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First published online 28 January 2009
doi: 10.1242/dev.032854
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1 Centro Andaluz de Biología del Desarrollo (CABD), CSIC-Universidad
Pablo de Olavide, Sevilla 41013, Spain.
2 IPATIMUP, Universidade do Porto, Porto 4200-465, Portugal.
3 IBMC, Universidade do Porto, Porto 4150-180, Portugal.
* Author for correspondence (e-mail: fcasfer{at}upo.es)
Accepted 28 December 2008
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Drosophila, SoxF, Organ growth, Wg, Wing imaginal disc
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Normal organ growth not only needs mitogens, but also mechanisms to control
their production, transport, reception and/or transduction to ensure that
proliferation is limited in space and time. Alterations in these control
mechanisms often lead to disease.
The Wnt/β-catenin signaling pathway promotes cell proliferation during
normal development and disease (Polakis,
2000). Wnts are lipid-modified glycosylated signaling molecules
that can reach distant cells. Binding of Wnts to the receptor complex
[composed of a Frizzled family receptor and an Arrow (LRP) co-receptor]
results in the stabilization of the transcriptional co-factor β-catenin
[armadillo (arm) in Drosophila]. Thereby,
β-catenin/Arm accumulates in the nucleus, where it associates with
Tcf/LEF DNA-binding transcription factors to regulate the expression of Wnt
target genes (Gordon and Nusse,
2006). Research in a number of model organisms has demonstrated
that the Wnt/β-catenin pathway controls cell proliferation in a variety
of tissues, including the nervous system
(Chenn and Walsh, 2002;
Chesnutt et al., 2004;
Dickinson et al., 1994) and
the progenitors of the intestine and hematopoietic systems
(Pinto et al., 2003;
Willert et al., 2003) in
mammals, and during imaginal disc development in Drosophila
(Giraldez and Cohen, 2003;
Johnston and Sanders, 2003;
Neumann and Cohen, 1996). It
is also known that most colorectal tumors, and a number of other tumor types,
are caused by aberrant Wnt/β-catenin signaling
(de Lau et al., 2007;
Polakis, 2000), which
underlines the necessity of tight regulation of this pathway.
The range and intensity of the signaling elicited by Wnt molecules have
been shown to be regulated by many different mechanisms, including
negative-feedback loops. These have been particularly well studied for the
main Drosophila Wnt gene, wingless (wg).
wg is required in the imaginal discs for the growth and patterning of
the adult body structures (Giraldez and
Cohen, 2003; Johnston and
Sanders, 2003). wg signaling results in the
downregulation of its two receptors, Dfz-2 (fz2 - FlyBase)
and fz (Cadigan et al.,
1998; Muller et al.,
1999) and in the upregulation of Dfz-3 (fz3 -
FlyBase), a non-productive low-affinity receptor, and of the extracellular Wg
inhibitor Notum (wingful)
(Gerlitz and Basler, 2002;
Giraldez et al., 2002;
Sato et al., 1999;
Sivasankaran et al., 2000).
Intracellularly, high levels of wg/Wnt signaling induce the
expression of two inhibitors of the pathway: naked cuticle
(Rousset et al., 2001;
Zeng et al., 2000) and
nemo (Zeng and Verheyen,
2004). All these feedback loops result in an attenuation of the
signal at the sites of maximal wg production and are generally
implicated in all processes in which wg is required.
The Drosophila wing disc gives rise to the wing blade, the notum
(body wall) and the hinge, which joins the wing blade to the body wall and
articulates its movements (see Fig.
1A-D). wg is expressed in two concentric rings in the
hinge domain (Baker, 1988) and
has been shown to be required for the proliferation of hinge cells
(Neumann and Cohen, 1996;
Zirin and Mann, 2007).
Moreover, wg overexpression is sufficient to drive hinge overgrowths
without causing major repatterning
(Neumann and Cohen, 1996;
Whitworth and Russell, 2003).
