Institut Jacques Monod, (UMR 7592 - CNRS/Université Pierre et Marie Curie/Université Denis Diderot), Laboratoire de Génétique du Développement et Evolution, 2 place Jussieu, 75251 Paris Cedex 05, France
Author for correspondence (e-mail:
bussond{at}ijm.jussieu.fr)
Accepted 25 November 2003
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SUMMARY |
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Key words: Oogenesis, polyhomeotic, Polycomb group, follicle cells, Drosophila
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Introduction |
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In the course of a gain-of-function screen aimed at identifying genes with
somatic function during oogenesis, we found that overexpression of
polyhomeotic (ph) leads to a significant reduction in female
fecundity. The ph gene is a member of the Polycomb group
(Pc-G) genes. Genes of the Pc-G and the
trithorax-group (trx-G) have been shown to control early
embryonic development, as well as imaginal disc morphogenesis, through
maintenance, but not initiation, of the spatial pattern of homeotic and
segment polarity gene expression during embryonic and imaginal disc
development (Dura and Ingham,
1988; Ingham and
Martinez-Arias, 1992
; Simon et
al., 1992
; Paro,
1993
; Randsholt et al.,
2000
; Francis and Kingston,
2001
). Several lines of evidence indicate that Pc-G
proteins associate in multimeric complexes associated with chromatin, thereby
ensuring gene silencing throughout development
(Fauvarque and Dura, 1993
;
Chan et al., 1994
), while
trx-G proteins counteract the silencing effect of Pc-G proteins
(Poux et al., 2002
). Mutations
in Pc-G genes exhibit synergistic and dose-dependent effects,
suggesting cooperative interactions between proteins encoded by these genes.
In addition, each Pc-G protein recognizes approximately 100 sites on
polytene chromosomes from Drosophila salivary glands, but these sites
do not fully overlap between these proteins
(Zink and Paro, 1989
;
DeCamillis et al., 1992
;
Lonie et al., 1994
).
Two-hybrid and immunoprecipitation studies have shown that the Pc-G
proteins form at least two non-overlapping protein complexes. One complex,
named PRC1, includes Polycomb (Pc), Posterior Sex Combs (Psc), Polyhomeotic
(PH), dRing/Sex Combs Extra (Sce) and Sex Comb on Midleg (Scm)
(Shao et al., 1999
;
Saurin et al., 2001
;
Fritsch et al., 2003
), while
the other contains Extra Sex combs (Esc) and Enhancer of zeste (E(z))
(Ng et al., 2000
;
Tie et al., 2001
).
Nevertheless, it has been shown that Pc, Psc and PH are differentially
distributed on regulatory sequences of the engrailed-related gene
invected, suggesting that there may be multiple Pc-G protein
complexes with different compositions that function at different target sites
(Strutt and Paro, 1997
). Since
some Pc-G proteins are not part of these complexes, for example Polycomb-like
(Pcl), it is supposed that there are other as yet unidentified complexes of
Pc-G proteins (for a review, see Otte and
Kwaks, 2003
).
The ph locus is located on the X chromosome at 2D2-3 position and
consists of two different transcription units, termed
polyhomeotic-distal (ph-d) and
polyhomeotic-proximal (ph-p), which correspond to a tandem
duplication of DNA (Dura et al.,
1987; Deatrick et al.,
1991
). Mutants homo- or hemizygous for lesions in either one of
the two units display the same phenotypes during embryonic development,
suggesting that these two molecular units encode redundant functions
(Dura et al., 1987
).
Furthermore, inactivation of one unit is transcriptionally compensated by the
other, thereby maintaining a nearly wild-type level of ph product
(Fauvarque et al., 1995
).
We show here that overexpression of the polyhomeotic gene leads to specific defects in follicle formation. Complementary analysis revealed that ph function is required within germarial somatic cells for their differentiation and proliferation. Removal of ph function leads to production of follicles with more or less than 16 germ cells, abnormal accumulation of cysts in the germarium, expansion of the interfollicular stalks between adjacent follicles, and polar cell differentiation defects. In addition, loss-of-function mutations reduce prefollicular and follicular cell proliferation in the germarium. Finally, we show that two other Pc-G members, Sce and Scm, are also implicated in early oogenesis.
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Materials and methods |
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We isolated by reverse PCR the genomic sequences flanking the selected P{y+}UAS insertions. Genomic DNA was extracted, digested by MspI and then circularized by ligation. The PCRs were performed under the following regime: (1) denaturation at 92°C for 5 minutes; (2) 40 cycles at 89°C for 5 seconds, 92°C for 10 seconds, 51°C for 45 seconds and 72°C for 2 minutes; and (3) a final 2-min extension at 72°C. The primer sequences correspond to the 5' P end. Primer sequences used to amplify the fragment were: OUY311 (5'-TTGATTCACTTTAACTTGCAC-3') and OUY521 (5'-ACACAACCTTTCCTCTCAACAA-3'). The PCR products were used directly in a nested PCR with the following primer sequences: OUY521 and OUY32 (5'-GCTTTCGCTTAGCGACGTG-3'). The cycle conditions were: (1) 92°C for 2 minutes; (2) 20 cycles at 89°C for 5 seconds, 92°C for 10 seconds, 56°C for 45 seconds and 72°C for 2 minutes; and (3) 72°C for 2 minutes. Amplification products were then sequenced directly by using the primer sequence OUY53 (5'-ATACTTCGGTAAGCTTCGGCTATCGACG-3').
Drosophila stocks
The control fly strains used were: wc and
w1118. The ph504 (noted
ph0) amorphic allele carries lesions in both ph-p
and ph-d units (Dura et al.,
1987). All other ph alleles are hypomorphic alleles. The
phlac strain was induced by PlacW-element
mutagenesis and corresponds to an insertion into the proximal unit, close to
the first intron-splicing site (Fauvarque
et al., 1995
). The A101 enhancer-trap insertion
(Ruohola et al., 1991
) was
used for tissue-specific ß-galactosidase staining of polar cells. For
testing ph overexpression, patched-GAL4 (Bloomington Stock
number 2017), da-GAL4 (Wodarz et
al., 1995
), e22c-GAL4 (Bloomington Stock number 1973) and
hs-GAL4 (Bloomington Stock number 3738) lines were used in
combination with PH-C7 (gift from F. Maschat), m20 and
m35 (Netter et al.,
2001
) P[UAS:cDNA ph] lines. Flies were raised on standard
media at 25°C.
