Department of Biological Chemistry, Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD, USA
* Author for correspondence (e-mail: dmontell{at}jhmi.edu)
Accepted 22 August 2002
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Eyes absent (EYA), Polar cell, Hedgehog, Oogenesis, Drosophila melanogaster
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Recent studies have demonstrated that the specification of all somatic
follicle cells requires the activation of Notch signaling
(Deng et al., 2001;
Grammont and Irvine, 2001
;
Lopez-Schier and St Johnston,
2001
). Early in oogenesis, Notch is essential for differentiation
of the polar cells and stalk cells. In the absence of polar cells that is
caused by the loss of Notch (N) function, cyst encapsulation fails and stalk
formation does not occur. As a result, neighboring egg chambers fail to
separate from each other. This gives rise to compound egg chambers that
contain multiple germline cysts in a single follicle epithelium. Notch
signaling also plays an essential role later in oogenesis in promoting the
differentiation of the epithelial follicle cells from an immature,
undifferentiated precursor state. Although Notch is indispensable for polar
cell specification, activation of Notch is not sufficient to induce polar cell
fate in the epithelial layer (Grammont and
Irvine, 2001
).
Another signal that affects somatic cell fates is Hedgehog (HH).
Overexpression of HH generates ectopic polar cells throughout the follicle
epithelium. The HH signaling pathway has been well characterized, and the same
pathway is employed in many different tissues
(Murone et al., 1999). Upon
binding of HH to its receptor, Patched (PTC), the activity of a second
transmembrane protein, Smoothened (SMO) is derepressed, ultimately leading to
the activation of Cubitus interruptus (CI). CI is a transcription factor that
transmits the HH signal from the cytoplasm to the nucleus, where it is
primarily responsible for the activation and repression of HH target genes
(Aza-Blanc and Kornberg, 1999
).
CI exists in at least two forms. In the absence of the HH signal, CI is
proteolytically cleaved to a 75 kDa repressor form. In the presence of HH,
this cleavage is prevented, and CI instead functions as a transcriptional
activator. Consistent with this model, effects of HH signaling are observed
only in the presence of CI (Methot and
Basler, 2001
). The regulation of CI is complex and involves the
function of positive as well as negative regulators. The negative regulators
required for repression of this pathway include the transmembrane protein PTC
(Hooper and Scott, 1989
;
Nakano et al., 1989
), protein
kinase A (PKA) (Lepage et al.,
1995
; Li et al.,
1995
; Pan and Rubin,
1995
), and a kinesin-related protein, Costal2 (COS2; COS
FlyBase) (Robbins et al.,
1997
; Sisson et al.,
1997
). Loss of either ptc, cos2 or Pka
stimulates intracellular HH signaling, even in the absence of HH. In the
ovary, this results in ectopic polar cells, as does overexpression of HH
(Forbes et al., 1996
;
Liu and Montell, 1999
;
Zhang and Kalderon, 2000
). The
mechanism by which overexpression of HH signaling promotes ectopic polar cell
fate is not known. Nor is it completely clear how polar cell, stalk cell and
epithelial follicle cell fates are normally specified.
We have isolated a new allele of the gene eyes absent
(eya), which, when mutated, generates ectopic polar cells throughout
the follicular epithelium, a phenotype similar to that caused by ectopic HH
signaling. EYA encodes a nuclear protein best known for its essential role in
the formation of the adult eye in Drosophila
(Bonini et al., 1993;
Bonini et al., 1998
;
Boyle et al., 1997
). Our
results indicate that EYA protein is normally absent from polar and stalk
cells, and EYA is a key repressor for polar cell fate. Furthermore, the
absence of EYA is sufficient to cause epithelial follicle cells to develop as
polar cells. Thus, EYA expression must be repressed in polar and stalk cells
in order for these fates to be determined. Finally, we show that the mechanism
by which ectopic HH signaling is able to transform other follicle cells into
ectopic polar cells is through suppressing EYA expression.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Mutant clones were generated by mitotic recombination using the
FLP/FRT system (Xu and Rubin,
1993) either by an X-chromosome hs-flp or by T155,
UAS-flp (Duffy et al.,
1998
). T155, UAS-flp directs expression of the
recombinase flp in follicle cell precursors and some imaginal discs,
including the eye-antenna, wing and leg discs. Clones were marked using the
tub84BlacZ (`tublacZ') transgenes on 2L and 2R
(Harrison et al., 1995
) or
ubi-nlsGFP (Davis et al.,
1995
) (Bloomington Stock Center). Three to five-day-old female
flies were dissected when clones were produced by T155, UAS-flp.
hs-flp induced clones were generated by heat-shocking newly hatched
females for 1 hour at 37°C, twice a day, for 3 days in a row. Adult
females were dissected 2-8 days after the last heat shock. The following
alleles were used:
ci clones marked by the loss of GFP were generated using flies of
the genotype: hs-flp;FRT42D P[Ci+]
P[hsp70-GFP]/FRT42D; ci94/ci94
(Methot and Basler, 1999).
Ectopic expression of constitutively activated CI was performed by excision of
an FRT-flanked CD2 gene from an act5C>CD2>GAL4 transgene
(Jiang and Struhl, 1998
) in
the presence of the activated UAS-Ci5m transgene from line T5m-s1
(Price and Kalderon, 1999
).
Overexpression of eya in the ovary was performed using either
hs-GAL4 or C306 (Manseau
et al., 1997
) or upd-Gal4 (from Dr Harrison) in animals
carrying either UASeya1 or UASeya2 (from Dr Nancy Bonini).
