InteractiveFly: GeneBrief

SoxNeuro: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

Gene name - SoxNeuro

Synonyms -

Cytological map position - 29E6

Function - transcription factor

Keywords - CNS, neuroblasts, pro-neural genes

Symbol - SoxN

FlyBase ID: FBgn0029123

Genetic map position -

Classification - HMG1/2 (high mobility group) box

Cellular location - nuclear

NCBI link: Entrez Gene

SoxN orthologs: Biolitmine
Recent literature
Carl, S. H. and Russell, S. (2015). Common binding by redundant group B Sox proteins is evolutionarily conserved in Drosophila. BMC Genomics 16: 292. PubMed ID: 25887553
Group B Sox proteins are a highly conserved group of transcription factors that act extensively to coordinate nervous system development in higher metazoans while showing both co-expression and functional redundancy across a broad group of taxa. In Drosophila melanogaster, the two group B Sox proteins Dichaete and SoxNeuro show widespread common binding across the genome. While some instances of functional compensation have been observed in Drosophila, the function of common binding and the extent of its evolutionary conservation is not known. This study used DamID-seq to examine the genome-wide binding patterns of Dichaete and SoxNeuro in four species of Drosophila. Through a quantitative comparison of Dichaete binding, the rate of binding site turnover was evaluated across the genome as well as at specific functional sites. The presence of Sox motifs was examined within binding intervals, along with the correlation between sequence conservation and binding conservation. To determine whether common binding between Dichaete and SoxNeuro is conserved, a detailed analysis was performed of the binding patterns of both factors in two species. This study found that, while the regulatory networks driven by Dichaete and SoxNeuro are largely conserved across the drosophilids studied, binding site turnover is widespread and correlated with phylogenetic distance. Nonetheless, binding is preferentially conserved at known cis-regulatory modules and core, independently verified binding sites. The strongest binding conservation was observed at sites that are commonly bound by Dichaete and SoxNeuro, suggesting that these sites are functionally important. This analysis provides insights into the evolution of group B Sox function, highlighting the specific conservation of shared binding sites and suggesting alternative sources of neofunctionalisation between paralogous family members.
Rizzo, N. P. and Bejsovec, A. (2017). SoxNeuro and shavenbaby act cooperatively to shape denticles in the embryonic epidermis of Drosophila. Development [Epub ahead of print]. PubMed ID: 28506986
During development, extracellular signals are integrated by cells to induce the transcriptional circuitry that controls morphogenesis. In the fly epidermis, Wingless (Wg)/Wnt signaling directs cells to produce either a distinctly-shaped denticle or no denticle, resulting in a segmental pattern of denticle belts separated by smooth, or 'naked', cuticle. Naked cuticle results from Wg repression of shavenbaby (svb), which encodes a transcription factor required for denticle construction. This study has discovered that although the svb promoter responds differentially to altered Wg levels, Svb alone cannot produce the morphological diversity of denticles found in wild-type belts. Instead, a second Wg-responsive transcription factor, SoxNeuro (SoxN), cooperates with Svb to shape the denticles. Co-expressing ectopic SoxN with svb rescued diverse denticle morphologies. Conversely, removing SoxN activity eliminated the residual denticles found in svb mutant embryos. Furthermore, several known Svb target genes are also activated by SoxN, and two novel target genes of SoxN were discovered that are expressed in denticle-producing cells and that are regulated independently of Svb. Thus it is concluded that proper denticle morphogenesis requires transcriptional regulation by both SoxN and Svb.
Schilling, T., Ali, A. H., Leonhardt, A., Borst, A. and Pujol-Marti, J. (2019). Transcriptional control of morphological properties of direction-selective T4/T5 neurons in Drosophila. Development. PubMed ID: 30642835
In the Drosophila visual system, T4/T5 neurons represent the first stage in which the direction of visual motion is computed. T4 and T5 neurons exist in four subtypes, each responding to motion in one of the four cardinal directions and projecting axons into one of the four lobula plate layers. However, all T4/T5 neurons share properties essential for sensing motion. How T4/T5 neurons acquire their properties during development is poorly understood. This study reveals that SoxN and Sox102F transcription factors control the acquisition of properties common to all T4/T5 neuron subtypes, i.e. the layer specificity of dendrites and axons. Accordingly, adult flies are motion blind after disrupting SoxN or Sox102F in maturing T4/T5 neurons. It was further foud that the transcription factors Ato and Dac are redundantly required in T4/T5 neuron progenitors for SoxN and Sox102F expression in T4/T5 neurons, linking the transcriptional programs specifying progenitor identity to those regulating the acquisition of morphological properties in neurons. This work will help to link structure, function and development in a neuronal type performing a computation conserved across vertebrate and invertebrate visual systems.

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 Sox3 proteins, 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 neural hypoplasia 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 the dorsoventral 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).

Drosophila neuroblast selection is gated by Notch, Snail, SoxB, and EMT gene interplay

In the developing Drosophila central nervous system (CNS), neural progenitor (neuroblast [NB]) selection is gated by lateral inhibition, controlled by Notch signaling and proneural genes. However, proneural mutants still generate many NBs, indicating the existence of additional proneural genes. Moreover, recent studies reveal involvement of key epithelial-mesenchymal transition (EMT) genes in NB selection, but the regulatory interplay between Notch signaling and the EMT machinery is unclear. This study finds that SoxNeuro (SoxB family) and worniu (Snail family) are integrated with the Notch pathway, and constitute the missing proneural genes. Notch signaling, the proneural, SoxNeuro, and worniu genes regulate key EMT genes to orchestrate the NB selection process. Hence, this study has uncovered an expanded lateral inhibition network for NB selection and demonstrate its link to key players in the EMT machinery. The evolutionary conservation of the genes involved suggests that the Notch-SoxB-Snail-EMT network may control neural progenitor selection in many other systems (Arefin, 2019).

Based upon previous studies, and the findings in this study, SoxN is necessary for NB generation. Regarding the Snail family, previous studies did not find apparent reductions of NBs numbers in sna, esg, and wor triple mutants. However, these studies focused on early stages of neurogenesis and may not have covered the complete span of NB formation. Analyzing embryos at Stage 11, after most, if not all, NBs have formed, this study found that wor mutants simultaneously removing one gene copy of sna and esg do indeed display a significant reduction in NB numbers. In addition, while AS-C and wor mutants both show a partial loss of NBs, this study found that AS-C;wor double mutants display significantly more severe loss than either single mutant alone. Moreover, misexpression of SoxN or wor, from optimized transgenes, reveals sufficiency for these genes in generating ectopic NBs in the ectoderm. Strikingly, SoxN/ wor co-misexpression can generate extensive numbers of ectopic NBs even in a genetic background lacking any AS-C proneural gene activity. Finally, both SoxN and wor are regulated by, and regulate, the Notch pathway. Based upon these findings, it is proposed that SoxN and wor constitute the missing proneural genes (Arefin, 2019).

What are the connections between the Notch pathway, SoxN, and wor? Logically, in NICD and m8 misexpression, which result in fewer NBs, a reduction was observed in SoxN and wor expression. However, counterintuitively, Notch and neur mutants, which display more NBs, also displayed reductions of SoxN and wor expression. One reason for this effect may pertain to the Notch signaling being important for Crb expression, and that crb is important for both SoxN and wor expression. The reciprocal connection, i.e., between SoxN/wor and the Notch pathway, as measured by m8-GFP expression, is also partly dichotomous. Specifically, SoxN and wor mutants display fewer NBs, a NotchON effect, but reduced m8-GFP expression, a NotchOFF effect. Similarly, misexpressions of SoxND and worD trigger more NBs, a NotchOFF effect, but increased m8-GFP expression, a NotchON effect. However, these findings mirror the activation of E(spl) genes by the proneural genes and point to a balancing act between the proneural, SoxN, and wor genes, as well as the Notch pathway (Arefin, 2019).

Previous studies found that the proneural genes, i.e., ac, sc, and l'sc, are upstream of wor. In line with this, in AS-C mutants loss of wor cells (NBs) and reduction of wor expression levels were observed in the NBs still generated. Similarly, it was previously found that ase misexpression increased wor expression in NBs. Previous studies revealed that SoxN mutants show reduced Ase expression in NBs, and that misexpression of SoxN could activate wor expression, whereas neither wor nor ase GoF or LoF affected SoxN expression in NBs (Bahrampour et al., 2017). In addition, SoxN mutants were found to display loss of ac, l'sc, and wor expression. This indicates that SoxN, which is expressed in the entire early neuroectoderm, acts upstream of the proneural genes, while proneural genes act upstream of wor. However, this SoxN->proneural->wor regulatory flow is complex: while both SoxN and wor misexpression can trigger NB generation, l'sc does not have this potency. Moreover, while SoxN expression may occur first, both AS-C and wor appear to be important for maintained and elevated SoxN expression in NBs. Hence, SoxN, AS-C, and wor appear to be involved in a mutually reinforcing interplay, which ensures robust NB selection once the Notch pathway balanced is tipped (Arefin, 2019).

DNA-binding studies for the factors studied herein, and analysis of related family members (D, Sna, esg, and Ase), suggest that the elaborate transcriptional interplay between all of the aforementioned TDs/co-factors, i.e., NICD/Su(H)/Mam, E(spl), proneural, SoxN, wor, and their respective genes, may result from direct transcriptional regulation (Arefin, 2019).

In Drosophila, crb and sdt control the epithelial polarity in a number of tissues. Recent studies revealed that Crb stabilizes Notch, and accordingly crb mutants show more NBs. This study furthermore found that crb overexpression results in fewer NBs and, interestingly, that crb overexpression can partly rescue Notch mutants. The reduction of NB numbers in crb mutants is logically accompanied by reduced m8-GFP expression, while, surprisingly, crb misexpression also triggered reduced m8-GFP expression. Notch signaling was found to activate Crb expression, evident by downregulation of Crb in Notch and neur mutants, and upregulation of Crb in NICD and m8 misexpression. Hence, with the exception of crb misexpression on m8-GFP, a clear-cut interplay between canonical Notch signaling and crb/Crb emerges. However, this interplay would constitute a runaway loop, with Notch activating crb, and Crb supporting Notch activation. Perhaps the finding that crb overexpression reduces m8-GFP points to a nebulous brake pedal in this loop (Arefin, 2019).

Regarding the connection between crb with SoxN, wor, and the proneural genes, clear interplay, albeit with a reverse logic was found for mutants versus misexpression. Specifically, misexpression of SoxN, wor, or proneural genes, which generates more NBs, with encompassing delamination, also results in reduced Crb expression. However, surprisingly, SoxN, wor, and proneural mutants, which display fewer NBs, also show reduced Crb expression, underscoring the balancing act of these gene regulations. It is envisioned that this may reflect a role for SoxN, wor, and AS-C in the proneural clusters (prepattern), where they may act to ensure Crb expression in the equivalence regions, thereby ensuring an efficient lateral inhibition process (Arefin, 2019).