Clonal analysis
Induction of Flipase expression by heat shock was done using either an
X-chromosomal or a II chromosomal hs-flp construct by heat shocking
females just after the eclosion at 38°C for 1 hour, once. Somatic
overexpression of ph was performed by generating Flip-out/GAL4 clones
in females hs-flp/+; UAS-phx/Act>CD2>Gal4 UAS-GFP
(Pignoni and Zipursky, 1997;
UAS-phx denotes either PH-C7, m20 or
m35). Flies were dissected 8 or 9 days after eclosion. Clones were
detected by the presence of GFP expression. Mutant clones for
ph were generated by mitotic recombination using the FLP/FRT system
(Xu and Rubin, 1993
) in
females hs-flp tub-lacZ FRT101/ph504 w FRT101 or
ubi-nls-GFP FRT101/ph504 w FRT101;
hsflp38/+. hs-flp tub-lacZ FRT101 and ubi-nls-GFP
FRT101; hs-flp38 lines were gifts from S. Goode (unpublished) and A.
Guichet, respectively; the ph504 w FRT101 was previously
described (Beuchle et al.,
2001
). Flies were dissected 2, 4 or 8 days after eclosion. Clones
were detected by the loss of lacZ or GFP expression.
FRT-mediated recombination events were induced specifically in the germline
using an ovo-flp transgene and revealed by loss of nuclear
GFP expression (I. Brun and C. Desplan, unpublished results) in
ubi-nls-GFP FRT101/ ph504 w FRT101; ovo-flp/+ females.
Flies were dissected 4 or 8 days after eclosion. Mutant clones for other
Pc-G genes were also generated and the following strains were used:
w; FRT82B ScmD1/TM6B, w; FRT82B Sce1/TM6B, yw;
FRT42D Su(z)2I.b8/SM6b, yw FRT42D PclD5/CyO, yw; FRT42B
sca AsxXT129/CyO, yw hs-flp; FRT42B hs-nGFP
(Beuchle et al., 2001
), yw
hs-flp; FRT42D ubi-nls-GFP/CyO and yw hs-flp; FRT82B ubi-nls-GFP
(gift from A. Guichet). AsxXT129 and
PclD5 have been reported to be null mutations,
Su(z)2I.b8 is a deficiency that removes Psc and
Su(z)2 (Soto et al.,
1995
), Sce1 has been reported to be a null
mutation (Breen and Duncan,
1986
) and ScmD1 is a frameshift mutation that
genetically behaves as a null allele
(Bornemann et al., 1998
). Flies
were dissected 8 or 9 days afer eclosion. In all cases, flies were kept at
25°C on standard media.
Staining ovaries
For antibody staining, ovaries were dissected in PBS and fixed in 3:1
heptane:4% formaldehyde in PBT (0.1% Tween-20 in PBS) or in 4%
paraformaldehyde in PBT for 25 minutes. The ovaries were washed three times in
PBT, blocked in 2% BSA for 1 hour, then incubated with primary antibody for 3
hours at room temperature or at 4°C overnight. The primary antibodies used
in this study were rabbit polyclonal anti-ß-galactosidase (1:200,
Boehringer), mouse monoclonal anti-ß-galactosidase 40-1a [1:200,
Developmental Studies Hybridoma Bank (DSHB)], mouse monoclonal anti-Orb 6H4
(1:30, DSHB), mouse monoclonal anti-Fasciclin III 7G10 (1:10, DSHB), rabbit
polyclonal anti--Spectrin (1:1000)
(Byers et al., 1987
), rabbit
polyclonal anti-phospho-histone H3 (1:1000, Upstate Biotechnology), mouse
monoclonal anti-Hts 1B1 (1:5, DSHB), mouse monoclonal anti-Armadillo N27A1
(1:100, DSHB) and mouse monoclonal anti-Eyes Absent eya10H6 (1:200, DSHB). The
fluorescence-conjugated secondary antibodies were purchased either from
Molecular Probes or Jackson Immunoresearch and used at a 1:200 dilution. All
samples were mounted in Cytifluor (Kent). For DNA labeling, the ovaries were
additionally incubated either for 20 minutes with 0.4 mg/ml RNase A and were
subsequently stained with 50 µg/ml propidium iodide (Molecular Probes) or
with 1 µg/ml of DAPI (Sigma).
For DAPI staining, tissues were fixed in 4% formaldehyde in PBS for 25 minutes and rinsed twice, first in PBT then in PBS. Ovaries were placed for one night in PBS:glycerol (1:1), with 1 µg/ml of DAPI (Sigma). Samples were examined either with a Leica DMR microscope or by confocal microscopy using a Leica DMR-BE microscope.
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Results |
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In order to confirm that somatic overexpression of ph perturbs
oogenesis, we took advantage of available P[UAST:cDNA-ph] lines
(PH-C7, m20 and m35; see Materials and methods). All the
da-, ptc-, e22c- and hs-GAL4/UAS-ph combinations we
tested were lethal before adult eclosion. We thus used the heat-shock
inducible flip-out/GAL4 system to generate mosaic follicles containing
overexpressing ph somatic cell clones. As previously observed in
4061/w; da-GAL4/+ females, we found both compound
(Fig. 2E) and apposed follicles
(data not shown), which were associated with the presence of large clones
marked by GFP protein expression (Fig.
2E'). As visualized after anti-Orb staining (Orb protein
accumulates specifically in the oocyte;
Lantz et al., 1992), compound
follicles contained two germline cysts developing together within a single
follicle (Fig. 2E'',
arrowheads). We next looked earlier for anomalies during germline cyst
encapsulation in the germarium. In a wild-type germarium, prefollicular cells
in region 2b (see Fig. 1)
extend long and thin processes, which separate individual linearly arranged
germline cysts. These cell extensions accumulate several cytoskeletal
proteins, including Fasciclin III (Fas III)
(Fig. 2F'), which is a
homophilic cell adhesion molecule (Ruohola
et al., 1991
). When ph was overexpressed, germaria
appeared long with multiple mature germline cysts in an extended region 2b/3
(Fig. 2G-G'', abnormal
germarium in which most of the germarial cells overexpressed ph;
Fig. 2G, same scale as
Fig. 2F). These nascent
follicles were only partially individualized by somatic cells, as visualized
by anti-Fas III staining (Fig.
