. Following extensive washing, the bound
antibodies were eluted with elution buffer (200 mM glycine and 1 mM EDTA, pH
2.8). The eluate was neutralized immediately with one-tenth volume of 1 M Tris
(pH 8.0). This antibody was used on western blots and for
immunohistochemistry.
Immunohistochemistry and X-gal staining
Staining of egg chambers with X-gal or antibodies were performed as
described previously (Lasko and Ashburner,
1990; Suzanne et al.,
2001
; Tanentzapf et al.,
2000
). The following primary antibodies have been used: rabbit
anti-Dome (1:200), mouse anti-Fas3 (1:40; 7G10, Developmental Studies
Hybridoma Bank-DSHB), mouse anti-ß-galactosidase (1:1000, Promega), mouse
anti-Crumbs (1:50; Cq4, DSHB); rat anti-DE-cadherin (1:50, a gift from Hiroki
Oda), rabbit anti-Stat92E (1:500, a gift from Steven Hou). Secondary
antibodies used in this study were anti-rabbit FITC (fluorescein)-tagged
(1:400), anti-mouse CY3 (1:400; Molecular Probes). Confocal images were taken
with a Leica TCS-SP1 confocal microscope. Other images were taken using a
Nikon Coolpix 990 digital camera and processed with Photoshop 6.0 (Adobe).
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RESULTS |
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In dome mosaic egg chambers, some BC clusters are not formed, migrate aberrantly and/or contain less cells than normal (Fig. 1D,E). In these cases, dome mutant cells are always found in the anterior region of the egg chamber, indicating that they cannot be determined to become migratory BCs. In mosaic BC clusters, wild-type cells usually migrate normally or ahead of dome mutant cells (Fig. 1E; data not shown), suggesting differences in the migratory ability and adhesiveness of mutant and wild-type cells.
Interestingly, in heterozygous females the BC cluster shows a slow border cell migration phenotype, aberrant shape and/or incomplete number of cells in the cluster due to the detachment of some BCs (Fig. 1C; data not shown). This suggests a semi-dominant, dose-sensitive effect of dome in this process and a possible role of dome during migration itself.
In addition to an aberrant migration of the BCs, we also found defects in the encapsulation of early egg chambers (Fig. 1F). In some cases, this can lead to the fusion of adjacent egg chambers (Fig. 1G), suggesting that dome may also be involved in the assembly of egg chambers during early oogenesis (see below).
domeless interacts with members of the JAK/STAT pathway in
BC migration
dome is a maternal effect gene controlling tracheal development
and segmentation in embryos where it participates in JAK/STAT signaling
(Brown et al., 2001;
Chen et al., 2002
).
Interestingly, members of the JAK/STAT pathway, including upd, hop
and Stat92E, have recently been shown to be involved in oogenesis for
proper stalk and BC differentiation (Baksa
et al., 2002
; Beccari et al.,
2002
; McGregor et al.,
2002
; Silver and Montell,
2001
). However, it is not known whether Dome is a receptor for
JAK/STAT signaling during oogenesis and whether it is activated by the Upd
ligand. In order to address these questions, we tested for genetic
interactions between dome and other members of the JAK/STAT pathway
using BC migration as an assay. Since dome, upd and hop
genes are all located on the X chromosome, we have only been able to test
interactions with Stat92E (Hou et
al., 1996
; Yan et al.,
1996
) and the recently identified dpias (a.k.a.
Su(var)2-10) gene (Betz et al.,
2001
), which are positive and negative regulators of the JAK/STAT
pathway, respectively. Removing one copy of Stat92E aggravates
dome/+ BC migration phenotypes, while removing one copy of
dpias suppresses them (Fig.
1H). This supports a model in which dome, Stat92E and
dpias would participate in the same pathway. In order to test the
role of dome in JAK/STAT signaling more directly, we analysed Stat92E
expression in follicle cells. Upon activation, Stat proteins translocate into
the nucleus to regulate target gene transcription. In dome mutant
follicle cells, nuclear Stat92E is dramatically reduced
(Fig. 1I,J), as it is in cells
mutant for hopC111 (data not shown). Since the active,
nuclear form of Stat92E is strongly affected, these data indicate that
dome is required cell autonomously to activate JAK/STAT signaling
during oogenesis.
