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First published online 3 May 2006
doi: 10.1242/dev.02369
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Department of Biology, McGill University, 1205 Doctor Penfield Avenue, Montreal, QC H3A 1B1, Canada.
* Author for correspondence (e-mail: laura.nilson{at}mcgill.ca)
Accepted 17 March 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Drosophila, capicua, mirror, Follicle cells, Patterning
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Roth, 2003;
Van Buskirk and Schübach,
1999). At the end of oogenesis, the follicle cells secrete the
eggshell, which displays pronounced axial asymmetries and locally specialized
structures that represent the outcome of the patterning process
(Waring, 2000). For example,
the anterior eggshell exhibits two prominent appendages flanking the dorsal
midline, reflecting a complex pattern of follicle cell fates along the
dorsoventral (DV) axis. Defects in epithelial patterning can therefore be
recognized through the resulting eggshell abnormalities.
DV asymmetry within the follicular epithelium is established during
mid-oogenesis by the product of the gurken (grk) gene, a
transforming growth factor (TGF) 
-like molecule restricted to the
future dorsal side of the oocyte
(Neuman-Silberberg and Schübach,
1993; Neuman-Silberberg and
Schübach, 1996). Grk acts as a localized ligand for the
Drosophila epidermal growth factor receptor (Egfr) in the overlying
follicle cells, and the resulting localized Egfr activity defines the dorsal
region of the epithelium (Nilson and
Schübach, 1999; Roth,
2003; Van Buskirk and
Schübach, 1999). As oogenesis proceeds, this initial
distinction of dorsal from ventral follicle cells is refined to generate a
more complex pattern of cell fates, including two dorsolateral appendage
primordia separated by a population of dorsal midline cells. The resolution of
primordia and midline fates within the dorsal domain is achieved through
feedback modulation of Egfr activity. Gurken-Egfr signaling induces expression
of rhomboid (rho), which leads to autocrine production of
the active form of another Egfr ligand, Spitz (Spi), presumably resulting in
amplification of the initial signal. Egfr activation also induces expression
of Argos (Aos), a direct antagonist of Spi, in the dorsal-most follicle cells,
thereby resolving the initial broad region of Egfr signaling into two
dorsolateral peaks (Klein et al.,
2004; Peri et al.,
1999; Shvartsman et al.,
2002; Wasserman and Freeman,
1998). The position of the primordia is also regulated along the
anteroposterior (AP) axis by Decapentaplegic (Dpp), a member of the Tgfß
family of signaling molecules, produced by the anterior-most follicle cells
(Deng and Bownes, 1997;
Peri and Roth, 2000;
Twombly et al., 1996).
The dorsal midline domain between the appendage primordia defines their
dorsal boundary, but how the ventral boundary of each primordium is determined
is not understood. Computational models of Aos function in the embryonic
ventral ectoderm have suggested that Aos may act at a short range to sequester
Spi and restrict its diffusion (Klein et
al., 2004; Reeves et al.,
2005). However, a ventral expansion of appendage-producing
follicle fates is not observed upon loss of aos function
(Wasserman and Freeman, 1998),
indicating that the ventral limit of the appendage primordia is not defined by
Aos-mediated regulation of Spi distribution. Alternatively, a graded dorsal
distribution of Egfr ligand could define the ventral limit of the primordia,
establishing dorsal fates only where levels exceed a critical threshold.
However, it is not clear whether additional factors function to translate this
potentially graded signal into a sharp boundary between primordium and
non-primordium fates (Shilo,
2005). Moreover, how the dorsal patterning process is integrated
with anterior positional information, which is present throughout the DV axis,
is not understood.
DV patterning of the follicular epithelium requires capicua
(cic) function. cic mutant females produce `dorsalized' eggs
that exhibit a ventral expansion of dorsal pattern elements. Although similar
eggshell phenotypes can result from the mislocalization of Grk
(Neuman-Silberberg and Schübach,
1993; Neuman-Silberberg and
Schübach, 1996), the expression and distribution of
grk are unaffected in cic mutant ovaries, suggesting that
cic acts downstream of grk
(Goff et al., 2001).