Therefore, the precise regulation of the wg pathway is crucial to
control the growth of the hinge. The mitogenic effect of wg on hinge
cells contrasts with its effect on the neighboring wing pouch cells which,
upon similar wg overexpression, are mostly driven into sensory organ
differentiation (Neumann and Cohen,
1996; Sanson et al.,
1996). One prediction from these results is that the
hinge-specific proliferative function of wg needs dedicated control
mechanisms to ensure normal hinge size and shape. To identify these
mechanisms, we searched genes that are differentially expressed in the hinge
territory for a role in wg-mediated proliferation. SoxF
(Sox15) belongs to the family of sequence-specific HMG Sox
transcription factors and has been shown to be expressed in the prospective
hinge of third larval stage (L3) wing discs
(Cremazy et al., 2001). The
functions of Sox genes have been extensively studied in mammals, in which they
play essential roles during development
(Kiefer, 2007). In addition,
misregulation of Sox genes is often associated with cancer
(Dong et al., 2004).
Only two of the eight Sox family genes present in the Drosophila
genome have been studied in detail: Dichaete (D) and
SoxNeuro (SoxN). They belong to the SoxB group and have
prominent roles in embryonic segmentation and nervous system development
(Overton et al., 2002). In
addition, it has recently been shown that both genes negatively regulate the
activity of the wg/Wnt pathway during cell fate specification in the
embryonic epidermis (Chao et al.,
2007; Overton et al.,
2007).
Here, we report that SoxF, which is the sole member of this Sox group in Drosophila, is also required to restrain wg signaling, but using a novel mechanism: the transcriptional repression of wg. In the absence of SoxF, wg transcription spreads through the hinge causing its overproliferation. SoxF is itself under the control of the canonical wg/Wnt pathway such that wg and SoxF regulate each other's transcription through a feedback loop. Moreover, the expression of rotund (rn), which is part of the proximodistal patterning mechanism of the wing disc, allows the exclusion of SoxF from a thin rim of cells, allowing them to express wg. Thereby, this rim becomes a spatially well-defined mitogen-producing center necessary to ensure normal hinge growth. This novel mode of action of a Sox gene on the Wnt pathway - the transcriptional repression of a Wnt gene - might be relevant to human disease, as loss of human SoxF genes has been implicated in colon carcinoma.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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)]. The SoxFKG09145 allele carries an
insertion of the P[SUPor-P] transposon in an intronic region of the gene,
which also harbors the CG30071 transcript (see Fig. S2 in the
supplementary material). Most homozygous SoxFKG09145 flies
die as pharate adults, and escapers are weak with held-out wings (see Fig. S2D
in the supplementary material). This latter phenotype is indicative of hinge
defects. In fact, these flies show abnormal proximal hinge structures: the
sclerites, the alula and the costa are affected (see Fig. S2E,F in the
supplementary material). Although the insertion does not affect SoxF
coding sequence, we observed by RT-PCR (data not shown) and in situ
hybridization (see Fig. S2B,C in the supplementary material) that
SoxF expression is completely lost in the wing disc of mutant L3
larvae. As this P-element carries insulator sequences, we also checked by
RT-PCR that expression of CG30071 and of the 5' neighboring
gene, RpS23, was not affected by the insertion, which was indeed the
case (see Fig. S2G in the supplementary material). We also generated new
alleles by imprecise excision of the P transposon from the original allele. In
addition to full revertants, we isolated more than ten mutant lines in which
different lengths of intron sequences were deleted, without affecting the
coding region, and which showed a range of phenotypic severity. These results
suggest that this intronic region carries crucial elements for the regulation
of SoxF expression. We also isolated some alleles that disrupt the
coding sequence. Among them, SoxF26 is specific to the
SoxF gene and deletes the first exon and part of the first large
intron, and is therefore likely to be a null allele (see Fig. S2A in the
supplementary material). This allele has the same phenotype as the initial
insertion. In addition, the phenotype and escaper rates of individuals
carrying SoxFKG09145 over a deficiency uncovering the
SoxF locus, Df(2R)Exel7130, are the same as for homozygous
SoxFKG09145 flies. Therefore,
SoxFKG09145 behaves as a genetic null allele. Cremazy and
co-workers (Cremazy et al.,
2001) reported that SoxF is expressed in the embryonic
peripheral nervous system (PNS). We obtained adult escapers of the molecular
null allele SoxF26. These animals, in addition to their
abnormally folded wings, are weak and die shortly after eclosion. Other hinge
mutants, such as wg spd-fg, are much healthier. Therefore, it is
possible that the larval lethality and weakness of adult escapers is due to
abnormal PNS development.