2G'', asterisks) and budding off from the germarium was
severely delayed or deficient. This partial individualization of the germline
cysts by prefollicular cells could explain the `curved' aspect of the
follicular epithelium systematically observed in multicyst follicles
(Fig. 2B,E). As it has been
shown that the absence of polar cell specification leads to formation of
multicyst follicles (Grammont and Irvine,
2001
), we next looked for the distribution of polar cells in
multicyst follicles associated with overexpression of ph. We always
found four groups of polar cells when two germline cysts were encapsulated
together (Fig.
2H',H'',H''' are three different focal planes of
the same multicyst follicle). In this example, two pairs of polar cells are
present at each pole of the follicle (Fig.
2H',H''', solid arrowheads), and two groups of polar
cells in the middle of the follicular epithelium
(Fig. 2H'',H''',
empty arrowheads). Thus, ph overexpression affects the behavior of
somatic germarial cells, leading to encapsulation of multiple germline cysts
and delayed budding, but polar cell specification is not altered.
ph is required specifically in somatic cells for follicle formation
In order to assess the endogenous requirement of ph, we next
looked for ovarian defects associated with induction of mitotic cell clones
using the ph504 (noted ph0) amorphic
allele, which carries lesions in both ph-p and ph-d units
(Dura et al., 1987). Since it
has been previously reported that ph is transcribed and translated in
both somatic and germline cells, in the germarium and in the vitellarium until
stage 10, except in the oocyte (DeCamillis
and Brock, 1994
), we aimed at determining whether ph
function is required in both cellular compartments, or restricted to one of
them. Therefore, we first generated ph mutant germline clones using
the Flp/FRT system and the ovo promoter to drive flipase
expression specifically in the germline. Among more than 150 ovarioles with
ph0 germline clones observed, we never detected any
ovarian defects (data not shown), showing that ph is not required in
the germline for early oogenesis.
We next generated amorphic ph0 somatic clones using a
heat-shock promoter to drive flipase expression. flipase
expression was induced at eclosion and 4-, 6- and 8-day-old females were
dissected in order to recover clones in the follicular epithelium of different
stage follicles. Surprisingly, no or few ph0 cells,
detected by lack of -lacZ or -GFP reporter expression, were
observed in the great majority of dissected ovarioles, while large twin clones
and wild-type clones generated in parallel were readily observed (data not
shown). Indeed, ph0 cell clones being expulsed from the
somatic epithelium were frequently observed (data not shown), which probably
explains the low number of clones recovered. Similar observations were
reported for ph null clones induced in wing imaginal discs
(Santamaria et al., 1989). In
addition, it is possible that the division rate of ph0
cells may be affected.
Despite the presence of very few ph0 cells
(Fig. 3A'), follicles
with more than 16 germline cells were observed
(Fig. 3A). These follicles
result from the encapsulation of several germline cysts together as a constant
15:1 ratio between nurse cells and oocyte was always observed using anti-Orb
staining (Fig. 3A'').
Apposed follicles were also observed (data not shown). In order to explain
these encapsulation defects, we examined prefollicular cell behavior in the
germarium. In the germaria recovered bearing both ph+ and
ph0 cells in region 2b/3
(Fig. 3B,
ph0 cells marked by an asterisk in inset), staining with
Hu-li tai shao (Hts) antibodies (Hts is present on lateral membranes of
prefollicular cells; Lin et al.,
1994) showed that while ph+ prefollicular
cells had encapsulated germline cysts by extending long, thin cell processes
centripetally (Fig. 3B',
arrow), ph0 cells remained at the periphery of the
germarium with a relatively round morphology in comparison
(Fig. 3B', asterisks in
inset). These results suggest that ph function is necessary in
prefollicular cells for these cells to be able to acquire their specific
adhesive and/or migratory properties necessary for proper encapsulation of
individual germline cysts.
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We next examined expression of polar cell markers. In wild-type follicles,
polar cells are arranged as two groups of somatic cells at each follicle
extremity, as detected by various markers including Eyes Absent
(Fig. 3E'', arrowheads),
Fas III (see Fig. 5A'',
arrow) and several enhancer trap lines (such as A101, see
Fig. 5F', inset). It has
been previously described that in wild-type females the number of cells
expressing these markers is greater than two at the extremities of young
follicles (in particular stage 2 and 3 follicles), then progressively
restricted to two by stage 5 (Besse and
Pret, 2003). The Eyes Absent (Eya) protein is normally expressed
in prefollicular cells then specifically turned off in polar cells, while
maintained in follicular cells (Bai and
Montell, 2002
; Torres et al.,
2003
) (Fig.
3E'', arrowheads). When ph0 clones were
induced, two groups of anterior and posterior polar cells were invariantly
observed both in normal and multicyst follicles (data not shown). However, the
presence of more than two polar cells at the extremities of stage 5 or older
follicles was observed in rare ph0 polar cell clusters,
marked by the absence of Eya expression, indicating an excess compared to
wild-type follicles at these stages (Fig.
3D,D') (five polar cells do not express Eya). Importantly,
ph0 follicular cells correctly express Eya protein
(Fig. 3E-E'', bracket),
like neighboring ph+ follicular cells
(Fig. 3E-E'').