Female flies were heat shocked for 1 hour twice a day for 3 days and then
their ovaries were dissected, stained and examined either immediately or 1-3
days later. Under these conditions, the frequency and severity of the
formation of compound egg chambers increased with longer exposure to heat
shock. If dissected 3 days after a 3-day heat-shock,
30% of the ovary was
composed of compound egg chambers. Overexpression of activated Notch was
performed by using FO64 line (Act5Cflip-out Gal4, UAS-GFP/CyO;
hsFLP,MKRS/TM6B) (from Dr Y. Hiromi) and UAS-Nintro
(from Dr Norbert Perrimon).
The following fly stains were also used: w1118 as wild
type controls; 93F/TM3 (Ruohola
et al., 1991); A101/TM3
(Bier et al., 1989
),
so5-lacZ (from Dr Pignoni), eya3cs/CyO;
E(P)10/CyO (Bonini et al.,
1998
); ub-Cadherin-GFP (from Dr Oda).
Immunohistochemistry and immunofluorescence
Ovary dissections were performed in Grace's medium plus 10% fetal calf
serum. For ß-galactosidase staining, whole ovaries were fixed in PBS
containing either 0.1% glutaraldehyde or 3.7% formaldehyde for 5 minutes, and
rinsed once with PBT (PBS+0.1% Triton X-100) followed by incubation of the
ovaries with 0.2% X-gal in staining solution (10 mM phosphate buffer pH7.2,
150 mM NaCl, 1 mM MgCl2, 3 mM K4[FeII(CN)6],
3 mM K3[FeIII(CN)6], 0.3% Triton X-100) for 10 minutes
to overnight. After staining, the ovaries were rinsed twice with PBT and
mounted in PBS containing 50% glycerol.
For fluorescence staining, the egg chambers were typically fixed in 3.7% formaldehyde (Polysciences) in 0.1 M phosphate buffer (pH 7.4) containing 0.5% NP40 for 20 minutes and rinsed three times in NP40 wash buffer (50 mM Tris pH 7.4, 150 mM NaCl, 0.5% NP40, 1 mg/ml BSA). Egg chambers were then blocked in NP40 wash buffer plus 5% normal goat serum for 30 minutes and were incubated in block solution containing the primary antibodies overnight at 4°C. After four washes for 20 minutes each in NP40 wash buffer, the egg chambers were incubated in block solution containing fluorescence-conjugated secondary antibodies at a dilution of 1:200 for 2 hours at room temperature. For rhodamine-phalloidin (Molecular Probes) and DAPI (Molecular Probes) staining, egg chambers were incubated with rhodamine-phalloidin in NP40 wash buffer at a dilution of 1:200 for 25 minutes followed by adding DAPI to a final concentration 0.5 µg/ml and incubation of egg chambers for another 5 minutes. After three washes for 20 minutes in NP40 wash buffer, the egg chambers were mounted in VECTASHIELD® (Vector Laboratories) or Aqua Poly/Mount (Polysciences). All images were captured either on a Noran OZ laser confocal microscope or on a LEICA TCSNT laser confocal microscope, or on a Zeiss Axioplan microscope.
The primary antibodies used were mouse anti-FAS3 (1:3), mouse anti-DAC
(1:250) (Developmental Studies Hybridoma Bank), mouse anti-EYA (10H6 1:1000)
(Bonini et al., 1993), rat
anti-full length form of CI (2A1 1:5)
(Slusarski et al., 1995
),
rabbit anti-CI (CIN, which recognizes both the full length and degraded form;
1:250) (Aza-Blanc and Kornberg,
1999
), rabbit anti-EY (1:300)
(Kurusu et al., 2000
), mouse
anti-ß-galatosidase (1:500) (Promega) and rabbit anti-ß-galatosidase
(1:2000) (Cappel). The secondary antibodies used were horse fluorescein
anti-mouse (Vector Laboratories), CyTM5-conjugated donkey anti-rabbit,
CyTM5-conjugated donkey anti-mouse; fluorescein (FITC)-conjugated donkey
anti-rabbit, FITC-conjugated donkey anti-rat, rhodamine
redTM-X-conjugated donkey anti-mouse and rhodamine
redTM-X-conjugated donkey anti-rabbit (Jackson ImmunoResearch).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
The extra border cell clusters found in cos2 or ptc
mutant egg chambers result from overproduction of polar cells
(Liu and Montell, 1999;
Zhang and Kalderon, 2000
)
(Fig. 1G,J). Polar cells can be
detected by staining with an antibody against Fasciclin III
(Ruohola et al., 1991
) (FAS3,
Fig. 1E-G), or by expression of
ß-galactosidase from the enhancer trap line A101
(neuralized-lacZ) (Bier et al.,
1989
), which is a marker for mature polar cells
(Fig. 1H-J). FAS3 is a
homophilic cell adhesion molecule that accumulates to the highest levels in
immature follicle cells in the germarium
(Fig. 1K) and at the interface
between the two polar cells from stage 3 to stage 10A of oogenesis in
wild-type egg chambers (Fig.
1K,L). There are two pairs of polar cells in wild-type egg
chambers, one pair located at the anterior pole of the egg chamber and another
at the posterior (Fig. 1H,L).
In 54C2 mutant egg chambers, the extra border cell clusters that
formed contained extra polar cells (Fig.
1F,I). In addition, ectopic polar cells were observed in early
stage egg chambers (not shown) and were found in many positions throughout the
follicle epithelium in egg chambers containing mutant clones
(Fig. 1I).
We identified the gene mutated in the 54C2 line based on
deficiency and meiotic recombination mapping. 54C2 was found to
reside in the 26D-27A region (see Materials and Methods for details).
Mutations in one known gene in this region, eya, failed to complement
the 54C2 mutation with respect to lethality, whereas all other
mutations in this region complemented. In addition, two independent
eya alleles, cliE11 and
cliD1, caused ovarian phenotypes in mosaic clones that
were similar to those of 54C2, including ectopic polar cells and
overproduction of border cells (data not shown). The phenotype of
54C2 in other tissues also resembled that of eya
(Hazelett et al., 1998) (data
not shown). 54C2 clones in the head were characterized by a loss of
eye tissue and its replacement with head cuticle containing orbital bristles,
whereas we observed no abnormality in either legs or wings. Both the eye and
ovarian phenotypes appeared to result from a single mutation, because the two
phenotypes co-segregated in recombination experiments. Therefore, we concluded
that 54C2 was a new allele of the eya gene.