Dorsalization can also result from increased Egfr levels and signaling, as
observed upon loss of the D-Cbl (Cbl –FlyBase) ubiquitin-protein ligase
in the follicle cells (Pai et al.,
2000). However, a general expansion of Egfr target gene expression
is not observed in cic mutant ovaries
(Goff et al., 2001),
indicating that the cic eggshell phenotype does not result from
generally increased Egfr activity. Loss of cic therefore has a unique
effect on eggshell patterning, suggesting that Cic functions at a novel step
in the regulation of Egfr-mediated cell fate determination.
cic encodes an evolutionarily conserved HMG-box transcription
factor that functions as a transcriptional repressor during embryogenesis and
wing development (Jimenez et al.,
2000; Roch et al.,
2002). In general, downregulation of Cic levels through receptor
tyrosine kinase (RTK) signaling activates gene expression. For example,
downregulation of Cic by Torso activity at the poles of the early embryo
establishes the embryonic termini, and loss of Cic in the wing imaginal disk
through localized Egfr signaling specifies the presumptive wing vein tissue
(Jimenez et al., 2000;
Roch et al., 2002). In the
ovary, Cic is downregulated in dorsal follicle cells during DV patterning
(Goff et al., 2001),
suggesting that Cic could negatively regulate RTK target genes in this tissue
as well.
We show that cic is required cell autonomously in ventral and lateral follicle cells to repress appendage-producing fates, and that this fate transformation correlates with and depends on the ectopic expression of mirror (mirr), an Egfr target normally expressed in dorsal anterior follicle cells. We further demonstrate that the ectopic expression of Mirr, a homeodomain transcription factor, at any position within the follicular epithelium is sufficient to determine appendage-producing fate. Interestingly, the ectopic mirr expression and appendage-producing fates observed in cic mutant ovaries are restricted to the anterior half of the follicular epithelium. Collectively, these data suggest that Cic regulates the specification of the appendage primordia by inhibiting the induction of dorsal fate determinants by anterior positional cues.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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),
cicBA53, mirre48
(McNeill et al., 1997),
P{lacW}l(3)mirr6D1 (mirr-lacZ)
(Ruohola-Baker et al., 1993),
fng13 (Grammont and
Irvine, 2001), UAS-mirr
(McNeill et al., 1997),
P{lacW}kek1AN296
(Sapir et al., 1998),
grkHK, grk2B6
(Thio et al., 2000),
rhomboid7M43 and rhomboid6
(Wasserman et al., 2000). Loss
of function clones were generated using y w P{hsFLP}122; P{Ubi-GFPnls}
P{neoFRT}82B or y w P{hsFLP}122; P{hs-N-myc} P{neoFRT}82B. The
stock y w; P{GAL4-Act5C(FRT.CD2).P}S, P{UAS-GFP} was used to generate
gain-of-function clones.
Sequencing of the cicBA53 allele
The cicBA53 allele was identified in a genetic screen
for patterning defects associated with follicle cell clones. Overlapping
fragments of approximately 800 base pairs each from the cic locus
were amplified by PCR from genomic DNA of homozygous
cicBA53 embryos, and from flies homozygous for the
parental chromosome on which the cicBA53 mutation was
induced, and then sequenced by the Genome Québec Innovation Centre
(Montréal, QC). The only sequence change was a base pair transition
from C to T at position 2563 of the genomic sequence (with position 1 being
the first base of the 5' UTR), which introduces a stop codon near the
end of the third exon.
Generation of mosaic ovaries
Loss-of-function follicle cell clones were generated by FLP-FRT-mediated
mitotic recombination as described, except that recombination was induced in
adults (Caceres and Nilson,
2005; Xu and Rubin,
1993). Homozygous mutant clones were marked by the absence of
nuclear green fluorescent protein (GFP) or N-Myc
(Xu and Rubin, 1993). To
generate GFP-marked gain-of-function clones, females bearing the y w
P{hsFlp}122 chromosome, the Flp-out cassette
P{GAL4-Act5C(FRT.CD2).P}S, P{UAS-GFP}
(Pignoni and Zipursky, 1997),
and a gene of interest under UAS control were incubated at 37°C for 2
minutes and 15 seconds.