Other fly stocks and genetic manipulations
UAS-SoxF was generated by cloning the full-length SoxF
coding region from the cDNA clone IP09065 as an EcoRI/XhoI
fragment into the pUASt plasmid. Transgenic flies were generated by standard
methods. To analyze, comparatively, the effects of gene overexpression using
the GAL4/UAS system (Brand and
Perrimon, 1993), the genotypes were synthesized to contain the
same number of UAS sequences by including a `neutral'
UAS-GFP if needed.
GAL4 lines
wg-GAL4, BxMS1096-GAL4 (FlyBase);
zfh2MS209-GAL4
(Whitworth and Russell,
2003).
UAS lines
UAS GFP-Wg (Pfeiffer et al.,
2002), UAS-ArmS10 (UAS-Arm*),
UAS-dTCFDeltaN (TCFDN) and
UAS-GFP (FlyBase); UAS-rn
(St Pierre et al., 2002) was a
gift from J. P. Couso; UAS-dsSoxF (number 45482, Drosophila
Genetic Resource Center, Kyoto, Japan).
Reporter lacZ lines
puckered-lacZ (pucE69), wg-lacZ
(FlyBase), SpFlag-lacZ (Neumann
and Cohen, 1996), rn-lacZ
(St Pierre et al., 2002).
Mutant strains
wgspd-fg (Neumann
and Cohen, 1996), wgCX3
(Klein and Arias, 1998)
w1118; Df(2R)Exel7130, P+Pbac[XP5.WH5] Exel7130/CyO (FlyBase).
Clonal analysis
The allele SoxFKG09145 was recombined onto an
FRT42D chromosome using standard genetic procedures. Mitotic
recombination SoxFKG09145 clones were generated by the
FRT/FLP method (Xu and Rubin,
1993) in L1-2 larvae from the cross between FRT42D
SoxFKG09145/CyO males to hsFLP122;;FRT42D ubi-GFP
females. To induce the clones, 24-72 hours after egg laying (AEL) larvae were
heat shocked at 37°C for 30 minutes. Mutant tissue was detected by the
absence of the GFP marker.
SoxF and dTCFDeltaN overexpression clones were
obtained by incubating yw hs-FLP122; act>y+>Gal4, UAS-GFP/+
UAS-SoxF/+ or UAS-dTCFDeltaN/+ larvae for 10 minutes
at 35.5°C at two developmental times (48-72 hours and 48-96 hours AEL).
wg, rn and arm overexpression clones were obtained by
crossing males of their respective UAS lines to yw122,
act>hsCD2>GAL4 females (Basler
and Struhl, 1994). Larvae from the crosses were heat shocked for
10-20 minutes at 35.5°C between 48 and 96 hours AEL. To mark the clones,
CD2 was induced by subjecting late L3 (wandering) larvae to a 30-minute heat
shock at 37°C, followed by a 30 minute recovery period at 25°C just
prior to dissection.
Immunostaining, in situ hybridization and BrdU incorporation
The antibodies used for immunostaining were: mouse
anti-β-galactosidase (Sigma, 1/1000) and rabbit anti-β-galactosidase
(Cappell, 1/1000), mouse anti-GFP (Invitrogen, 1/1000), mouse anti-CD2
(Serotec, 1/400), mouse anti-Nub (Ng et
al., 1995), rabbit anti-Tsh
(Wu and Cohen, 2000), mouse
anti-Wg (4D4, Developmental Studies Hybridoma Bank, Iowa University, 1/100),
guinea pig anti-Hth (Casares and Mann,
1998), mouse anti-Arm (N27A1, Developmental Studies Hybridoma
Bank, 1/50), rabbit anti-cleaved Caspase 3 (Cell Signaling, 1/500).