In addition, further complexity regarding the role of crb stems from recent findings revealing that Neur, an E3 ligase critical for Dl endocytosis, also controls the stability of Sdt, and thereby affects Crb protein levels. It is tempting to speculate that NotchOFF cells (NBs), which maintain proneural gene expression and hence activate neur expression, will have increased Neur, and hence increased endocytosis of Sdt, and thereby decreased Crb levels, leading to reduced Notch receptor activation. Because Neur also increases endocytosis of Dl, high Neur levels would help drive Notch activation in the neighboring cells (epidermal cells). Moreover, since Notch (NICD and m8CK2) activates Crb expression and represses neur expression, this should ensure more Crb in the NotchON cells (epidermal cells), thereby further supporting Notch activation. By these mechanisms, the transcriptional regulation of crb/neur gene expression and the stability/endocytosis control of Crb/Sdt/Dl levels and localization, and thereby Notch activation, act as a hitherto undiscovered loop providing additional thrust to the lateral inhibition decision (Arefin, 2019).

Similar to the TF interplay described above, the gene-specific and/or genome-wide DNA-binding studies indicate that the gene regulation of crb and neur may be mediated by direct transcriptional regulation of the Notch pathway TFs (NICD/Su(H)/Mam, E(spl), proneural), as well as the SoxB and Snail family TFs (Arefin, 2019).

EMT has been extensively studied in mammals and has revealed roles for the Crb, Scribble (Scrib), and Par complexes, as well as for Notch signaling and the Snail and SoxB families. Previous studies, and the current findings, demonstrate that the majority of these genes also play key roles during Drosophila NB selection and delamination. This supports the notion that NB selection and delamination could be viewed as an EMT-like process. However, the NB-type EMT differs from canonical EMT in several aspects. In canonical EMT, all apical polarity complexes (Crb, Par, and Scrib complexes) and Cadherins are turned off, and there is no activation of asymmetry genes. Hence, delamination is followed by symmetric cell division and cell migration. In contrast, in 'NB-type EMT,' while, similarly, the Crb complex and Cadherins are turned off, the Par complex (baz/par-3, par-6, and aPKC) and the Scribble complex (scrib, dlg, and lgl) remain expressed, and asymmetric genes, e.g., mira, insc, and pros, are turned on. In addition, within NBs, SoxN, wor, and ase activate key cell-cycle driver genes, i.e., Cyclin E and stg, and repress expression of the cell-cycle inhibitor dacapo. These gene expression changes result in NB delamination, but retain apico-basal polarity in the NB, and ensure repetitive rounds of asymmetric cell divisions, generating the unique features of CNS lineages (Arefin, 2019).

Based upon these findings and those previously published, a model emerges wherein SoxB acts early to govern neuroectodermal competence, intersecting with the early transient wave of proneural gene expression in the proneural clusters. SoxN and proneural genes engage in interplay with the Notch-mediated lateral inhibition process, which is also gated by Crb-Sdt-Neur membrane-localized control of Notch receptor activity and Dl ligand endocytosis. The outcome of these interactions is that early NBs become NotchOFF and elevate their SoxN and proneural expressions, as well as activate wor and Ase expression. This results in the downregulation of a subset of EMT genes (i.e., Crb complex and Cadherins), while the Scrib and Par complexes are maintained. The combined action of SoxN, proneural, wor, and Ase triggers activation of asymmetric cell division genes and cell-cycle driver genes, the outcome of which is NB delamination, followed by asymmetric cell divisions and lineage generation. In contrast, the surrounding NotchON cells continue expressing E(spl) genes, downregulating the SoxN, proneural, neur, and Dl genes. This results in the continued expression of the Crb, Scrib, and Par complexes, as well as failure to activate wor, ase, asymmetric, and cell-cycle genes. The combined effect of these regulatory decisions is that these cells remain in the ectoderm and do not divide (Arefin, 2019).

The process of NB selection bears many similarities to the process of peripheral SOP selection. However, while one Snail family gene, esg, is indeed important also for PNS precursor development, there is no study linking sna or wor, nor the SoxB genes SoxN and D, to SOP selection. Hence, while both SOP and NB formation requires AS-C and esg, NB formation additionally requires Sna, wor, SoxN, and Dichaete. It is tempting to speculate that this may relate to two clear differences between SOPs and NBs: EMT and proliferation. Specifically, while NBs undergo a complete EMT-like process, SOPs remain associated with the ectoderm. Moreover, NBs can divide up to 20 times, making 40-cell lineages, while most, if not all, SOPs make 5-cell lineages. The connections between the SoxB and Snail families with NB and GMC proliferation and the EMT pathway (this paper as well as Bahrampour et al., 2017, 2019) suggest that both of these NB-specific properties are driven by the expanded TF code specific to NBs (Arefin, 2019).

In mammals, the neuroepithelial-to-radial glia cell (NE-RGC) transition is in many aspects analogous to the NB selection and delamination process in Drosophila. Intriguingly, recent studies suggest that NE-RGC can perhaps also be viewed as an EMT-like process, although in this case the process has been modified even further, and the RGC retains an apical connection throughout neurogenesis and undergoes interkinetic nuclear migrations. Strikingly, in two recent studies the NE-RGC transition was found to involve the mouse Snail and Scratch factors, both of which are members of the Snail family. Other players in the NB-selection program outlined above also play key roles in the early development of the mammalian CNS and in the NE-RGC transition, although the direct comparison of gene function and cell behavior becomes nebulous. Hence, it would appear that the neuroectoderm->neural progenitor selection and delamination process has undergone several evolutionary modifications, perhaps becoming less and less akin to a canonical EMT process in more derived animals. Nevertheless, it is tempting to speculate that several of the basic principles of EMT are utilized in the mammalian NE-RGC process and that viewing it as such may be helpful for future studies (Arefin, 2019).


Transcriptional Regulation

Determining the position of ventro-lateral neuroectoderm versus dorsal non neural ectoderm is controlled by maternal (dorsal) and zygotic genes (dpp, sog, brk, sna, twi). SoxN expression is specifically affected in these mutants. dl mutants lack early SoxN expression. Embryos mutants for dpp show a dorsal expansion of SoxN expression, as also observed when misexpressing sog by means of the Gal4 system. Inversely, misexpressing dpp early in embryogenesis leads to severe reduction of SoxN expressing-cells, as observed in sog mutants and sog, brk double mutants. Finally, twi mutants are characterized by a ventral expansion of SoxN expression into the presumptive mesoderm. These experiments are consistent with a role for the D/V patterning genes in the control of SoxN expression, with SoxN being negatively regulated dorsally and ventrally by dpp and mesoderm genes, and positively by sog and brk in the neuroectodermal region. A similar situation has been reported in Xenopus, with SoxD, an essential mediator of neural induction, being negatively regulated by BMP4 and positively by chordin (the vertebrate homologs of dpp and sog, respectively) (Cremazy, 2000).

Targets of Activity

The differential loss of NBs and their progeny in the DV axis in SoxN mutants may result from the failure of neuroectodermal cells to be specified to a neural fate. Since SoxN is expressed throughout the neuroectoderm prior to neuroblast delamination and Dichaete is reported to have effects on proneural gene expression (Zhao, 2002), proneural gene expression was examined in SoxNU6–35 mutants. In SoxNU6–35 a loss of lateral column achaete (ac) expression is seen as well as an overall reduction in ac levels. When the double mutants are examined for ac expression, a synergistic and an additive effect is seen. As with SoxN, the overall level of ac expression is lower compared with heterozygous siblings and there is a marked loss of lateral column ac expression. In addition, the double mutants display the Dichaete phenotype since ectopic intermediate column expression is seen in some rows (6%). However, in 21% of segments no ac expression is seen, suggesting that both Sox genes are principally able to positively regulate ac expression. Taken together, it is concluded that in the neuroectoderm, the elimination of group B Sox expression results in an early failure in neural specification and subsequent loss of neural progenitors (Overton, 2002).

ac is a marker for certain medial and lateral proneural clusters but is not normally expressed in the intermediate column. A striking reduction was observed in the SoxN mutant in the number of lateral column proneural clusters expressing ac. In 70% of these clusters, ac expression is no longer detected, compared with 12% of medial column proneural clusters. The loss of lateral column ac expression suggests that SoxN functions early in the neuroectoderm to specify proneural clusters correctly. In addition to this, there is an overall reduction in ac expression levels in the medial proneural clusters compared with the heterozygous sibling embryos stained in the same reaction. This implies that SoxN is required more generally in the neuroectoderm to establish the appropriate level of ac expression. The expression was examined of the related proneural gene l'sc in the neuroectoderm prior to neuroblast delamination and, in contrast to the results with ac, no appreciable effect was seen. Thus it appears that SoxN is selectively required in the neuroectoderm for the regulation of some proneural gene expression. The loss of ac expression in lateral proneural clusters partly explains why such a dramatic loss of lateral NBs is seen in SoxNU6–35 mutants. However, all lateral NBs are strongly affected in SoxNU6–35 embryos, including those that express l'sc and not ac. Hence, the normal expression of l'sc in SoxNU6–35 mutant embryos argues against a simple linear pathway in which SoxN acts only upstream of proneural genes, and suggests a mechanism in which SoxN functions both upstream and in parallel to the proneural genes to promote neuroblast formation. This parallel function of SoxN is additionally supported by the observation that the severe hypoplasia of SoxN mutant embryos resembles the phenotype in AS-C mutants, and is more severe than can be accounted for by the effect on ac expression, as loss of ac alone does not produce severe phenotypes (Overton, 2002).

A derepression of ac expression in the intermediate column has been reported in Dichaete mutants has been reported (Zhao, 2002) and those observations were confirmed. Therefore, whereas both SoxN and Dichaete mutants show loss of neuroblasts, the effect in the neuroectoderm differs: SoxN mutants display loss of ac expression but Dichaete mutants show some ac derepression (Overton, 2002).

Loss of SoxN results in a severe loss of NBs. Expression studies show that SoxN protein is present in the NE before and during the entire process of neurogenesis. Hence, the expression pattern provides no clue as to which step(s) depend on SoxN function. To approach this question, two key steps in neurogenesis were studied: (1) the establishment cell clusters with neural potential and (2) the 'singling out' of NBs (Buescher, 2002).

The proneural genes of the AS-C have been shown to be essential for the promotion of NB formation and deletion of the entire gene complex results in the loss of ~75% of all NBs. Many NBs that normally derive from clusters of neuroectodermal cells, which express either ac, sc, l'sc or a combination of these genes, fail to form in SoxN mutant embryos. This raises the question of whether proneural genes are still expressed in a SoxN mutant background in clusters of ectodermal cells, and, if so, do they still confer neural potential to these cells? In wild-type embryos, prior to NB segregation (stage 8), Ac protein is found in cell clusters in rows 3 and 7 in the ventral and lateral column of the NE, while L'sc is found in stripes of two to three cell widths that transverse the entire NE. Staining of stage 8 SoxN mutant embryos with anti-L'sc antibody revealed no appreciable difference from wild-type L'sc expression. Staining with anti-Ac antibody showed that Ac expression is initiated in both ventral and lateral clusters, but expression levels appear reduced and show significant variation in lateral cell clusters (Buescher, 2002).