Dome is expressed in all follicle cells and localizes to apicolateral
membranes
In egg chambers, upd expression is restricted to two pairs of
polar cells, which are located at the most anterior and posterior parts of the
follicle cell layer (Fig. 2A,B)
(Beccari et al., 2002;
McGregor et al., 2002
;
Silver and Montell, 2001
).
Interestingly, we found that the upd mRNA is concentrated at the
apical side of the polar cells, which is suggestive of a polarized synthesis
and secretion of this ligand during oogenesis
(Fig. 2A,B). Upd is proposed to
signal to neighboring cells, committing them to the BC fate
(Beccari et al., 2002
;
Silver and Montell, 2001
). It
is thus important to determine the expression pattern of dome
relative to its putative ligand. In situ hybridization suggests that
dome is expressed at low level in all germline and follicle cells
(Fig. 2C). Indeed,
dome transcripts can only be detected over background level after
overexpression using the UAS-GAL4 system
(Fig. 2D)
(Brand and Perrimon, 1993
). To
determine the subcellular localization of the Dome protein in follicle cells,
we raised antibodies directed against an intracellular fragment of Dome (aa
918-1113; see Materials and Methods). Consistent with in situ data,
immunostaining of egg chambers reveals that Dome is expressed in all germline
and follicle cells. Dome is a membrane protein whose localization is
restricted to apicolateral regions (Fig.
3). The membrane staining is specific, since it is absent in cells
that are mutant for dome (Fig.
3A,B).
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The subcellular localization of Dome protein was further analysed using a
Dome-GFP construct which retains wild-type activity (see below;
Fig. 5D,G,H). Expression of
Dome-GFP in follicle cells targets the fusion protein to apicolateral
membranes, in a pattern similar to wild-type Dome (compare
Fig. 3B and 3C). In addition,
the fusion protein accumulates in intracellular vesicles
(Fig. 3C). This specific
vesicular pattern may reflect an enhanced trafficking of Dome in the secretory
and/or endocytic pathway(s) due to overexpression. Consistently, the
overexpression of the wild-type Dome protein, or a truncated form
(DomeEXT, a deletion of the extracellular domain; see
Fig. 5D) also gives rise to
similar Dome-containing vesicles (Fig.
3D,E).
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Upd-dependent internalization of the Dome receptor
The presence of Dome in intracellular vesicles after overexpression
prompted us to check whether Dome could undergo detectable trafficking in
normal conditions. Indeed, detailed analysis of Dome protein localisation in
follicle cells reveals a specific accumulation of endogenous Dome in
intracellular vesicles, starting from stages 2-3 of oogenesis. In early stages
(Fig. 4A,B), Dome vesicles are
present in all follicle cells, but later they become restricted to regions
where the Upd ligand is most abundant, i.e., close to the polar cells
(Fig. 4C,F-I). These vesicles
contain Dome protein, since they are absent in dome mutant follicle
cell clones (Fig. 4B,H,I).
Dome-containing vesicles are present in BCs before and during migration
(Fig. 4C-E), thus following the
migration pattern of upd-expressing polar cells
(Fig. 1A). In the posterior
region where polar cells also express upd
(Fig. 2A,B), the same pattern
of vesicles is visible, though these cells do not migrate
(Fig. 4F,H).
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Interestingly, the abundance and distribution of Dome vesicles depend on
the distance from the polar cells, which are the source of the Upd ligand
(Fig. 4F,G). To test whether
vesicle formation is Upd dependent, we generated flies in which upd
was ectopically expressed. Cells newly expressing upd accumulate a
high level of Dome vesicles (Fig.
4J,K). In addition, overexpression of upd in the BCs
leads to an elevated number of vesicles
(Fig. 4L), as compared to
wild-type cells (Fig. 4C).