Consistent with these findings, select eya allelic combinations
are female sterile (Bonini et al.,
1998). In addition, in eya3cs/E(P)10 mutant
females, the polar cell markers FAS3 and A101 were expressed in virtually all
of the follicle cells (Fig.
1L,O). These egg chambers usually contained abnormal germline
cells (Fig. 1M,P). In extreme
examples with this genotype (eya3cs/E(P)10), ovarian
development ceased in the germarium and the germarium appeared swollen
compared with the wild type (Bonini et al.,
1998
) (Fig. 1K,N).
This extreme phenotype was not observed in ovaries containing eya
mosaic clones, presumably because it resulted from a reduction (but not
elimination) of EYA expression in all cells, whereas the mosaic clone
phenotype resulted from more focal loss of EYA expression.
EYA protein is normally absent from both polar cells and stalk
cells
As loss of eya in follicle cells led to ectopic polar cells in the
ovary, we postulated that expression of EYA might normally be repressed in the
polar cells. Alternatively, EYA might be repressed via a post-translational
modification in polar cells. To distinguish between these possibilities, we
examined the expression pattern of the EYA protein in the ovary. Egg chambers
were double stained with antibodies against EYA
(Bonini et al., 1993) and
anti-ß-galactosidase antibodies in order to identify either polar cells,
in the A101 enhancer trap line, or stalk cells in the enhancer trap line 93F
(Ruohola et al., 1991
)
(Fig. 2).
|
The earliest expression of EYA was observed in follicle cells in region 2b of the germarium (Fig. 2A,B). EYA continued to be expressed in all follicle cells with the exception of polar and stalk cells until late stage 8 (Fig. 2C,D). After stage 8, EYA protein was restricted to the anterior follicle cells, including border cells, squamous cells and centripetal cells (Fig. 2E,F). EYA was not expressed detectably in the germ cells of any stage. Thus, the absence of EYA protein in the polar cells was consistent with a role as a repressor of polar cell fate.
In order to define when the EYA protein was first lost in the polar/stalk cell lineage in the germarium, we examined germaria for cells that did not express EYA. In this experiment, Cadherin-GFP expression driven by the ubiquitin promoter was used to outline all cells in the germarium, and the cells were double stained with antibodies for EYA. We found that EYA protein was absent from a group of cells along the border between regions 2b and 3 of the germarium (Fig. 2A). This was more obvious in the cells between region 2 of the germarium and the stage 1 egg chamber (Fig. 2B).
Loss of eya is sufficient to transform epithelial follicle
cells into polar cells
To examine the autonomy of the effects of eya in repressing polar
cell fate, we examined egg chambers carrying marked mosaic clones. In this
experiment, wild-type follicle cells expressed GFP, whereas eya
mutant cells lacked GFP (Fig.
3). EYA protein was only detected in wild-type follicle cells, and
not in the mutant clones, indicating that eya54C2 was a
protein null allele (Fig.
3A-C). In addition, both polar cell markers, Fasciclin III
(Fig. 3D-I) and A101
(Fig. 3G-I), were ectopically
expressed in all eya mutant follicle cells. Thus, loss of
eya is sufficient to cause ectopic expression of polar cell markers
in the follicle epithelium in a cell autonomous fashion.
|
As EYA is also normally excluded from stalk cells
(Fig. 2D), we wondered whether
loss of eya could lead to ectopic stalk cells. We stained
eya mosaic egg chambers with the stalk cell marker 93F
(Ruohola et al., 1991). We
never observed ectopic 93F expression in eya mutant cells (data not
shown). Nor did we observe any long stalks between egg chambers in
eya mosaic ovaries. Therefore, loss of eya only leads to
ectopic polar cells but not stalk cells.
EYA is downstream of ptc, pka and cos 2 in regulating polar
cell fate
Ectopic polar cells are produced either by overexpression of HH or by loss
of negative regulators of the HH pathway, such as ptc, Pka and
cos2 (Forbes et al.,
1996; Liu and Montell,
1999
; Zhang and Kalderon,
2000
). To test whether eya expression was under the
control of HH signaling, we examined the expression of EYA in follicle cell
clones lacking ptc, Pka or cos2. When Pka mosaic
egg chambers were double stained for EYA and for the polar cell marker A101,
EYA protein was missing from all of the ectopic polar cells found in mutant
clones (Fig. 4D-F). The same
was true of ptc and cos2 mosaic egg chambers (data not
shown). Thus, EYA expression is influenced by ectopic HH signaling, and
eya is downstream of ptc, cos2 and Pka in terms of
polar cell fate specification. However, not all Pka mutant follicle
cells expressed FAS3 and not all of the FAS3-positive cells lacked EYA
(Fig. 4A-C). FAS3 is expressed
not only by polar cells, but also by immature or undifferentiated follicle
cells, whereas A101 labels only mature polar cells
(Grammont and Irvine, 2001
).
Thus, the cells that expressed FAS3 and EYA and lacked A101 may have been
cells in which differentiation had been delayed or prevented. Besides ectopic
polar cells and undifferentiated, immature follicle cells, there were also
EYA-positive, FasIII-negative cells, a combination normally found in
differentiated epithelial follicle cells
(Fig. 4C).
|
Mutual repression of EYA and CIAC
The effects of HH signaling require the function of CI
(Methot and Basler, 2001). In
the absence of HH, CI is processed to produce a transcriptional repressor, a
short form referred to as CIR. In the presence of HH, the
processing is prevented and the full-length form of CI, CIAC,
activates transcription. Polar cell fate might either be repressed by
CIR or promoted by CIAC, or both. To test the
possibility that the CIR represses polar cell fate, egg chambers
containing clones homozygous for a null mutation in ci were analyzed.
