Department of Biological Chemistry, Johns Hopkins School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205-2185, USA
* Author for correspondence (e-mail: dmontell{at}jhmi.edu)
Accepted 14 September 2004
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SUMMARY |
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Key words: extra macrochaetae, Oogenesis, Differentiation, Follicle cells, Drosophila
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Introduction |
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Although the progeny of the somatic stem cells ultimately receive a complex
series of cues that cause them to differentiate into multiple spatially
organized cell types, many of these differentiation events occur relatively
late in egg chamber development. The early differentiation of follicle cells
within the germarium is rather simple and involves the choice between two cell
lineages: epithelial follicle cells (here termed `main body cells') that
contact the developing cyst; and cells that comprise the precursors of the
polar and stalk cells (here termed `polar/stalk precursors')
(Larkin et al., 1996;
Tworoger et al., 1999
).
Mutants that eliminate the formation of the polar and stalk cells, which
results in the formation of fused egg chambers, have been identified
(Grammont and Irvine, 2001
;
Lopez-Schier and St Johnston,
2001
); one mutant, eyes absent (eya),
autonomously changes the fate of the main body cells into polar cells
(Bai and Montell, 2002
).
However, it is not clear which signals limit EYA expression to main body
cells.
The Notch (N) pathway is involved at multiple steps in the differentiation
of follicle cells (Gonzalez-Reyes and St
Johnston, 1998), including at mid-oogenesis, when main body
follicle cells normally cease proliferation, differentiate and undergo several
cycles of genomic endoreplication. Notch mutant follicle cells fail
to differentiate and, instead of endoreplicating, they overproliferate
(Deng et al., 2001
;
Lopez-Schier and St Johnston,
2001
). In addition, Notch appears to be required for the
differentiation of the polar cells themselves
(Grammont and Irvine, 2001
).
One important ligand for Notch signaling in the ovary is Delta, which is
expressed in the germline at very low levels in the germarium
(Deng et al., 2001
;
Lopez-Schier and St Johnston,
2001
) and at high levels at stage 6, when the main body cells
differentiate (Bender et al.,
1993
). Delta signals through the Notch receptor, resulting in a
presenilin-dependent cleavage with release of the intracellular domain
(N-intra). N-intra binds to Suppressor of Hairless [Su(H)], the
Drosophila equivalent of CBF1
(Lopez-Schier and St Johnston,
2001
), and, together with Mastermind, activates transcription of
downstream targets. In neural development, major downstream transcriptional
targets of Notch are members of the Enhancer of Split family, which suppress
transcription of Achaete and Scute (Van
Doren et al., 1991
). However, the Enhancer of Split complex does
not appear to be required for follicle cell differentiation
(Deng et al., 2001
;
Lopez-Schier and St Johnston,
2001
), and it is unclear what the functional downstream targets of
Notch are in these cells. Achaete and Scute are members of a tissue-specific
class of basic helix-loop-helix (bHLH) genes, which act with Daughterless, a
ubiquitously distributed bHLH, to suppress transcription of Delta, and
possibly other targets. Daughterless is involved in Notch signaling in the
follicle cells (Cummings and Cronmiller,
1994
; Smith et al.,
2002
), but its bHLH partners have not been identified.
Extra macrochaetae (EMC) is a helix-loop-helix protein that is unable to
activate transcription because it lacks a DNA-binding basic domain
(Ellis et al., 1990;
Garrell and Modolell, 1990
).
Instead, it inhibits the transcriptional activity of bHLH proteins by binding
to them and sequestering them (Martinez et
al., 1993
; Van Doren et al.,
1991
; Van Doren et al.,
1992
). EMC is a transcriptional target of Notch in the wing
(Baonza et al., 2000
) and in
the eye (Baonza and Freeman,
2001
). In addition to its functions in Notch signaling, EMC is
required for cell proliferation (Baonza and
Garcia-Bellido, 1999
). The mammalian counterparts of EMC, the Id
(Inhibitor of DNA binding, and Inhibitor of Differentiation) family, are
involved in myriad differentiation events, primarily as inhibitors of
differentiation. Id proteins interact with the cell cycle machinery
(Lasorella et al., 2001
;
Ruzinova and Benezra, 2003
;
Zebedee and Hara, 2001
) and
are upregulated in a number of metastatic cancers
(Duncan et al., 1992
). Here we
identify a role for EMC in follicle cell differentiation, and identify it as
an effector of Notch signaling in the ovary.