Thus, Upd can promote the internalization of the Dome receptor. Vesicles may
likely reflect active endocytosis of the Dome receptor after binding to its
ligand, a known mechanism by which receptor-ligand complexes are inactivated
or recycled. Consistently, the intracellular domain of Dome contains several
tyrosine-based and di-leucine motifs, which have been shown to work as
internalization sorting codes in several vertebrate receptors
(Trowbridge et al., 1993). It
is noteworthy that both endogenous and ectopic Dome vesicles are found
preferentially at the apical side of the cells, a region where the
upd mRNA, and probably the Upd protein, are most abundant. In this
respect, the pattern of endogenous Dome vesicles may well reflect the level
and pattern of Dome and JAK/STAT pathway activation in follicle cells.
Both the intracellular and extracellular domains of Dome are
essential for its function
To assess the contribution of specific domains in Dome activity, we
performed a structure-function analysis by expressing truncated Dome proteins
in BCs using slbo-GAL4 as a driver. In this study we used
UAS-dome, UAS-domeGFP, UAS-domeCYT (deletion
of the cytoplasmic domain) (Brown et al.,
2001
), and UAS-dome
EXT (deletion of the
extracellular domain; see Fig.
5D). The fate and migratory phenotype of BCs were assessed using
the slbo-lacZ marker (Fig.
5A) (Montell et al.,
1992
). Overexpression of Stat92E or hop leads to
migration defects, while expression of the upd gene causes the
formation of extra BCs (Fig.
5B,C,M) (Beccari et al.,
2002
; Silver and Montell,
2001
). Excess of Dome or DomeGFP in BCs blocks their migration
(Fig. 5E-H), which never takes
place, as indicated by the presence of slbo-lacZ-positive cells at
the tip of old egg chambers, and female sterility (data not shown). This
phenotype indicates a dose-sensitive effect of dome on BC migration,
reminiscent of the dose sensitivity of the slbo gene
(Rorth et al., 2000
), another
gene that is crucial for BC migration.
In egg chambers expressing DomeEXT or Dome
CYT, outer BCs are
absent and only polar cells are formed and express the slbo-lacZ
marker. This phenotype, which is similar to a complete loss of dome
in BCs (Fig. 1), is consistent
with these truncated Dome proteins behaving as dominant negative forms. We
confirmed this conclusion by looking at the embryonic cuticle phenotypes
induced in early embryos using a maternal GAL4 line
(Brown et al., 2001
; data not
shown).
To date, Dome is the only known receptor for the JAK/STAT pathway in
Drosophila. In order to test the absolute requirement of
dome in upd signaling during oogenesis, we used an epistasis
test in egg chambers. When the upd gene is overexpressed, extra BCs
are recruited and the level of slbo-lacZ expression is strongly
enhanced, indicating that slbo is positively regulated in conditions
of high JAK/STAT activity (compare Fig. 5A
and 5M) (Silver and Montell,
2001). The co-expression of upd and a dome
dominant negative construct (UAS-Dome
CYT) completely
suppresses the gain-of-function phenotypes associated with upd
overexpression, including recruitment of extra BCs and enhanced
slbo-lacZ expression (compare Fig.
5M and 5N). The resulting phenotype is similar to the simple
expression of Dome
CYT (Fig.
5I). These results demonstrate that dome is downstream of
upd to activate JAK/STAT signaling in follicle cells, and that
dome is essential for upd function.
Dome expression is autoregulated in follicle cells
Either reduction, as in dome heterozygotes
(Fig. 1C), or elevation, as in
overexpression experiments (Fig.
5E-H), of dome expression leads to BC migration defects,
suggesting that dome function is tightly regulated in normal egg
chambers. To further test this hypothesis, we examined Dome expression in
cells that are mutant for JAK/hop. In hop mutant follicle
cell clones, the level of Dome protein is higher than in the neighboring
wild-type cells (Fig. 6A),
suggesting that hop normally downregulates Dome expression. Since
this effect could be due to either transcriptional or post-transcriptional
regulation, we tested whether dome gene expression itself is
affected. For this purpose, and because endogenous dome transcripts
are barely detectable (Fig.
