BIOLOGICAL OVERVIEW

Sox proteins form a family of HMG-box transcription factors related to SRY, the mammalian testis determining factor. Sox-mediated modulation of gene expression plays an important role in various developmental contexts. Drosophila SoxNeuro, a putative ortholog of the vertebrate Sox1, Sox2 and Sox3proteins, is one of the earliest transcription factors to be expressed pan-neuroectodermally. SoxNeuro is essential for the formation of the neural progenitor cells in the central nervous system. Loss of function mutations of SoxNeuro are associated with a spatially restricted hypoplasia: neuroblast formation is severely affected in the lateral and intermediate regions of the central nervous system, whereas ventral neuroblast formation is almost normal. Evidence is presented that a requirement for SoxNeuro in ventral neuroblast formation is masked by a functional redundancy with Dichaete, a second Sox protein whose expression partially overlaps that of SoxNeuro. SoxNeuro/Dichaete double mutant embryos show a severe neuralhypoplasia throughout the central nervous system, as well as a dramatic loss of achaete expressing proneural clusters and medially derived neuroblasts. Genetic interactions of SoxNeuro and thedorsoventral patterning genes ventral nerve chord defective (vnd) and intermediate neuroblasts defective (ind) underlie ventral and intermediate neuroblast formation. Expression of the Achaete-Scute gene complex suggests that SoxNeuro acts upstream and in parallel with the proneural genes. The finding that Dichaete and SoxN exhibit opposite effects on achaete expression within the intermediate neuroectoderm demonstrates that each protein also has region-specific unique functions during early CNS development in the Drosophila embryo (Buescher, 2002 and Overton, 2002).

Sox genes encompasses a group of transcriptional regulators, related by an HMG1-type DNA-binding domain, to the mammalian testis-determining factor SRY. The Sox gene family is large and diverse. Many members of the Sox gene family have dynamic tissue-specific expression patterns during embryogenesis, suggesting that they may play a variety of roles during development. On the basis of sequence similarity, both in the DNA-binding domain and in other, group-specific conserved motifs, Sox proteins have been divided into at least seven subgroups (A-G). Group B Sox are most closely related to SRY, sharing over 85% sequence identity between their DNA-binding domains and recognising virtually identical DNA sequences. In flies, frogs, chicks and mammals, group B Sox genes are expressed in the neuroectoderm from the earliest stages of neurogenesis (Collignon, 1996; Uwanogho, 1995; Rex, 1997; Russell, 1996; Nambu and Nambu, 1996; Wood, 1999; Cremazy, 2000). In these animals, related group B genes are co-expressed in the neuroectoderm, leading to the idea that they may function redundantly or influence each other's activity. In mice and chicks, three group B genes (Sox1, Sox2 and Sox3) are widely co-expressed in the neuroectoderm and neural tube; in Drosophila, only two group B genes, Dichaete and SoxNeuro (Cremazy, 2000), are expressed early in the CNS (Overton, 2002 and references therein).

Although well characterised in terms of expression, in vivo functional studies of Sox genes in early CNS development are less well established. In the mouse, Sox1 null mutants survive until adulthood, where some role in CNS function is suggested by a spontaneous seizure phenotype. However, the fact that homozygous mutants survive without significant defects in CNS development suggests that any major role in early CNS development is dispensable (Nishiguchi, 1998). In mice, Sox2 mutants are reported to die prior to implantation (Collignon, 1996) therefore the role of Sox2 in CNS development has not been described. Sox3 mutations have not been reported. Direct evidence for the involvement of Sox genes in CNS development comes from in vitro stem cell studies, where it was shown that the Sox1 gene can induce neural fate in competent ectodermal cells (Pevny, 1998). Furthermore, a Sox2-ßGeo insertion construct has been used to select neural precursors from stem cell populations, suggesting that Sox2 is a marker for early neural fate (Li, 1998). In Xenopus, the SoxD gene is first expressed in the prospective neuroectoderm and then later throughout the neural plate. Injection of SoxD mRNA into early embryos can induce ectopic neural tissue and injection of a dominant negative form of SoxD leads to loss of neural tissue, establishing a role for this Sox gene in Xenopus neuralization (Mizuseki, 1998a). Additionally, the Xenopus Sox2 gene, which acts downstream of SoxD, appears to be required for establishing neural competence in neuroectodermal cells (Mizuseki, 1998b; Overton, 2002 and references therein).