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Materials and methods |
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UAS-emc 5.3 and emcP5C (an enhancer trap in
emc) were obtained from A. Garcia-Bellido
(Baonza et al., 2000;
Ellis et al., 1990
) and were
either crossed to C306 in parallel with EP3620 (UAS-emc
5.3) or dissected as 2-day-old homozygotes
(emcP5C).
To examine EMC or EYA expression in FLP-OUT clones, EP3620 or
UAS-N34A
[(Doherty et al., 1996
)
obtained from Ken Irvine] were crossed to hsFLP; ayGal4,
P[w+; UAS-mCD8GFP]/TM3 [ayGal4
(Ito et al., 1997
) is a
transgene in which Gal4 is expressed in clones of cells when FLP is induced by
heat shock; expression of UAS-mCD8GFP
(Lee and Luo, 1999
) marks the
clones; both were obtained from the Bloomington Stock Center
(http://fly.bio.indiana.edu)].
Non-balancer females were heat-shocked in a water bath for 1 hour at 37°C,
then placed at 25°C or 29°C with wet yeast for 2 or 3 days before
dissecting.
For analysis of loss-of-function phenotypes, mutant clones were generated
using the FLP/FRT system (Xu and Rubin,
1993) and hs-FLP; adult females were heat shocked in a
37°C water bath twice in 1 day (4 to 6 hours between heat-shocks), and
then placed on wet yeast at 25°C for 1 to 7 days. Clones were marked using
Ubi-nlsGFP (emc and N)
(Davis et al., 1995
) or using
anti-EYA antibody (eya54C2)
(Bonini et al., 1993
).
N55E11, FRT18A/FM7
(Grammont and Irvine, 2001
)
and emc1, FRT80B/TM6b [a strong hypomorphic allele of
emc (Ellis et al.,
1990
)] were obtained from Ken Irvine and Bloomington,
respectively. eya54C2, FRT40A was described previously
(Bai and Montell, 2002
).
emcAP6, FRT2A/TM6b [emcAP6 is a null
allele obtained from Pascale Heitzler
(Ellis, 1994
;
Heitzler et al., 1996
)] and
emcEP3620, FRT2A/TM6b were generated by standard methods.
Clones of emc1, FRT80B were generated in parallel with
clones of N55E11, FRT18A, and with clones of a wild-type
FRT80B chromosome. They were analyzed in a PZ80-containing
background 3 days after heat shock, since they exhibited no phenotype 2 days
after heat shock. We were unable to observe clones of emc1
mutant cells more than 3 days after heat shock. Clones of
emcAP6, FRT2A, and of emcEP3620, FRT2A
were analyzed in parallel with clones of a wild-type FRT2A chromosome
and were found as many as 7 days after heat shock.
In order to examine EMC overexpression using Upd-Gal4, a driver
expressed only in polar cells and their precursors
(Bai and Montell, 2002)
(obtained from Doug Harrison), Upd-Gal4; UAS-mCD8GFP/CyO was crossed
to w; PZ80/CyO; EP3620/TM6b or UAS-LacZ or w; PZ80/CyO;
UAS-mCD8GFP. Non-balancer flies were placed at 29°C for 3 to 5 days
before dissection. This was modified for Upd-Gal4 temperature shifts:
females and several males of the appropriate genotypes in a vial were fed with
wet yeast overnight at 18°C, incubated in a 33°C water bath for 6
hours, then returned to 18°C with fresh wet yeast for 24 hours before
dissecting, fixing and staining with anti-ßGal antibody and
4,6-diamidino-2-phenylindole (DAPI). Each egg chamber from stages 4 through 9
was scored for the number of polar cells in the expected anterior and
posterior polar cell groups.
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Immunofluorescence and immunohistochemistry |
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For immunofluorescence, the following primary antibodies were used: mouse
anti-Fas3 7G10 1:10 (DSHB); rabbit anti-ß-galactosidase 1:400 (Cappel;
NC); rabbit anti-EMC 1:1000 [a gift from Y. Jan
(Baonza et al., 2000)]; mouse
anti-EYA 10H6 (Bonini et al.,
1993
) 1:50 (DSHB); rabbit anti-Phosphohistone H3 1:1000 (Upstate
Biotechnology, NY); mouse anti-Cyclin B 1:10 (DSHB). Ovarioles were fixed for
10 minutes at room temperature in 4% formaldehyde in 0.1 mmol/l phosphate
buffer, rinsed twice in NP-40 wash, stained in the appropriate antibody
diluted in NP-40 wash overnight at 4°C, washed for 1 hour at room
temperature in NP-40 wash, and stained with appropriate secondary antibodies
diluted in NP-40 wash for 1 hour at room temperature. Secondary antibodies
used (Molecular Probes, OR) were Alexa 488 anti-rabbit, Alexa 488 anti-mouse,
Alexa 568 anti-rabbit, Alexa 568 anti-mouse, Alexa 647 anti-rabbit and Alexa
647 anti-mouse, each at 1:400. DAPI was added to the secondary incubation at
1:400. After incubation with secondary antibody, samples were rinsed twice in
NP-40 wash and mounted in Vectashield (Vector Laboratories, CA). Fluorescent
images were captured on an Ultraview spinning disk confocal microscope, a
Zeiss Axioplan, or a Zeiss Apotome.