If polar cell fate were repressed by CIR, we would expect loss of
both forms of CI to lead to ectopic polar cells. However, ectopic polar cells
were not observed in clones lacking CI, and EYA expression was normal
(Fig. 5A). These results
suggest that loss of CIR is not sufficient to cause ectopic polar
cells and CI activity is not normally required for polar cell fate
specification.
|
To test the possibility that CIAC promotes polar cell fate, we
induced the expression of a constitutively active derivative of CI
(Price and Kalderon, 1999)
(see Materials and Methods). Three days after induction of constitutively
active CI by heat shock, recovered egg chambers were double stained with
anti-ß-galactosidase and antibodies against EYA. In such egg chambers,
ß-galactosidase marked cells expressing constitutively active CI. Similar
to the situation for PKA mutant clones, there were three types of cells in the
CIAC flipout clones. The phenotypes were very similar to
pka mutant clones. Those ß-galactosidase-positive cells that
lacked EYA expression (Fig. 5D)
ectopically expressed the polar cell marker FAS3 (not shown). This result
suggests that EYA expression can be repressed, and polar cell fate promoted,
by CIAC.
Expression of ptc is regulated directly by CIAC in
response to HH signaling in all tissues examined, thus ptc-lacZ is a
reporter for strong activation of the HH pathway
(Aza-Blanc and Kornberg, 1999).
We found one ptc-lacZ line that shows expression in one cell of the
polar cell pair (Fig. 5B). If
eya were strictly downstream of CI in polar cell fate determination,
we would not expect ptc-lacZ expression to be affected in
eya mosaic clones. However, 10% of the ectopic polar cells produced
in eya mutant clones (n>50) expressed ptc-lacZ
(Fig. 5C). To examine this
apparent activation of CI further in eya mutant mosaic clones, we
stained mutant egg chambers with 2A1, an antibody that recognizes only the
full-length, activated form CIAC. In wild-type egg chambers,
CIAC is expressed at equal levels in all follicle cells
(Forbes et al., 1996
). By
contrast, CIAC protein was upregulated in eya mutant
follicle cells, compared with the surrounding heterozygous cells
(Fig. 5G-I). The upregulation
level of CIAC in eya mutant cells was not quite as great
as that in pka mutant cells (Fig.
5E,F,H,I), but nevertheless different from wild type. Thus, EYA
and CIAC exhibited mutual repression.
Upregulation of full-length CI in eya mutant clones could result
either from an increase in transcription of the ci gene, or from
inhibition of processing of CI protein from the full-length CIAC to
the shorter CIR. To examine the regulation of ci
transcription in the ovary, we used a ci-lacZ enhancer trap line,
which is expressed in the same pattern as the endogenous gene
(Eaton and Kornberg, 1990). We
found no significant increase in ci-lacZ expression in eya
mutant follicle cells (data not shown). We also stained eya mosaic
egg chambers with an antibody CIN, which recognizes all forms of CI, to
monitor total CI protein (Aza-Blanc and
Kornberg, 1999
). There was no detectable increase in CIN staining
in eya mutant cells (Fig.
5G). Thus, total CI levels remained constant in eya
mutant clones, whereas CIAC levels rose, suggesting that processing
of full-length CI to CIR was inhibited in the eya mutant
cells.
Ectopic overexpression of eya is sufficient to suppress
stalk/polar cell fates
As eya loss of function caused ectopic polar cell differentiation,
and EYA is normally absent from both polar cells and stalk cells, we wanted to
know if over- or mis-expression of eya would suppress these cell
fates. We used flies containing hs-Gal4 and UAS-eya
transgenes to examine the effects of ectopic eya expression in the
ovary. Ectopic expression of eya resulted in compound egg chambers
with more than 16 germ cells, which were not separated by interfollicular
stalks and were not always completely enveloped by the somatic epithelial
layer (Fig. 6A,C,E). The stalk
cell marker 93F was missing from these compound egg chambers, although some
pairs of FAS3-positive cells were clearly observed
(Fig. 6B,F,G), indicating that
the formation of compound egg chambers might result from loss of stalk cells.
The frequency and severity of the compound egg chamber phenotype varied with
heat-shock conditions (see Materials and Methods). To rule out the possibility
that formation of compound egg chambers could result from extra germline cell
divisions, we examined mutant ovaries with rhodamine-conjugated phalloidin to
visualize actin. We found that no germ cells were connected to their neighbors
by more than four ring canals (data not shown). In addition, within compound
egg chambers, individual sets of germ cells were recognizable as groups of 15
similarly sized nurse cells and a single oocyte nucleus. Therefore,
overexpression of eya suppressed stalk cell fate.
|
Because loss of EYA promoted polar cell differentiation, over- or mis-expression would be expected to interfere with polar cell fate. Indeed, when a polar cell specific GAL4 line, upd-GAL4, was used to force EYA expression exclusively in the polar cells, one-third of the egg chambers (n=99) were found to be missing polar cells from one end (Fig. 6D). Anterior polar cells appeared to be more affected than those at the posterior. We did not observe egg chambers lacking polar cells from both ends, and compound egg chambers were rare in this genotype. In the few egg chambers from hs-GAL4;UAS-eya females that were well separated by stalk cells, polar cells were also often missing from one end as shown by absence of FAS3 and A101 expression (not shown). In both hs-GAL4;UAS-eya and upd-GAL4;UAS-eya, a border cell migration defect was observed in >50% of stage 10 egg chambers (Fig. 6H), suggesting that the normal function of polar cells in promoting border cell migration had been compromised.