Immunohistochemistry
Immunohistochemistry was performed as described previously
(Caceres and Nilson, 2005),
except incubation with secondary antibodies was performed overnight at
4°C. The following primary antibodies were used, each at a 1:50 dilution:
anti-BR-C (25E9, gift of Greg Guild); anti-ß-galactosidase (40-1a,
Developmental Studies Hybridoma Bank); anti-DE-Cadherin (DCAD2, gift of
Tadashi Uemura, Kyoto University); anti-c-Myc (A-14, Santa Cruz
Biotechnology). Secondary antibodies (Molecular Probes) were used at the
following dilutions: goat anti-mouse Alexa Fluor 568, 1:500; goat anti-rabbit
Alexa Fluor, 1:800; goat anti-rat Alexa Fluor 546, 1:800.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The
dorsalmost region remains free of appendage material (see
Fig. 1D). We defined five
phenotypic classes to describe the range of eggshell phenotypes observed,
which seem to increase in severity with the thickness of the ventral
collar.
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).
cicBA53 is a novel allele with a premature stop codon
upstream of the cicT6 and cicU6
mutations and is therefore predicted to produce the most severely truncated
protein (see Materials and methods).
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) to generate follicle cell clones homozygous for the
cicfetU6 allele. Eggs from mosaic females exhibited
ventral and lateral foci of ectopic appendage-like material
(Fig. 1G-H'), and
analysis of follicle cell clones marked with the defective chorion 1
eggshell marker (Nilson and Schübach,
1998) confirmed that these foci were associated with ventral and
lateral clones of cic mutant follicle cells (data not shown). Ectopic
appendage material was not observed on the dorsal side. This mosaic analysis
indicates that cic is required in the follicle cells for eggshell
patterning. This ectopic appendage material was restricted to the anterior
half of the eggshell, while posterior clones exhibited no detectable phenotype
(data not shown), consistent with previous observations that only anterior
follicle cells are competent for appendage production
(Peri and Roth, 2000;
Queenan et al., 1997).
Interestingly, ectopic appendage material at the extreme anterior of the
eggshell often appears as appendage-like projections
(Fig. 1H'), suggesting
that only cic mutant follicle cells in the corresponding region of
the columnar follicular epithelium can form an appendage-producing epithelial
tube.
Mutant eggshell phenotypes reflect a change in follicle cell fate
The ventral expansion of dorsal eggshell structures suggests a
corresponding shift in the DV pattern of follicle cell fates. To visualize
follicle cell fate directly, we examined the expression of the
Broad-Complex (BR-C) locus, which encodes a group of four
alternatively spliced zinc finger transcription factor isoforms whose elevated
expression after stage 10 of oogenesis marks the dorsal appendage primordia
(Bayer et al., 1996;
Deng and Bownes, 1997;
DiBello et al., 1991;
Tzolovsky et al., 1999). In
wild-type ovaries, BR-C is first detected at uniform levels in all follicle
cells, but beginning at stage 10 BR-C expression is lost from dorsal anterior
and dorsal midline follicle cells, and then becomes elevated in two
populations of anterior follicle cells flanking the dorsal midline
(Fig. 2A,B). These cells
comprise the majority of each primordium and will form the roofs of the
epithelial tubes that secrete the appendages
(Dorman et al., 2004). The
remaining posterior and ventral cells exhibit low BR-C levels, which diminish
as oogenesis proceeds.
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Ectopic high BR-C levels were also observed after stage 10A in cic
mutant follicle cell clones (Fig.
2D-F). Consistent with the mosaic eggshell phenotype, this effect
was restricted to cells in the anterior half of the columnar epithelium
(Fig. 2D). Dorsal clones had no
effect on the BR-C expression pattern (data not shown), consistent with the
lack of Cic in this domain at this stage
(Goff et al., 2001). In the
vast majority of clones, the ectopic expression of high BR-C levels was cell
autonomous (Fig. 2E), although
we did observe rare cases of nonautonomy, where high BR-C levels were also
observed in a few adjacent wild-type cells
(Fig. 2F), typically near the
endogenous appendage primordia.
