InteractiveFly: GeneBrief

Sox box protein 15: Biological Overview | References

Wnt molecules act as mitogenic signals during the development of multiple organs, and the aberrant activity of their pathway is often associated with cancer. Therefore, the production of Wnts and the activity of their signaling pathway must be tightly regulated. This study has investigated the mechanisms of this regulation in the Drosophila hinge, a domain within the wing imaginal disc that depends on the fly Wnt1 ortholog wingless (wg) for its proliferation. The results uncover a new feedback loop in the wg pathway in which the spatially restricted activation of the Sox gene SoxF (Sox15) by wg represses its own transcription, thus ensuring tight regulation of growth control. rotund, a wing proximodistal patterning gene, excludes SoxF from a thin rim of cells. These cells are thus allowed to express wg and act as the source of mitogenic signal. This novel mode of action of a Sox gene on the Wnt pathway -- through transcriptional repression of a Wnt gene -- might be relevant to human disease, as loss of human SoxF genes has been implicated in colon carcinoma (Dichtel-Danjoy, 2009).

One of the long-standing questions in biology is how organ growth is coordinated with tissue patterning. Research during recent decades has shown that a limited set of signals and signaling pathways control this coordination. Some of these signals are mitogenic, and their production at specific sites, called signaling centers, links spatial information to cell proliferation within developing organs. 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 (Dichtel-Danjoy, 2009).

The Wnt/β-catenin signaling pathway promotes cell proliferation during normal development and disease. 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. 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 and the progenitors of the intestine and hematopoietic systems in mammals, and during imaginal disc development in Drosophila. It is also known that most colorectal tumors, and a number of other tumor types, are caused by aberrant Wnt/β-catenin signaling, which underlines the necessity of tight regulation of this pathway (Dichtel-Danjoy, 2009).

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. wg signaling results in the downregulation of its two receptors, Dfz-2 (fz2 - FlyBase) and fz and in the upregulation of Dfz-3 (fz3 - FlyBase), a non-productive low-affinity receptor, and of the extracellular Wg inhibitor Notum (wingful). Intracellularly, high levels of wg/Wnt signaling induce the expression of two inhibitors of the pathway: naked cuticle and nemo. 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 (Dichtel-Danjoy, 2009).

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. wg is expressed in two concentric rings in the hinge domain and has been shown to be required for the proliferation of hinge cells. Moreover, wg overexpression is sufficient to drive hinge overgrowths without causing major repatterning. 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. 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, genes were sought 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, 2001). The functions of Sox genes have been extensively studied in mammals, in which they play essential roles during development. In addition, misregulation of Sox genes is often associated with cancer (Dichtel-Danjoy, 2009).

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. 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, 2007; Overton, 2007; Dichtel-Danjoy, 2009 and references therein).

This paper reports 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 (Dichtel-Danjoy, 2009).

In order to determine the role played by SoxF during hinge development, a SoxF allele, Sox15^KG09145 (now renamed SoxF^KG09145) was characterized. The SoxF^KG09145 allele carries an insertion of the P[SUPor-P] transposon in an intronic region of the gene, which also harbors the CG30071 transcript. Most homozygous SoxF^KG09145 flies die as pharate adults, and escapers are weak with held-out wings. 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. Although the insertion does not affect SoxF coding sequence, it was observed by RT-PCR and in situ hybridization that SoxF expression is completely lost in the wing disc of mutant L3 larvae. Sice this P-element carries insulator sequences, it was 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. This study has also generated new alleles by imprecise excision of the P transposon from the original allele. In addition to full revertants, more than ten mutant lines were isolated 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. Some alleles were isolated 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. This allele has the same phenotype as the initial insertion. In addition, the phenotype and escaper rates of individuals carrying SoxF^KG09145 over a deficiency uncovering the SoxF locus, Df(2R)Exel7130, are the same as for homozygous SoxF^KG09145 flies. Therefore, SoxF^KG09145 behaves as a genetic null allele. Cremazy (2001) reported that SoxF is expressed in the embryonic Peripheral nervous system (PNS). Adult escapers of the molecular null allele SoxF²⁶ exhibit, in addition to their abnormally folded wings, are also 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 (Dichtel-Danjoy, 2009).

This study describes a novel negative-feedback mechanism in the wg pathway that is required to restrain the expression of wg itself, and which is essential to control organ growth. 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, and for nemo, which acts intracellularly to block the signal transduction pathway. 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 in this study, 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 inner ring (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. 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. One of the salient features of D discs is the repression of the wg IR, which is reminiscent of the wg repression by SoxF described in this study. 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 (Dichtel-Danjoy, 2009).