The effect of activated Notch on EYA expression
One signal that is known to be essential for polar cell formation is Notch
(N). To investigate the relationship between Notch and EYA, we examined EYA
expression in N mutant clones. Loss of Notch leads to the disappearance of
polar cells (Grammont and Irvine,
2001; Lopez-Schier and St
Johnston, 2001
). Consistent with this, we found that all N mutant
cells expressed EYA (not shown). To test whether activated N is sufficient to
repress EYA expression, we made flip-out clones of UAS-Nintra in
somatic follicle cells, and triple stained the egg chambers for GFP, A101 and
EYA. All A101-positive cells were EYA negative, and all EYA-negative cells
expressed A101 (Fig. 7).
However, not all GFP-positive cells were A101 positive. Consistent with other
reports, we found that ectopic, A101-positive, polar cells only formed near
the two poles of egg chambers. Surprisingly, activated Notch was able to cause
ectopic polar cells to form non-cell-autonomously
(Fig. 7).
|
Function of eye development genes in Drosophila
oogenesis
The eya gene is essential for Drosophila eye development
where it functions as part of a cascade of regulatory genes including
eyeless (ey) (Quiring et
al., 1994), twin of eyeless (toy)
(Czerny et al., 1999
), sine
oculis (so) (Cheyette et al.,
1994
) and dachshund (dac)
(Mardon et al., 1994
). To
determine whether this group of genes functions together to specify follicle
cell fates, we examined the expression of each gene in the ovary. Expression
of EY is found in the nuclei of all follicle cells as assessed by antibody
staining (data not shown). We examined the expression of SO in the ovary using
a reporter, so5-lacZ. The so5 reporter
was expressed in stalk cells, but not in polar cells
(Fig. 8A). Consistent with this
expression pattern, stalk cells were missing in so mutant egg
chambers, whereas the correct number of polar cells was present
(Fig. 8B-D). Neither
toy nor dac was expressed in the ovary (data not shown).
These results indicate that although some of the eye specification genes
function in the ovary, the regulatory network is not the same in this tissue.
Rather, the individual retinal determination genes function independently of
the network in the ovary.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
EYA protein is first lost in the cells along the border between regions 2b and 3 of the germarium. Those cells are likely to be the cells that separate cysts and may be the early progeny of the polar/stalk cell precursors. Therefore, loss of EYA expression appears to be the earliest available marker for this lineage.
Although EYA is normally missing from both polar cells and stalk cells, the
expression of the mature stalk cell marker 93F was never observed in
eya mosaic clones. Hence, loss of eya transformed the
epithelial follicle cells only into polar cells, not into stalk cells.
However, mis-expression of eya early in oogenesis leads to the
absence of stalk cells and generates compound egg chambers, sometimes
containing normal pairs of polar cells. It seems that the formation of
compound egg chambers depends more directly on the loss of stalk cells than on
the loss of polar cells. This was also the case when EYA expression was forced
exclusively in the polar cells. Although the function of polar cells was
compromised, the stalk cells still formed compound egg chambers were not
observed. Further evidence for the crucial role of stalk cells in separating
egg chambers is the compound egg chamber phenotype resulting from loss of
so, a gene that is only expressed in the stalk cells. Therefore,
repression of EYA appears to be required for stalk cell formation, which is in
turn essential to separate egg (Dobens and
Raftery, 2000).
Why does loss of eya lead only to ectopic polar cells, not to stalk cells in the epithelial follicle layer? One possible reason is that the stalk cells, as opposed to polar cells and epithelial follicle cells, normally form in the absence of direct contact with germline cell. Thus, signals from the germline might prevent stalk cell fate in cells that directly contact the germline.
One germline signal that is known to play a role in polar cell
specification is Delta, which signals from the germline to Notch in the soma
to control the differentiation of polar cells
(Lopez-Schier and St Johnston,
2001). Epithelial follicle cells do not respond to Delta in the
same way, presumably because, unlike polar cells, they do not express
fringe (Grammont and Irvine,
2001
; Zhao et al.,
2000
). fringe encodes a glucosyltransferase that
potentiates the ability of the Notch receptor to be activated by its ligand,
Delta. Mutation of either Notch or fringe leads to the
disappearance of polar cells (Grammont and
Irvine, 2001
; Lopez-Schier and
St Johnston, 2001
). As a result, EYA-negative cells are not found
in the follicles. Mis-expression of either FNG or activated Notch produces
ectopic polar cells only at the poles of the egg chamber, whereas loss of EYA
can cause polar cells to form throughout the follicle epithelium. Thus Notch
signaling appears to be necessary, but not sufficient to repress EYA
expression and lead to polar cell formation. Surprisingly, activated Notch
also can produce ectopic polar cells cell-nonautonomously at the poles of the
egg chamber. The reason for this could be that activated Notch signaling might
activate the expression of Delta, which, in turn, can activate Notch signaling
in the adjacent cells.
Another signaling pathway that impinges on polar and stalk cell fates is
the JAK/STAT pathway (McGregor et al.,
2002). The ligand for this pathway is unpaired (UPD), which is
expressed specifically in polar cells. The ligand interacts with a receptor,
which in turn activates the tyrosine kinase known as Hopscotch (HOP). HOP
activity results in phosphorylation and nuclear translocation of the
transcription factor STAT92E. In the ovary, UPD secreted from polar cells
functions to suppress polar cell fate in stalk cells. It has been proposed
that N signaling specifies a pool of cells competent to become polar and stalk
cells and the UPD/JAK/STAT pathway distinguishes polar versus stalk fates
(McGregor et al., 2002
). Thus,
whatever signal normally represses EYA in the polar/stalk lineage presumably
acts prior to UPD/JAK/STAT as EYA repression occurs in both polar and stalk
cells, possibly in the common precursor cell. The observation that forcing EYA
expression in polar cells under the control of upd-GAL4 can repress
polar cell fate suggests that this fate remains malleable for some time after
its normal specification. The relatively low penetrance of this phenotype
(
30%) might be due to the late expression of upd-GAL4 relative
to the normal timing of EYA repression.