2C), we used a dome-Gal4 line as a reporter of
dome expression. dome-Gal4 is a pGAL4 enhancer trap element
that is inserted in the promoter region of the dome gene (20 bp
upstream of the transcription start site;
Fig. 6E). dome-GAL4 is
expressed weakly in anterior and posterior follicle cells, but much more
highly in polar cells and BCs (Fig.
6B). Expression of UAS-dome leads to a strong reduction
of dome-GAL4 driven GFP expression in BCs
(Fig. 6C), suggesting that
elevated dome activity can downregulate its own expression. In
contrast, expression of the dominant negative form DomeEXT did not
significantly affect the level of dome-GAL4 activity
(Fig. 6D). These data suggest
that in normal egg chambers, dome expression is negatively controlled
by the JAK/STAT pathway. In agreement with this proposal, the analysis of the
dome promoter region (12 kb upstream to the transcription start site)
revealed the presence of 2 short sequences that perfectly match the consensus
Stat92E binding site, TTCNNNGAA (Fig.
6E) (Yan et al.,
1996
). These putative Stat92E sites are similar to those present
in the eve promoter, which have been shown to respond to JAK/STAT
signaling (Yan et al.,
1996
).
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Role of dome in follicle cell morphogenesis
In order to better understand how dome may control egg chamber
development, we examined the expression of different follicle cell markers in
dome mosaic egg chambers. Previous work has shown that the JAK/STAT
pathway controls the accumulation of DE-cadherin and Fas3 in follicle cells
(Baksa et al., 2002;
McGregor et al., 2002
;
Silver and Montell, 2001
). In
dome mutant clones, DE-cadherin is strongly reduced
(Fig. 7A,B) suggesting that
dome participates in the formation of a correctly differentiated
epithelium throughout oogenesis. Up until stage 3, Fas3 is abundant in all
follicle cells (Ruohola et al.,
1991
), while later, Fas3 level drops sharply in all follicle cells
except in the polar cells (data not shown). Therefore, Fas3 serves as a
differentiation marker to monitor the maturation of developing egg chambers.
The removal of dome leads to an abnormally high accumulation of Fas3
in older egg chambers, indicating that these cells did not differentiate
normally and remained immature (Fig.
7C-F).
The formation of incompletely encapsulated egg chambers in dome
mosaic females (Fig. 1F,G) is
reminiscent of the loss-of-function phenotype of crumbs mutations
(Tanentzapf et al., 2000).
Indeed, crumbs is required for the initial polarization of follicle
cells, to control their mesenchymal-epithelial transition. In the absence of
crumbs, precursors of the follicle cells do not encapsulate the
germline cells, leading to egg chambers with epithelial discontinuities. In
contrast, crumbs mutant cells that have been generated after the
formation of the follicular epithelium have no apparent abnormalities,
indicating that Crumbs is required for initial polarization of the epithelium,
but not for its maintenance (Tanentzapf et
al., 2000
). Interestingly, dome mutant cells do not
express the apical determinant Crumbs in follicle cells
(Fig. 7G-J). We thus conclude
that dome is necessary for the initial polarization of the follicular
epithelium in the germarium through the control, direct or indirect, of
crumbs expression.
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DISCUSSION |
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We show here that dome interacts genetically with the
Stat92E and dpias genes during BC migration, and that
dome phenotypes in ovaries are similar to those found in
Stat92E and hop mutants
(Baksa et al., 2002;
Beccari et al., 2002
;
McGregor et al., 2002
;
Silver and Montell, 2001
).
Furthermore, Stat92E nuclear localization is lost in dome mutant
follicle cells (Fig. 1I,J),
indicating that the mechanisms leading to Stat02E activation and subsequent
nuclear translocation require dome. Since dome is epistatic
to upd (Fig. 5M,N), our data indicate that dome is required downstream of upd
and upstream of Stat92E for JAK/STAT signaling in egg chambers.
Altogether, these results provide strong evidence that Dome is a receptor
molecule for Upd during oogenesis.