Appropriate secondary antibodies were conjugated to Alexa 488, 568 or 647
(Invitrogen, 1/800). After dissection and fixation, larvae were incubated with
primary antibodies overnight at 4°C or for 2 hours at room
temperature.
Rhodamine-phalloidin staining (Invitrogen, 1/400) was performed during secondary antibody incubation or was added directly to the mounting medium.
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). Discs
were incubated for 30 minutes in a 10 mM BrdU (Roche) solution, and BrdU was
detected with a mouse anti-BrdU antibody (Roche, 1/400).
Fluorescent in situ hybridization was performed as described
(Vanzo and Ephrussi, 2002)
with minor modifications. SoxF antisense RNA probes were synthesized
from a plasmid that contains the coding sequence of SoxF or the probe
described by Cremazy et al. was used
(Cremazy et al., 2001), with
incubation at 65°C or 55°C, respectively. Probes were labeled with
digoxigenin (Dig), and detected with an alkaline phosphatase-conjugated
anti-Dig antibody (1/1000), both from Roche. Signal was developed using Fast
Red tablets (Roche) followed by standard immunostaining
(Vanzo and Ephrussi, 2002).
Confocal image acquisition was performed on a SP2-AOBS confocal system
(Leica). Stacks of (x,y) sections were recorded along the
z-axis every 1 µm. Single z-sections (`cross-sections')
were recorded as (x,z) confocal sections, with a z-step of 1
µm. In some cases, maximum or average projections of the z-series
were produced in order to visualize the total signals in the samples. Confocal
data processing was performed using Adobe Photoshop.
RNA extraction and RT-PCR
RNA extraction was performed using the RNeasy Kit (Qiagen). For each
genotype, eight larvae were collected in lysis buffer (RLT, RNeasy, Qiagen)
and ground with a pestle in an Eppendorf tube. The lysate was passed through a
QIAshredder column (Qiagen) to optimize extraction and DNA digestion was
performed during the process of extraction.
For the RT-PCR reactions, 5-7 µg of RNA was used for the first-strand cDNA synthesis (SuperScript First-Strand Synthesis Kit, Invitrogen). PCR was performed using 2 µl of the first-strand synthesis reaction with GoTaq polymerase (Promega). PCR conditions were: 30 cycles of 30 seconds at 95°C, 30 seconds at 55°C, 30 seconds at 42°C. Primers were: L1-SoxF (5'-TGCAACTGCAACAACATCAA-3') and R1-SoxF (5'-GTCAGATAGCCACCGTGCTC-3'), which amplify a fragment specific to the SoxF transcript; L1RpS23 (5'-AGATCTTGGGCGTTCCTTCT-3') and R1Rps23 (5'-TTGCAATCCAAATCACAGGA-3') for the RpS23 gene; L1CG30071 (5'-AGAAGCTGGAGCAGAAGCTG-3') and R1CG30071 (5'-GCTGCTGAATTCTTGGAAGG-3') for the CG30071 gene; L1-8394 (5'-GCGATGGCGAGTATAGGAAC-3') and R1-8394 (5'-CAGCGATACGATGAACATGC-3') for the CG8394 gene. For the SoxF, CG8394 and RpS23 genes, amplification was specific for the corresponding messenger RNAs, as the primers were designed against coding sequences that are separated by introns in the pre-mRNAs.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Casares and Mann, 2000;
del Alamo Rodriguez et al.,
2002; Neumann and Cohen,
1996).
During L2, prospective distal hinge cells express the POU gene
nubbin (nub) (Ng et al.,
1995; Zirin and Mann,
2007), while proximal hinge cells express the zinc-finger gene
teashirt (tsh) (Azpiazu
and Morata, 2000; Fasano et
al., 1991; Soanes and Bell,
2001) (summarized in Fig.