Ectopic HH signaling and polar cell formation
Loss of EYA results in the production of ectopic polar cells virtually
anywhere in the egg chamber. At first glance, this phenotype looks very
similar to that of ectopic activation of the HH pathway, either by
overexpression of HH or by loss of the negative regulators PTC, PKA or COS2.
Indeed the ectopic polar cells that form in ptc, Pka or cos2
mutant clones lack EYA. However, ectopic HH signaling has additional effects
besides ectopic polar cell formation, whereas loss of EYA does not. We
observed several different cell types in the ptc, Pka or
cos2 mutant clones. There were EYA-positive but FAS3-negative cells,
which may correspond to differentiated epithelial follicle cells. There were
also cells expressing both EYA and FAS3, which could be immature,
undifferentiated precursor cells. Finally, there were the EYA-negative but
FAS3-postive polar cells. In this study, we show that the production of
ectopic polar cells caused by ectopic activation of the HH pathway occurs by
repression of EYA. But what is the normal relationship between HH signaling
and polar cell formation, and why does excessive HH signaling generate ectopic
polar cells as well as other cell types?
To address these questions, we have to consider the normal role of HH
signaling in the ovary. Expression of HH protein has only been observed in the
terminal filament and cap cells at the extreme anterior tip of the germarium
(Forbes et al., 1996). The
normal function of HH appears to be to regulate somatic stem cell fate and
proliferation (Zhang et al.,
2001
). Loss of HH signaling in somatic stem cells results in the
loss of stem cell fate. Conversely, overexpression of HH leads to
overproduction of stem-cells. Despite the fact that ectopic expression of HH
leads to ectopic polar cells, HH signaling does not appear to specify polar
cell fate normally. The best direct evidence for that is that smo
mutant cells, which cannot transduce HH signals, are still capable of
generating normal polar cells at normal positions
(Zhang and Kalderon, 2000
). In
addition, normal polar cells can develop in the absence of ci (this
study).
Why, then, does ectopic HH signaling produce ectopic polar cells? It has
been previously argued that excessive HH signaling might maintain follicle
cells, and the polar/stalk cell lineage in particular, in a precursor state
for an abnormally long period of time
(Tworoger et al., 1999;
Zhang and Kalderon, 2000
).
Thus, delayed specification of polar cells would permit more proliferation
than usual in this lineage. This model might explain the presence of extra
polar cells at the two poles of the egg chamber, where the polar cells
normally reside. However, it does not explain the presence of ectopic polar
cells elsewhere in the egg chamber, or why there are three different cell
types present in the ptc, Pka and cos2 mutant clones. Based
on the normal role of HH in regulating proliferation and maintenance of stem
cells and their immediate progeny, the prefollicle cells, we propose that
ectopic HH signaling might cause ectopic prefollicle cell fates within the
epithelial follicle layer of early egg chambers. As these cells undergo
further proliferation, and then differentiation, they produce the various
follicle cell types observed in the ptc, Pka and cos2
clones. Additional, as yet unknown, signals might determine which specific
fates the differentiating cells adopt. However, the normal mechanisms that
function to coordinate follicle cell fates spatially are obviously lacking in
the mutant clones, as the three types of cells appear in random locations
relative to each other. This provides an explanation for how ectopic HH
signaling might produce polar cells all over the egg chamber, rather than only
at the two poles of the egg chamber, where the polar/stalk precursors normally
reside.
The relationship between CI and EYA
Ectopic HH signaling produces numerous effects in the Drosophila
ovary, which include regulating proliferation of somatic cells as well as
specification of polar cells (Forbes et
al., 1996; Zhang and Kalderon,
2000
). Both of these effects appear to be achieved through the
cell autonomous action of CI. This raises the question of how different
effects are elicited by the same signal. The data presented here indicate that
ectopic HH activates polar cell fate by repressing eya expression,
the function of which is required to repress polar cell fate. As loss of
eya does not mimic ectopic hedgehog in causing extra proliferation,
it is not yet clear what factors act downstream of ectopic HH to affect
proliferation.
The relationship between EYA and CI is not a simple linear one. Although
EYA expression is repressed by CIAC, mutations in eya also
alter the balance between CIAC and CIR, without
affecting overall ci expression. CIAC is upregulated in
eya mutant follicle cells. In addition, some of the ectopic polar
cells in eya mosaic egg chambers express ptc-lacZ, which is
an indicator for activation of CI
(Aza-Blanc and Kornberg, 1999).
Thus, there appears to be mutual repression between CIAC and EYA.
One place in the mammalian embryo where a similar relationship between CI and
EYA might exist is in patterning the eye field. HH is normally expressed at
the midline where it represses eye development. In the absence of HH, a single
cyclopic eye forms at the midline
(Macdonald et al., 1995
). The
three mammalian homologs of EYA are all expressed in the eye primordium
(Xu et al., 1997
) and
therefore it may be that the antagonism between HH and EYA revealed in this
study is also employed in the mammalian embryo to repress midline eye
development.