Mutations in the Drosophila gene Dichaete have specific defects in the specification or differentiation of glial lineages in the midline of the CNS, a structure in which Dichaete is a uniquely expressed Sox gene (Sanchez-Soriano, 1998; Ma, 2000). Outside of the midline, in the ventral neuroectoderm where Dichaete and SoxN are co-expressed (Cremazy, 2000), neural phenotypes are relatively weak (N. Sanchez-Soriano, PhD thesis, University of Cambridge, 1999). Dichaete is involved in the specification of cell fate in the neuroectoderm and in NB formation via interactions with the homeodomain-encoding genes vnd and ind (Zhao, 2002; Overton, 2002 and references therein).

Interestingly, the manner in which NBs are lost in SoxN and AS-C mutants appears mechanistically different. In AS-C mutants only a small proportion of NBs fails to be singled out and fails to delaminate from the NE (~25% of early NBs). The majority of NBs still segregate and later may be subject to cell death. By contrast, in SoxN mutant embryos, neuroectodermal cells fail to be singled out as NBs and delamination does not take place. Thus, it appears that loss of SoxN affects NBs formation at an earlier step than the loss of proneural genes. Proneural gene expression is regulated largely independently of SoxN, since loss of SoxN does not affect the neuroectodermal expression of L'sc and does not abolish that of Ac. It is suggested that SoxN acts upstream and in parallel to the proneural genes. Comparison of the NB phenotypes of AS-C mutant and SoxN mutant embryos has revealed that overlapping but not identical subsets of NBs were affected. This result suggests that SoxN function -- as it is understood it at this time -- does not explain why some NBs do not require the proneural genes of the AS-C. The binary decision of neuroectodermal cells to adopt the neural or the epidermal fate requires Notch signaling (Buescher, 2002).

It would be interesting to determine if neuroectodermal cells in SoxN mutants are still able to adopt the epidermal fate. However, owing to the lack of appropriate markers, which would indicate early epidermal differentiation, the formation of the ventral denticle belts was examined at the first instar larval stage. Denticle belt formation is severely impaired in SoxN mutant embryos, indicating that epidermal development is disturbed. Hence, in the absence of SoxN, the ability of neuroectodermal cells to undergo neural and/or epidermal development may be compromised (Buescher, 2002).

The SoxN mutant phenotype shows a strong spatial aspect with respect to the DV axis: loss of SoxN severely affects the formation of NBs that derive from the lateral and intermediate regions of the NE but have little effect on ventral NB formation. This DV effect of SoxN mutations is not mirrored in a corresponding DV SoxN expression pattern. Thus, the mutant phenotype rather reflects a differential requirement for SoxN in different regions. Analysis of ventral NB formation in SoxN;Dichaete double mutant embryos provides at least a partial explanation for these regional differences because the concomitant loss of SoxN and Dichaete results in a strong loss of ventral NBs. This suggests that SoxN and Dichaete may functionally substitute for each other. A functional redundancy of SoxN and Dichaete is not unexpected since the proteins have structural similarities and overlapping expression patterns. Like SoxN, Dichaete has also been classified as a group B Sox protein; the HMG domains of these two proteins show 87% amino acid identity. Since the ability of sequence-specific DNA binding resides within the HMG domain, it is likely that SoxN and Dichaete bind to the same DNA motif present in an identical set of target genes. This is supported by studies that have shown that various vertebrate Sox proteins can bind to the same DNA sequence. Neuroectodermal Dichaete and SoxN expression overlaps in the ventral and intermediate region and therefore a functional redundancy would be expected to occur in ventral and intermediate NB formation. However, the severe phenotype of SoxN single mutants in intermediate NB formation suggests that Dichaete cannot always substitute for SoxN function. Additional evidence that SoxN and Dichaete function is not equivalent stems from the observation that loss of Dichaete or SoxN has different effects on Ac expression in the intermediate region of the NE: in Dichaete, but not in SoxN mutant embryos, Ac expression is partially derepressed in the intermediate column (Zhao, 2002; Buescher, 2002).