1I). At the beginning of L3, a ring of wg expression
appears in the prospective distal hinge, the so-called wg inner ring
(IR). The wg IR expression is included within the nub domain
in cells that also express the Kruppel-like transcription factor rn
(del Alamo Rodriguez et al.,
2002), and is driven by a specific regulatory element, the
spade-flag (spd-fg) enhancer
(Neumann and Cohen, 1996).
Starting in early L3, wg IR drives intercalary proliferation between
the nub and tsh domains generating a region that expresses
neither of the two, the so-called gap domain
(Zirin and Mann, 2007). By
late L3, a second ring of wg, called the wg outer ring (OR),
appears in the prospective proximal hinge, and abuts the distal limit of
tsh expression (see Fig.
1I). In this paper, we focus on the regulation of the expression
and function of the wg IR domain, as it has a major role in
controlling hinge proliferation.
|
). GFP+ (hinge) and GFP- (body wall plus
wing blade) cells were FACS sorted and their transcriptome profiles compared
(a full account of this analysis will be published elsewhere). This experiment
identified Drosophila Sox15 (CG8404) as the transcript most
over-represented in hinge cells. Recently, Bowles and co-workers reassigned
this gene to the SoxF group of the Sox family, making it the sole
Drosophila member of the group, which in mammals includes Sox7,
Sox17 and Sox18 (Bowles et
al., 2000). We adopt their nomenclature and hereafter refer to
this gene as SoxF.
SoxF had been reported to be transcribed in the hinge of late L3
wing discs (Cremazy et al.,
2001). We further mapped the SoxF domain relative to
wg and rn reporters by in situ hybridization. The
SoxF domain abuts rn and the wg IR on its distal
border and extends into the proximal hinge overlapping the late wg OR
(Fig. 1E-H; see Fig. S1 in the
supplementary material; data not shown). Therefore, the realm of SoxF
expression straddles the gap domain (Zirin
and Mann, 2007). This adjacent, non-overlapping expression between
SoxF and the domains of rn and wg is also observed
at earlier stages (see Fig. S1 in the supplementary material).
The loss of SoxF function leads to hinge-specific overproliferation without loss of hinge identity
In order to determine SoxF function, we analyzed wing imaginal
discs from larvae homozygous for the null allele
SoxFKG09145 (see Materials and methods). SoxF
mutant wing discs showed hinge overgrowths
(Fig. 2) that caused misfolding
of both its dorsal and ventral regions. However, the wing pouch and body wall
regions seemed unaffected. To determine the origin of these overgrowths within
the hinge, we mapped them relative to the expression of nub, tsh and
the intervening gap domain (Fig.
2A,B). In SoxF mutant discs, both the prospective distal
hinge, which expresses nub, and the gap domain were significantly
enlarged (Fig. 2A,B). In
addition, the overgrown hinge still expressed high levels of
homothorax (hth) (not shown), which is a hinge marker
(Azpiazu and Morata, 2000;
Casares and Mann, 2000).
Therefore, the tissue overgrowth observed in the SoxF mutants
correlates with the SoxF expression domain, suggesting that
SoxF has an autonomous effect on the control of hinge proliferation.
In addition, the overgrowth cannot be explained by changes in cell fate
because we still detected normal expression of hinge-specific markers.
In SoxF mutant discs, we detected elevated levels of incorporation of the S-phase marker BrdU specifically in the hinge, indicating that the overgrowths were in fact caused by increased cell proliferation (Fig. 2C,D). In addition, we noted an increase in apoptotic cell death, as detected by activated Caspase 3 (Decay - FlyBase) staining (see Fig. 4D). This apoptosis is associated with activation of the Jnk pathway, as indicated by the upregulation of a transcriptional reporter of the Jnk target puckered (not shown).