In addition to ectopic expression of BR-C, loss of Cic results in
morphological changes characteristic of appendage-producing follicle cells. In
wild-type egg chambers, appendage morphogenesis begins with the apical
constriction of the high BR-C expressing cells of the appendage primordia
(Fig. 2G)
(Dorman et al., 2004). With
the exception of those at the dorsal midline, cic mutant follicle
cells undergo apical constriction reminiscent of that observed in the
endogenous appendage-producing populations
(Fig. 2H). As observed for
ectopic high BR-C levels, this effect is restricted to cic mutant
follicle cells in the anterior half of the epithelium. In addition, the
follicle cell nuclei appear more closely packed in both the endogenous
appendage roof primordia and in regions of anterior cic mutant cells,
presumably as a consequence of this apical constriction
(Fig. 2B,C). These molecular
and morphological data indicate that cic mutant follicle cells within
the ventral and lateral anterior region of the follicular epithelium adopt an
appendage-producing follicle cell fate, suggesting that Cic normally functions
cell autonomously to repress this fate.
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;
McNeill et al., 1997). In
midoogenesis, mirr is expressed in dorsal anterior follicle cells and
this expression depends on Egfr signaling
(Jordan et al., 2000;
Zhao et al., 2000b).
Disruption of mirr function has been associated with dorsal appendage
defects, but the function of mirr in follicle cell fate determination
is not well understood.
We used a mirr-lacZ transgenic reporter
(Jordan et al., 2000;
Ruohola-Baker et al., 1993) to
characterize mirr expression in wild-type and cic mutant egg
chambers. In wild-type egg chambers, mirr expression is restricted to
dorsal anterior follicle cells, and the boundary of this domain aligns with
that of the high BR-C domain (Fig.
3A,A'). In cic mutants, the dorsal anterior
mirr expression pattern is established normally, but, by stage 10B,
mirr expression expands ventrally and is detected throughout the DV
circumference of the epithelium, although dorsal levels of mirr
appear to be consistently higher at this stage
(Fig. 3B)
(Goff et al., 2001). Similar
to the wild-type pattern, this ectopic mirr expression is restricted
to the anterior portion of the epithelium.
Clones of cic mutant follicle cells also exhibit ectopic mirr expression, indicating that the regulation of mirr requires cic function in the follicle cells (Fig. 3C-E'). Consistent with the cic homozygous phenotype, this effect differs along the AP axis; ectopic mirr expression is observed in cic mutant follicle cells in roughly the anterior half of the columnar epithelium (Fig. 3C,C', arrowheads), but not in those in the posterior half (Fig. 3C,C', arrows). Although ectopic mirr expression may appear to extend further to the posterior in mosaics than in homozygous epithelia (compare Fig. 3B with 3C'), this apparent difference can be attributed to the morphological changes in the follicle cells in the homozygotes, which result in the clustering of their nuclei and shift their apparent position anteriorly (see also Fig. 2C). Although ectopic mirr was detected in a few wild-type cells adjacent to rare clones near the endogenous mirr domain, the ectopic expression of mirr-lacZ in the vast majority of cic mutant clones was cell autonomous (Fig. 3C-D'). In late-stage egg chambers, these mirr-expressing cells surround foci of ectopic appendage material (Fig. 3E-E''), further supporting a correlation between mirr expression and the determination of appendage-producing fate.
Reduction of mirr function suppresses the cic phenotype
The correlation between loss of cic function and ectopic
mirr expression suggested that deregulation of mirr may
contribute to the patterning defects in cic mutant egg chambers. To
test this hypothesis, we asked whether a reduction in mirr function
would suppress the cic eggshell phenotype. For all combinations of
cic alleles tested, heterozygosity for a hypomorphic mirr
allele (Jordan et al., 2000;
Ruohola-Baker et al., 1993)
resulted in greater percentages of eggs falling into weaker phenotypic
categories (Table 1). For
cicfetU6/cicBA53 and
cicfetU6/cicfetE11, these shifts were highly
statistically significant (
2-tests, P<0.0001).