The loss of one copy of vnd or ind in a SoxN homozygous mutant background dominantly enhances the SoxN phenotype, suggesting that SoxN genetically interacts with vnd and ind. Since the expression of Vnd and Ind does not require SoxN function, it is concluded that SoxN does not act upstream of vnd and ind, but rather in parallel. In ind mutant embryos, Ac expression in the NE is derepressed in the intermediate region. Nevertheless, NBs fail to form within this region. vnd is required for Ac expression in the ventral NE. However, there seems to be no causal relationship between the loss of Ac expression and the subsequent loss of NBs, since ectopic expression of Ac does not rescue NB formation. Thus, it appears that expression of the genes of the AS-C can confer neural potential to the NE only when SoxN, vnd and ind expression is intact (Buescher, 2002).

It is presumed that the differences between Dichaete and SoxN may well reflect interactions between each Sox protein and a different partner mediated by protein domains outside the highly conserved DNA-binding domain. In accordance with this, Zhao suggests that, in the neuroectoderm, Dichaete interacts with the product of the ind gene to mediate repression of ac. Since ind is specifically expressed within the intermediate neuroectoderm, it is tempting to speculate that this protein might interact specifically with Dichaete to repress ac while it does not interact with SoxN in the same way if indeed at all. However, Zhao (2002) provide evidence for interactions between Dichaete and both ind and vnd in the context of NB specification. Since the data suggest that SoxN and Dichaete function is at least redundant within the vnd-positive medial row, it is very likely that Vnd interacts with SoxN as well as Dichaete (Overton, 2002).

The interaction of HMG-domain proteins and homeodomain containing proteins has been recognized for some time, and appears to be a general feature of HMG1-type DNA-binding domains. More specific interactions between Sox-domain proteins and homeodomains have been demonstrated in mouse, where Sox2 interacts with Oct4, and in flies, where Dichaete interacts with Vvl. Therefore it is considered likely that SoxN can interact with the DV patterning protein Vnd, and also with Ind and Msh to regulate expression in the AS-C; the observation that vnd, ind and msh transcript levels are unaffected in the SoxN mutant suggests that SoxN is likely to act in parallel with rather than upstream of these DV patterning genes. The loss of ac in vnd mutant embryos is insufficient to explain the loss of S1 NBs since restoration of ac expression in this background does not rescue the phenotype, which itself is more severe than would be expected if the sole action of vnd was through AS-C. In the SoxN mutant, expression of l'sc is unaffected in lineages that fail to delaminate. It is considered likely that the role of SoxN parallel to AS-C in the neuroectoderm is to act alongside the DV patterning genes in controlling this activity. However, given that an overall reduction is seen in ac expression throughout the neuroectoderm in the SoxN mutant, it is possible that SoxN plays a more general role in regulating ac, perhaps acting as a factor for modulating chromatin structure (Overton, 2002 and references therein).

One feature that stands out in the studies of Sox activity in Drosophila is the structure of the target genes that have been identified to date. During embryonic segmentation, prior to the establishment of neuroectoderm, Dichaete is required for the correct expression of the primary pair-rule genes even skipped, hairy and runt. In the midline, slit is a direct target; in the neuroectoderm ac is a likely target, and in the hindgut hedgehog and decapentaplegic are likely targets (Sanchez-Soriano, 2000). Each of these genes is characterized by having complex structure and complex regulation. This is most apparent for the pair-rule genes, whose regulatory sequences extend over many kilobases. Where Dichaete is the only Sox gene involved in the regulation of these genes (e.g., at cellular blastoderm), a variable phenotype is observed in Dichaete mutants, both at the gross morphological level and at the molecular level. It has been suggested that this reflects an architectural role for Sox proteins in gene regulation, since Sox-domain proteins bind DNA in the minor groove and are capable of modulating chromatin structure. It is noted with interest that the regulatory sequences that control the expression of ac are also extremely complex and the ac expression phenotype observed in SoxN-Dichaete mutant embryos is also variable. This suggests that at least one of the functions of Sox proteins may be modulating chromatin structure at complex regulatory regions, allowing the integration of many different regulatory inputs. In this view, the loss of Sox function would destabilize gene expression but would not be expected to completely eliminate it (Overton, 2002 and references therein).

