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First published online 16 May 2007
doi: 10.1242/dev.02856
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Institute of Developmental Biology, University of Cologne, Gyrhofstr.17, D-50931, Germany.
* Author for correspondence (e-mail: siegfried.roth{at}uni-koeln.de)
Accepted 23 March 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Oogenesis, Follicle cells, EGF signaling, Gurken, Proto-oncogene, dSno, brinker, Drosophila
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Dobens and Raftery, 2000;
Berg, 2005). The anterior egg
pole is marked by a micropyle required for sperm entry, and the
dorsal-anterior side of the egg carries the dorsal appendages (DAs) with a
respiratory function and the operculum, a lid-like structure that opens when
the larva hatches. The eggshell layers are secreted by the somatically derived
follicle cells.
Two major signaling pathways control the specification of the dorsal
chorion structures: the epidermal growth factor (EGF)- and the transforming
growth factor ß (TGF-ß)-pathway
(Nilson and Schupbach, 1999;
Twombly et al., 1996;
Dobens et al., 2000;
Peri and Roth, 2000;
Roth, 2003;
Berg, 2005). The
EGF/TGF-
-like ligand Gurken (Grk)
(Neuman-Silberberg and Schupbach,
1993) is produced at the dorsal-anterior cortex of the oocyte
where grk mRNA is localized. From this position Grk protein is
secreted and activates the Drosophila EGF receptor (Egfr) in
overlying dorsal follicle cells. These cells later produce the operculum and
the two DAs.
Whereas Grk signaling also spreads to more posterior regions of the
follicular epithelium, the operculum and DAs are only derived from anterior
follicle cells. This restriction depends on a second, anterior signal that is
likely to be provided by Decapentaplegic (Dpp), one of the TGF-ß family
members in Drosophila. Dpp was shown to influence the specification
of the DAs and the operculum (Twombly et
al., 1996; Deng and Bownes,
1997; Dobens et al.,
2000; Peri and Roth,
2000). Partial loss-of-function alleles of dpp and/or Dpp
pathway components lead to the production of eggs with a reduced operculum and
mis-positioned DAs. Conversely, increasing the dpp levels causes an
expansion of anterior eggshell structures
(Twombly et al., 1996;
Dobens et al., 2000;
Ward and Berg, 2005).
Recently, it has been shown that brinker (brk), a repressor
of Dpp target genes (Campbell and
Tomlinson, 1999; Jazwinska et
al., 1999; Minami et al.,
1999), is essential for DA formation
(Chen and Schupbach, 2006).
In this study we deal with two aspects of Dpp signaling within the follicular epithelium: first, with the global function of Dpp in the Grk-dependent specification of all dorsal follicle cell fates, and second, with the interplay of Dpp and EGF signaling gradients in specifying distinct dorsal follicle cell fates.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Antibodies: anti-Broad Z1 [mouse monoclonal, Developmental Studies Hybridoma
Bank (DSHB); 1:100 dilution], anti-Fasciclin 3 (Fas3) (mouse monoclonal, DSHB;
1:50), anti-pMAD (rabbit polyclonal, 1:3000, gift of Dr Morata, Centro de
Biologia Molecular, Madrid, Spain), anti-ß-gal (rabbit polyclonal, MP
Biomedicals; 1:750), anti-green fluorescent protein (GFP) (rabbit polyclonal,
Molecular Probes; 1:3000), anti-Medea (1:1000)
(Sutherland et al., 2003),
anti-Grk (ID12, mouse monoclonal, DSHB; 1:100), Alexa Fluor-555-conjugated
anti-mouse, Alexa Fluor-555-conjugated anti-rabbit, Alexa Fluor-488-conjugated
anti-rabbit and Alexa Fluor-488-conjugated anti-mouse (Molecular Probes;
1:400).
Genetic mosaics in the follicular epithelium
All loss-of-function clones genetically marked by the absence of GFP were
generated by the FRT/FLP recombination technique
(Xu and Rubin, 1993). Adult
females were heat shocked for 1 hour at 37°C and dissected after 4 days.
The ovaries were then processed for antibody staining. The following stocks
were used: tkva12 FRT40A(neo)/CyO;
Med13neo82B FRT/TM3, Sb;
brkM68 FRT18A/FM7; and
pip-lacZ.
Cloning of Drosophila sno (dSno)
A CG7233 cDNA was amplified from a Drosophila ovarian cDNA library
(Stroumbakis et al., 1994)
using the following primers: Sno1 (5'-CTTATATTAAACCCAAC-3') and
Sno2 (5'-GCCGTCGGCAAAATGCAAATGC-3'). This cDNA closely resembles
dSnoI as described by Takaesu et al.