SoxF blocks wg transcription in the hinge through the spd-fg enhancer
Since expression of wg at the IR is necessary for, and sufficient
to induce, the proliferation of hinge cells
(Neumann and Cohen, 1996;
Zirin and Mann, 2007), we
asked whether its expression was altered in SoxF mutants. We compared
the expression of a wg-lacZ transcriptional reporter in wild-type and
SoxFKG09145 mutant wing discs. In wild-type discs,
wg-lacZ is expressed in two distinct rings in the hinge, IR and OR,
separated by a non-expressing region (Fig.
3A). However, in SoxF mutant discs, wg
transcription spread throughout the hinge and no wg-negative
territory remained (Fig. 3B).
When we examined the effect of removing SoxF function in clones, we
observed effects on wg expression only in clones spanning the hinge.
In these clones, wg expression filled the domain between the IR and
OR rings, which thus became connected (Fig.
3C). Sox mutant clones in the wing pouch or prospective notum had
no effect on wg (not shown). These results indicate that
SoxF is required cell-autonomously to repress wg
transcription in the domain that separates the IR and OR.
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|
). In
wild-type discs, spd-fg-lacZ expression was seen as a narrow stripe
centered in the prospective distal hinge fold
(Fig. 3D,D'). However, in
discs in which the expression of SoxF had been knocked down by RNAi
(zfh-2MS209-GAL4; UAS-dsSoxF), the expression of this
reporter was considerably wider (Fig.
3E,E'), now filling the distal hinge fold and abutting the
proximal hinge fold (Fig.
3E'). Therefore, the repression of wg by
SoxF is likely to occur through the wg spd-fg enhancer. As wg expression was derepressed in SoxF mutant conditions, we tested whether SoxF was sufficient to block wg transcription. We observed that SoxF-expressing clones were able to repress wg expression in the IR at both the protein (Fig. 3G,G') and transcriptional (Fig. 3F,F') level. According to our previous observations, the expression of SoxF also blocks the expression of the spd-fg-lacZ enhancer (Fig. 3G) in a cell-autonomous manner, reinforcing the idea that the regulation of wg IR by SoxF works through the spd-fg enhancer. Clones overlapping the OR showed no effects on wg expression, in agreement with the co-expression of SoxF and wg OR found in normal discs (data not shown).
The derepression of wg is required for the hinge overgrowth of SoxF mutant discs
The correlation between wg derepression and hinge overgrowth,
together with the known role of wg as an essential mitogen in the
hinge, led us to test whether wg was itself required for the
overgrowths. We recombined the SoxFKG09145 allele into a
wgspd-fg background. wgspd-fg is a
regulatory mutation that deletes the enhancer that drives wg
expression in the IR (Couso et al.,
1994; Neumann and Cohen,
1996; Tiong and Nash,
1990). Accordingly, in spd-fg mutant discs, the
prospective distal hinge underproliferates and spd-fg mutant adults
lack distal hinge structures (Neumann and
Cohen, 1996). We verified that in spd-fg mutant discs
there is no increase in apoptosis, as monitored by anti-activated Caspase 3
staining (not shown), which confirms that reduced hinge proliferation is the
major cause of the spd-fg adult phenotype. In
wgspd-fg, SoxFKG09145 double mutants, the
distal hinge was not overgrown (Fig.
4C). This result indicates that wg is required for the
overproliferation observed in SoxF mutant discs. In fact, the
reduction of the distal hinge was even stronger in the double mutant discs
than in wgspd-fg discs, as indicated by the width of the
distal hinge fold and the almost complete loss of the wg IR
(Fig. 4B,C). This stronger
phenotype can be accounted for by the apoptosis that we still detect in the
hinge region of wgspd-fg, SoxFKG09145 discs
(Fig. 4E), which is similar to
that observed for SoxF single mutants and which does not occur in
wgspd-fg discs.