Our study shows that Dome is not uniformly distributed at the membrane but
is restricted to apicolateral regions. Other receptor molecules have been
shown to preferentially localize to apicolateral membranes, such as the EGF
and Notch receptors (Lopez-Schier and St
Johnston, 2001; Sapir et al.,
1998
), suggesting that the apical region is an active signaling
interface for several receptors in follicle cells. Indeed, the apical
localization of upd mRNA, membrane Dome and Dome-containing vesicles
support a model in which ligand-receptor interactions take place apically in
follicle cells, to activate the JAK/STAT pathway.
Dome is a transmembrane protein with both extracellular and intracellular
domains whose functions are unknown. The extracellular part contains a
cytokine-binding module (CBM) and 3 fibronectin-type III domains likely
participating in ligand binding, while the intracellular domain presumably
interacts with Hop, through binding to one or several potentially
phosphorylated tyrosines (Brown et al.,
2001; Chen et al.,
2002
; unpublished results). Using truncated forms of Dome we show
that both the extracellular and intracellular domains are essential for BC
migration and signal transduction (Fig.
5). The dominant negative phenotypes that are observed are
consistent with a model in which Dome
CYT would titrate the ligand Upd,
and Dome
EXT would titrate Hop, therefore inducing a dramatic reduction
in signaling strength. Both constructs may also lead to the formation of
non-functional Dome-Dome dimers by capturing the wild-type Dome protein in an
inactive complex. Further biochemical work will be necessary to understand the
molecular mechanisms underlying Dome signal transduction.
dome is transcriptionally and post-transcriptionally
regulated in follicle cells
Previous studies have shown that the migration of BCs is sensitive to gene
dosage, making it useful for genetic screens
(Beccari et al., 2002;
Duchek and Rorth, 2001
;
Duchek et al., 2001
). The
reduction or elevation of slbo, a gene encoding a C/EBP homolog
(Montell et al., 1992
), is
sufficient to produce BC migration defects. Consistently, recent work has
shown that Slbo protein levels are tightly regulated by the ubiquitination
pathway (Rorth et al., 2000
).
Our results show that the BCs are also sensitive to changes in Dome protein
levels. Indeed, either a decrease (Fig.
1) or an increase (Fig.
5) of Dome causes BC migration defects. There are several
mechanisms by which gene activity can be regulated, including
post-translational regulation, as with the Slbo protein
(Rorth et al., 2000
), or
transcriptional regulation. Our data suggest that dome expression is
regulated in part by a transcriptional negative feedback loop
(Fig. 6). Two consensus STAT
binding sites (Yan et al.,
1996
) present in the promoter region of the dome gene may
prove to be important for this regulation. Interestingly, it has been shown
that vertebrate STAT proteins can have both positive and negative regulatory
functions (Ramana et al.,
2000
). Further work will be necessary to determine whether Stat92E
is a direct repressor of dome.
In addition to a transcriptional control of dome, there are also
post-translational mechanisms regulating Dome function in follicle cells,
through a dynamic pattern of intracellular vesicles. We show that these
Dome-containing vesicles are located at a relevant distance from Upd-producing
cells, and that Upd can promote the formation of de novo vesicles upon ectopic
expression. These results, together with the presence of several
tyrosine-based and di-leucine motifs known to sort proteins for
internalization (Trowbridge et al.,
1993), are consistent with Dome-containing vesicles being the
result of endocytosis upon ligand binding. Endocytosis is an important process
controlling several signaling pathways during development
(Seto et al., 2002
), acting on
the recycling or desensitization of ligand-receptor complexes. Our work thus
provides the first experimental evidence that JAK/STAT signaling in flies may
be regulated by endocytosis.