(Takaesu et al., 2006). The
1360 bp amplified fragment was cloned in pCR-TOPO 2.1 and sequenced. A
KpnI and NotI fragment from pCR-TOPO-dSno was
cloned into blueskript for making antisense RNA probes and into pUAST to
produce transgenic lines.
Generating dSno mutants
Mutations in dSno were generated by imprecise excisions of the
P-element insertion, l(2)Sh1402 (see Fig. S1 in the supplementary material).
The breakpoints of the deletions were mapped by PCR. We uncovered a total of
17 different lines that had deletions in the dSno region and all were
homozygous viable. One of the lines, dSno174, was fine
mapped and turned out to be a deletion removing CG7233 (see Fig. S1 in the
supplementary material).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Dobens et al.,
2000). These cells secrete Dpp protein that forms an
anterior-to-posterior gradient as suggested from the distribution of the Dpp
signal transducer pMAD (Tanimoto et al.,
2000) and the expression of the direct Dpp target gene,
dad-lacZ (Tsuneizumi et al.,
1997; Muzzopappa and Wappner,
2005) (Fig. 1A-F).
When the anterior follicle cells flatten, this gradient moves closer to the
oocyte where Grk is already asymmetrically localized
(Fig. 1A-F). At stage 10A the
follicle cell rearrangement is completed. Thinly stretched-out follicle cells
cover the nurse cells [nurse cell follicle cells (NFCs)]. Tall follicle cells
cover the oocyte [columnar follicle cells (CFCs)]. dpp transcription
is largely restricted to the NFCs (Twombly
et al., 1996; Dobens et al.,
2000), whereas decreasing levels of pMAD can be detected in a five
cell-wide stripe and dad expression in a four cell-wide stripe of
anterior CFCs (Fig. 1C,F). At
the dorsal side these CFCs are exposed to high levels of Grk signaling
(Fig. 1C, dorsal view;
Fig. 1F, lateral view). Because
Dpp and Grk signaling function together in specifying dorsal follicle cell
fates, it is possible that at stage 10A a crosstalk between the two pathways
ensues that affects the cytoplasmic signal transducers or primary Dpp target
genes. This might result in a dorsoventral (DV) asymmetry of nuclear pMAD
and/or dad expression at stage 10A. However, these patterns are
symmetric along the DV axis (Fig.
1C,F). The same was observed for the distribution of the common
Smad Medea (data not shown) (Wisotzkey et
al., 1998; Parker et al.,
2004; Muzzopappa and Wappner,
2005). We conclude that at least with regard to high and medium
signaling levels (see below) there is no influence of Grk on Dpp signaling and
primary Dpp target gene expression. These observations are in variance with
the suggestion that Dpp signaling is significantly enhanced in dorsal follicle
cells (Araujo and Bier, 2000;
Carneiro et al., 2006).
Dpp has a direct long-range function in specifying the DAs
At stage 10A the presumptive operculum cells occupy a triangular-shaped
region positioned at the anterior-dorsal side of the CFCs. They express high
levels of Fas3 (Fig. 1K)
(Kose et al., 1997;
Ward and Berg, 2005). The DAs
are largely derived from cells expressing high levels of BR-C
(Fig. 1G)
(Deng and Bownes, 1997;
Dorman et al., 2004;
Ward and Berg, 2005). In the
following we will refer to these cells as BR cells.
The posterior-most BR cells are approximately nine-cell rows away from the
NFCs in which Dpp is expressed and dad expression and pMAD expand
only to the fourth and fifth rows of CFCs, respectively. Therefore, it is
possible that the function of dpp in DA specification is mediated via
a secondary signal (Dobens et al.,
2000). To address this question we produced large follicle cell
clones mutant for Med, a common Smad shared by the activin and Dpp
signaling pathways (Wisotzkey et al.,
1998; Parker et al.,
2004) and thickveins (tkv), the type I receptor specific
for Dpp (Raftery and Sutherland,
1999; Parker et al.,
2004). Med and tkv mutant clones gave identical
results.
