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).
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).
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).
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).
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