|
; Zeng and
Verheyen, 2004). The fact that the domain of SoxF
expression in the hinge coincides with the region under wg
proliferative control prompted us to ask whether SoxF itself could be
induced by the wg pathway. Indeed, clones of
Arm*-expressing cells, in which the pathway is constitutively
active, caused cell-autonomous activation of SoxF expression in
regions of the notum close to the hinge
(Fig. 5A). Although we have not
performed a detailed study of where Arm* clones activate
SoxF using markers for distinct domains within the notum, we noted
that SoxF induction does not occur in the dorsal-most notal region,
suggesting that factors such as pannier and/or the iro-C
genes, which are expressed in this region
(Calleja et al., 2000;
Diez del Corral et al., 1999),
might be limiting the competence to activate SoxF in response to Wnt
signaling. In agreement with this restricted competence, SoxF is not
found associated with two other domains of wg expression in the disc
(not shown) that map to the prospective tegula of the wing
(Casares and Mann, 2000) and
the medial notum (Calleja et al.,
1996). Similar to Arm*, clones expressing Wg also led
to ectopic expression of SoxF, although this time the induction was, in part,
non-autonomous, reflecting the diffusible nature of the Wg ligand
(Fig. 5B). Conversely, clones
in which the wg pathway is blocked by the expression of a
dominant-negative form of TCF (Pangolin - FlyBase),
dTCFDeltaN, resulted in autonomous repression of
SoxF (Fig. 5C).
Similar results were observed when the expression of
dTCFDeltaN was driven in the dpp domain that
intersects SoxF expression (Fig.
5D). Therefore, these results reveal a cross-regulatory loop
between wg and SoxF: wg induces SoxF,
which in turn acts as a wg antagonist by blocking the spreading of
its transcription throughout the hinge and, potentially, by attenuating its
pathway. We noted that SoxF is expressed in late L2 wing discs, well
before the rings of wg expression in the hinge are established (see
Fig. S1 in the supplementary material). In order to determine whether
wg is required for SoxF early in development, and not only
in the hinge, we examined SoxF transcription in late L2 discs of
wgCX3 mutants. wgCX3 is a regulatory
mutant that lacks the earliest wg expression in the wing disc, and,
as a consequence, wgCX3 discs fail to establish the wing
field (Klein and Arias, 1998).
These mutant discs lack SoxF expression (see Fig. S1A,B in the
supplementary material), indicating that wg is required throughout
development for SoxF transcription in the wing disc.
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The activation of wg expression in the hinge is coupled to the
mechanisms that pattern the wing disc along its proximodistal (PD) axis
(Azpiazu and Morata, 2000;
Casares and Mann, 2000;
del Alamo Rodriguez et al.,
2002; Terriente Felix et al.,
2007; Whitworth and Russell,
2003; Wu and Cohen,
2002). One of the genes required for wg IR expression is
rn, a transcription factor that is expressed in the prospective
distal hinge and wing pouch. The wg IR, which abuts the SoxF
expression, appears at the edge of the rn domain
(del Alamo Rodriguez et al.,
2002). Therefore, the rn and SoxF expression
domains are mutually exclusive (Fig.
1E,G; see Fig. S1 in the supplementary material). We checked
whether rn could be repressing SoxF and thus polarizing its
activation along the PD axis in the hinge. Ectopic clones of rn
repressed SoxF expression autonomously
(Fig. 5E,E'), suggesting
that this is indeed the case. The reciprocal repression, of SoxF on
rn, did not seem to take place, as the domain of the rn-lacZ
reporter did not change in SoxF mutant discs (not shown). Therefore,
SoxF is linked to the mechanism of PD axis formation of the disc in a
way that ensures its directional activation by wg specifically
straddling the gap domain of the hinge, the cell population whose
proliferation is controlled by IR wg. Here, SoxF performs a
key role in restricting the activation of the wg pathway and, by
doing so, controls hinge growth.
|
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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During Drosophila development, the wg pathway often leads
to the activation of genes that attenuate its signaling pathway. This is the
case, for example, for Notum and Dfz-3, which are expressed
in the wing disc in response to peak levels of signaling to reduce ligand
availability for the Wg receptors
(Sivasankaran et al., 2000),
and for nemo, which acts intracellularly to block the signal
transduction pathway (Zeng and Verheyen,
2004). In all cases described, these negative-feedback components
act in all domains of wg expression and none regulates wg
expression at the transcriptional level. However, in the case investigated
here, the putative transcription factor SoxF is activated
non-autonomously by wg in a hinge-specific manner. SoxF in
turn represses wg transcription driven by the wg spd-fg
enhancer, thus restricting the production of wg to the thin IR
domain. Interestingly, the SoxF phenotype is similar to those of
dominant Dichaete (D) mutations. D is a SoxB gene
not normally expressed in the wing disc
(Mukherjee et al., 2000).
