BACKGROUND
during the process of development, precise changes in gene expression are critical for correctly timed differentiation and body plan development.
tissue-specific gene expression is often highly conserved across distantly related species and changes in gene expression are thought to contribute to speciation and the evolution of phenotypic diversity  <cit> .
however, the search for direct targets of the transcription factors regulating developmental processes and the identification of regulatory networks remains challenging.
in particular, it is difficult to differentiate between downstream effects, functional compensation by related genes and genuine direct targets.
in the context of conserved families of transcriptional regulators, research in model organisms utilizing a number of different genetic tools can be helpful in elucidating the precise regulatory mechanisms at play in specific tissues with the hope that such insights are generalisable to a broader range of species.
group b sox domain factors represent such a family of conserved transcription factors with roles in a number of important developmental processes, including the regulation of stem cell pluripotency  <cit> , eye development  <cit> , and drosophila segmentation  <cit>  among others.
in the context of the central nervous system, early expression of group b sox genes in the neuroectoderm is detected in all metazoans examined to date, and in at least some cases it is known that this expression is functionally important  <cit> .
however, a degree of functional redundancy between different sox genes expressed in the same tissues makes this a difficult gene family to study and means that comparatively few direct regulatory targets are known despite the importance of this class of transcription factor  <cit> .
in drosophila there are four group b genes: soxneuro , dichaete, sox21a and sox21b, the last three organised in a genomic cluster that is conserved in insects  <cit> .
soxn and dichaete encode group b <dig> proteins corresponding to mammalian sox <dig> and sox <dig>  respectively  <cit> .
we have previously shown that the mouse sox <dig> gene is able to effectively rescue dichaete mutant phenotypes, highlighting the considerable functional conservation exhibited by sox proteins  <cit> .
dichaete has an early role in embryonic segmentation, where it acts to regulate the expression of primary pair rule genes, and is then active in the anlage of the central nervous system  <cit> .
in the early stages of drosophila cns specification, dichaete and soxn are required for the correct expression of the proneural gene achaete and in this role they cooperate with the dv-patterning homeodomain proteins intermediate neuroblasts defective  and ventral neuroblasts defective   <cit> .
after gastrulation, dichaete is dynamically expressed in many segregated neuroblasts and their progeny  <cit> .
in the developing cns, dichaete has a defined phenotype in the ventral midline where it is a uniquely expressed sox gene early in embryogenesis.
it is required for the correct specification and development of midline glial cells, directly regulating the slit gene via interaction with the pou domain protein ventral veins lacking  and the pas domain protein encoded by single minded <cit> .
interestingly, a sox-pou interaction is also a critical part of early mouse development, where sox <dig> partners with oct <dig>  <cit> .
while the precise role dichaete plays during the later stages of embryonic cns development has not yet been clarified, a recent study suggests a role in maintaining the stem cell-like neuroblasts in a self-renewing state, with downregulation of dichaete leading to exit from the cell cycle and premature differentiation, again showing similarity to the function of sox <dig> in mammals  <cit> .
dichaete and soxn are both known to be active in the early neuroectoderm and, similarly to mammalian group b genes, display a degree of functional redundancy or compensation.
mutations in either of these genes exhibit relatively weak phenotypes in cells where they are co-expressed but double mutants show severe neural hypoplasia, suggesting that at least one group b gene is necessary for the correct specification or differentiation of early neural progenitors  <cit> .
however, while it is clear the fly group b sox proteins can functionally compensate in regions of the neuroectoderm, a careful analysis of the phenotypes suggests that each protein has a unique role, even in cells that they are co-expressed in.
this is most apparent in the intermediate column of the neuroectoderm where dichaete acts to repress the expression of the proneural gene achaete  whereas soxn appears to have activating functions  <cit> .
to date, the number of characterised direct targets of dichaete in the developing cns is comparatively small, including the proneural gene ac in the neuroectoderm and slit in the midline  <cit> .
the early embryo genome-wide binding profiles published by the berkeley drosophila transcription network project  and the modencode consortium identify dichaete binding at thousands of genomic locations, suggesting a considerable number of direct target genes  <cit> .
however, since the binding profiles were obtained using whole embryos, they do not give insight into tissue-specific aspects of dichaete function.