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ph mutant germaria exhibit abnormal encapsulation by prefollicular cells and abnormal budding of germline cysts
We next examined ovaries associated with a whole-fly ph partial
loss-of-function context. We found ovarian defects associated with one of the
previously described homozygous viable phlac mutations
(Dura et al., 1987)
(Fig. 2D) and showed that these
defects are specifically due to this mutation, since they are maintained after
outcrossing over six generations (data not shown). As visualized after DAPI
staining, phlac ovarioles exhibited follicles with either
more (Fig. 4A,B; compare to
wild-type ovariole in Fig. 2A)
or fewer (Fig. 4C, arrow) than
16 germ cells. When a follicle contained more than 16 germ cells (23%
ovarioles contained at least one compound follicle over 248 ovarioles
observed), we found groups of nurse cells with either equivalent
(Fig. 4A, arrow) or different
(Fig. 4B, arrows) degrees of
polyploidy. All compound follicles were multicyst, since they contained
several oocytes (anti-Orb staining, data not shown). In addition, apposed
chambers were also observed in phlac females (data not
shown). When a follicle contained fewer than 15 nurse cells (5.6%,
n=248), one of the adjacent chambers contained the complementary
number of nurse cells or degenerating nurse cells
(Fig. 4C; empty arrowhead
points to a pycnotic nucleus). Thus, perturbing ph function in the
phlac mutant leads to defects in follicle formation.
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Differentiation of interfollicular and polar cells is affected in phlac mutants
As differentiation of specialized germarial somatic cells are affected in
ph0 clones, we next examined expression of stalk and polar
cell differentiation markers in phlac females. In
wild-type ovarioles, interfollicular stalk cells express high levels of
-Spectrin (Fig.
5A', inset), and low levels of Fas III
(Fig. 5A'', inset). We
found that phlac mutant stalks contained an excess number
of cells (10 to 50) frequently organized into two or three lines (32%,
n=231) (Fig. 5B,C).
Such structures were comprised of cells with at least some characteristics of
interfollicular stalk cells because they strongly expressed
-Spectrin
(Fig. 5B'). However,
since they also strongly express Fas III
(Fig. 5C'), their
differentiation state was ambiguous.
Second, we examined polar cell differentiation. In wild-type follicles,
polar cells are detected using either anti-Fas III antibody or the enhancer
trap line A101 (neuralized-lacZ), which are both markers for mature
polar cells from stage 2 onward (data not shown and
Fig. 5A'', arrow). In
phlac ovarioles, whether follicles contained more or fewer
than the normal number of germline cells, there were always two groups of
polar cells, one at each extremity, anterior and posterior
(Fig. 5D,D', follicle
with fewer than 15 nurse cells; Fig.
5E,E', multicyst follicle). However, these groups often
contained more than two polar cells (Fig.
5F,F', insets; 11 polar cells can be seen at the posterior
pole of a stage 4 multicyst follicle). Although it has been shown previously
that early stage 2 and 3 follicles in wild-type ovarioles also frequently
contain more than two polar-cell marker expressing cells, a maximum of five of
these cells was observed (Besse and Pret,
2003) (Fig. 5G). In
addition, by stage 4, in wild-type ovarioles, the restriction to two polar
cells at each pole was almost complete and from stage 5 onwards there were
never more than two observed (Besse and
Pret, 2003
) (Fig.
5G). In contrast, phlac mutant ovarioles
exhibited a significant proportion of stage 4, as well as some stage 5 and up
to stage 9, follicles with more than two polar cells
(Fig. 5G and data not shown).
The presence of groups with more than two polar cells after stage 4 was not
strictly correlated with an abnormal number of germline cells within the
follicle but was always associated with an abnormal interfollicular stalk.
However, abnormal interfollicular stalks associated with two polar cells at
each extremity were also observed. In addition, ph mutant ovarioles
exhibited a shift in the dynamics of A101 staining: the appearance of this
staining is delayed since 100% (n=112) of stage 2
phlac follicles exhibited no marked cells compared to 34%
(n=78) of stage 2 wild-type follicles. In conclusion, ph
mutations perturb interfollicular stalk cell differentiation and stalk
morphogenesis, delay the polar cell differentiation program and generate an
excess of both of these cell types. Since stalk and polar cells arise from the
same precursors (Tworoger et al.,
1999
), the differentiation of these precursors seems specifically
affected in phlac mutants.
Proliferation of germarial somatic cells is reduced in phlac mutant females
We next wanted to know whether the excess in number of polar and
interfollicular stalk cells was associated with an alteration in proliferation
of precursor cells. We investigated the proliferative activity of
phlac germarial somatic cells using antibodies against
phospho-histone 3 (PH3), which specifically stain cells undergoing mitosis
(Hendzel et al., 1997), and
anti-Fas III antibodies to identify the different regions of the germarium
(e.g. Zhang and Kalderon,
2001
) (Fig.
2F'). As no positive marker for ovarian somatic stem cells
has been described so far, and as inner germarial sheath cells in 2a/2b
junction have been recently shown to be mitotically active
(Song and Xie, 2002
;
Song and Xie, 2003
), any
PH3+ somatic cells found just anteriorly to Fas III staining were
considered to be somatic stem cells and inner sheath cells. Control
phlac/+ and mutant phlac ovarioles
were double-stained with antibodies against Fas III and PH3, and the number of
proliferating stem and inner sheath cells (between region 2a and 2b),
prefollicular cells (region 2b), and follicular cells (region 3) was scored
for each genetic background. Very abnormal ovarioles, as those shown in
Fig. 4G', were not
included in this study because the different germarial regions could not be
delimited easily. As shown in Table
1, the number of mitotic somatic cells is significantly lower for
phlac females compared to phlac/+
control sisters for prefollicular and follicular cells in regions 2b and 3 of
the germarium. Although there is also a difference between these two genetic
contexts for mitotic activity in the 2a/2b region, the low frequency of this
type of event and the presence of inner sheath cell mitotic activity may
explain the fact that it is not possible to demonstrate the statistical
significance of this difference. We also verified that, as in the wild-type
context, there is no proliferation of stalk and polar cells after stage 1 in
phlac ovarioles (data not shown). Therefore, the excess
number of stalk and polar cells observed in ph mutant ovarioles
cannot be explained by excess proliferation of precursors in the germarium.
Nonetheless, the reduced proliferation of prefollicular and follicular cells
in the germaria of ph mutant ovarioles probably contributes to the
germline cyst encapsulation and budding defects observed.