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Results |
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|
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We examined effects of emc mutations on development of polar and
stalk cells. Notch signaling is required for differentiation of all the
follicle cells, including main body, polar and stalk cells
(Grammont and Irvine, 2001;
Lopez-Schier and St Johnston,
2001
). As with Notch, egg chambers with emc
mutant clones lacked polar cells at one end or the other
(Fig. 4O,P, arrowheads),
demonstrating a requirement for EMC in polar cell specification or
differentiation. However, in contrast to Notch, the emc
polar cell phenotype was partially penetrant (31.3%: 21 out of 67 clones).
Notch mutant egg chambers can be fused to their neighbors
(Grammont and Irvine, 2001;
Lopez-Schier and St Johnston,
2001
). Likewise, fused emc mutant egg chambers could be
found, possessing 32 germline nuclei (n=12). The presence of two
oocyte nuclei (Fig. 4, compare
Q and R, arrows) suggested that these egg chambers probably resulted from
fusion of two distinct cysts rather than from an extra round of germline
nuclear division. Fused egg chambers in ovarioles containing emc
mutant clones, like those observed in ovarioles containing Notch
mutant clones, possessed polar cells only at the termini of the fused cysts
(Fig. 4 compare S and T,
arrowheads), suggesting that fusion resulted from defects in polar cell
differentiation or polar/stalk precursor specification, rather than from
defects in stalk cell differentiation or morphogenesis.
If EMC affected polar cell precursor specification in addition to polar
cell differentiation, then we would expect that clones generated in the
germarium (i.e. those observed 4 days after heat shock) would produce
fused egg chambers more frequently than those generated in existing egg
chambers (i.e. those observed 1-2 days after heat shock). However, fused egg
chambers were observed at the same low frequency in both cases. Thus, current
evidence does not support a role for EMC in generating polar/stalk cell
precursors.
Relationship between EMC, Notch and EYA
EMC functions downstream of Notch in some tissues, and emc and
Notch mutant clones share some phenotypic characteristics, so we
examined whether expression of EMC in the main body cells depended upon Notch.
EMC expression was greatly reduced in large Notch clones on the main
body at stage 6 (n=36) (Fig.
5A,B). By contrast, EMC expression was normal in mutant clones of
STAT, another gene required for follicle cell differentiation (data
not shown).
|
The similarity between the expression patterns of EMC and EYA, the dependence of EYA on EMC in main body cells, and the formation of fused egg chambers when each is overexpressed, suggest that EMC is a regulator of EYA expression in all the follicle cells. Therefore we tested whether forcing EMC expression in the polar cells could induce EYA expression and affect polar cell formation. We generated FLP-OUT Gal4 clones of cells that expressed EMC. The polar cells were rarely targeted and out of several thousand clones observed, we obtained just six that resulted in fused egg chambers or the elimination of a single polar cell pair from one end of the egg chamber (Fig. 6A-D, arrows in Fig. 6A,C, and data not shown).
|
|
In addition to loss of polar cells, extra polar cells were sometimes formed
when EMC was expressed either with Upd-Gal4
(Table 1) or other drivers such
as HS-gal4 (data not shown). In wild-type ovarioles, EMC is reduced
in immature polar cells from stage 1 through stage 4 but is expressed in
mature polar cells at stage 5 and above (see
Fig. 3). This coincides with
stages in which a reduction of polar cell number occurs in wild-type
ovarioles: up to four or five polar cell precursors are observed in polar cell
groups belonging to stage-2 and stage-3 egg chambers, but two to three of
these precursor cells die upon maturation into polar cells at stage 4 or 5
(Besse and Pret, 2003). We
hypothesized that the ability of EMC to induce loss or gain of polar cells was
a consequence of differing roles of EMC at different times during polar cell
development. Specifically, we thought that EMC might be a negative regulator
of polar cells in stage 1 through stage 4, and a positive regulator subsequent
to this. In order to test this hypothesis, we took advantage of the
temperature-dependence of Gal4 activity. We shifted flies expressing
UAS-emc under the control of Upd-Gal4 to high (Gal4-active)
temperature for 6 hours in order to express EMC in immature polar cells, and
then down to low (Gal4-inactive) temperature for 24 hours to allow these cells
to mature. We then determined in which egg chambers polar cells were lost, and
in which egg chambers extra polar cells were formed. Based on the known
durations of various stages (Spradling,
1993
), we can estimate at what stage the egg chambers expressed
EMC, and thus determine at what stages polar cell development was inhibited or
enhanced by EMC. Egg chambers that were in stage 2 or 3 during EMC expression,
and were in stage 4-8 when they were analyzed, showed more frequent loss of
polar cells (Fig. 7A). By
contrast, extra polar cells were observed most frequently in egg chambers that
were at stage 9 at analysis (Fig.