Follicle cell clones homozygous mutant for either Med13 or tkva12 and localized in dorsal-anterior regions of the CFCs show downregulation of both Fas3 and BR-C in a cell-autonomous manner (Fig. 1H-J,L-N). In rare cases in which the clone borders are highly irregular, single Med+ cells can be found surrounded by Med mutant cells. These Med+ cells express normal levels of BR-C. In addition, large anterior clones do not prevent BR-C expression in wild-type (wt) cells positioned posterior to the clone (Fig. 1H, arrows). Thus, Dpp protein spreads beyond the region that lacks Dpp signaling components to induce BR-C expression. These data suggest that a Dpp gradient exists in stage 10A egg chambers that extends at least nine-cell rows from the source of dpp expression and patterns the dorsal chorion structures. It also implies that our pMAD and dad-lacZ stainings can only be used to detect high-to-medium levels of Dpp signaling in the follicular epithelium (Fig. 1C,F).
Grk requires Dpp to induce DV eggshell polarity
A certain portion of the eggs laid by females in which large clones of
Med or tkv mutant follicle cells were induced (henceforth
termed tkv-/Med- eggs) lack, as
expected, both the operculum and the two DAs. Surprisingly, these eggs are
totally symmetric along the DV axis with regard to their overall egg
structure; i.e. the chorion imprints and the region surrounding the micropyle
show no DV asymmetries (Fig.
1U; Fig. 2G,H).
This suggests that Dpp is required to induce all aspects of DV eggshell
polarity. The tkv-/Med- eggs closely
resemble eggs in which the second round of Grk signaling and thus the
induction of DV polarity fails to occur
(Micklem et al., 1997;
Newmark et al., 1997;
Peri and Roth, 2000;
Guichet et al., 2001)
(Fig. 1T). This suggests that
Dpp is essential for Grk to induce DV eggshell polarity.
Therefore, we wondered whether Dpp is essential for all aspects of DV Grk
signaling. Besides induction of dorsal follicle cell types, Grk is also
involved in establishing the embryonic DV axis by repressing pipe
(pip) (Sen et al., 1998).
pip codes for a sulfotransferase that causes the sulfation of an
unknown extracellular matrix component essential for embryonic DV axis
formation, but not for eggshell polarity. pip expression was analyzed
in egg chambers carrying large Med13 or
tkva12 mutant follicle cell clones
(Fig. 1O-R). To detect
pip expression we used a pip-lacZ reporter construct that
contains 3 kb of genomic DNA upstream of the pip transcriptional
start site (M.T. and S.R., unpublished data). No change in pip-lacZ
expression was detected. Thus, Dpp is essential for those aspects of Grk
signaling that lead to establishment of DV eggshell polarity, but does not
affect follicle cell patterning required for embryonic axis formation.
Anterior-most eggshell structures require Dpp, but not Grk, signaling
Margarits et al. have distinguished three regions of the operculum with
distinct cell imprints: one directly surrounding the micropyle, a second
extending from there to the DAs and a third occupying the area between the DAs
(Fig. 2A,B) (Margarits et al.,
1980). In ventral and lateral regions the operculum is separated from the
remaining chorion by a raised structure, the collar
(Fig. 2B, white arrow). In eggs
derived from grk mutant females only regions II and III are deleted,
whereas region I symmetrically surrounds the micropyle, followed by a
symmetric collar (Fig. 2D,E).
In Med- eggs, in addition to the deletion of regions II
and III, the chorionic region surrounding the micropyle is reduced in size and
lacks chorion imprints, indicating that a particular follicle cell type is
absent (Fig. 2G,H). In
agreement with this assumption, both wt and grk mutant stage 14 egg
chambers possess a group of follicle cells surrounding the micropyle that
express high levels of Fas3 (Fig.
2C,F). This group of Fas3-positive cells is absent in
tkv-/Med- eggs
(Fig. 2I). Thus, Dpp apparently
specifies the anterior-most region of the operculum in the absence of Grk
signaling. This analysis also implies that only regions II and III of the
operculum are true dorsal structures, whereas region I is an anterior
structure.
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).
Grk mutant eggs have micropyles at both ends, separated from the
remaining chorion by a symmetric collar. Overexpression of dpp in a
grk mutant background results in misshaped micropyles, the loss of
the collar and a reduction of the egg diameter
(Fig. 2J). These phenotypes
were usually seen only at the egg pole, which harbors the nurse cells. They
are accompanied by an expansion of the anterior Fas3 domain and patches of
ectopic Fas3 expression in more central positions of the egg
(Fig. 2K). Thus, Dpp
overexpression in the absence of grk leads to the expansion of a
follicle cell population that may correspond to region I of the operculum. In
summary, these data suggest that Dpp is both required and sufficient to
specify a subtype of anterior follicle cells in the absence of Grk
signaling.
Combined overexpression of Grk and Dpp transforms all mainbody follicle cells into operculum fate and reveals a crosstalk between both pathways
To gain more information on how the two pathways interact in specifying
different follicle cell types, we manipulated the levels of Dpp signaling
within the follicular epithelium and at the same time increased the levels of
Grk signaling from the oocyte.