Comparative studies of the key steps in neural development have revealed a remarkable conservation across a wide range of species. Common features include early neural determination, which depends on the antagonistic action of positive (Sog, Chordin) and negative (Dpp, BMP) acting factors and the singling out of neural progenitor cells and aspects of DV patterning. The results presented in this study suggest that conservation extends to the function of Sox proteins in neural development. Based on sequence homology, the closest vertebrate relatives of SoxN and Dichaete are Sox1, Sox2 and Sox3. These proteins are closely related in structure throughout their entire length and are expressed in overlapping patterns in developing neural tissues (Collignon, 1996). These features, taken together with the observation that mice carrying a homozygous Sox1 mutation display rather mild defects in neural development, have led to the hypothesis that the functions of Sox1, Sox2 and Sox3 function is at least partially redundant (Nishiguchi, 1998). Analysis of SoxN;Dichaete double mutant embryos confirms this hypothesis in Drosophila, since SoxN and Dichaete function is indeed redundant with respect to the formation of a subset of NBs (Buescher, 2002).

Interestingly, the regulation of SoxN and Sox2 expression appears to be conserved in Drosophila and Xenopus: both are negatively regulated by Dpp (BMP4) and positively regulated by the Dpp antagonist Sog (Chordin). Experiments using dominant-negative forms of Sox2 in animal cap ectoderm have shown that Sox2 is required for the maintenance rather than the initial induction of neural tissue (Kishi, 2000). This is in agreement with the observations that loss of SoxN does not alter the early expression of Brk, Sog and Dpp and thus does not seem to promote neurogenesis through the determination of the ventrolateral region in the blastoderm embryo. Despite indications for a role for vertebrate Sox genes in neural differentiation, Sox gene mode of action remains unclear since neither target genes nor CNS interaction partners have been identified. Observations that SoxN genetically interacts with vnd and ind suggest the vertebrate homologs of Vnd [Nkx2.2 family] and Ind [Gsh1/2] are potential CNS partners for Sox1, Sox2 and Sox3. Like Vnd and Ind, Nkx2.2 and Gsh1 are expressed in developing neural tissue and govern aspects of regional specification. Further studies will demonstrate whether Sox gene function represents a neuralizing pathway that is conserved across species (Buescher, 2002).

GENE STRUCTURE

cDNA clone length - 1914

Bases in 5' UTR - 185

Exons - 1

Bases in 3' UTR - 580

PROTEIN STRUCTURE

Amino Acids - 382

Structural Domains

Proteins of the Sox family are transcriptional regulatorsthat all contain a conserved motif, the HMG box, with morethan 50% amino acid identity with that of the sex determining protein SRY. The Sox HMG domain is responsible forsequence specific DNA binding, and bending of the targetDNA. These proteins appear to have important roles during development, like sex determination, CNS differentiation, cartilage formation or lens formation. SoxN contains an HMG box with more than 90% aminoacid identity and 95% similarity to the human group B SOX1/2/3 proteins. This proteincontains poyalanine stretches, as in mammalian Sox1/3 andchicken Sox21 proteins and in other transcriptional modulators, and a short sequence immediately C-terminal to the HMG domain, that fits relativelywell with a consensus sequence found at the same positionin group B Sox proteins. The gene is intronless, as observed for most of the Sox genes, and maps at position 29F1-F2 on chromosome 2L (Cremazy, 2000).

EVOLUTIONARY HOMOLOGS

See Dichaete for information about SoxNeuro evolutionary homologs.

date revised: 15 September 2002

Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.