In wild-type embryos, the process of lateral inhibition results in the singling out of one cell per proneural cluster that will enter the neural pathway. This process is accompanied by an upregulation of proneural gene expression, delamination of the NB from the neuroectodermal layer and the initiation of expression of a set of neuronal precursor genes. In stage 9 SoxN mutant embryos, a failure in the upregulation of Ac expression was frequently observed in lateral proneural clusters. In those instances in which Ac was still upregulated, expression was less robust than in wild type and varied significantly among different hemisegments. Variation of Ac expression levels was also apparent in ventrally delaminating cells. The failure to upregulate Ac expression was accompanied by a failure in cell delamination. Moreover, the expression of neuronal precursor genes was severely affected: in wild type, one of the earliest precursor genes to be expressed is asense (ase); ase is expressed in all delaminating NBs. In SoxN mutant embryos, ase expression was strongly reduced. These results suggest that in SoxN mutant embryos the establishment of proneural clusters is impaired but not abolished. The subsequent process of singling out NBs is severely defective (Buescher, 2002).

The Drosophila HMG-domain proteins SoxNeuro and Dichaete direct trichome formation via the activation of shavenbaby and the restriction of Wingless pathway activity

Trichomes are cytoplasmic extrusions of epidermal cells. The molecular mechanisms that govern the differentiation of trichome-producing cells are conserved across species as distantly related as mice and flies. Several signaling pathways converge onto the regulation of a conserved target gene, shavenbaby (svb, ovo), which, in turn, stimulates trichome formation. The Drosophila ventral epidermis consists of the segmental alternation of two cell types that produce either naked cuticle or trichomes called denticles. The binary choice to produce naked cuticle or denticles is affected by the transcriptional regulation of svb, which is sufficient to cell-autonomously direct denticle formation. The expression of svb is regulated by the opposing gradients of two signaling molecules - the epidermal growth factor receptor (Egfr) ligand Spitz (Spi), which activates svb expression, and Wingless (Wg), which represses it. It has remained unclear how these opposing signals are integrated to establish a distinct domain of svb expression. This study shows that the expression of the high mobility group (HMG)-domain protein SoxNeuro (SoxN) is activated by Spi, and repressed by Wg, signaling. SoxN is necessary and sufficient to cell-autonomously direct the expression of svb. The closely related protein Dichaete is co-regulated with SoxN and has a partially redundant function in the activation of svb expression. In addition, SoxN and Dichaete function upstream of Wg and antagonize Wg pathway activity. This suggests that the expression of svb in a discreet domain is resolved at the level of SoxN and Dichaete (Overton, 2007).

In the embryonic ventral epidermis of Drosophila, two alternative cell fates are specified: smooth cells and trichome-producing cells. These binary cell fates are distinguished by the expression of svb, the most-downstream effector of epidermal morphogenesis. svb is necessary and sufficient to cell-autonomously direct trichome formation. The expression of svb is regulated by the opposing gradients of two signaling molecules: Spi, which activates, and Wg, which represses, svb expression. svb is expressed in segmentally reiterated, epidermal stripes, which invariantly encompass six rows of cells. This raises the question of how is opposing extrinsic information integrated to establish a distinct domain of svb expression with a sharp posterior border (Overton, 2007)?

This study demonstrates that the HMG-domain proteins SoxN and Dichaete represent a molecular link between the expression of svb and the upstream Der- and Wg-signaling cascades. SoxN and Dichaete are expressed in the ventral epidermis at the time when epidermal cell fates are specified. The late phase of SoxN and Dichaete expression is stimulated by Der- and repressed by Wg-pathway activity. These regulatory mechanisms result in the expression of SoxN and Dichaete in those six rows of cells within each abdominal segment that differentiate to produce trichomes. SoxN and, to a lesser extent, Dichaete, are necessary and sufficient to activate the expression of svb. Furthermore, these results show that the well-described repression of svb by Wg is due to the repression of SoxN, which, in turn, results in the loss of svb activation. Likewise, the Spi-mediated activation of svb expression relies on the activation of SoxN, which, in turn, activates svb. This indicates that the competition of Der- and Wg-pathway activities for the specification of trichome-producing versus smooth cell fates is resolved at the level of SoxN and Dichaete (Overton, 2007).

These results do not provide much insight into the issue of how opposing extrinsic information is integrated such that a sharp posterior border of svb expression is achieved. Instead, they raise the question of how is a sharp posterior border of SoxN and Dichaete expression established/maintained? The findings suggest that this is achieved by a combination of negative- and positive-feedback loops. (1) Evidence is provided that SoxN and Dichaete negatively regulate Wg pathway activity. This negative-feedback loop provides a likely mechanism for the establishment and maintenance of a sharp posterior border of SoxN and Dichaete expression. The issue arises of how robust this system might be in the face of fluctuating levels of Wg pathway activity. The efficiency with which SoxN and Dichaete restrict Wg pathway activity will crucially rely on the levels of SoxN and Dichaete protein. In this context, it is noteworthy that the levels of SoxN protein, but not Dichaete, are several-fold higher in the two posterior-most rows of the SoxN stripe compared with the anterior four rows. The regulatory mechanisms that underlie the different levels of SoxN expression are currently unclear. (2) Evidence is provided that the maintenance of SoxN and Dichaete expression is supported by a positive-feedback loop: svb, the expression of which is activated by SoxN and Dichaete, is itself required for the maintenance of SoxN and Dichaete expression. Together, these mechanisms contribute to an invariant read-out of cell identity from opposing Der- and Wg-pathway activities (Overton, 2007).

In Drosophila, SoxN and Dichaete are necessary and sufficient to activate the expression of svb, which in turn directly regulates the expression of genes involved in trichome morphogenesis. Is a function in hair formation of the Sox proteins conserved in other species, including vertebrates? A previous study has shown that the mouse Sox9 protein is required for the differentiation of hair-producing epidermal cells and acts genetically downstream of sonic hedgehog pathway activity (Vidal, 2005). This study did not address whether Sox9 regulates the expression of movo1 (Ovol1), the mouse ortholog of svb. Nevertheless, the demonstrated roles of SoxN, Dichaete and Sox9 raise the exciting question of do Sox proteins have an essential function in the activation of an epidermal differentiation program that is conserved across species as distantly related as mice and flies (Overton, 2007).

Protein Interactions

SUMO represses transcriptional activity of the Drosophila SoxNeuro and human Sox3 central nervous system-specific transcription factors

Sry high mobility group (HMG) box (Sox) transcription factors are involved in the development of central nervous system (CNS) in all metazoans. Little is known on the molecular mechanisms that regulate their transcriptional activity. Covalent posttranslational modification by small ubiquitin-like modifier (SUMO) regulates several nuclear events, including the transcriptional activity of transcription factors. This study demonstrates that SoxNeuro, an HMG box-containing transcription factor involved in neuroblast formation in Drosophila, is a substrate for SUMO modification. SUMOylation assays in HeLa cells and Drosophila S2 cells reveal that lysine 439 is the major SUMO acceptor site. The sequence in SoxNeuro targeted for SUMOylation, IKSE, is part of a small inhibitory domain, able to repress in cis the activity of two adjacent transcriptional activation domains. These data show that SUMO modification represses SoxNeuro transcriptional activity in transfected cells. Overexpression in Drosophila embryos of a SoxN form that cannot be targeted for SUMOylation strongly impairs the development of the CNS, suggesting that SUMO modification of SoxN is crucial for regulating its activity in vivo. Finally, evidence is presented that SUMO modification of group B1 Sox factors was conserved during evolution, because Sox3, the human counterpart of SoxN, is also negatively regulated through SUMO modification (Savare, 2005).

This report shows that the SoxN and its human counterpart Sox3, both involved in CNS development, are SUMO modified in vivo. Ootential SUMOylation sites (ψKXE motif) were sought in all mammalian and Drosophila Sox proteins. One or several ψKXE motifs are present in some but not all Sox genes, these motifs being usually conserved within a given subgroup between Drosophila and humans. These include group B1 (H.s Sox1/2/3 and D.m SoxN), group C (H.s Sox11 and D.m SoxC), group D (H.s Sox5/6/13), group E (H.s Sox8/9/10 and D.m Sox100B), group F (H.s Sox17), and group H (H.s Sox30). Recently, Sox9 was shown to be SUMO modified, and SUMO modification was associated with transcriptional repression. In all the other groups (B2, F, and G), no ψKXE motif is present (except Drosophila SoxB2-2, human group C Sox11 and group F Sox17), suggesting that these proteins are not SUMO modified. To confirm this, the same SUMOylation assay was used as described in this report for SoxN and Sox3, and no SUMO modified human Sox7, mouse Sox15 and Drosophila Dichaete (respectively, group F, G, and B) was detected. Thus, based on the data and the presence of ψKXE motif in various Sox, one can postulate that SUMO modification might be used to regulate several Sox group genes (Savare, 2005).

The results show that SUMO modification of the CNS-specific group B1 SoxN and Sox3 proteins was conserved during evolution to regulate their transcriptional capacity. Based on the presence of ψKXE motif in group B1 proteins (SoxN in Drosophila and Sox1/2/3 in humans), and its absence in group B2 (Dichaete in Drosophila and Sox14/21 in humans), it is tempting to speculate that these two subgroups differ in their ability to be regulated by SUMOylation. This is particularly interesting because in Drosophila, SoxN and Dichaete were shown to partially overlap in their expression and function within the neuroectoderm, suggesting that these genes are to some extent functionally redundant in the developing CNS but that there must exist molecular mechanisms responsible for their specificity of action in restricted areas of the CNS (interactions with specific partners? posttranslational modifications?). Furthermore, it has been shown in chick that group B2 Sox14/21 could bind and differentially regulate δ1-crystallin gene regulatory sequences, known to be regulated by group B1 Sox1/2/3 factors in vivo. These observations suggested that target of group B genes might be regulated by the counterbalance of activating and repressing Sox proteins in restricted sites of the developing CNS. In light of these results, SUMOylation might be one of the mechanisms used for this purpose (Savare, 2005).

As shown in this study, substitution of lysine 439 to arginine within SoxN IKSE motif impaired SoxN SUMO modification in both transfected HeLa and S2 cells. SoxN transcriptional activity was dramatically enhanced in three conditions: in the substitution mutant K439R, in the deletion mutants where the IKSE motif was deleted, and when the dominant negative form of Ubc9 was used to interfere with the endogenous SUMO machinery. This correlation between transcriptional repression and the ability of SoxN to be SUMOylated strongly suggests that SUMO conjugation to SoxN results in transcriptional repression. Similar results were obtained for its human counterpart Sox3. Many of the SUMO-modified proteins identified to date are transcription factors, and in most cases, SUMO modification has been associated with transcriptional repression. Nevertheless, the molecular mechanisms underlying this repression are still a matter of debate. In some cases, SUMO modification was associated with the relocalization of the targeted factor to specialized repressive subnuclear structures such as PML bodies. In SoxN and Sox3, data in HeLa and S2 cells suggest that SUMOylation is apparently not associated with major changes in the nuclear localization of these proteins. This was also evident in vivo, because the wild-type and K439R SoxN forms both localized similarly in the nuclei (Savare, 2005).