Two different Gal4 driver lines, GR1Gal4 and CY2Gal4, were utilized to
drive moderate and high levels, respectively, of a UASdpp transgene
in all CFCs (Queenan et al.,
1997; Goentoro et al.,
2006). Moderate Dpp overexpresssion (GR1Gal4 x
UASdpp) leads to uniform pMAD levels in all follicle cells at stage 9
(data not shown), but levels decrease in early stage 10A (except for the
posterior pole) and result in an extended gradient with randomly variegated
pMAD levels (Fig. 3A). Higher
levels of Dpp expression (Cy2Gal4 x UASdpp) result in uniform
levels of pMAD throughout stages 9 (data not shown) and 10A
(Fig. 3B).
|
). This
driver line leads to a strong increase in the amount of Grk protein in stage 9
and 10 oocytes. The protein remains asymmetrically distributed, but higher
concentrations are found at the ventral side compared with wt (compare
Fig. 3C with
Fig. 3A,B). Grk overexpression
does not change the shape or anteriorposterior (AP) extension of the pMAD
gradient (Fig. 3C), and
combined overexpression of Grk and high amounts of Dpp results in the same
uniform levels of pMAD expression as overexpression of Dpp alone (compare
Fig. 3B with
Fig. 3D). This confirms the
assumption that Grk signaling does not influence the levels of Dpp
signaling.
EGF signaling within the CFCs was monitored with the help of the primary
EGF target gene kekkon (kek)
(Queenan et al., 1997;
Ghiglione et al., 1999;
Peri and Roth, 2000;
Guichet et al., 2001;
Goentoro et al., 2006). In wt
stage 10A egg chambers kek is expressed in a wedge-shaped domain of
anterior-dorsal CFCs overlying the oocyte nucleus
(Fig. 3Ed). It covers
approximately 25% of the DV axis at the position of the nucleus and
approximately 50% of the AP axis along the dorsal midline of the CFCs.
Moderate Dpp overexpression leads to eggs with enlarged DAs that are
shifted towards posterior (Fig.
3Fa). Accordingly, the BR-C domains are dramatically
enlarged, mainly by expanding towards posterior, whereas the lateral limits
are only slightly shifted to the ventral side as compared with wt
(Fig. 3Fc). Higher levels of
Dpp expression lead to an expansion of the operculum at the expense of the DAs
(Dobens et al., 2000)
(Fig. 3Ga). BR-C
expression is reduced to a narrow stripe encircling the expanded domain of
Fas3 expression (Fig. 3Gb,c).
Compared with moderate overexpression of dpp, the posterior and
lateral limits of the total region giving rise to the dorsal follicle cells
has only slightly changed. This region does not extend into the posterior-most
20% and is restricted to the dorsal-most 40% of the CFCs.
Surprisingly, kek expression expands laterally and posteriorly depending on the level of ectopic Dpp signaling (compare Fig. 3Ed with Fig. 3Fd and Fig. 3Gd). With Cy2Gal4 high levels of kek are found in a domain comprising 70% of AP and 40% of the DV axis. This expanded kek domain closely corresponds to the region that produces the enlarged operculum. If one assumes that kek expression directly reflects the levels of EGF signaling, this observation suggests that the activation of the Dpp pathway enhances the levels of EGF pathway activity.
The lateral borders of the region giving rise to dorsal follicle cells
appear to be strictly defined by a certain threshold of Grk signaling.
Increased Grk signaling leads to a ventral shift of the DA and an expansion of
the operculum (Neuman-Silberberg and
Schupbach, 1994) (data not shown). Strong Grk overexpression
results in 30% of eggs with operculum as well as Fas3 expression along the
entire DV axis, whereas BR-C transcription is suppressed
(Fig. 3Ha-c). However, the AP
width of the operculum and Fas3 expression are not altered. Accordingly,
kek is expressed at high levels around the entire CFC circumference,
although its expression remains restricted along the AP axis as in wt egg
chambers (Fig. 3Hd). This is
surprising because under the same conditions pip is repressed
throughout the CFCs, indicating that Grk signals along the entire AP axis
(Sen et al., 1998) (data not
shown). This discrepancy is likely to be because of different thresholds of
Grk signaling required for kek activation and pipe
repression. We assume that even after strong Grk overexpression, high levels
of EGF signaling remain restricted to the anterior half of the CFCs.