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Discussion |
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Surprisingly, a similar phenotype to that observed in the ph overexpression study, multicyst follicles (several cysts within one follicle), was observed with loss-of-function ph alleles (discussed further below). Importantly, in contrast to ph overexpression, multicyst follicles in ph loss-of-function mutant ovaries always have only two groups of polar cells, one at each pole. Therefore, it seems that, unlike for overexpression of ph, delayed or deficient polar cell specification in ph mutants contributes to inclusion of several cysts within a single follicle. Thus, ph overexpression and loss-of-function phenotypes are distinguishable, indicating that the origin of the phenotypes is probably different.
ph function is necessary in somatic cells of the germarium for both their proliferation and differentiation
The implication of the ph gene in ovarian somatic cells was also
studied using two different loss-of-function mutations: the hypomorphic
phlac mutation, which consists of a PlacW
transposon inserted in the first intron of ph-p
(Fauvarque et al., 1995); and
via clonal analysis of the amorphic ph504 (noted
ph0) allele, which eliminates the functions of both
ph-p and phd (Dura et
al., 1987
). The origin of the multicyst phenotype caused by
ph loss-of-function mutations was characterized more precisely by
analysis of the process of follicle formation in the germarium. This study
showed that several early aspects of the somatic cell developmental program
(including proliferation, morphogenesis and differentiation) are perturbed by
these ph mutations.
On the one hand, the rate of division of germarial somatic cells is reduced in a ph hypomorphic mutant background as assayed by immunohistochemical analysis of the mitosis-specific PH3. This probably contributes to delayed follicle encapsulation and budding, evidenced by the accumulation of mature germline cysts in germarial region 3 of ph mutant ovarioles, and, consequently, by the formation of multicyst follicles. Although the same type of analysis was not possible upon induction of clones of the ph0 amorphic mutation in somatic ovarian cells, the fact that these clones are very small or absent compared with control clones suggests that a proliferation defect may also be associated with this ph mutation.
On the other hand, the morphogenetic properties of prefollicular cells and their differentiation into polar cells, interfollicular stalks and follicular epithelia are also specifically perturbed in ph mutants, which also probably contributes to formation of multicyst follicles. Our ph0 clonal analysis showed that ph function is necessary specifically in somatic cells, and not in the germline, for proper follicle formation. Almost all the phenotypes observed in ph mutant ovaries were reproduced upon induction of ph0 clones in prefollicular cells and their descendants.
First, prefollicular cell individualization of germline cysts is compromised in germarial regions 2a and 2b of ph mutant ovarioles. Fas III, which is specifically upregulated in prefollicular cells in wild-type germaria, is expressed normally in ph mutant prefollicular cells (phlac and ph0), but these cells remain at the periphery of the germarium and fail to undergo normal morphological changes necessary for germline cyst encapsulation, allowing multiple cysts, not individualized by somatic cells, to accumulate in region 3. In other ph mutant germaria in which prefollicular cells have migrated between germline cysts, encapsulation is disorganized, and germline cysts can be split and follicle budding significantly delayed. Therefore, in the germarium, interaction between prefollicular cells and the germline is defective.
Second, prefollicular cell differentiation into polar cells,
interfollicular stalk cells and follicular epithelial cells is delayed and/or
incomplete in the presence of ph mutations. Using a polar cell
specific marker (A101/neu-lacZ), we showed that specification of
polar cells, normally appearing by stage 2 in wild-type follicles, was delayed
in ph mutant ovarioles until stage 3. In addition, multicyst
follicles produced in ph mutant ovarioles contained two pairs of
polar cells, one at each extremity of the anterior/posterior axis. Therefore,
polar cell specification, which is necessary for individualization of germline
cysts, is perturbed by ph mutations. Interfollicular stalk cell
differentiation was assayed by expression of the stalk-specific marker,
-Spectrin, and by loss of Fas III, which is normally expressed at high
levels in precursor prefollicular cells. In stalks of both ph mutant
and ph0/ph+ mosaic ovarioles, although
-Spectrin expression was normally upregulated, Fas III was also present
at high levels, indicating an ambiguous state of differentiation. Poor
differentiation of stalk cells was further substantiated by the abnormally
long and disorganized interfollicular stalks that showed intercalation
defects. Abnormal perdurance of the early prefollicular cell marker Fas III
was also observed at the level of the follicular epithelium in very affected
ph mutant ovarioles, as well as in ph0 epithelial
cell clones. Taken together, these results suggest that ph mutations
result in the prolongation of a precursor state for polar, stalk and
follicular epithelial cells.
Third, ph mutant ovarioles exhibit an excess of polar cells (up to
11) at both the anterior and posterior poles of follicles, which persists
beyond stage 9, accompanied by an excess of adjacent interfollicular stalk
cells (from 10 to 50). ph0 clones induced in the polar
cell lineage also produced an excess of polar cells after stage 5, and the
presence of ph0 cells was also associated with abnormally
long stalks. Overproliferation of the pool of precursor cells common to both
polar and stalk cells would result in an excess of these cells. However, the
mitotic activity of germarial somatic cells was assayed by PH3 staining and a
reduction compared to wild-type was observed in regions 2a, 2b and 3. It has
recently been shown that an excess of polar cells is normally present in early
stage follicles in wild-type females, and that the final pair of polar cells
is selected from this group via apoptosis-induced cell death
(Besse and Pret, 2003).
However, in this study, a maximum of six such pre-polar cells was observed
when apoptosis was specifically blocked. Since ph mutant ovarioles
exhibit up to 11 polar cells, it would seem that apoptosis of pre-polar cells
is probably not the only aspect of polar cell development affected. Finally,
it is possible that the process by which the pool of polar and stalk cell
precursors, distinct from the progenitors of the epithelial follicle cells, is
set aside may be affected by ph mutations, leading to both problems
in their number and differentiation
(Tworoger et al., 1999
).
Determination of this pool probably involves several cell-cell signaling
pathways in region 2b of the germarium, implicating Delta/Notch and EGFR
signaling initiating from the germline and Hedgehog signaling from anterior
terminal filament somatic cells (Goode et
al., 1996b
; Zhang and
Kalderon, 2000
; Grammont and
Irvine, 2001
; Lopez-Schier and
St Johnston, 2001
; Besse et
al., 2002
). ph may participate, in parallel or within one
(or several) of these signaling pathways, to the regulation of the somatic
cell differentiation program in the germarium. So far, attempts to uncover
genetic or molecular interactions between ph and genes of these
signaling pathways have proven unfruitful (K.N., unpublished results).