7B), which were at stage 5 during EMC expression. Thus,
UAS-emc inhibits polar cell development at stages 2 and 3, but
promotes it at stage 5.
|
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Discussion |
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Notch and EMC are both involved in separation of adjacent egg chambers.
Follicles mutant for Notch or emc include egg chambers
containing multiple germline cysts and lacking intervening polar and stalk
cells. Recent work has shown that Notch signaling is not required for early
packaging (Torres et al.,
2003), but its role in specifying polar/stalk precursors, which
might occur subsequent to packaging, has not been addressed specifically.
However, Notch is required autonomously for polar cell differentiation
(Grammont and Irvine, 2001
;
Lopez-Schier and St Johnston,
2001
), and at least one pair of differentiated polar cells is
required to induce a stalk. Therefore, the packaging defects observed in
Notch mutants may be due solely to either the failure of polar cells
to differentiate or to a particular role in specifying polar/stalk precursors.
The fused egg chambers we observed in emc mutants could also be due
to a role in one or both processes. If emc were involved separately
in both processes, then we would expect to see a dramatic increase in the
frequency of fused egg chambers when clones were generated in the germarium,
where precursors are formed. Since we did not see such an increase, we do not
posit a role for emc in polar/stalk precursor formation; however,
without a better understanding of polar/stalk precursors, this cannot be
confirmed.
emc is probably not the only effector downstream of Notch in the ovariole, since emc mutant clones exhibit some differences from Notch mutant clones. In general the effects of emc were less penetrant than those of Notch. For example, emc mutant cells, like Notch mutant cells, exhibited persistent Phosphohistone H3 (data not shown) and Cyclin B labeling but at low frequency and at low levels. In addition, although emc is involved in polar cell specification or differentiation, it is only partially required for this, since normal polar cells could be observed even in clones of a null emc allele. Thus, there are likely to be additional downstream effectors of Notch in the ovary.
Molecular epistasis indicates that Notch signals through EMC to induce or
maintain EYA expression in the main body follicle cells. EYA expression is
lost in large Notch mutant follicle cell clones and can be induced by
forced expression of activated Notch in the follicle cells. EYA is involved in
inhibiting polar and stalk cell fate, so one might expect that Notch
or emc loss-of-function mutants would make extra polar cells. This is
not the case, however; it seems likely that the reason Notch and
emc mutant cells do not become polar cells is that they fail to
differentiate. EYA does not appear to be required for differentiation, but
rather for main body cell fate, since eya mutant cells differentiate
into polar cells (Bai and Montell,
2002). Thus, the Notch pathway must branch downstream of EMC, with
one pathway leading to EYA expression and repression of polar cell fate, and a
separate pathway leading to differentiation
(Fig. 8).
|
Three lines of evidence suggest that EMC may be a key regulator of EYA expression. First, EMC and EYA expression are similar in multiple respects. Both are upregulated in follicle cells in region 2B of the germarium, and in main body follicle cells from stages 2 through 6. Both are downregulated in the polar/stalk lineage from the germarium through stage 3, and in the oocyte-associated follicle cells at stages 7 through 9. Second, EMC is required for EYA expression in the main body. Third, EMC and EYA produce similar overexpression phenotypes, including fused egg chambers and the loss of polar cells, and, when EMC is overexpressed in the polar cells, EYA expression is induced. Taken together, these data suggest that the expression of EYA in the follicle cells is largely regulated by EMC. EMC is a helix-loop-helix protein that lacks the basic DNA-binding domain of the bHLH transcription factors. It normally opposes the activity of bHLH transcription factors by sequestering them in non-productive complexes. Thus, the dependence of EYA on EMC is likely to be indirect. Presumably, EMC inhibits a bHLH protein that inhibits expression of EYA. The identity of this protein remains an interesting subject for further study.
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ACKNOWLEDGMENTS |
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