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; Peri and Roth,
2000). Within the region giving rise to dorsal follicle cells,
high levels of Grk and high levels of Dpp have similar consequences: they
promote operculum at the expense of the DA fates. A positive influence of Dpp
signaling on the levels of EGF pathway activity might explain why both
pathways are interchangeable with regard to certain cell fate decisions. Such
a positive influence is suggested by changes of kek expression upon
ectopic Dpp pathway activation. However, kek transcription is only an
indirect indicator of EGF pathway activity, in contrast to, for example, the
detection of activated MAPK. Thus, currently we cannot exclude alternative
explanations for the observed effect of Dpp on kek expression.
However, we favor a direct crosstalk between both pathways (see
Discussion).
The functions of dSno and brinker in dorsal follicle cell patterning
To understand the read-out of the Dpp gradient we analyzed inhibitors of
Dpp signaling and their interaction. In particular, we characterized the
ovarian function of the Drosophila homolog of the Sno oncogene.
dSno has recently been shown to antagonize TGF-ß signaling in
nervous system and in wing development
(Takaesu et al., 2006;
Ramel et al., 2007).
During ovarian development high levels of dSno are expressed at
the tip of the germarium and lower levels in germline cells from stage 1 to
stage 3 egg chambers (Fig. 4A).
No obvious expression of dSno was observed from stage 4 to stage 9
(Fig. 4A and data not shown).
Interestingly, at stage 10A dSno is expressed in a 3-4 cell-wide
oblique stripe, which runs from an anterior-lateral to a posterior-dorsal
position within CFCs (Fig.
4B,C; Fig. 5A). The
dSno enhancer trap line, l(2)Sh1402
(Oh et al., 2003), reproduces
this expression pattern and was used for colocalization studies
(Fig. 4D-F). The posterior
limit of dSno expression coincides with the posterior limits of the
BR cells. Thus, the dSno stripe apparently encircles the entire
follicle cell region, giving rise to dorsal chorion structures suggesting that
it is involved in spatially limiting the range of Dpp signaling.
To test this hypothesis we generated loss-of-function mutations in the dSno gene (see Materials and methods). dSno174 is a 2.2 kb deletion that removes the domain of dSno essential for its interaction with Smad proteins in vertebrates (see Fig. S1 in the supplementary material). dSno174 homozygous mutant flies are viable and produce viable progeny. However, a 50% reduction in viability was observed compared with wt. Also, the number of fertilized eggs was significantly decreased as compared with controls (65% as compared with 97% in control crosses). Interestingly, eggs deposited by dSno174/dSno174 females exhibit mild, but consistent patterning defects of dorsal chorion structures (Fig. 4H,K,N): the operculum is slightly enlarged and the DAs are shifted toward the posterior and are wider apart from each other and shorter than in wt eggs. Accordingly, the dorsal region between the BR-C domains is enlarged and the number of BR cells is decreased (Table 1; compare Fig. 6A,B with Fig. 6C,D). Overexpression of dSno using the strong driver line Cy2GAL4 leads to a reduction in the size of the operculum and a shift of the DAs towards anterior (Fig. 4I,L,O). Neither loss-of-function nor overexpression of dSno results in a change in dpp mRNA expression or pMAD distribution (see Fig. S2 in the supplementary material). Nevertheless, the loss-of-function phenotype of dSno is reminiscent of enhanced Dpp signaling, whereas the overexpression phenotype resembles reduced Dpp signaling. It is therefore likely that dSno acts directly or indirectly as a Dpp pathway inhibitor within the follicular epithelium.
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). brk is, like dSno, largely absent in the
region giving rise to the operculum, but its expression shows more extensive
overlap with the region giving rise to the DAs
(Chen and Schupbach, 2006)
(Fig. 5B). In addition,
brk expression decreases towards the ventral side and lacks a sharp
posterior boundary (Chen and Schupbach,
2006) (Fig. 5B).
Like dSno, brk is also required to limit the size of the operculum.
However, in contrast to dSno, loss of brk leads to a
complete deletion of the DAs (Chen and
Schupbach, 2006) (Fig.
4P-R). The operculum does not increase its AP dimension, but
expands laterally at the expense of the DA anlagen. Large dorsally located
brk clones lead to a broad dorsal domain of uniform Fas3 expression
(Fig. 4Q,R).
dSno and brinker are dual targets of Dpp and EGF signaling in the follicular epithelium
Like vertebrate sno, dSno is activated by TGF-ß signaling
(Luo, 2004). This follows from
cell clones mutant for Med. In such clones, in which Dpp signaling is
abolished, dSno is not expressed
(Fig. 5O,P). By contrast,
brk has recently been shown to be repressed by Dpp signaling.