Moreover, mirr mutant follicle cell clones in a homozygous
cic mutant background do not exhibit the high BR-C levels
characteristic of the cell fate transformation observed in cic mutant
egg chambers. This effect is cell autonomous
(Fig. 4A,A'). The
suppression of the cic phenotype by a loss of mirr function
indicates that deregulation of mirr expression underlies the
cic phenotype and suggests that Mirr is an important determinant of
appendage-producing fate.
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;
Yang et al., 1999). Follicle
cells expressing ectopic mirr exhibit elevated levels of BR-C
(Fig. 4B,B'), and display
the apical constriction and nuclear organization characteristic of the
endogenous appendage primordia and cic mutant follicle cells in the
anterior half of the epithelium (Fig.
4C,C'; data not shown). In later stages, these cells were
associated with ectopic appendage material (data not shown). These data
indicate that expression of mirr is sufficient to determine multiple
characteristics of appendage-producing fate. This effect is less dramatic than
that observed in cic mutant cells, however, and is likely to reflect
either lower levels of mirr expression driven by the UAS element, or
a contribution of other Cic targets. Interestingly, although the cell fate
transformation resulting from loss of cic function was confined to
the anterior half of the epithelium, the fate changes associated with
UAS-mirr expression were observed even in posterior clones
(Fig. 4C,C'), suggesting
that the competence to adopt this fate is restricted at the level of
mirr regulation.
A fng expression boundary is not sufficient for induction of appendage-producing fate
Our data implicate mirr as an important Cic target and
appendage-producing fate determinant. One candidate regulatory target of Mirr
in this process is fringe (fng), which encodes a
glycosyltransferase that regulates cell fate decisions through the modulation
of Notch signaling (Blair,
2000; Bruckner et al.,
2000; Hicks et al.,
2000; Moloney et al.,
2000). Mirr regulates the expression of fng in a variety
of developmental contexts. For example, repression of fng by Mirr in
the Drosophila eye imaginal disc establishes a fng
expression boundary, which in turn generates a center of Notch signaling
activity that defines the equator of ommatidial polarity
(Cho and Choi, 1998;
Dominguez and de Celis, 1998;
Papayannopoulos et al., 1998;
Yang et al., 1999). Mirr also
represses fng in the ovary, but a function for the dorsal anterior
fng expression boundary in midoogenesis has not been demonstrated
(James et al., 2002;
Jordan et al., 2000;
Peri et al., 2002;
Zhao et al., 2000b).
Given our observation that ectopic mirr is sufficient to determine
an appendage-producing fate, we hypothesized that Mirr may induce this fate
through the repression of fng and the creation of a boundary of
fng expression. To test this idea, we generated fng mutant
follicle cell clones, reasoning that the resulting ectopic fng
expression boundaries should be equivalent to those resulting from ectopic
mirr expression and might therefore induce the same change in cell
fate. Although fng clones at the egg chamber termini result in
defective egg chambers that degenerate in midoogenesis, clones in the main
body of the follicular epithelium do not affect egg chamber development and
can be analyzed for patterning defects
(Grammont and Irvine, 2001;
Grammont and Irvine, 2002;
Zhao et al., 2000b). Ventral
and lateral fng clones do not exhibit high BR-C levels, changes in
cell shape, or production of ectopic appendage material
(Fig. 4D,D'; data not
shown), indicating that a fng expression boundary is not sufficient
to induce an appendage-producing fate and that mirr acts through
other targets in this cell fate decision.
Ectopic mirr expression in cic mutant egg chambers is independent of grk and rhomboid
Although the localization of Grk restricts high levels of Egfr activity to
the dorsal side of the egg chamber, low levels of Egfr ligand are also present
ventrally, as revealed by the observation that loss of the ubiquitin ligase
Cbl from ventral follicle cells results in grk-dependent ventral Egfr
activity (Pai et al., 2000).