Requirements for the Polycomb group in oogenesis
The ph gene was first characterized as one of the Drosophila
Pc-G genes, which encode transcriptional repressors required for
maintaining the spatial pattern of homeotic gene expression during embryonic
and larval development (Christen and
Bienz, 1994; McKeon et al.,
1994
; Soto et al.,
1995
). ph has additional functions during development,
since it has also been implicated in restriction of anterior compartment
expression of engrailed and hedgehog in the wing imaginal
disc (Maschat et al., 1998
;
Maurange and Paro, 2002
). Our
present results, which implicate ph function and that of two other
Pc-G genes (Sce and Scm) in somatic cell
development during early oogenesis, thus suggest that Pc-G function
may be more generalized than previously thought. One other study reported
ovarian defects associated with two temperature-sensitive alleles
(pcoox736hs and pcomy939hs) of the
E(z) gene, but the defects observed do not resemble those of
ph (degeneration of nurse cells and little growth in the size of the
follicle beyond stage 3 or 4) (Philips and
Shearn, 1990
).
We also examined ovarian phenotypes associated with mutations in several
other Pc-G genes. The product of the Scm gene interacts
directly with PH (Peterson et al.,
1997), and, with PH, forms part of the same complex, PRC1, in
Drosophila embryos (Shao et al.,
1999
). We found that the presence of large somatic cell clones of
ScmD1 (amorphic mutation) leads to similar, though not
completely overlapping, phenotypes than those observed for mutations in the
ph gene. In particular, our results suggest that somatic cells are
poorly differentiated and this leads to formation of multicyst follicles and
abnormal interfollicular stalks with an excess number of cells. We also tested
other Pc-G members, some which also belong to the PRC1 complex
(Sce and Psc), while others do not (Asx, Su(z)2 and
Pcl). Ovarian defects were observed only with a mutation in the
Sce gene, and these defects closely resembled those obtained with
ScmD1. Since follicle cell clones mutant for
ScmD1 and Sce1 covered large areas of
the follicular epithelium, like wild-type clones, we can conclude that, unlike
for ph0, ScmD1 and
Sce1 somatic cells are not affected in their proliferative
property and/or viability. In addition, ScmD1 and
Sce1 mutations did not affect polar cell number or
differentiation. These results indicate that these anomalies were specific to
mutations in the ph gene. Thus, we show that several components of
the PRC1 complex, but not all, seem to be implicated in follicle formation and
their functions do not seem to overlap fully. In addition, none of the genetic
interactions between Pc-G genes known to exist for embryonic segment
identity were reproduced in the ovary system (data not shown). We can make two
hypotheses concerning the role of these Pc-G genes in ovarian
folliculogenesis: (1) either each Pc-G gene acts specifically on its
own specific subset of target genes in somatic cells of the ovary, possibly
regulating the transcriptional machinery directly rather than forming
particular Pc-G complexes that alter chromatin structure; or (2) repression of
target genes in somatic cells of the ovary occurs via Pc-G complexes
in a chromatin-dependent manner, but the complexes involved differ markedly in
composition from those identified for embryonic cell identity. Further
experiments will be needed to distinguish between these two possibilities.
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ACKNOWLEDGMENTS |
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Footnotes |
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REFERENCES |
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---|
Bai, J. and Montell, D. (2002). Eyes absent, a
key repressor of polar cell fate during Drosophila oogenesis.
Development 129,5377
-5388.
Baksa, K., Parke, T., Dobens, L. and Dearolf, C. (2002). The Drosophila STAT protein, stat92E, regulates follicle cell differentiation during oogenesis. Dev. Biol. 243,166 -175.[CrossRef][Medline]
Besse, F. and Pret, A. M. (2003).
Apoptosis-mediated cell death within the ovarian polar cell lineage of
Drosophila melanogaster. Development
130,1017
-1027.
Besse, F., Busson, D. and Pret, A. M. (2002).
Fused-dependent Hedgehog signal transduction is required for somatic cell
differentiation during Drosophila egg chamber formation.
Development 129,4111
-4124.
Beuchle, D., Struhl, G. and Muller, J. (2001).
Polycomb group proteins and heritable silencing of Drosophila Hox
genes. Development 128,993
-1004.
Bornemann, D., Miller, E. and Simon, J. (1998).
Expression and properties of wild-type and mutant forms of the
Drosophila sex comb on midleg (SCM) repressor protein.
Genetics 150,675
-686.
Breen, T. R. and Duncan, I. M. (1986). Maternal expression of genes that regulate the bithorax complex of Drosophila melanogaster. Dev. Biol. 118,442 -456.[Medline]
Byers, T. J., Dubreuil, R., Branton, D., Kiehart, D. P. and Goldstein, L. S. (1987). Drosophila spectrin. II. Conserved features of the alpha-subunit are revealed by analysis of cDNA clones and fusion proteins. J. Cell Biol. 105,2103 -2110.[Abstract]
Chan, C. S., Rastelli, L. and Pirrotta, V. (1994). A Polycomb response element in the Ubx gene that determines an epigenetically inherited state of repression. EMBO J. 13,2553 -2564.[Abstract]
Christen, B. and Bienz, M. (1994). Imaginal disc silencers from Ultrabithorax: evidence for Polycomb response elements. Mech. Dev. 48,255 -266.[CrossRef][Medline]
Deatrick, J., Daly, M., Randsholt, N. B. and Brock, H. W. (1991). The complex genetic locus polyhomeotic in Drosophila melanogaster potentially encodes two homologous zinc-finger proteins. Gene 105,185 -195.[CrossRef][Medline]
DeCamillis, M. and Brock, H. W. (1994). Expression of the polyhomeotic locus on development of Drosophila melanogaster. Roux's Arch. Develop. Biol. 203,429 -438.