Accordingly, strong overexpression of Dpp leads to uniform expression of
dSno, whereas it completely abolishes brk expression
(Fig. 5E,F). Mild
overexpression of Dpp causes an irregular expansion of dSno
expression and an enlargement of the anterior region of brk
repression (Fig. 5C,D).
The regulation of dSno and brk expression by EGF signaling is complex. Egg chambers lacking Grk signaling do not express dSno (Fig. 5M). However, they show weak expression of brk in a central region of the CFCs (Fig. 5N). Thus, EGF signaling is required for dSno, but not for brk activation. Intermediate levels of EGF signaling seem to promote both dSno and brk expression whereas high levels repress both genes (compare Fig. 5A,G for dSno with Fig. 5B,H, including inset, for brk). Thus, in the case of brk mild EGF signaling counteracts the repressive effect of Dpp, whereas in the case of dSno mild EGF collaborates with Dpp. This subtle balance between Grk and Dpp signaling leads to the semi-circular expression domain in the case of dSno, whereas it provides a sharp anterior on-off boundary in the case of brk.
A paradoxical situation with regard to brk expression is observed upon mild overexpression of Dpp: Dpp appears to enhance rather than repress brk (Fig. 5D,J). However, given our observation that Dpp signaling promotes kek expression, suggesting that it enhances EGF signaling, these results can be explained by an indirect effect via EGF signaling: Dpp leads to additional EGF signaling and this, in turn, activates brk. With regard to brk, the regulatory input of EGF and Dpp signaling appears to be balanced such that brk expression always correlates with the formation of DAs.
Taken together, brk and dSno show similarities and
differences in their regulation by Dpp and Grk. Therefore, they possess
different, but partially overlapping, expression domains within the region
giving rise to dorsal follicle cells. Moreover, the expression of both genes
partially overlaps with dad expression
(Fig. 1F). Dad is a negatively
acting Smad protein (Tsuneizumi et al.,
1997). The fact that dSno expression overlaps with that
of two other Dpp inhibitors might explain its weak loss-of-function
phenotype.
Dose-dependent interactions of Dpp inhibitors with dSno
To address this point we asked whether the homozygous dSno
phenotype (compare Fig. 6C,D
with Fig. 6A,B) was enhanced by
removing one or two copies of the other inhibitors. Females homozygous for the
semilethal P-insertion dad(sl)
(Tsuneizumi et al., 1997) lay
eggs that resemble wt eggs and show normal BR-C expression (compare
Fig. 6E,F with
Fig. 6A,B). Eggs laid by double
homozygous dSno174; dad(sl) mutants
(Fig. 6G) have DAs that were
thicker than those of dSno single mutants (compare with
Fig. 6C) and had highly
irregular ends in addition to an enlarged operculum. In accordance with this,
the BR-C expression domains have irregular borders and are pushed
laterally in stage 10B egg chambers (Fig.
6B,H). The dorsal distance between the BR-C domains is
approximately twofold larger than in wt
(Table 1).
We also generated a mutant line, which was homozygous for dSno and lacked one copy of brk. Eggs laid by such females are shorter in length and have a significantly larger operculum as compared with dSno homozygous or brk heterozygous mutants (Fig. 6C,I,K). Furthermore, the DAs are stout and massively thick at their ends (Fig. 6K) and BR-C expression is pushed more posteriorly and laterally (Fig. 6D,J,L; Table 1). Both in dSno; dad(sl) and in brk; dSno double mutants the number of BR cells is only slightly reduced. Thus, the massive expansion of the operculum does not occur at the expense of the DAs, but instead leads to lateral and posterior expansion of the total region giving rise to dorsal chorion fates. This suggests that dSno functions in concert with dad and brk in confining the region of the Dpp gradient that gives rise to dorsal chorion fates.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Guichet et al., 2001) and
repress pipe (Fig.
1O-R). Thus, Dpp is not required for Grk signaling per se. We
suggest that Dpp signaling rather activates transcription factors or causes
chromatin modifications that allow Grk to induce dorsal target genes involved
in follicle cell specification.