In both SoxN and Sox3, it was found that the ψKXE motif is targeted for SUMOylation, and constitutes an inhibitory domain able to affect the activity of adjacent TADs. Interestingly, this motif is surrounded by conserved proline residues, reminiscent of the SC synergy domain (consensus P-X0-4-ψKXE-X0-3-P) found in several transcription factors, including SP3, c-myb, C/EBP, and Sox9. Potential SC motifs also are found in other Sox: H.s Sox6, H.s Sox8, and H.s Sox30. SC motif is both necessary and sufficient to limit transcriptional synergy, because its disruption selectively enhances synergistic activation at compound response elements without altering the activity driven from a single site. Thus, SUMOylation of the SC domain is believed to modulate higher order interactions among transcriptional regulators. This motif in Sox proteins might behave as SC domain, because these factors are known to pair off with specific partners to exert full and synergistic activity in a context dependent manner. Because SUMO modification is believed to modulate protein-protein interactions, it will be of interest to examine whether Sox SUMOylation is able to interfere with their ability to interact with their partners (Savare, 2005).

Using transgenic Drosophila lines, strong evidence was obtained that SUMOylation regulates the activity of SoxN in vivo. Indeed, overexpressing the SUMO-deficient K439R SoxN form resulted in strong defects in embryonic CNS. Because the GAL4 driver used for embryonic overexpression is ubiquitous, these results are interpreted as the capacity of the nonSUMOylable form to interfere with endogenous SoxN in the cells were SoxN is expressed (neuroblasts and neurons). In addition, the experiments where the wild-type and K439R SoxN proteins were overexpressed in larvae clearly showed that the two forms display different activity in vivo, further demonstrating the functional relevance of SoxN SUMOylation in vivo. Because the K439R form is a strong transcriptional activator as observed in luciferase assays in transfected cells, it can be postulated that the repressing activity of SoxN is important for the proper development of embryonic CNS. Further work will be required to demonstrate whether SUMOylation regulates SoxN activity in all the different cell types where the protein is expressed (embryonic, larval and adult CNS, larval and adult eyes, and larval leg imaginal discs) (Savare, 2005).



SoxN displays a highly dynamic expression pattern during Drosophila embryogenesis. A head-specific stripe of expression is detected in early syncitial blastoderm. Shortly after, SoxN transcripts are detected laterally in the trunk, to end up in late blastoderm with two lateral stripes of expression in the presumptive ventro-lateral neuroectoderm. During gastrulation, expression is observed in the cephalic and ventral neurogenic regions. From stage 8 onwards, SoxN expression remains associated with the developing CNS. During late embryogenesis, a few cells in the ventral nerve cord express SoxN, but most of the staining is observed in the ventral and lateral epidermis, the chordotonal organs of the PNS and the brain (Cremazy, 2000).

Another related Sox gene, Dichaete, is also expressed in the developing CNS. At gastrulation, SoxN expression domain extends more laterally than that of Dichaete. While SoxN is nuclearly localized in all cells, Dichaete displays both cytoplasmic and nuclear localization, as already observed. Later, waves of forming neuroblats initiate both Dichaete and SoxN expression, or Dichaete only or SoxN only. The two proteins also co-localize in the chordotonal organs of the PNS. In the brain, the proteins are expressed in mostly distinct areas (Cremazy, 2000).

To determine the SoxN protein expression pattern, a polyclonal antibody raised to SoxN protein. Immunostaining with this antibody has shown that RNA and protein expression patterns in the NE are virtually identical. Maintenance of SoxN expression was not observed in delaminating NBs; rather, SoxN protein levels in NBs are low and transient; they may represent a 'carry-over' of neuroectodermally expressed protein. However, a small number of neural progenitor cells in the intermediate region continue to express SoxN and give rise to SoxN-positive progeny. It is noteworthy that anti-SoxN staining in stage 9-11 NE appears patchy, suggesting that protein expression, although ubiquitous, is not uniform (Buescher, 2002).

Dual role for Drosophila lethal of scute in CNS midline precursor formation and dopaminergic neuron and motoneuron cell fate: lethal of scute, tailup and SoxNeuro act together to control midline cell fate

Dopaminergic neurons play important behavioral roles in locomotion, reward and aggression. The Drosophila H-cell is a dopaminergic neuron that resides at the midline of the ventral nerve cord. Both the H-cell and the glutamatergic H-cell sib are the asymmetric progeny of the MP3 midline precursor cell. H-cell sib cell fate is dependent on Notch signaling, whereas H-cell fate is Notch independent. Genetic analysis of genes that could potentially regulate H-cell fate revealed that the lethal of scute [l(1)sc], tailup and SoxNeuro transcription factor genes act together to control H-cell gene expression. The l(1)sc bHLH gene is required for all H-cell-specific gene transcription, whereas tailup acts in parallel to l(1)sc and controls genes involved in dopamine metabolism. SoxNeuro functions downstream of l(1)sc and controls expression of a peptide neurotransmitter receptor gene. The role of l(1)sc may be more widespread, as a l(1)sc mutant shows reductions in gene expression in non-midline dopaminergic neurons. In addition, l(1)sc mutant embryos possess defects in the formation of MP4-6 midline precursor and the median neuroblast stem cell, revealing a proneural role for l(1)sc in midline cells. The Notch-dependent progeny of MP4-6 are the mVUM motoneurons, and these cells also require l(1)sc for mVUM-specific gene expression. Thus, l(1)sc plays an important regulatory role in both neurogenesis and specifying dopaminergic neuron and motoneuron identities (Stagg, 2011).

In insects, dopaminergic neurons are found in both the nerve cord and brain. One of the best-characterized insect dopaminergic neurons is the H-cell (named for its 'H'-like axonal projections), which is present in the CNS midline cells of the nerve cord. The H-cell was first described in grasshopper as one of the two progeny of the Midline Precursor 3 (MP3) cell, and shown in the moth Manduca sexta to be dopaminergic. The H-cell midline interneuron is also present in Drosophila, and similar to other dopaminergic neurons expresses a set of genes encoding dopamine biosynthetic enzymes, including pale (ple; which encodes tyrosine hydroxylase) and dopa decarboxylase (Ddc). The H-cell also expresses a vesicular monoamine transporter (Vmat), dopamine membrane transporter (DAT) and neurotransmitter receptors that receive input for serotonin (5-HT1A), glutamate (Glu-RI) and neuropeptide F (NPFR1). This characteristic pattern of gene expression and its 'H' axonal projection, to a large degree, constitute the unique character of the H-cell (Stagg, 2011).

Recent work has provided insight into the origins of midline neurons and glia (see Formation of midline precursors (MPs) and MP neurons in Drosophila). Around the time of gastrulation, the single-minded midline master regulatory gene activates the midline developmental program, and soon after 3 MP equivalence groups (MP1, MP3, MP4) of five or six cells/each form. Notch signaling selects one cell from the MP1 group to become an MP1 and the others become midline glia (MG). The same occurs for the MP3 group, with one cell becoming an MP3 and the others MG. Development of the MP4 group is more complex, with sequential Notch-dependent formation of MP4 followed by MP5, MP6 and the median neuroblast (MNB). Each MP undergoes a single division that leads to two neurons. For MP3-6, this involves binary cell fate decisions: MP3 gives rise to the dopaminergic H-cell and glutamatergic H-cell sib interneurons, and MP4-6 each gives rise to a GABAergic iVUM interneuron and glutamatergic/octopaminergic mVUM motoneuron pair. The differences in MP3-6 neuron cell fate are due to the asymmetric localization of the Numb protein, which is high in H-cell and mVUMs, but low in H-cell sib and iVUMs, and differential Sanpodo localization. Although Notch signaling directs H-cell sib and iVUMs to their fates, it is blocked in H-cell and mVUMs due to the presence of Numb. Thus, H-cell sib and iVUM cell fate and gene expression are dependent on Notch signaling, and a different regulatory program governs H-cell and mVUM fates (Stagg, 2011).

This paper asks the question: what regulatory proteins govern Notch-independent H-cell and mVUM fate and gene expression? Also addressed is how the two types of midline precursors, MPs and MNB, form. Proneural genes of the bHLH transcription family have been implicated in controlling neural precursor formation and neuron-specific transcription in both vertebrates and invertebrates. The Drosophila bHLH proneural genes, achaete (ac), scute (sc), lethal of scute [l(1)sc] and atonal have been implicated in the formation of either sensory cell or CNS neuroblast precursors. Proneural bHLH genes can also direct the formation of specific neuronal cell types, as exemplified by studies in the vertebrate spinal cord. Neuronal cell type specification is commonly due to the combinatorial action of proneural and homeodomain-containing proteins. This study demonstrates that three transcription factors: the L(1)sc bHLH protein, Tailup (Tup; Islet) Lim-homeodomain protein and the Sox family protein SoxNeuro (SoxN), work together to control overlapping aspects of H-cell gene expression. In addition, l(1)sc regulates mVUM motoneuron gene expression. All three proneural members of the Drosophila achaete-scute complex (AS-C) [ac, l(1)sc and sc] are expressed in MPs in distinct patterns, and l(1)sc is required for the formation of MP4-6 and the MNB. Thus, l(1)sc controls both midline precursor formation and, in combination with SoxN and tup, controls H-cell-specific gene expression and cell fate. Both the l(1)sc and tup genes may also function together more broadly and control non-midline dopaminergic neuron gene expression (Stagg, 2011).

The formation of midline neural precursors (five MPs and the MNB) is a dynamic, yet stereotyped process. The MPs undergo cellular changes in which their nuclei delaminate from an apical position within the ectoderm and move to the basal (internal) surface. There they divide after orienting their spindles. The precursors arise in a distinct order: MP4/MP3>MP5>MP1>MP6>MNB (Wheeler, 2008). The l(1)sc gene is required for the formation of the MP4-6 and MNB precursors and their neuronal progeny. Since MP4 could not be definitively distinguished from MP5 in Df(1)sc-B57, there is some uncertainty whether both cell types are regulated by l(1)sc. However, as most segments only possess two VUMs, and those are VUM6s in over 60% of segments, it is likely that both MP4 and MP5 are commonly affected in Df(1)sc-B57, in addition to MP6. The ac and sc genes are both expressed in MPs and MNB, yet do not appear to play a significant role in MP and MNB formation. Although l(1)sc is the major proneural gene that controls formation of embryonic neuroblasts, relatively little is known about how it functions and the identity of relevant target genes. In one study, it was shown that morphological changes that accompany neuroblast formation were dependent on l(1)sc function. This is likely to be the case for l(1)sc and MP4-6 and MNB development, as MP4-6 and MNB delamination or division was commonly absent in Df(1)sc-B57. One key question is what activates or maintains l(1)sc expression in MP3-6 and MNB? Signaling by hedgehog (hh) is likely to be important, as no midline l(1)sc expression is present in hh mutant embryos (Stagg, 2011).