DeCamillis, M., Cheng, N. S., Pierre, D. and Brock, H. W. (1992). The polyhomeotic gene of Drosophila encodes a chromatin protein that shares polytene chromosome-binding sites with Polycomb. Genes Dev. 6,223 -232.[Abstract]
Dura, J. M. and Ingham, P. (1988). Tissue- and stage-specific control of homeotic and segmentation gene expression in Drosophila embryos by the polyhomeotic gene. Development 103,733 -741.[Abstract]
Dura, J. M., Brock, H. W. and Santamaria, P. (1985). Polyhomeotic: a gene of Drosophila melanogaster required for correct expression of segmental identity. Mol. Gen. Genet. 198,213 -220.[Medline]
Dura, J. M., Randsholt, N. B., Deatrick, J., Erk, I., Santamaria, P., Freeman, J. D., Freeman, S. J., Weddell, D. and Brock, H. W. (1987). A complex genetic locus, polyhomeotic, is required for segmental specification and epidermal development in D. melanogaster. Cell 51,829 -839.[Medline]
Fauvarque, M. O. and Dura, J. M. (1993). polyhomeotic regulatory sequences induce developmental regulator-dependent variegation and targeted P-element insertions in Drosophila. Genes Dev. 7,1508 -1520.[Abstract]
Fauvarque, M. O., Zuber, V. and Dura, J. M. (1995). Regulation of polyhomeotic transcription may involve local changes in chromatin activity in Drosophila. Mech. Dev. 52,343 -355.[CrossRef][Medline]
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.
Francis, N. J. and Kingston, R. E. (2001). Mechanisms of transcriptional memory. Nat. Rev. Mol. Cell Biol. 2,409 -421.[CrossRef][Medline]
Fritsch, C., Beuchle, D. and Muller, J. (2003). Molecular and genetic analysis of the Polycomb group gene Sex combs extra/Ring in Drosophila. Mech. Dev. 120,949 -954.[CrossRef][Medline]
Goode, S., Melnick, M., Chou, T. B. and Perrimon, N.
(1996a). The neurogenic genes egghead and
brainiac define a novel signaling pathway essential for epithelial
morphogenesis during Drosophila oogenesis.
Development 122,3863
-3879.
Goode, S., Morgan, M., Liang, Y. P. and Mahowald, A. P. (1996b). brainiac encodes a novel, putative secreted protein that cooperates with Grk TGF alpha in the genesis of the follicular epithelium. Dev. Biol. 178, 35-50.[CrossRef][Medline]
Goode, S., Wright, D. and Mahowald, A. P.
(1992). The neurogenic locus brainiac cooperates with
the Drosophila EGF receptor to establish the ovarian follicle and to
determine its dorsal-ventral polarity. Development
116,177
-192.
Grammont, M. and Irvine, K. D. (2001). fringe and Notch specify polar cell fate during Drosophila oogenesis. Development 128,2243 -2253.[Medline]
Hendzel, M. J., Wei, Y., Mancini, M. A., Van Hooser, A., Ranalli, T., Brinkley, B. R., Bazett-Jones, D. P. and Allis, C. D. (1997). Mitosis-specific phosphorylation of histone H3 initiates primarily within pericentromeric heterochromatin during G2 and spreads in an ordered fashion coincident with mitotic chromosome condensation. Chromosoma 106,348 -360.[CrossRef][Medline]
Ingham, P. W. and Martinez-Arias, A. (1992). Boundaries and fields in early embryos. Cell 68,221 -235.[Medline]
Kim, J., Sebring, A., Esch, J. J., Kraus, M. E., Vorwerk, K., Magee, J. and Carroll, S. B. (1996). Integration of positional signals and regulation of wing formation and identity by Drosophila vestigial gene. Nature 382,133 -138.[CrossRef][Medline]
King, R. C. (1970). Ovarian Development in Drosophila melanogaster. New York: Academic Press.
Lantz, V., Ambrosio, L. and Schedl, P. (1992). The Drosophila orb gene is predicted to encode sex-specific germline RNA-binding proteins and has localized transcripts in ovaries and early embryos. Development 115, 75-88.[Abstract]
Lin, H. (2002). The stem-cell niche theory: lessons from flies. Nat. Genet. 3, 931-940.[CrossRef]
Lin, H., Yue, L. and Spradling, A. C. (1994).
The Drosophila fusome, a germline-specific organelle, contains
membrane skeletal proteins and functions in cyst formation.
Development 120,947
-956.
Lonie, A., D'Andrea, R., Paro, R. and Saint, R.
(1994). Molecular characterisation of the Polycomblike
gene of Drosophila melanogaster, a trans-acting negative regulator of
homeotic gene expression. Development
120,2629
-2636.
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.
Margolis, J. and Spradling, A. (1995).
Identification and behavior of epithelial stem cells in the
Drosophila ovary. Development
121,3797
-3807.
Maschat, F., Serrano, N., Randsholt, N. B. and Geraud, G.
(1998). engrailed and polyhomeotic interactions
are required to maintain the A/P boundary of the Drosophila
developing wing. Development
125,2771
-2780.
Maurange, C. and Paro, R. (2002). A cellular
memory module conveys epigenetic inheritance of hedgehog expression
during Drosophila wing imaginal disc development. Genes
Dev. 16,2672
-2683.
McGregor, J., Xi, R. and Harrison, D. (2002).
JAK signaling is somatically required for follicle cell differentiation in
Drosophila. Development
129,705
-717.
McKeon, J., Sinclair, D. A., Cheng, N., Couling, M. and Brock, H. W. (1994). Mutations in some Polycomb group genes of Drosophila interfere with regulation of segmentation genes. Mol. Gen. Genet. 244,474 -483.[Medline]
Monnier, V., Girardot, F., Cheret, C., Andres, O. and Tricoire, H. (2002). Modulation of oxidative stress resistance in Drosophila melanogaster by gene overexpression. Genesis 34,76 -79.[CrossRef][Medline]
Netter, S., Faucheux, M. and Theodore, L. (2001). Developmental dynamics of a polyhomeotic-EGFP fusion in vivo. DNA Cell Biol. 20,483 -492.[CrossRef][Medline]
Ng, J., Hart, C. M., Morgan, K. and Simon, J. A.
(2000). A Drosophila ESC E(Z) protein complex is
distinct from other polycomb group complexes and contains covalently modified
ESC. Mol. Cell Biol. 20,3069
-3078.