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. It is proposed 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, 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. 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. The 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. Therefore, SoxF joins SoxN and SoxD (Sox102F - FlyBase) (Chao, 2007; Overton, 2007) as the third Drosophila Sox known to antagonize the wg pathway. The vertebrate Sox proteins Sox9 (Mori-Akiyama, 2007), XSox3 (Zorn, 1999) and XSox17 (Sinner, 2004) have also been shown to downregulate the Wnt/β-catenin pathway. Therefore, this antagonism seems evolutionarily conserved (Dichtel-Danjoy, 2009).

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, 2007). Moreover, inactivation of the SoxE gene Sox9 leads to increased cell proliferation and hyperplasia in the mouse intestine (Bastide, 2007). The authors concluded that Sox9 is essential for the fine-tuning of the transcriptional activity of the Wnt pathway (Bastide, 2007). Interestingly, the expression of Sox9 is regulated by the Wnt pathway itself (Blache, 2004). These 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 (Dichtel-Danjoy, 2009).

Search PubMed for articles about Drosophila Sox15

Bastide, P., Darido, C., Pannequin, J., Kist, R., Robine, S., Marty-Double, C., Bibeau, F., Scherer, G., Joubert, D., Hollande, F. et al. (2007). Sox9 regulates cell proliferation and is required for Paneth cell differentiation in the intestinal epithelium. J. Cell Biol. 178: 635-648. PubMed Citation: 17698607

Blache, P., van de Wetering, M., Duluc, I., Domon, C., Berta, P., Freund, J. N., Clevers, H. and Jay, P. (2004). SOX9 is an intestine crypt transcription factor, is regulated by the Wnt pathway, and represses the CDX2 and MUC2 genes. J. Cell Biol. 166: 37-47. PubMed Citation: 15240568

Chao, A. T., Jones, W. M. and Bejsovec, A. (2007). The HMG-box transcription factor SoxNeuro acts with Tcf to control Wg/Wnt signaling activity. Development 134: 989-997. PubMed Citation: 17267442

Cremazy, F., Berta, P. and Girard, F. (2001). Genome-wide analysis of Sox genes in Drosophila melanogaster. Mech. Dev. 109: 371-375. PubMed Citation: 11731252

del Alamo Rodriguez, D., Terriente, J., Galindo, M. I., Couso, J. P. and Diaz-Benjumea, F. J. (2002). Different mechanisms initiate and maintain wingless expression in the Drosophila wing hinge. Development 129: 3995-4004. PubMed Citation: 12163403

Dichtel-Danjoy, M. L., Caldeira, J. and Casares, F. (2009). SoxF is part of a novel negative-feedback loop in the wingless pathway that controls proliferation in the Drosophila wing disc. Development 136(5): 761-9. PubMed Citation: 19176582

Mori-Akiyama, Y., van den Born, M., van Es, J. H., Hamilton, S. R., Adams, H. P., Zhang, J., Clevers, H. and de Crombrugghe, B. (2007). SOX9 is required for the differentiation of paneth cells in the intestinal epithelium. Gastroenterology 133: 539-546. PubMed Citation: 17681175

Overton, P. M., Chia, W. and Buescher, M. (2007). The Drosophila HMG-domain proteins SoxNeuro and Dichaete direct trichome formation via the activation of shavenbaby and the restriction of Wingless pathway activity. Development 134: 2807-2813. PubMed Citation: 17611224

Sinner, D., Rankin, S., Lee, M. and Zorn, A. M. (2004). Sox17 and beta-catenin cooperate to regulate the transcription of endodermal genes. Development 131: 3069-3080. PubMed Citation: 15163629

Sinner, D., Kordich, J. J., Spence, J. R., Opoka, R., Rankin, S., Lin, S. C., Jonatan, D., Zorn, A. M. and Wells, J. M. (2007). Sox17 and Sox4 differentially regulate beta-catenin/T-cell factor activity and proliferation of colon carcinoma cells. Mol. Cell. Biol. 27: 7802-7815. PubMed Citation: 17875931

Zorn, A. M., Barish, G. D., Williams, B. O., Lavender, P., Klymkowsky, M. W. and Varmus, H. E. (1999). Regulation of Wnt signaling by Sox proteins: XSox17 alpha/beta and XSox3 physically interact with beta-catenin. Mol. Cell 4: 487-498. PubMed Citation: 10549281

date revised: 20 June 2009

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