As dorsal anterior expression of mirr during DV patterning requires
Grk-Egfr signaling (Jordan et al.,
2000; Zhao et al.,
2000b), we considered the possibility that the ectopic expression
of mirr in the absence of cic might result from an increased
sensitivity of follicle cells to these low levels of ventral Egfr activity. To
test this hypothesis, we asked whether loss of grk function could
suppress the ectopic expression of mirr in cic mutant egg
chambers.
Control egg chambers from grk mutant females that were also
heterozygous for cic exhibited no mirr expression in dorsal
anterior follicle cells, consistent with previous observations
(Jordan et al., 2000;
Zhao et al., 2000b) and
confirming that this allelic combination eliminates grk function
(Fig. 5B). In egg chambers that
were homozygous for mutant alleles of both cic and grk, the
high dorsal levels of mirr observed in cic mutant egg
chambers (Fig. 5A) were
abolished, but ectopic mirr expression was still detected throughout
the anterior circumference (Fig.
5D,D'). Consistent with this mirr expression
pattern, the resulting eggshells exhibited the loss of appendages
characteristic of loss of grk function
(Fig. 5C) but retained a
pronounced collar of appendage material
(Fig. 5E). These data indicate
that, although the dorsal high point of mirr expression requires
grk, the ectopic mirr expression resulting from the loss of
cic is grk independent.
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).
However, follicle cell clones ectopically expressing rhomboid have a
nonautonomous effect on cell fate (Ward
and Berg, 2005), consistent with the production of a diffusible
molecule like Spi, and therefore differ markedly from cic mutant
clones, suggesting that rhomboid is not a Cic target. To address this issue, we generated rhomboid mutant follicle cell clones in a cic mutant background. If the cic mutant phenotype were mediated through ectopic rhomboid, these clones should suppress the associated cell fate changes. Visualization of BR-C expression revealed that rhomboid mutant clones have no effect on the ectopic appendage-producing fates in cic mutant egg chambers (Fig. 5F,F'), indicating that ectopic rhomboid expression is not the cause of the cic phenotype. Taken together, the failure of loss of grk or rhomboid to suppress the cic phenotype suggests that the ectopic induction of mirr and of appendage-producing fates in cic mutant ovaries is not a result of ectopic ligand-mediated Egfr activation.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), this observation suggests that downregulation of Cic by
Egfr signaling establishes a dorsal domain of follicle cells competent to
adopt an appendage-producing fate. This interpretation is consistent with the
function of Cic in the developing wing, where Egfr-mediated downregulation of
Cic is required for determination of wing vein fate
(Roch et al., 2002). Cic is
not required, however, to determine the fate of the dorsal midline follicle
cells, which separate the appendage primordia, as this domain is established
normally in the absence of cic.
Specification of appendage roof versus floor cell fates
High BR-C expressing cells make up the majority of the appendage primordium
and form the roof of the appendage-producing epithelial tube, whereas the
cells in a single row at the dorsal and anterior edges of the high BR-C domain
form the tube floor (Dorman et al.,
2004). The ectopic expression of high BR-C levels and apical
constriction exhibited by cic mutant follicle cells are
characteristic of the roof cell fate
(Dorman et al., 2004), and the
production by cic mutant clones of knob-like foci of appendage
material, rather than elongated appendage-like structures, suggests that
ectopic floor cell fates are not coordinately specified
(Ward and Berg, 2005).
However, ectopic appendage-like projections are observed at the extreme
anterior of eggs from cic mosaic egg chambers, suggesting that both
roof and floor cells are determined in the more anterior clones, and cooperate
to form an appendage-producing tube. This distinction may reflect the
proximity of such clones to the source of Dpp, which is produced by the
anterior-most follicle cells and specifies the AP position of the appendage
primordia (Deng and Bownes,
1997; Dobens et al.,
2000; Peri and Roth,
2000; Twombly et al.,
1996), and therefore are could suggest a requirement for Dpp in
floor cell fate determination.
