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).
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).
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).
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date revised: 10 April 2008
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