However, flies carrying dominant D mutations show reduced hinge
structures. This phenotype is caused by ectopic D expression in the
prospective hinge region of the disc
(Russell, 2000). One of the
salient features of D discs is the repression of the wg IR
(Russell, 2000), which is
reminiscent of the wg repression by SoxF we have described.
Therefore, and taking into account the similarity between Sox proteins in
their HMG DNA-binding domain, the ectopic D might be mimicking the
repression of wg that is normally exerted by SoxF.
The tight regulation of the growth of the hinge depends critically on the
wg-induced activation of SoxF in the growing territory.
Nevertheless, this activation is `polarized' along the PD axis, taking place
only in cells adjacent and proximal to the IR. We propose that this
directionality in SoxF activation results from the mechanisms that
pattern the wing disc along its PD axis. It has been suggested that
wg is activated non-autonomously by a signal produced by the
vg-expressing wing pouch cells, but excluded from them
(del Alamo Rodriguez et al.,
2002). This would generate a circular domain of wg
expression surrounding the wing pouch. However, in the absence of
SoxF, the domain of wg is abnormally broad and causes hinge
overgrowth. This ectopic wg expression does not seem to result from a
misregulation of hinge-specific genes: the expression of nub, tsh,
hth and rn and their relative positioning in the hinge are
unaffected in SoxF mutant discs (Figs
2 and
4; data not shown). Therefore,
it seems that in the absence of SoxF, hinge cells cannot respond to
the wg activating signals with enough precision to give rise to a
thin ring of wg expression. Our results show that this precision is
achieved through a double repression mechanism. First, wg activates
its own transcriptional repressor, SoxF. This would lead to the
extinction of wg expression if it were not for rn, which
acts as a repressor of SoxF. Second, rn, by repressing
SoxF, permits wg transcription. The result is that
wg expression becomes restricted to a narrow circular stripe at the
edge of the rn domain that provides a highly localized source of Wg.
This signal activates, simultaneously and in the same cells, proliferation and
the upregulation of SoxF, which restricts the production of the
signal (Fig. 6). Therefore,
SoxF joins SoxN and SoxD (Sox102F -
FlyBase) (Chao et al., 2007;
Overton et al., 2007) as the
third Drosophila Sox known to antagonize the wg pathway. The
vertebrate Sox proteins Sox9 (Mori-Akiyama
et al., 2007), XSox3 (Zorn et
al., 1999) and XSox17 (Sinner
et al., 2004) have also been shown to downregulate the
Wnt/β-catenin pathway. Therefore, this antagonism seems evolutionarily
conserved.
The relationship between SoxF genes, the wg/Wnt pathway and the
control of tissue proliferation seems to extend to disease. The SoxF
Sox17 is normally expressed in the gut epithelium where it
downregulates Wnt signaling via degradation of β-catenin and TCF. In
colon carcinomas, the expression of the SoxB gene Sox17 is often
reduced, and this is associated with tissue overproliferation
(Sinner et al., 2007).
Moreover, inactivation of the SoxE gene Sox9 leads to increased cell
proliferation and hyperplasia in the mouse intestine
(Bastide et al., 2007). The
authors concluded that Sox9 is essential for the fine-tuning of the
transcriptional activity of the Wnt pathway
(Bastide et al., 2007).
Interestingly, the expression of Sox9 is regulated by the Wnt pathway
itself (Blache et al., 2004).
Our results in Drosophila point to the possibility that the
transcriptional regulation of Wnt expression by Sox genes might be a common
feature of this proliferation-associated feedback loop.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/136/5/761/DC1
|
Footnotes |
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