Concluding remarks
It is clear that the effect of the ectopic HH signaling on the
specification of the polar cell fate is through the repression of EYA. What
still remains unknown is the spatially localized signal that normally
represses EYA expression in polar and stalk cells. As Notch signaling is
necessary, but not sufficient, to define polar cells, it is likely that there
is an additional, spatially localized signal required for specifying polar
cell fate. Clearly, EYA is a key regulator that represses polar and stalk cell
fates. Whatever the regulatory signal that normally specifies polar cell fate,
it must regulate EYA expression to determine a polar versus non-polar cell
fate in the follicular epithelium.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Aza-Blanc, P. and Kornberg, T. B. (1999). Ci: a complex transducer of the hedgehog signal. Trends Genet. 15,458 -462.[CrossRef][Medline]
Bai, J., Uehara, Y. and Montell, D. J. (2000). Regulation of invasive cell behavior by Taiman, a Drosophila protein related to AIB1, a steroid receptor coactivator amplified in breast cancer. Cell 103,1047 -1058.[Medline]
Bier, E., Vaessin, H., Shepherd, S., Lee, K., McCall, K., Barbel, S., Jan, L. Y. and Jan, Y. N. (1989). Searcing for pattern and mutation in the Drosophila genome with a P-lacZ vector. Genes Dev. 3,1288 -1300.[Abstract]
Bonini, N. M., Leiserson, W. M. and Benzer, S. (1993). The eyes absent gene: genetic control of cell survival and differentiation in the developing Drosophila eye. Cell 72,379 -395.[Medline]
Bonini, N. M., Leiserson, W. M. and Benzer, S. (1998). Multiple roles of the eyes absent gene in Drosophila. Dev. Biol. 196,42 -57.[CrossRef][Medline]
Boyle, M., Bonini, N. and DiNardo, S. (1997).
Expression and function of clift in the development of somatic gonadal
precursors within the Drosophila mesoderm. Development
124,971
-982.
Cheyette, B. N., Green, P. J., Martin, K., Garren, H., Hartenstein, V. and Zipursky, S. L. (1994). The Drosophila sine oculis locus encodes a homeodomain-containing protein required for the development of the entire visual system. Neuron 12,977 -996.[Medline]
Czerny, T., Halder, G., Kloter, U., Souabni, A., Gehring, W. J. and Busslinger, M. (1999). twin of eyeless, a second Pax-6 gene of Drosophila, acts upstream of eyeless in the control of eye development. Mol. Cell 3, 297-307.[Medline]
Davis, I., Girdham, C. H. and O'Farrell, P. H. (1995). A nuclear GFP that marks nuclei in living Drosophila embryos; maternal supply overcomes a delay in the appearance of zygotic fluorescence. Dev. Biol. 170,726 -729.[CrossRef][Medline]
Deng, W. M., Althauser, C. and Ruohola-Baker, H.
(2001). Notch-Delta signaling induces a transition from mitotic
cell cycle to endocycle in Drosophila follicle cells.
Development 128,4737
-4746.
Dobens, L. L. and Raftery, L. A. (2000). Integration of epithelial patterning and morphogenesis in Drosophila ovarian follicle cells. Dev. Dyn. 218, 80-93.[CrossRef][Medline]
Duffy, J. B., Harrison, D. A. and Perrimon, N.
(1998). Identifying loci required for follicular patterning using
directed mosaics. Development
125,2263
-2271.
Eaton, S. and Kornberg, T. B. (1990). Repression of ci-D in posterior compartments of Drosophila by engrailed. Genes Dev. 4,1068 -1077.[Abstract]
Forbes, A. J., Lin, H., Ingham, P. W. and Spradling, A. C.
(1996). hedgehog is required for the proliferation and
specification of ovarian somatic cells prior to egg chamber formation in
Drosophila. Development
122,1125
-1135.
Grammont, M. and Irvine, K. D. (2001). fringe
and Notch specify polar cell fate during Drosophila oogenesis.
Development 128,2243
-2253.
Harrison, D., Binari, R., Nahreini, T., Gilman, M. and Perrimon, N. (1995). Activation of a Drosophila Janus kinase (JAK) causes hematopoietic neoplasia and developmental defects. EMBO J. 14,2857 -2865.[Abstract]
Hazelett, D. J., Bourouis, M., Walldorf, U. and Treisman, J.
E. (1998). decapentaplegic and wingless are regulated by eyes
absent and eyegone and interact to direct the pattern of retinal
differentiation in the eye disc. Development
125,3741
-3751.
Hooper, J. E. and Scott, M. P. (1989). The Drosophila patched gene encodes a putative membrane protein required for segmental patterning. Cell 59,751 -765.[Medline]
Jiang, J. and Struhl, G. (1998). Regulation of the Hedgehog and Wingless signalling pathways by the F- box/WD40-repeat protein Slimb. Nature 391,493 -496.[CrossRef][Medline]
King, R. C. (1970). Ovarian Development in Drosophila melanogaster. London: Academic Press.
Kurusu, M., Nagao, T., Walldorf, U., Flister, S., Gehring, W. J.
and Furukubo-Tokunaga, K. (2000). Genetic control of
development of the mushroom bodies, the associative learning centers in the
Drosophila brain, by the eyeless, twin of eyeless, and Dachshund genes.
Proc. Natl. Acad. Sci. USA
97,2140
-2144.
Lepage, T., Cohen, S. M., Diaz-Benjumea, F. J. and Parkhurst, S. M. (1995). Signal transduction by cAMP-dependent protein kinase A in Drosophila limb patterning. Nature 373,711 -715.[CrossRef][Medline]
Li, W., Ohlmeyer, J. T., Lane, M. E. and Kalderon, D. (1995). Function of protein kinase A in hedgehog signal transduction and Drosophila imaginal disc development. Cell 80,553 -562.[Medline]
Liu, Y. and Montell, D. J. (1999).
Identification of mutations that cause cell migration defects in mosaic
clones. Development 126,1869
-1878.
Lopez-Schier, H. and St Johnston, D. (2001).
Delta signaling from the germ line controls the proliferation and
differentiation of the somatic follicle cells during Drosophila oogenesis.
Genes Dev. 15,1393
-1405.
Macdonald, R., Barth, K. A., Xu, Q., Holder, N., Mikkola, I. and
Wilson, S. W. (1995). Midline signalling is required for Pax
gene regulation and patterning of the eyes.