Although all MPs and MNB express l(1)sc, only MP4-6 and MNB were affected in mutants - formation of MP1 and MP3 were unaffected. These differences are unlikely to be solely due to different levels of L(1)sc protein or to a combination of Ac, L(1)sc and Sc. L(1)sc protein levels are relatively constant among all five MPs and MNB, both L(1)sc and Sc are present in all MPs and MNB, and Ac, L(1)sc and Sc are present in MP1 as well as MP5,6 and MNB, yet no defects in MP1 and MP3 delamination or cell division were observed. Instead, the ability of l(1)sc to direct development of some MPs and not others may reflect the different cell states (and distinct co-factors) of the precursor populations from which each MP arises. Similarly, l(1)sc controls expression of different genes in the H-cell compared with mVUMs, probably based on their different origins (MP3 versus MP4-6). Variability in the genetic control of midline MP formation extends to the non-midline MP2 cells. The MP2s require both ac and sc for MP formation and differentiation, whereas l(1)sc does not play a role. Thus, MP2 and midline MPs (MP4-6) each require AS-C gene activity for proneural and differentiation functions, but use different AS-C family members (Stagg, 2011).

At least two distinct genetic programs control H-cell gene expression: (1) H-cell-specific gene expression is controlled by l(sc), tup and SoxN, and (2) unknown factors control gene expression that is present in both the H-cell and H-cell sib. All H-cell-specific gene expression requires l(1)sc function. tup acts in parallel to control important aspects of H-cell gene expression, including the DAT, ddc and ple genes. SoxN acts downstream of l(1)sc to control NPFR1 expression. H-cell neural function gene expression begins at stage 13, well after l(1)sc expression is absent, indicating that l(1)sc is unlikely to directly regulate these genes. However, Tup is present after stage 13 and could directly regulate DAT, ddc and ple; SoxN is also present and could directly regulate NPFR1. The l(1)sc gene regulates mVUM gene expression in a manner similar to its control of H-cell expression, but does so independently of tup, which is not expressed in mVUMs. It is noted that L(1)sc protein is present at higher levels in H-cell than mVUMs, although the significance of this is unclear. Expression of genes common to both H-cell and H-cell sib cells, including 5-HT1A, Glu-RI and tup, were not affected in l(1)sc or Notch pathway mutants, indicating a second distinct regulatory pathway. This was also observed for genes expressed in common between mVUMs and iVUMs (Stagg, 2011).

The relationship between l(1)sc and tup in controlling H-cell-specific gene expression is complex. Both genes are initially expressed in the H-cell and H-cell sib after MP3 division, but expression of both is soon restricted to the H-cell. Misexpression of l(1)sc resulted in the ectopic expression of tup in the H-cell sib, similar to other H-cell-specific genes. However, in l(1)sc mutants, tup expression was not absent in the H-cell, but instead tup expression remained present in the H-cell and sometimes in two cells: one was the H-cell and the other was (probably) the H-cell sib. In addition, l(1)sc expression was not affected in tup mutants. These results indicated that: (1) l(1)sc and tup act in parallel in the H-cell to regulate dopaminergic pathway gene transcription; and (2) l(1)sc downregulates tup in the H-cell sib, indicating a role for l(1)sc in H-cell sib development. The best marker for the H-cell sib is CG13565, although it is expressed in wild type in only 54% of segments. In Df(1)sc-B57 mutant embryos, CG13565 was expressed in 46% of segments, similar to wild type. However, given its variability of gene expression in Df(1)sc-B57 mutants and the normal variability of CG13565 expression, it remains possible that l(1)sc (and tup) may play roles in H-cell sib development. Additional experiments are necessary to determine how l(1)sc and tup function together to control H-cell-specific gene expression (Stagg, 2011).

Within midline cells, l(1)sc plays important roles in controlling H-cell and mVUM gene expression, while playing relatively insignificant roles in MP1, H-cell sib and iVUM neuronal gene expression. Whether non-midline neuronal gene regulation is regulated by l(1)sc is currently being addressed. Significantly, Df(1)sc-B57 mutant embryos show a strong reduction in DAT and ple expression in the non-midline dorsal lateral dopaminergic neurons. It has been shown that ple expression in these cells is also reduced in tup mutant embryos. Although more detailed cellular and genetic studies are required to bolster these observations, these data raise the possibility that both l(1)sc and tup may regulate gene expression in both midline and non-midline dopaminergic neurons. More generally, l(1)sc control of neuron-specific gene expression is likely to be uncommon. This is based on the observation that in the developing CNS, there is little L(1)sc protein colocalizing with newly divided Elav+ neurons or GMCs (Stagg, 2011).

Because of the key neurobiological and medical importance of dopaminergic neurons, there has been intensive analysis of the regulatory factors that control their development in vertebrates and C. elegans. Are the regulatory programs involved in dopaminergic neuron differentiation conserved between insects, worms, and mammals? The two key regulatory proteins that control Drosophila H-cell dopamine differentiation are l(1)sc and tup. In vertebrates the bHLH genes mouse achaete-scute homolog [Mash1; homolog of l(1)sc] and neurogenin 2 (Ngn2) play roles in midbrain dopaminergic neuron development, although the role of Mash1 is secondary to Ngn2, which has a key function in dopaminergic differentiation. However, Mash1 (as well as Ngn2) can initiate neurogenic programs of other neuronal cell types. This was emphatically demonstrated in recent work in which forced expression of Mash1 and two other transcription factor genes converted murine fibroblast cells to neurons. The mammalian orthologs of Drosophila tup, Isl1 and Isl2, play important roles in motoneuron differentiation, but have not been reported to influence dopaminergic neuron development and gene expression. Recently, C. elegans and vertebrate ETS family transcription factor genes were shown to directly regulate dopamine pathway gene expression. It will be important to identify the transcription factors in Drosophila that directly regulate dopaminergic neural function genes and connect them to the regulatory genes identified in this paper (Stagg, 2011).


Three mutant alleles of the SoxN gene (GA1192, C463 and C2139) were generated by EMS-mutagenesis in a large screen that was aimed at the identification of novel genes which play a role in axon guidance. Mutations in SoxN are associated with multiple defects in axon morphology, as evidenced by thinner, interrupted connectives and incompletely formed commissures. Moreover, mutant embryos show severe defects in head formation and gut constrictions. This study has focused on the role of SoxN in neurogenesis. All experiments were performed using null mutation GA1192 (Buescher, 2002).

Analysis of SoxN mutant embryos with antibodies that recognize marker gene expression in subsets of neurons revealed a drastic loss of neurons. During late stages of embryonic development, the protein Even-skipped (Eve) is expressed in ~20 neurons per hemisegment: the aCC/pCC and the CQ neurons, which derive from the ventral part of the NE; the RP2 neuron, which derives from the intermediate region, and the El neuron cluster, which arises in a more lateral region. SoxN mutant embryos show a near complete loss of Eve-positive RP2 neurons (98% loss) and EL neuron clusters (100% loss), whereas the aCC/pCC neurons are only slightly affected (3% loss) and the CQ neurons remain unaffected (0% loss). As all Eve-positive neurons derive from GMCs that themselves express Eve, early mutant embryos were examined for the presence of Eve-positive GMCs. A loss of Eve-expressing parental GMCs was observed occurring with frequencies comparable with that of the loss of their respective neuronal progeny. To determine if the observed loss of neurons is specific only for Eve-expressing cells, SoxN mutant embryos were stained with an antibody against Fushi tarazu (Ftz), a protein that is transiently expressed in large number of GMCs and neurons. Anti-Ftz staining revealed a severe loss of Ftz-positive GMCs/neurons. Strikingly, the loss occurs predominantly in the intermediate and lateral regions of the CNS while the ventralmost region forms almost normally (Buescher, 2002).

The failure to form specific GMCs/neurons could be explained by loss or mis-specification of the respective parental NBs. To assess NB formation in SoxN mutant embryos, an antibody against worniu (Wor), a protein expressed in all NBs, was used. In wild-type embryos, ~30 NBs delaminate from the NE during embryonic stages 8-11 in five waves (SI-SV). SI NBs form three discrete columns: the ventral column which is made up of three NBs and the MP2 precursor; the intermediate column with two NBs; and the lateral column, which comprises four NBs. At later stages (SII-SV) additional NBs fill the space between these columns (Buescher, 2002).

Anti-Wor staining of stage 9 SoxN mutant embryos has indicated that SI NB formation in the lateral and intermediate columns is severely impaired. In the lateral column instead of the wild-type set of four NBs per hemisegment only one or two NBs are formed. Different lateral NBs are differentially affected. For example, NB3-5 fails to form in 82% of the hemisegments, whereas NB2-5 fails to form in only 22% of the hemisegments (for all NB between 50-100 hemisgements scored). Similar observations were made with respect to NB formation in the intermediate column, which in wild type is composed of NB-5-3 and NB3-2. Both NBs frequently fail to form in SoxN mutant embryos (NB5-3, 14% loss; NB3-2, 67% loss). By contrast, the four NBs of the ventral column form almost normally. Analysis of older mutant embryos with anti-Wor has revealed that SoxN is also required for the formation of late arising NBs. Stage 11 embryos exhibit drastically reduced numbers of NBs; NBs that do form, appear predominantly in the ventral region. These results were confirmed using antibodies against three additional NB marker genes: hunchback, snail and klumpfuss. Staining of stage 11 SoxN mutant embryos with anti-Engrailed antibody has revealed no difference to the wild-type Engrailed expression pattern, suggesting that the loss of NBs is not due to segmentation defects (Buescher, 2002).

To characterize the SoxN phenotype with respect to the formation of late arising NBs, mutant embryos were stained with antibodies that label subsets of NBs. Anti-Vnd labels all ventral NBs, anti-Eagle labels four late forming NBs in the lateral region, and anti-Huckebein-lacZ labels early- and late-forming NBs in the ventral, intermediate and lateral regions. In addition, anti-Odd-skipped and anti-Repo antibodies were used to score the MP2 precursor and the lateral glioblast, respectively (Buescher, 2002).

The loss of SoxN causes a severe hypoplasia. However, specific spatial and temporal aspects are observed. (1) SoxN is required for the formation of NBs that derive from the lateral and intermediate regions of the NE, but does not appear to play a major role in ventral NB formation. (2) Late arising NBs are more severely affected than early arising NBs. (3) NBs that arise at the same time and in the same column are differentially affected by the loss of SoxN: compare the loss of intermediate SI NBs NB3-2(67%) and NB 5-3(14%) (Buescher, 2002).