Cic restricts appendage-producing fate through repression of mirr
We have shown that loss of mirr function suppresses the cell fate
transformation caused by the loss of cic, providing evidence that
mirr is a crucial regulatory target of Cic and is required for
appendage-producing fate determination. We have also found that mirr
is required to specify high BR-C expressing fates in the endogenous appendage
primordia (M.R.A. and L.A.N., unpublished) and that ectopic expression of
mirr in clones of follicle cells is sufficient to induce
characteristics of this fate. These observations are consistent with previous
evidence that decreased mirr function is correlated with reduced
dorsal appendages and that ectopic mirr expression throughout the
follicular epithelium can generate extra appendage material
(Jordan et al., 2000;
Zhao et al., 2000b). However,
our direct analysis of follicle cell fate markers in clones with defined
boundaries demonstrates that the expression of mirr is sufficient to
induce both molecular and morphological markers of appendage-producing fate,
and that this effect is cell autonomous. Such a role for mirr would
be consistent with its function in other developmental contexts, where the
localized expression of Iro-C genes defines cell identity
(Cavodeassi et al., 2000;
Cavodeassi et al., 2001;
Cavodeassi et al., 2002;
Dominguez and de Celis,
1998).
Cells engineered to express ectopic mirr have been reported to
express ectopic rhomboid (Jordan
et al., 2000; Schweitzer et
al., 1995), which could in principle generate active Spi and lead
to ectopic Egfr activity and changes in follicle cell fate. Our data show
clearly that mirr-mediated induction of rhomboid is not the
primary cause of the ectopic appendage-producing fates in cic mutant
ovaries, as this phenotype is not suppressed by loss of rhomboid
function. However, some level of rhomboid induction by ectopic
mirr could lead to active Spi production, particularly near the
endogenous mirr domain, which may in turn explain the rare cases of
nonautonomy associated with cic mutant clones in this region.
cic mutant ovaries also fail to express pipe
(pip), which is normally expressed in ventral follicle cells and
defines the embryonic DV axis (Goff et
al., 2001; Sen et al.,
1998). Although neither mirr nor fng
loss-of-function clones affect pip expression
(Peri et al., 2002), ectopic
overexpression of mirr has been reported to repress pip
(Jordan et al., 2000).
Multiple lines of evidence, however, indicate that ectopic expression of
mirr in cic mutant ovaries does not account for their lack
of pip expression. For example, ectopic mirr expression does
not occur in cic mutant ovaries until stage 10B, well after the
absence of pip is detected (Goff
et al., 2001). In addition, loss of cic in ventral
posterior follicle cells results in loss of pip without induction of
ectopic mirr (see Fig.
3) (Goff et al.,
2001). Taken together with the evidence that rhomboid is
not required for the cic mutant phenotype (see above), these data
indicate that Cic regulates pip independently of its effect on
mirr expression.
Although our data demonstrate a role for mirr as an important
determinant of appendage-producing fate, the relevant transcriptional targets
of Mirr in this process remain to be identified. Although Mirr represses
fng in dorsal anterior follicle cells
(Jordan et al., 2000;
Zhao et al., 2000a), the
resulting fng boundary is not involved in DV patterning of
appendage-producing fates. Recent microarray analysis has identified a number
of genes that are regulated by mirr overexpression or alterations in
Egfr signaling and that therefore represent potential Mirr targets
(Jordan et al., 2005). Mirr
also appears to regulate cell affinity, as mirr mutant clones in the
eye exhibit a round shape (Yang et al.,
1999). In dorsal anterior follicle cell patterning, this proposed
function might contribute to the smooth boundary observed between the roof and
floor cells of the appendage primordia
(Ward and Berg, 2005),
possibly contributing to appendage morphogenesis.