Roles of dome in follicle and border cells
Our analysis of dome reveals several important functions in
follicle cells. First, we show that during early oogenesis, dome is
required for the encapsulation of germline cells into a functional egg
chamber. dome mutant follicle cells made in the germarium are unable
to assemble into the nascent follicular epithelium, thus leading to
incompletely encapsulated egg chambers at stage 2-3
(Fig. 1F,G). The fusion of some
egg chambers seen in our and other studies
(Fig. 1G and data not shown)
(Baksa et al., 2002;
McGregor et al., 2002
),
suggests that follicle cells normally separating adjacent egg chambers in the
germarium have rapidly degenerated. This conclusion is supported by the fact
that mutant cells cannot be observed in early egg chambers
(Fig. 1). This is an
alternative to the model in which the formation of fused egg chamber would be
associated to stalk cell defects (McGregor
et al., 2002
).
The dramatic, early follicle cell phenotype contrasts with the essentially
normal phenotype of dome mutant cells observed in later stage egg
chambers. In this case, follicle cells are viable and divide normally (Figs
3,
4,
6,
7). A similar, dual phenotype
has been reported in crumbs mutant chambers. After initial
polarization of the follicle cells in the germarium, Crumbs is no longer
required and its loss has no visible effects
(Tanentzapf et al., 2000).
Importantly, we show here that dome controls Crumbs expression in
follicle cells, thus providing a novel link between the JAK/STAT signaling
pathway and epithelial polarity.
In addition to its early function in the germarium, dome is required for the normal expression of several follicle cell markers, including DE-cadherin and Fas3. It is important to note that despite a clear defect in the expression of these markers, dome mosaic egg chambers are morphologically normal. However, because completely mutant egg chambers cannot be obtained because of the early effect of dome in the germarium, one cannot rule out the possibility that large mutant clones would lead to abnormal development of egg chambers.
The pattern of epithelial markers in dome mutant cells indicates
that the JAK/STAT pathway is active in all follicle cells
(Fig. 7), a notion that is
reinforced by the wide expression of nuclear Stat92E
(Fig. 1). How is Dome activated
during egg chamber development and does this activation follow the same
profile at all stages? Given the restricted pattern of upd expression
in the egg chamber and its dramatic effect upon overexpression, it is unlikely
that Upd is able to signal long distances in the follicular epithelium of late
stage egg chambers. Rather, we favor a model by which the JAK/STAT pathway
plays a pre-patterning function, acting early during egg chamber development
to activate DE-cadherin and Crumbs expression (see also
McGregor et al., 2002). This
view is consistent both with the expression pattern of upd and the
distribution of Dome-containing vesicles described in this study. We have
shown that the formation of endogenous vesicles can be promoted by Upd, and
that a gradient of such vesicles is present around polar cells. Strikingly,
these vesicles, which likely indicate active signaling through Dome, are
widespread at early stages and become more restricted later on. We propose
that during early development, the Upd signal produced by anterior and
posterior polar cells contributes to the differentiation of all follicle
cells. At this stage, Upd would be more diffusible than later, as suggested by
the pattern of Dome intracellular vesicles
(Fig. 4). The study of the
mechanisms controlling Dome activation and Upd activity will require
additional tools to directly detect Upd, as, for example, Upd-GFP fusion
proteins.
In contrast to the situation in main body follicle cells, the role of
dome in BCs is essential. dome and other JAK/STAT pathway
components (Beccari et al.,
2002; Silver and Montell,
2001
) promote the differentiation of a selected group of follicle
cells into a cohesive migratory cluster, a process requiring several other
inputs (Montell, 2001
).
Mutations in dome induce phenotypes ranging from a complete absence
of BCs to non-cohesive BC clusters. This suggests that dome may be
required during migration itself, in addition to its role in the recruitment
of BCs at stages 8-9. Although only conditional mutants could help to address
this question, the pattern of Dome vesicles in the BCs before and during
migration supports a sustained activation of the JAK/STAT pathway. Such a
requirement could also explain the semi-dominant migration phenotype of
dome heterozygous egg chambers
(Fig. 1).
Our study has revealed several new findings about the function of dome and the JAK/STAT pathway during oogenesis. Future work will help to understand how Upd and Dome initially interact at the cell surface and transduce the signal to downstream JAK/STAT pathway members.
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ACKNOWLEDGMENTS |
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Footnotes |
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