In addition to the CNS, the NE gives rise to the ventral epidermis. To study possible defects of the ventral epidermis, the cuticle of unhatched SoxN larvae were examined. In wild-type first instar larvae, denticle belts are formed on the ventral side of the eight abdominal segments. Each denticle belt is made up of five rows of setae. In SoxN mutant larvae, a severe loss of anterior setae, which results in a reduction of the AP expansion of the denticle belts, is observed. These results indicate that SoxN mutations lead to defects in both tissues that derive from the NE: the CNS and the ventral epidermis (Buescher, 2002).

From the same EMS stock collection, three lines (GA1192, C463 and C2139) were recovered that fail to complement each other and they all exhibit similar morphological defects. However, the morphological defects observed in C2139 mutant embryos are less severe than those of GA1192 and C463. All three alleles display similar CNS phenotypes either in homozygosity or in heterozygosity with one another. Using deficiencies, lethality and all phenotypic defects mapped to the cytological position 29F. The phenotype of a homozygous deficiency that removes 29F (DfN-22, breakpoints: 29C;30C) is identical to that of GA1192 and C463, while the weaker CNS phenotype of C2139 is enhanced in heterozygosity with DfN-22. These data strongly suggest that GA1192 and C463 represent amorphic alleles, while C2139 appears to be a hypomorphic allele (Buescher, 2002).

Sequencing of genomic DNA from homozygous C463 embryos has revealed an internal deletion of 311 bp (from position 1373-1684; AJ252124); this introduces a frame-shift. The deduced 234 amino acid mutant polypeptide shares the first 215 amino acids with wild-type SoxN protein followed by 19 amino acids of novel peptide sequence. This mutation removes the C-terminal part of the HMG box and all SoxN sequences C-terminal to it. This polypeptide is most probably non-functional (Buescher, 2002).

In addition to SoxN a second HMG box protein, Dichaete is expressed prior to and during NB formation. Within the NE, Dichaete is expressed from stage 7 to stage 12 in two longitudinal stripes that encompass the ventral and intermediate but not the lateral region (Cremazy, 2000). Dichaete mutant embryos display severe defects in CNS development. Dichaete plays a role in the formation of several late arising ventral and intermediate NBs (Zhao, 2002). However, as observed in SoxN mutants, Dichaete mutant embryos do not show significant defects in ventral SI NB formation. Do Dichaete and SoxN function redundantly with respect to early ventral NB formation? A double mutant stock Dichaete87;SoxNGA1192 was generated and stage 9 embryos were stained with anti-Wo. Since homozygous Dichaete mutants show severe segmentation defects in the abdomen, this analysis was restricted to the thoracic segments; in double mutant embryos, ventral SI NB formation is severely impaired: e.g. in SoxN and Dichaete single mutant embryos, the formation of NB1-1 is hardly affected (3% and 2% loss, respectively), while in double mutant embryos NB1-1 fails to form in 48% of the hemisegments. Thus, SoxN and Dichaete function is at least partially redundant with respect to early ventral NB formation (Buescher, 2002).

SoxN and Dichaete expression also overlaps in the intermediate region of the NE and therefore both proteins may contribute to early intermediate NB formation. The formation of the intermediate S1 NB5-3, which is moderately affected in SoxN single mutants (14% loss) and hardly affected in Dichaete single mutants (1%), was analyzed. In SoxNGA1192/Dichaete87 double mutant embryos, an enhanced loss of NB5-3 (25%) was observed, and thus it is concluded that SoxN and Dichaete both contribute to the formation of the intermediate NB5-3 (Buescher, 2002).

Prior to and during NB formation, three homeobox genes, vnd, ind and msh, are expressed in adjacent longitudinal columns and subdivide the NE along the DV axis. vnd and ind play a crucial role in NB formation: loss of vnd or ind results in the loss of ventral or intermediate NBs, respectively. To determine if SoxN plays a role in the initiation or maintenance of Vnd, Ind or Msh expression, stage 8 SoxN mutant embryos were stained with anti-Vnd and anti-Msh antibodies, or an ind-specific RNA probe. The staining patterns of these genes were found to be identical to that of wild-type embryos, indicating that SoxN is dispensable for their expression. Conversely, staining of vnd, ind or msh mutant embryos with an anti-SoxN antibody revealed no role for vnd, ind or msh in the maintenance of SoxN expression prior to and during NB formation (Buescher, 2002).

These results demonstrate that the expression of SoxN and the DV patterning genes is regulated independently. However, the vnd and ind mutant and the SoxN mutant phenotypes exhibit strikingly similar phenotypes with respect to ventral and intermediate NB formation. Moreover, SoxN and Vnd/Ind are co-expressed during NB formation. This prompted a study to see whether SoxN genetically interacts with vnd and/or ind in the NE. The SoxN allele C2139, which appears to be a hypomorph was chosen, and whether removal of one copy of vnd or ind dominantly enhances the phenotype of SoxN was tested. The stocks vndDelta38/+;SoxNC2139/SoxNC2139 and ind16.2/+; SoxNC2139/SoxNC2139 were generated, and the formation of NBs was scored using anti-Wor for the ventral SI NBs and the intermediate NB5-3. In addition, anti-Eve was used to score the RP2 neuron, the progeny of the intermediate SIII NB4-2. Anti-Wor staining of stage 9 vndDelta38/+;SoxNC2139/SoxNC2139 embryos revealed an enhanced loss of ventral SI neuroblasts, ranging from 12% to 18%. In ind16.2/+; SoxNC2139/SoxNC2139 mutant embryos an increased loss of NB5-3 was observed and an increased loss of the RP2 neuron. Thus, SoxN interacts genetically with vnd in ventral and with ind in intermediate NB formation (Buescher, 2002).

The lateral column of NBs derives from a stripe of msh-expressing NE. msh has been shown to play an important role in the specification of lateral NBs, but does not appear to play a role in NB formation. To analyze whether the loss of SoxN uncovers a function of msh in NB formation, SoxNGA1192;mshlttEMS double homozygous mutant embryos were generated and the formation of lateral S1 NBs was scored with anti-Wor antibody. No enhancement of the SoxNGA1192 homozygous phenotype was observed and therefore it is concluded that even in the absence of SoxN, msh has no role in NB formation (Buescher, 2002).

A large collection of chemically induced Drosophila mutations, isolated on the basis of abnormal CNS phenotypes, was screened for lines missing specific neuroblast lineages. One line (U6-35) was identified in which virtually all thoracic and abdominal eagle (eg)- and empty spiracles (ems)-expressing neurons and glia were missing from the CNS of homozygous embryos (e.g.m NB lineages 2-4, 3-3, 7-3 and 6-4 missing in over 99% of hemisegments; NB lineages 3-3, 3-5 and 4-4 missing in over 95% of hemisegments). The mutation was localized genetically by recombination and deficiency mapping and it was found to be uncovered by Df(2L)N22-5, a deletion encompassing the 29F region. A Sox-domain containing gene has also been noted in this region in the course of a molecular screen for new Drosophila Sox genes that was subsequently found to be identical to SoxNeuro (Cremazy, 2000). Since SoxN is known to be expressed early in CNS development, and the related gene Dichaete had previously been shown to have specific CNS phenotypes, SoxN was considered to be a candidate for the gene mutated in the U6-35 line. The SoxN gene was sequenced from the U6-35 stock and it was found to carry a C-T transition that changes a glutamine at position 133 of the protein to a stop codon. This premature stop occurs before the DNA-binding domain and is expected to eliminate the function of the gene. In support of this, the phenotype of U6-35 homozygotes is identical to that observed in U6-35/Df(2L)N22-5 embryos. Therefore, U6-35 represents a null mutation in the SoxN gene and shall be hereafter referred to as SoxNU6–35 (Overton, 2002).

At a gross level, SoxNU6–35 mutant embryos show a severely disrupted CNS. When examined with the global axonal marker BP102 and the more specific marker FasII a substantial reduction was observed in the longitudinal axon tracts. In 60% of the mutant hemisegments scored there is a complete loss of longitudinal tracts judged by BP102 staining. In addition, the anterior and posterior commissures are also affected; in 52% of mutant segments the commissures fail to separate and are sometimes absent (2%). With FasII staining a disruption was observed in the regular axonal fasciculation pattern and many cases of axons inappropriately crossing the midline were noted. There appears to be no difference in the phenotype along the anteroposterior axis. The PNS shows no major defects when examined with the PNS-specific 22C10 antibody. Thus the defects in SoxNU6–35 suggest a failure in the morphogenesis or differentiation of the CNS. Since SoxN expression is initiated after cellularization (Cremazy, 2000), no segmentation defects are observed in SoxNU6–35. In addition to these phenotypes, defects were observed in spacing in 68% of SoxNU6–35 mutant embryos; within the CNS, the spacing between two segments in the middle of the embryo, most often A3 and A4, is greatly increasedm while spacing in the neighbouring segments is reduced; in severe cases there are gaps in the neuroectoderm, however, no segments are lost. Since these defects were never observed before stage 12, it is thought that this phenotype is a result of mechanical defects during germband retraction; in support of this, a failure to complete germband retraction was observed in a small number of mutant embryos (Overton, 2002).

In order to examine the defects in the developing CNS associated with SoxNU6–35 in greater detail, mutant embryos were stained with markers for specific NBs and/or their progeny. These data can be summarized as follows: using Hunchback (Hb) and Even skipped (Eve), along with the Eg and Ems staining, a striking asymmetry in NB loss is observed in SoxNU6–35 mutants. The use of Hb as a marker for all NBs delaminating in SI shows that medial column NBs are less affected (between 4% [MP2] and 38% [NB5-2] missing) than those that form in the intermediate (52% of NB5-3 missing) and lateral columns (between 23% [NB7-4] and 69% [NB 5-6] missing). This observation is supported by using Eve as a marker for progeny of certain NBs. The CQ neurons (NB 7-1) and aCC/pCC (NB 1-1), which are progeny of NBs that delaminate in the medial column during the SI wave, are relatively unaffected (less than 4% missing). By contrast, the RP2 neuron, which is a progeny of NB 4-2, an intermediate column SII NB, and the cells of the Eve lateral cluster (ELC), which are progeny of the laterally delaminating SIV NB 3-3, are strongly affected (96% and 100% missing, respectively). Additionally, the antibody staining against Eg and Ems shows that NBs delaminating in the intermediate or lateral columns in SII-SV are often missing (e.g., 6-4, 7-3, 2-4 and 3-3), a greater than 90% loss (Overton, 2002).

Taken together, these data suggest that SoxN is required for the correct specification and/or formation of NBs in both the intermediate and lateral columns. It appears that there is much less of a requirement for SoxN in the medial column, at least for early delaminating NBs. SinceSoxN and Dichaete expression overlaps in the medial neuroectoderm and Dichaete mutants also have little effect on early medial NB lineages (Zhao, 2002), it is possible that the proteins are able to functionally compensate in this part of the developing CNS. However, the fact that later-born intermediate and lateral NBs are more affected than the S1 NBs delaminating from these regions additionally suggests a stronger requirement for SoxN function in these post S1 NBs and/or their progeny (Overton, 2002).