Otte, A. P. and Kwaks, T. (2003). Gene repression by Polycomb group protein complexes: a distinct complex for every occasion? Curr. Opin. Genet. Dev. 13,448 -454.[CrossRef][Medline]
Paro, R. (1993). Mechanisms of heritable gene repression during development of Drosophila. Curr. Opin. Cell Biol. 5,999 -1005.[Medline]
Peterson, A. J., Kyba, M., Bornemann, D., Morgan, K., Brock, H. W. and Simon, J. (1997). A domain shared by the Polycomb group proteins Scm and ph mediates heterotypic and homotypic interactions. Mol. Cell Biol. 17,6683 -6692.[Abstract]
Philips, M. D. and Shearn, A. (1990). Mutations
in polycombeotic, a Drosophila Polycomb-group gene, cause a
wide of maternal and zygotic phenotypes. Genetics
125,91
-101.
Pignoni, F. and Zipursky, S. L. (1997).
Induction of Drosophila eye development by decapentaplegic.
Development 124,271
-278.
Poux, S., Horard, B., Sigrist, C. J. and Pirrotta, V. (2002). The Drosophila trithorax protein is a coactivator required to prevent re-establishment of polycomb silencing. Development 129,2483 -2493.[Medline]
Randsholt, N., Maschat, F. and Santamaria, P. (2000). polyhomeotic controls engrailed expression and the hedgehog signaling pathway in imaginal discs. Mech. Dev. 95,89 -99.[CrossRef][Medline]
Rorth, P. (1996). A modular misexpression
screen in Drosophila detecting tissue-specific phenotypes.
Proc. Natl. Acad. Sci. USA
93,12418
-12422.
Rubsam, R., Hollmann, M., Simmerl, E., Lammermann, U., Schafer, M. A., Buning, J. and Schafer, U. (1998). The egghead gene product influences oocyte differentiation by follicle cell-germ cell interactions in Drosophila melanogaster. Mech. Dev. 72,131 -140.[CrossRef][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]
Santamaria, P., Deatrick, J. and Randsholt, N. B. (1989). Pattern triplications following genetic ablation on the wing of Drosophila. Roux's Arch. Develop. Biol. 198,65 -77.
Saurin, A. J., Shao, Z., Erdjument-Bromage, H., Tempst, P. and Kingston, R. E. (2001). A Drosophila Polycomb group complex includes Zeste and dTAFII proteins. Nature 412,655 -660.[CrossRef][Medline]
Shao, Z., Raible, F., Mollaaghababa, R., Guyon, J. R., Wu, C. T., Bender, W. and Kingston, R. E. (1999). Stabilization of chromatin structure by PRC1, a Polycomb complex. Cell 98, 37-46.[Medline]
Simon, J., Chiang, A. and Bender, W. (1992). Ten different Polycomb group genes are required for spatial control of the abdA and AbdB homeotic products. Development 114,493 -505.[Abstract]
Song, X. and Xie, T. (2002).
DE-cadherin-mediated cell adhesion is essential for maintaining somatic stem
cells in the Drosophila ovary. Proc. Natl. Acad. Sci.
USA 99,14813
-14818.
Song, X. and Xie, T. (2003). wingless
signaling regulates the maintenance of ovarian somatic stem cells in
Drosophila. Development
130,3259
-3268.
Soto, M. C., Chou, T. B. and Bender, W. (1995).
Comparison of germline mosaics of genes in the Polycomb group of
Drosophila melanogaster. Genetics
140,231
-243.
Spradling, A. (1993). Developmental genetics of oogenesis. In Development of Drosophila melanogaster (eds M. Bate and A. Martinez-Arias), pp. 1-70. New York: Cold Spring Harbor Laboratory Press.
Spradling, A. C., de Cuevas, M., Drummond, B. D., Keyes, L., Lilly, M., Pepling, M. and Xie, T. (1997). The Drosophila germarium: stem cells, germ line cysts, and oocytes. Cold Spring Harbor Symp. Quant. Biol. 62, 25-34.[Medline]
Spradling, A., Drummond-Barbosa, D. and Kai, T. (2001). Stem cells find their niche. Nature 414,98 -104.[CrossRef][Medline]
Strutt, H. and Paro, R. (1997). The polycomb group protein complex of Drosophila melanogaster has different compositions at different target genes. Mol. Cell. Biol. 17,6773 -6783.[Abstract]
Tie, F., Furuyama, T., Prasad-Sinha, J., Jane, E. and Harte, P.
J. (2001). The Drosophila Polycomb Group proteins
ESC and E(Z) are present in a complex containing the histone-binding protein
p55 and the histone deacetylase RPD3. Development
128,275
-286.
Torres, I. L., Lopez-Schier, H. and St Johnston, D. (2003). A Notch/Delta-dependent relay mechanism establishes anterior-posterior polarity in Drosophila. Dev. Cell 5,547 -558.[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.
Weatherbee, S. D., Halder, G., Kim, J., Hudson, A. and Carroll,
S. (1998). Ultrabithorax regulates genes at several levels of
the wing-patterning hierarchy to shape the development of the
Drosophila haltere. Genes Dev.
12,1474
-1482.
Wodarz, A., Hinz, U., Engelbert, M. and Knust, E. (1995). Expression of crumbs confers apical character on plasma membrane domains of ectodermal epithelia of Drosophila. Cell 82,67 -76.[Medline]
Xu, T. and Rubin, G. M. (1993). Analysis of
genetic mosaics in developing and adult Drosophila tissues.
Development 117,1223
-1237.
Zhang, Y. and Kalderon, D. (2000). Regulation
of cell proliferation and patterning in Drosophila oogenesis by
Hedgehog signaling. Development
127,2165
-2176.
Zhang, Y. and Kalderon, D. (2001). Hedgehog acts as a somatic stem cell factor in the Drosophila ovary. Nature 410,599 -604.[CrossRef][Medline]
Zink, B. and Paro, R. (1989). In vivo binding pattern of a trans-regulator of homoeotic genes in Drosophila melanogaster. Nature 337,468 -471.[CrossRef][Medline]
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