Development 121,3267
-3278.
Manseau, L., Baradaran, A., Brower, D., Budhu, A., Elefant, F., Phan, H., Philp, A. V., Yang, M., Glover, D., Kaiser, D. et al. (1997). GAL4 enhancer traps expressed in the embryo, larval brain, imaginal discs, and ovary of Drosophila. Dev. Dyn. 209,310 -322.[CrossRef][Medline]
Mardon, G., Solomon, N. M. and Rubin, G. M.
(1994). dachshund encodes a nuclear protein required for normal
eye and leg development in Drosophila. Development
120,3473
-3486.
Margolis, J. and Spradling, A. C. (1995).
Identification and behavior of epithelial stem cells in the Drosophila ovary.
Development 121,3797
-3807.
McGregor, J. R., Xi, R. and Harrison, D. A.
(2002). JAK signaling is somatically required for follicle cell
differentiation in Drosophila. Development
129,705
-717.
Methot, N. and Basler, K. (1999). Hedgehog controls limb development by regulating the activities of distinct transcriptional activator and repressor forms of Cubitus interruptus. Cell 96,819 -831.[Medline]
Methot, N. and Basler, K. (2001). An absolute
requirement for Cubitus interruptus in Hedgehog signaling.
Development 128,733
-742.
Murone, M., Rosenthal, A. and de Sauvage, F. J. (1999). Hedgehog signal transduction: from flies to vertebrates. Exp. Cell Res. 253,25 -33.[CrossRef][Medline]
Nakano, Y., Guerrero, I., Hidalgo, A., Taylor, A., Whittle, J. R. and Ingham, P. W. (1989). A protein with several possible membrane-spanning domains encoded by the Drosophila segment polarity gene patched. Nature 341,508 -513.[CrossRef][Medline]
Ohlmeyer, J. T. and Kalderon, D. (1998). Hedgehog stimulates maturation of Cubitus interruptus into a labile transcriptional activator. Nature 396,749 -753.[CrossRef][Medline]
Pan, D. and Rubin, G. M. (1995). cAMP-dependent protein kinase and hedgehog act antagonistically in regulating decapentaplegic transcription in Drosophila imaginal discs. Cell 80,543 -552.[Medline]
Pignoni, F., Hu, B., Zavitz, K. H., Xiao, J., Garrity, P. A. and Zipursky, S. L. (1997). The eye-specification proteins So and Eya form a complex and regulate multiple steps in Drosophila eye development. Cell 91,881 -891.[Medline]
Price, M. A. and Kalderon, D. (1999).
Proteolysis of cubitus interruptus in Drosophila requires phosphorylation by
protein kinase A. Development
126,4331
-4339.
Quiring, R., Walldorf, U., Kloter, U. and Gehring, W. J. (1994). Homology of the eyeless gene of Drosophila to the Small eye gene in mice and Aniridia in humans. Science 265,785 -789.[Medline]
Robbins, D. J., Nybakken, K. E., Kobayashi, R., Sisson, J. C., Bishop, J. M. and Therond, P. P. (1997). Hedgehog elicits signal transduction by means of a large complex containing the kinesin-related protein costal2. Cell 90,225 -234.[Medline]
Ruohola, H., Bremer, K. A., Baker, D., Swedlow, J. R., Jan, L. Y. and Jan, Y. N. (1991). Role of neurogenic genes in establishment of follicle cell fate and oocyte polarity during oogenesis in Drosophila. Cell 66,433 -449.[Medline]
Sisson, J. C., Ho, K. S., Suyama, K. and Scott, M. P. (1997). Costal2, a novel kinesin-related protein in the Hedgehog signaling pathway. Cell 90,235 -245.[Medline]
Slusarski, D. C., Motzny, C. K. and Holmgren, R.
(1995). Mutations that alter the timing and pattern of cubitus
interruptus gene expression in Drosophila melanogaster.
Genetics 139,229
-240.
Spradling, A. C. (1993). Developmental genetics of oogenesis. In The Development of Drosophila melanogaster (ed. M. Bate and A. Martinez-Arias), pp.1 -70. Cold Spring Harbor: Cold Spring Harbor Laboratory Press.
Tinker, R., Silver, D. and Montell, D. J. (1998). Requirement for the vasa RNA helicase in gurken mRNA localization. Dev. Biol. 199, 1-10.[CrossRef][Medline]
Tworoger, M., Larkin, M. K., Bryant, Z. and Ruohola-Baker,
H. (1999). Mosaic analysis in the drosophila ovary reveals a
common hedgehog-inducible precursor stage for stalk and polar cells.
Genetics 151,739
-748.
Xu, P. X., Woo, I., Her, H., Beier, D. R. and Maas, R. L.
(1997). Mouse Eya homologues of the Drosophila eyes absent gene
require Pax6 for expression in lens and nasal placode.
Development 124,219
-231.
Xu, T. and Rubin, G. M. (1993). Analysis of
genetic mosaics in developing and adult Drosophila tissues.
Development 117,1223
-1237.
Zhang, X. F., Wang, J. F., Matczak, E., Proper, J. A. and
Groopman, J. E. (2001). Janus kinase 2 is involved in stromal
cell-derived factor-1 alpha-induced tyrosine phosphorylation of focal adhesion
proteins and migration of hematopoietic progenitor cells.
Blood 97,3342
-3348.
Zhang, Y. and Kalderon, D. (2000). Regulation
of cell proliferation and patterning in Drosophila oogenesis by Hedgehog
signaling. Development
127,2165
-2176.
Zhao, D., Clyde, D. and Bownes, M. (2000).
Expression of fringe is down regulated by Gurken/Epidermal Growth Factor
Receptor signalling and is required for the morphogenesis of ovarian follicle
cells. J. Cell Sci. 113,3781
-3794.