To unambiguously demonstrate that the phenotype of U6-35 mutant embryos is due to the mutation in SoxN, attempts were made to rescue SoxNU6–35 phenotypes by driving UASSoxN expression in the developing CNS with KrGAL4. The Kr-Gal4 line expresses Gal4 at high levels in the neuroectoderm within the central domain of the embryo from stage 9 onwards. In SoxNU6–35/SoxNU6–35; KrGAL4/UASSoxN embryos, a substantial rescue of RP2 neurons and ELC cells (progeny of NB4-2 and NB3-3, respectively) is observed. Absence of the RP2 neuron is now observed in only 33% of hemisegments, while the ELC cells are absent in 67% of hemisegments compared with 96% and 100% respectively in embryos without UASSoxN. These data indicate that the CNS phenotype of U6-35 embryos results from a specific loss of SoxNeuro (Overton, 2002).

SoxN and Dichaete are both expressed early in the neuroectoderm. Dichaete is restricted to the ventral region, extending from the midline to the position of the intermediate column (Zhao, 2002), and SoxN is excluded from the midline and extends more dorsally to encompass the entire neuroectoderm (Cremazy, 2000). Dichaete mutants show strong phenotypes in the midline, where Dichaete is uniquely expressed (Sanchez-Soriano, 1998), and SoxN mutants exhibit strong phenotypes in the lateral half of the CNS where SoxN is uniquely expressed. In Dichaete mutants, SI medial NBs are not affected (Zhao, 2002) but there is a loss of later delaminating SII and SIII NBs from both medial and intermediate columns. SoxN and Dichaete overlap in the medial and intermediate neuroectodermal columns and in the medial column, SoxN phenotypes are weaker than those observed in the lateral columns. These data are consistent with the idea that the genes may be able to compensate functionally in the medial column neuroectoderm. To examine the consequences of removing group B Sox function from the early CNS, a double mutant combination was constructed, using null alleles for both Dichaete and SoxN. The overall structure of the CNS was examined as well as markers for specific NBs and/or progeny in the double mutant embryos (Overton, 2002).

Staining the double mutants with BP102 reveals a severe disruption in the organization and structure of the CNS. A complete loss of longitudinal axons is observed in many segments with frequent gaps in the neuropil. Commissures are often absent, and those that do form are virtually never separated. The phenotype of the double mutants is far more severe than observed with either single mutant and supports the idea that the genes can act redundantly or in related pathways. If this is the case then an enhanced effect is expected on medial NBs and their progeny when both SoxN and Dichaete function are removed, compared with each of the single mutants, since this is the region in which they are extensively co-expressed. In line with this expectation it has been observed that in the SI medial lineages of NB1-1 and NB7-1, identified by eve expression, there is a rather severe reduction in the number of aCC/pCC and CQ cells in double mutants compared with each of the single mutants. Note that these lineages are virtually unaffected in either of the single mutants. Additionally, in the intermediate column, the Hb expressing neuroblast 5-3 is absent at a higher frequency in double mutant embryos than in SoxN or D mutants (79% compared with 52% and 2%, respectively), indicating that Dichaete is to some extent able to compensate for a loss of SoxNeuro within this lineage. Although it is impossible to determine accurately the identity of the remaining Hb expressing SI NBs in the double mutants, the total number of cells in thoracic segments was counted, and in SoxNU6–35 homozygotes 30% of Hb expressing NBs are missing; in Dichaete mutants less than 1% are missing, whereas 56% are missing in the double mutants. Taken together, it is concluded that in the cases of overall CNS structure as well as medial and intermediate column SI NBs, evidence is seen for functional redundancy between related Group B Sox genes (Overton, 2002).

Both SoxN and Dichaete are expressed early in the neuroectoderm, SoxN expression being initiated slightly before that of Dichaete. It is therefore possible that SoxN regulates the expression of Dichaete and this possibility was examined by staining SoxNU6–35 mutant embryos for Dichaete. A rather unexpected phenotype was observed; in around half of the mutant embryos, Dichaete levels were apparently normal. However, in the remaining half Dichaete levels were reduced, but only in the anterior half of the neuroectoderm; the posterior appeared to be normal. This is not due to a staining artifact because in the affected embryos Dichaete is expressed normally in the midline all along the AP axis. Thus, it appears that SoxN does have an effect on Dichaete expression, but that this effect is variable and restricted along the AP axis. In any case the SoxN phenotypes cannot be explained by a loss of Dichaete expression in the neuroectoderm because ectopic expression of ac would be expected to be seen in SoxNU6–35 as is seen in Dichaete and the double mutants (Overton, 2002).

Therefore, it is concluded that in the neuroectoderm the two group B Sox proteins, SoxN and Dichaete, can functionally compensate but they also have antagonistic functions, particularly within the intermediate neuroectoderm (Overton, 2002).

SoxNeuro acts with Tcf to control Wg/Wnt signaling activity

Wnt signaling specifies cell fates in many tissues during vertebrate and invertebrate embryogenesis. To understand better how Wnt signaling is regulated during development, genetic screens were performed to isolate mutations that suppress or enhance mutations in the fly Wnt homolog, wingless (wg). This study finds that loss-of-function mutations in the neural determinant SoxNeuro (also known as Sox-neuro, SoxN) partially suppress wg mutant pattern defects. SoxN encodes a HMG-box-containing protein related to the vertebrate Sox1, Sox2 and Sox3 proteins, which have been implicated in patterning events in the early mouse embryo. In Drosophila, SoxN has been shown to specify neural progenitors in the embryonic central nervous system. This study shows that SoxN negatively regulates Wg pathway activity in the embryonic epidermis. Loss of SoxN function hyperactivates the Wg pathway, whereas its overexpression represses pathway activity. Epistasis analysis with other components of the Wg pathway places SoxN at the level of the transcription factor Pan (also known as Lef, Tcf) in regulating target gene expression. In human cell culture assays, SoxN represses Tcf-responsive reporter expression, indicating that the fly gene product can interact with mammalian Wnt pathway components. In both flies and in human cells, SoxN repression is potentiated by adding ectopic Tcf, suggesting that SoxN interacts with the repressor form of Tcf to influence Wg/Wnt target gene transcription (Chao, 2007).

SoxN downregulates the Wg/Wnt pathway to reduce target gene expression. Downregulation is a crucial process because it sensitizes the signal response to allow rapid on/off switching and also keeps the system off in cells that are not actively responding to signal. Many genes have been shown to negatively regulate Wg/Wnt pathway activity through the destabilization of Arm/beta-catenin. Far fewer are known to exert negative regulatory effects downstream of Arm. The vertebrate Sox proteins -- Sox9, XSox3, XSox17α and XSox17ß -- as well as Chibby, a conserved nuclear factor, antagonize Wg/Wnt signaling by binding to Arm/beta-catenin and preventing it from partnering with Tcf to activate target gene expression. SoxN, however, does not bind beta-catenin in cell-culture assays, and does not share strong homology with the C-terminal sequences through which vertebrate Sox proteins bind this protein. Furthermore, SoxN function is not influenced by Arm levels. No difference was observed in SoxN-mediated TOPflash repression when cells were induced by co-transfection with a constitutively stabilized beta-catenin versus with Wnt-induced medium. Instead, both TOPflash and genetic experiments indicate that SoxN function depends on Tcf and Gro, its co-repressor (Chao, 2007).

One way to explain these observations is that SoxN contributes to the assembly or stability of the Tcf repressor complex on DNA. The consensus-sequence recognition for HMG domains in the Sox and Tcf families is reported to be similar, although XSox3 and XSox17ß fail to bind a consensus Tcf DNA sequence. It is shown that SoxN does not compete for Tcf-binding sites as a means of repressing target gene transcription, but the data support a model in which SoxN might bind DNA elsewhere or might bind Tcf sites transiently to initiate or stabilize the assembly of a repressor complex (Chao, 2007).

A similar model may explain the results from Xenopus that showed that XSox3-mediated repression does not require interaction between XSox3 and beta-catenin. XSox3 strongly interferes with dorsal fate specification in Xenopus embryos and represses TOPflash-reporter activity in vitro. HMG-domain mutations render XSox3 inactive in embryos without affecting its interaction with beta-catenin or its repression in TOPflash assays. Thus, it is the DNA-binding domain, not the beta-catenin-interacting C-terminus, that is relevant to its in vivo function in dorsal determination in Xenopus. XSox3 represses the expression of the dorsal-specific Nodal-related gene Xnr5 through optimal core binding sequences adjacent to and partially overlapping with Tcf sites in the Xnr5 promoter (Zhang, 2003). By contrast, the fly SoxN shows no discrepancy between its behavior in TOPflash assays and its in vivo effects. This suggests that the synthetic Tcf-binding sites arranged in the TOPflash-reporter plasmid are sufficient to support SoxN repressor function (Chao, 2007).

Because adding Tcf-site competitor DNA does not diminish the repressive capacity of limiting amounts of SoxN, the role of SoxN in repression does not appear to be stoichiometric. Therefore, the idea is favored that Sox proteins may act in a catalytic fashion during repressor-complex assembly at Wnt target gene promoters, rather than forming a structural part of the repressor complex itself. It was not possible to detect direct binding of SoxN with either Tcf, Gro or Arm, raising the possibility that SoxN interacts with some as yet unidentified protein that chaperones assembly of the repressor complex. A SoxN-binding cofactor, SNCF, has been identified in Drosophila (Bonneaud, 2003), but this gene is expressed only in pre-gastrulation embryos. Because Wg signaling occurs exclusively post-gastrulation, and specification of naked cuticle begins more than 4 hours after gastrulation, it is not thought that SNCF is a likely candidate for mediating this aspect of SoxN function. Rather, it is likely to play a role in the neuronal specification events promoted by SoxN at earlier stages of embryogenesis (Chao, 2007).

It is curious that uniformly overexpressed SoxN represses Wg signal transduction in dorsal epidermal cells more severely than in ventral cells. This effect is evident in both cuticle pattern elements and in en expression, and is reminiscent of defects observed in the 'transport-defective' class of wg mutant alleles, which includes wgNE2. These mutations restrict Wg-ligand movement ventrally to promote only local signaling response while simultaneously abolishing all dorsal signaling, suggesting a fundamental difference in ventral and dorsal cell response. Perhaps it is not a coincidence that the NC14 mutation was isolated in the wgNE2 mutant background. Further analysis of SoxN function may help to determine the molecular basis for dorsoventral differences in Wg signal transduction (Chao, 2007).


See Dichaete for information about SoxNeuro evolutionary homologs.