Mirror might be such a transcription factor that is activated by Dpp and
confers the ability to adopt dorsal fates to a ring of anterior follicle cells
(Jordan et al., 2000;
Atkey et al., 2006).
mirror acts downstream of Grk and probably also downstream of Dpp in
specifying dorsal follicle cells (Atkey et
al., 2006). However, mirror expression alone leads only
to the formation of DA material. Thus, it is likely that mirror only
provides the general potential to produce dorsal follicle cells. Additional
inputs from Dpp and EGF signaling are needed to produce the full set of dorsal
follicle cell fates. This scenario suggests two phases of Dpp signaling. An
early phase demarcates the region in which Grk induces dorsal follicle cell
fates. This might require only one (low level) threshold of Dpp signaling and
is likely to be mediated through activation of mirror. A later phase
establishes distinct dorsal follicle cell fates. Here, Dpp acts as a morphogen
in combination with EGF signaling.
The correspondence of EGF signaling levels and dorsal follicle cell fates
The results presented in this study suggest that high levels of EGF and Dpp
signaling correspond to regions II and III of the operculum, whereas lower
levels of both pathways correspond to the DAs. With regard to region III of
the operculum that separates the two DAs, our assumption appears to contradict
the model of Wasserman and Freeman
(Wasserman and Freeman, 1998).
These authors showed that Grk signaling induces the expression of rhomboid
(rho), which in turn activates Spitz, a second TGF--like molecule.
This leads to an amplification of EGF signaling. Highest signaling levels
centered at the dorsal midline lead to the induction of the inhibitor
argos (aos), which antagonizes Spitz. This in turn lowers the levels
of EGF signaling along the dorsal mildline. According to this model, high
levels of EGF signaling promote DA, lower levels operculum region III
formation. However, the expression patterns of kek, which result from
Grk or Dpp overexpression, appear to contradict this model. Indeed, we believe
that the regulatory loop of rho and aos is not required to
establish the operculum or DA fates per se. The pattern of BR-C expression is
not significantly altered in rho or aos mutant follicle cell
clones (G.A., B.V.S. and S.R., unpublished data). However, rho and
aos might contribute to patterning processes that are required for
the morphogenesis and, as a result of this, for splitting of the DAs. Ward and
Berg have shown that DA extension (tube formation) requires the collaboration
of rho-expressing floor cells and BR-C-expressing roof cells
(Ward and Berg, 2005). The
rho-expressing floor cells are part of the Fas3 expression domain
that separates the BR-C domains. These rho-expressing cells have to
form a separate stripe on each side of the dorsal midline to allow the
splitting of the DAs (Ward and Berg,
2005). We suggest that the rho/aos regulatory
loop is required to generate two distinct stripes of late-rho
expression within the dorsal Fas3 domain. The result is a splitting of the DAs
accompanied by the establishment of a region of Fas3 cells that do not express
rho, and thus give rise to region III of the operculum.
The read-out of the EGF and Dpp signaling gradients in the follicular epithelium and the functions of Dpp inhibitors
The establishment of the region giving rise to dorsal follicle cells and
its subdivision into operculum and DA-producing cells is an intriguing problem
of two-dimensional patterning (Fig.
7). The pattern of cell fates depends on the
concentration-dependent read-out of two orthogonal signaling gradients (EGF
and Dpp). This read-out is complex because the signaling pathways themselves
appear to influence each other. First, there is evidence for a direct
influence of Dpp on EGF signaling; second, the Dpp inhibitors brk and
dSno are targets of both pathways; and third, rho is also a
target of both pathways (Peri and Roth,
2000), an aspect that will not be further discussed here.
Evidence for a direct crosstalk between both pathways is provided by the
analysis of kek expression. kek appears to be a primary
target gene of EGF signaling as basal levels of its expression are independent
of Dpp (Peri and Roth, 2000;
Guichet et al., 2001).
However, we observed an enhancement of kek expression upon
dpp overexpression in stage 10A prior to the activation of
rho and aos. Moreover, the stage 10B expression patterns of
rho and aos do not correlate with the observed changes in
kek expression (data no shown). Thus, these changes cannot be caused
by the secondary modulation of the EGF signaling profile. Therefore, we
suggest a direct crosstalk between both pathways. This could be because of a
Dpp receptor-dependent activation of the ras/MAPK cascade. A TGF-ß
receptor-dependent activation of the MAPK cascade has been observed in several
vertebrate cell types (for reviews, see
Derynck and Zhang, 2003;
Javelaud and Mauviel, 2005).
One could imagine that the triangular-shaped domain of Fas3 expression, which
defines the anterior and dorsal borders of the BR-C domain, is specified by
high levels of EGF signaling brought about by a Dpp-dependent enhancement of
MAPK signaling. A confirmation of this model would necessitate direct
monitoring of MAPK activity upon altered Dpp signaling.
The border between operculum and DAs is also crucially dependent on
brk. In brk mutant follicle cell clones, Fas3 expression
expands at the expense of the BR-C domains
(Fig. 4P-R)
(Chen and Schupbach, 2006).