Cic blocks induction of mirr by anterior positional cues
In both cic homozygous egg chambers and cic mutant
follicle cell clones, ectopic mirr expression is restricted to the
anterior half of the epithelium, indicating that mirr is also
regulated by positional information along the AP axis. Although in principle a
posterior repressor could account for this effect, on the basis of prevailing
models of follicular epithelium AP patterning (see below), we favor the
hypothesis that expression of mirr requires positive input from an
anterior positional cue. We propose that, in wild-type ovaries, Cic blocks the
induction of mirr by this anterior signal. However on the dorsal
side, where Cic becomes downregulated, this signal is not blocked, leading to
the induction of mirr expression and appendage-producing fate. In
cic mutant ovaries, the anterior signal induces mirr
expression throughout the DV axis.
A likely candidate for an anterior signaling molecule required for
mirr expression is Dpp, which is produced by the anterior-most
follicle cells and regulates gene expression along the AP axis
(Deng and Bownes, 1997;
Dobens et al., 2000;
Peri and Roth, 2000;
Twombly et al., 1996).
Coordinate regulation of mirr along the DV and AP axes provides a
molecular explanation for the observation that appendage-producing fates are
determined at the intersection of Egfr and Dpp signaling
(Deng and Bownes, 1997;
Peri and Roth, 2000).
Regulation of mirr by an anterior cue such as Dpp could also explain
the observation that mirr expression in cic mutant ovaries
is normal until stage 10B; although the cic mutant cells are
competent to express mirr, detectable levels may not be induced until
the posterior migration of anterior follicle cells in mid-oogenesis brings the
source of Dpp to the anterior margin of the oocyte
(Guichet et al., 2001).
In addition to invoking an anterior signal in the regulation of mirr, our data indicate that mirr is also positively regulated by dorsally restricted Egfr signaling, independent of Cic. cic mutant egg chambers exhibit ectopic mirr throughout their anterior circumference, but mirr levels remain highest dorsally. In grk;cic double mutant egg chambers this dorsal high point of mirr expression is abolished, suggesting that the wild-type dorsal anterior mirr expression pattern is the result of both dorsal and anterior inputs.
Model for patterning of the follicular epithelium along the DV axis
Collectively, our data support a model in which Cic blocks the induction of
mirr expression and appendage-producing fates in response to an
anterior signal, for example Dpp. Egfr-mediated downregulation of Cic in
dorsal anterior follicle cells therefore allows these cells to respond to Dpp,
contributing to the dorsal anterior mirr expression pattern
(Fig. 6A, top), whereas the
presence of Cic in ventral and lateral follicle cells blocks their response to
this cue (Fig. 6A, bottom).
Within the dorsal Cic-free domain, the Rhomboid/Spi/Aos autocrine-feedback
loop would regulate Egfr activity to resolve two distinct appendage primordia
(Fig. 6B)
(Peri et al., 1999;
Wasserman and Freeman, 1998).
In cic mutant egg chambers, all follicle cells would be competent to
respond to the anterior signal, resulting in ectopic mirr expression
and appendage-producing fate in the anterior follicle cells that receive the
signal.
|
;
Peri and Roth, 2000), and the
simplest interpretation has been that these signals function additively to
specify appendage-producing fate. Instead, however, the demonstration that the
distribution of Cic along the DV axis determines the competence of follicle
cells to respond to AP patterning signals reveals unexpected crosstalk between
DV and AP patterning signals, and indicates that Cic integrates these
pathways. Along the DV axis, we propose that the pattern of the follicular epithelium is determined by the function of two Egfr targets, Cic and Aos, in distinct domains (Fig. 6B). High levels of Egfr activity induce production of Aos at the dorsal midline, where it antagonizes Spi, thus splitting the initial dorsal domain of Egfr activity and defining the dorsal limits of the appendage primordia. Lower levels of Egfr signaling are sufficient to downregulate Cic, defining a dorsal domain that lacks Cic and is therefore competent to adopt dorsal fates. Cic remains present in ventral and lateral follicle cells, where it blocks the induction of crucial transcriptional targets, such as mirr, by Dpp. The dorsal limit of the Cic domain thus defines the ventral limit of the appendage primordia. Cic-mediated repression of target genes may represent a general mechanism for the integration of multiple spatial inputs in a developing tissue.
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ACKNOWLEDGMENTS |
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