The evolutionally-conserved function of group B1 Sox family members confers the unique role of Sox2 in mouse ES cells

In mouse ES cells, the function of Sox2 is essential for the maintenance of pluripotency. Since the Sox-family of transcription factors are well conserved in the animal kingdom, addressing the evolutionary origin of Sox2 function in pluripotent stem cells is intriguing from the perspective of understanding the origin of pluripotency. This question was approached using a functional complementation assay in inducible Sox2-null ES cells. Assaying mouse Sox proteins from different Groups, it was found that only Group B1 and Group G proteins were able to support pluripotency. Interestingly, invertebrate homologs of mammalian Group B1 Sox proteins were able to replace the pluripotency-associated function of mouse Sox2. Moreover, the mouse ES cells rescued by the Drosophila SoxNeuro protein are able to contribute to chimeric embryos. These data indicate that the function of mouse Sox2 supporting pluripotency is based on an evolutionally conserved activity of the Group B1 Sox family. Since pluripotent stem cell population in developmental process could be regarded as the evolutional novelty in vertebrates, it could be regarded as a co-optional use of their evolutionally conserved function (Niwa, 2016).

FGFR2 is required for airway basal cell self-renewal and terminal differentiation
Airway stem cells slowly self-renew and produce differentiated progeny to maintain homeostasis throughout the life-span of an individual. Mutations in the molecular regulators of these processes may drive cancer or degenerative disease, but are also potential therapeutic targets. Conditionally deleting one copy of FGF Receptor 2 (see Drosophila Breathless) in adult mouse airway basal cells results in self-renewal and differentiation phenotypes. This study shows that FGFR2 signalling correlates with maintenance of expression of a key transcription factor for basal cell self-renewal and differentiation, SOX2 (see Drosophila Sox Neuro). This heterozygous phenotype illustrates that subtle changes in Receptor Tyrosine Kinase signalling can have significant effects, perhaps providing an explanation for the numerous changes seen in cancer (Balasooriya, 2017).

Structural basis for nuclear import selectivity of pioneer transcription factor SOX2

SOX (SRY-related HMG-box) transcription factors perform critical functions in development and cell differentiation. These roles depend on precise nuclear trafficking, with mutations in the nuclear targeting regions causing developmental diseases and a range of cancers. SOX protein nuclear localization is proposed to be mediated by two nuclear localization signals (NLSs) positioned within the extremities of the DNA-binding HMG-box domain and, although mutations within either cause disease, the mechanistic basis has remained unclear. Unexpectedly, this study found that these two distantly positioned NLSs of SOX2 (Drosophila homolog: SoxN) contribute to a contiguous interface spanning 9 of the 10 ARM domains on the nuclear import adapter IMPalpha3 (Drosophila homolog: karyopherin alpha3). This study identified key binding determinants and show this interface is critical for neural stem cell maintenance and for Drosophila development. Moreover, this study identified a structural basis for the preference of SOX2 binding to IMPalpha3. In addition to defining the structural basis for SOX protein localization, these results provide a platform for understanding how mutations and post-translational modifications within these regions may modulate nuclear localization and result in clinical disease, and also how other proteins containing multiple NLSs may bind IMPα through an extended recognition interface (Jagga, 2021).


Search PubMed for articles about Drosophila Sox Neuro

Arefin, B., Parvin, F., Bahrampour, S., Stadler, C. B. and Thor, S. (2019). Drosophila neuroblast selection is gated by Notch, Snail, SoxB, and EMT gene interplay. Cell Rep 29(11): 3636-3651. PubMed ID: 31825841

Balasooriya, G., Goschorska, M., Piddini, E. and Rawlins, E. L. (2017). FGFR2 is required for airway basal cell self-renewal and terminal differentiation. Development [Epub ahead of print]. PubMed ID: 28348168

Bonneaud, N., Savare, J., Berta, P. and Girard, F. (2003). SNCF, a SoxNeuro interacting protein, defines a novel protein family in Drosophila melanogaster. Gene 319: 33-41. Medline abstract: 14597169

Buescher, M., Hing, F. S. and Chia, W. (2002). Formation of neuroblasts in the embryonic central nervous system of Drosophila melanogaster is controlled by SoxNeuro. Development 129: 4193-4203. 12183372

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(5): 989-97. Medline abstract: 17267442

Collignon, J., Sockanathan, S., Hacker, A., Cohen-Tannoudji, M., Norriss, D., Rastan, S., Stevanovic, M., Goodfellow, P. N. and Lovell-Badge, R. (1996). A comparison of the properties of Sox-3 with Sry and two related genes, Sox-1 and Sox-2. Development 122: 509-520. 8625802

Cremazy, F., Berta, P. and Girard, F. (2000). SoxNeuro, a new Drosophila Sox gene expressed in the developing central nervous system. Mech. Dev. 93: 215-219. 10781960

Jagga, B., Edwards, M., Pagin, M., Wagstaff, K. M., Aragao, D., Roman, N., Nanson, J. D., Raidal, S. R., Dominado, N., Stewart, M., Jans, D. A., Hime, G. R., Nicolis, S. K., Basler, C. F. and Forwood, J. K. (2021). Structural basis for nuclear import selectivity of pioneer transcription factor SOX2. Nat Commun 12(1): 28. PubMed ID: 33397924

Kishi, M., Mizuseki, K., Sasai, N., Yamazaki, H., Shiota, K., Nakanishi, S. and Sasai, Y. (2000). Requirement of Sox2-mediated signaling for differentiation of early Xenopus neuroectoderm. Development 127: 791-800. 10648237

Li, M., Pevny, L., Lovell-Badge, R. and Smith, A. (1998). Generation of purified neural precursors from embryonic stem cells by lineage selection. Curr. Biol. 8: 971-974. 9742400

Ma, Y., Certel, K., Gao, Y., Niemitz, E., Mosher, J., Mukherjee, A., Mutsuddi, M., Huseinovic, N., Crews, S. T., Johnson, W. A. and Nambu, J. R. (2000). Functional Interactions between Drosophila bHLH/PAS, Sox, and POU transcription factors regulate CNS midline expression of the slit gene. J. Neurosci. 20: 4596-4605. 10844029

Mizuseki, K. Kishi, M., Shiota, K., Nakansihi, S. and Sasai, Y. (1998a). SoxD: an essential mediator of induction of anterior neural tissues in Xenopus. Neuron 21: 77-85. 9697853

Mizuseki, K. Kishi, M., Matsui, M., Nakansihi, S. and Sasai, Y. (1998b). Xenopus Zic-related-1 and Sox-2, two factors induced by Chordin have distinct activities in the initiation of neural induction. Development 125: 579-587. 9435279

Nambu, P. A. and Nambu, J. R. (1996). The Drosophila fish-hook gene encodes an HMG domain protein essential for segmentation and CNS development. Development 122: 3467-3475. 8951062

Nishiguchi, S., Wood, H., Kondoh, H., Lovell-Badge, R. and Episkopou, V. (1998). Sox1 directly regulates the gamma-crystallin genes and is essential for lens development in mice. Genes Dev. 12: 776-781. 9512512

Niwa, H., Nakamura, A., Urata, M., Shirae-Kurabayashi, M., Kuraku, S., Russell, S. and Ohtsuka, S. (2016). The evolutionally-conserved function of group B1 Sox family members confers the unique role of Sox2 in mouse ES cells. BMC Evol Biol 16: 173. PubMed ID: 27582319

Overton, P. M., Meadows, L. A., Urban, J. and Russell, S. (2002). Evidence for differential and redundant function of the Sox genes Dichaete and SoxN during CNS development in Drosophila. Development 129: 4219-4228. 12183374

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(15): 2807-13 . PubMed citation

Pevny, L. H., Sockanathan, S., Placzek, M. and Lovell-Badge, R. (1998). A role for SOX1 in neural determination. Development 125: 1967-1978. 9550729

Rex, M., Orme, A., Uwanogho, D., Tointon, K., Wigmore, P. M., Sharpe, P. T. and Scotting, P. J. (1997). Dynamic expression of chicken Sox2 and Sox3 genes in ectoderm induced to form neural tissue. Dev. Dyn. 209: 323-332. 9215646

Russell, S. R. H., Sanchez-Soriano, N., Wright, C. R. and Ashburner, M. (1996). The Dichaete gene of Drosophila melanogaster encodes a Sox-domain protein required for embryonic segmentation. Development 122: 3669-3676. 8951082

Sanchez-Soriano, N. and Russell, S. (1998). The Drosophila Sox-domain protein Dichaete is required for the development of the central nervous system midline. Development 125: 3989-3996. 9735360

Sanchez-Soriano, N. and Russell, S. (2000). Regulatory mutations of the Drosophila Sox gene Dichaete reveal new functions in embryonic brain and hindgut development. Dev. Biol. 220: 307-321. 10753518

Savare, J., Bonneaud, N. and Girard, F. (2005). SUMO represses transcriptional activity of the Drosophila SoxNeuro and human Sox3 central nervous system-specific transcription factors. Mol, Biol, Cell 16: 2660-2669. PubMed Citation: 15788563

Stagg, S. B., Guardiola, A. R. and Crews, S. T. (2011). Dual role for Drosophila lethal of scute in CNS midline precursor formation and dopaminergic neuron and motoneuron cell fate. Development 138(11): 2171-83. PubMed Citation: 21558367

Uwanogho, D., Rex, M., Cartwright, E. J., Pearl, G., Healy, C., Scotting, P. J. and Sharpe, P. T. (1995). Embryonic expression of the Chicken Sox2, Sox3 and Sox11 genes suggests an interactive role in neuronal development. Mech. Dev. 49: 3847-3854. 7748786

Vidal, V. P., Chaboissier, M. C., Lutzkendorf, S., Cotsarelis, G., Mill, P., Hui, C. C., Ortonne, N., Ortonne, J. P. and Schedl, A. (2005). Sox9 is essential for outer root sheath differentiation and the formation of the hair stem cell compartment. Curr. Biol. 15: 1340-1351. PubMed citation: 16085486

Wheeler, S. R., Stagg, S. B. and Crews, S. T. (2008). Multiple Notch signaling events control Drosophila CNS midline neurogenesis, gliogenesis and neuronal identity. Development 135(18): 3071-3079. PubMed Citation: 18701546

Wood, H. B. and Episkopou, V. (1999). Comparative expression of the mouse Sox1, Sox2 and Sox3 genes from pre-gastrulation to early somite stages. Mech. Dev. 86: 197-201. 10446282

Zhang, C., Basta, T., Jensen, E. D. and Klymkowsky, M. W. (2003). The beta-catenin/VegT-regulated early zygotic gene Xnr5 is a direct target of SOX3 regulation. Development 130: 5609-5624. Medline abstract: 14522872

Zhao, G. and Skeath, J. G. (2002). The Sox-domain containing gene Dichaete/fish-hook acts in concert with vnd and ind to regulate cell fate in the Drosophila neuroectoderm. Development 129: 1165-1174. 11874912

Biological Overview

date revised: 17 August 2020

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