However, brk expression is upregulated within a broad domain at the
dorsal side that also includes the Fas3-expressing region separating the BR-C
domains (Fig. 5B). Although
brk represses Fas3 expression in lateral regions allowing BR-C
expression, brk is unable to repress Fas3 at the dorsal midline
(Fig. 7). This suggests that
Fas3 expression, which is predominantly dependent on high levels of EGF
signaling, cannot be repressed by brk, whereas Fas3 expression in
more lateral regions predominantly dependent on Dpp signaling is repressed by
brk.
The hemi-circular boundary of the total region giving rise to dorsal
chorion fates appears to be defined by a constant value reflecting the sum or
the product of EGF and Dpp signaling (Fig.
7). The cis-regulatory elements of dSno
represent a sensitive sensor for this dual input. At the dorsal midline, lower
amounts of Dpp signaling are required to activate dSno than in
lateral regions, and the opposite holds true for EGF signaling
(Fig. 7). During brain
development in flies (Takaesu et al.,
2006) and in several contexts in vertebrates (for a review, see
Luo, 2004) Sno is involved in
the control of cell proliferation that has been shown to be crucially
dependent on the relative levels of TGF-ß and EGF signaling (for a
review, see Siegel and Massague,
2003). It is conceivable that for spatial patterning of the
follicular epithelium dSno uses regulatory elements that are derived
from a more basic function in the control of cell proliferation in other
tissues. The follicle cell expression of dSno might provide a
convenient experimental setting to dissect such regulatory elements.
The fact that loss of dSno causes only mild defects is because of
redundancy. A combination of three Dpp inhibitors appears to be involved in
establishing the border between dorsal follicle cells and the remainder of the
mainbody follicular epithelium. brk clones alone have no effect on
the position of this border because they cause only a replacement of the DAs
by operculum (Fig. 4P-R)
(Chen and Schupbach, 2006).
dad mutant clones seem to lack patterning defects altogether
(Chen and Schupbach, 2006).
However, already removing one copy of these inhibitors in a homozygous
dSno mutant background leads to an enlargement of operculum and a
posterior shift of the DAs. Weak phenotypic effects of dSno have
recently been reported for wing vein formation
(Ramel et al., 2007). Wing
vein formation, too, represents a developmental context in which several Dpp
inhibitors collaborate.
Molecular function of dSno
The dSno mutation we have generated deletes a highly conserved
protein domain that is responsible for the interaction with Smad proteins in
vertebrates (for a review, see Luo,
2004) and with Medea in flies
(Takaesu et al., 2006). The
knockout mutations in mice are based on the deletion of this domain
(Shinagawa et al., 2000).
Thus, this dSno mutation should represent a null allele. However,
Takaesu et al. report an unusual complexity of the dSno locus and
describe a deletion that suggests that dSno is lethal, in variance to
our findings (Takaesu et al.,
2006). However, Ramel et al. describe a truncation allele lacking
an important part of the conserved Smad interaction domain that, like our
allele, is viable (Ramel et al.,
2007). Because the possibility exists that the deletion described
by Takaesu et al. affects other genes in the chromosomal region of
dSno (Takaesu et al.,
2006), the question of lethality of dSno requires further
analysis.
Loss of dSno in the follicular epithelium does not result in
changes in dpp expression or pMAD distribution. Whereas a feedback on
dpp expression was not expected, possible changes in pMAD
distribution might be below the level of detection with our staining protocol.
However, there are two other possible explanations. First, in brain
development dSno has been shown to be a mediator of Baboon (Activin),
rather than Dpp signaling (Takaesu et al.,
2006). To investigate whether this also holds true for the
follicular epithelium we have induced large baboon (Activin type I
receptor) mutant follicle cell clones. These clones did not show patterning
defects (data not shown), suggesting that dSno does not act via
Baboon signaling with regard to follicle cell patterning. Second, the failure
to detect changes in pMAD distribution might follow from the molecular
mechanism of Sno action. A core feature of the inhibitory function of Sno
proteins results from their ability to bind to the common Smad (Smad4). This
binding prevents (or modulates) the interaction with phosphorylated R-Smads
required for the transcriptional control of target genes
(Wu et al., 2002). If this
mechanism applies to DSno, the loss of dSno would not change the
phosphorylation state of MAD and, if the interaction between DSno and Medea
occurred predominantly in the nucleus, there would also be no significant
change in the nuclear accumulation of pMAD.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/12/2261/DC1
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ACKNOWLEDGMENTS |
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REFERENCES |
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