similarly, due to functional compensation and pleiotropy it is also difficult to tease apart tissue-specific aspects of dichaete function from the genomic analysis of loss-of-function mutants.
in this study, we probed tissue-specific dichaete function in vivo, using two newly developed dominant negative dichaete alleles.
we focused on specific neural tissues in the early embryo: the neuroectoderm and the ventral midline.
we first characterised the constructs using phenotypic analysis to determine whether their overall effect matches that of dichaete mutants.
we then used a mixture of phenotypic and binding analysis to characterise two new putative dichaete direct target genes, commisureless  in the midline and asense  in the neuroectoderm.
we showed that dichaete directly binds to regulatory elements associated with these genes.
finally, we expressed the dominant negatives in specific neural tissues and performed whole transcriptome expression profiling, comparing these data with results from existing genome-wide binding studies to identify a set of potential dichaete targets.
using these data, we also assess the appropriateness of dominant negatives as a tool for unravelling tissue-specific transcription factor function.
RESULTS
evaluation of dominant negative constructs
since dichaete is widely and dynamically expressed during drosophila development and null mutations have pleiotropic effects  <cit> , its function in specific tissues is difficult to study in detail.
to facilitate tissue-specific analysis of dichaete action, we generated dominant negative  dichaete proteins and expressed them in specific tissues via the gal4-uas system  <cit> .
we produced three types of construct: two dominant negative proteins, either lacking the dichaete hmg dna-binding domain  or a fusion between the full length dichaete and the strong repressor domain from the engrailed protein , we also produced a full length wild-type dichaete control.
both types of dominant negative protein have been previously reported in work with vertebrate group b sox proteins: the enrep in xenopus  <cit> , chick  <cit>  and zebrafish  <cit> , and the Δhmg in xenopus  <cit> .
enrep fusions are reported to turn transcription factors into strong repressors, effectively shutting down the expression of target genes  <cit> .
the way that Δhmg proteins act is less clear but it is likely they function by sequestering essential co-factors.
thus our expectations when using the dn constructs was that the enrep fusion would reveal sets of genes where dichaete function is required for activation but would not detect genes where dichaete normally acts as a repressor, whereas the Δhmg construct should uncover genes repressed or activated by dichaete via interactions with a cofactor.
since we have previously shown that sox <dig> effectively rescues dichaete mutant phenotypes, we also generated a similar set of dominant negative constructs using mouse sox <dig>  to test the generality of our approach.
to assess the effectiveness of the dn constructs we expressed each of them in the midline of wild type flies using a sim-gal <dig> driver and in neuroblasts using a pros-gal <dig> driver.
in the case of sim-gal <dig> we used a driver line heterozygous for sim-gal <dig> on the 2nd and homozygous for sim-gal <dig> on the 3rd chromosome crossed to homozygous uas lines.
for pros-gal <dig>  a 2nd chromosome homozygous driver line was crossed with homozygous uas lines.
in both cases, all progeny express the transgene.
we have previously shown that expressing wild type dichaete or msox <dig> with a midline driver rescues dichaete null mutant phenotypes  <cit> : both dichaete Δhmg and enrep constructs produced dichaete-like phenotypes in the cns when expressed with sim-gal <dig>  bp <dig> staining of dn-dichaete expressing embryos showed collapsed commissures, reduced separation between the longitudinal tracts, gaps in longitudinal connectives, misrouting and inappropriate midline crossing by longitudinal axons .
similar phenotypes were observed with the msox <dig> constructs  and in all cases 50-70% of embryos  exhibited some degree of cns disruption.
in agreement with our previous observations  <cit> , expression of the wild type sox proteins under the same conditions produced very mild phenotypes .
focusing on the longitudinal connectives as revealed by fasii staining, we observed strong cns phenotypes with both the fly and mouse dn constructs .
since slit is a dichaete target gene in the midline, we examined slit in dn expressing conditions and found, as expected, a reduction in expression .
this observation indicates that, at the level of an individual target gene, the dn constructs mimic dichaete loss of function.
however, with both fasii and bp <dig> staining, the phenotypes we observed were more severe than those exhibited by dichaete null mutants.
this suggests that the dn constructs affect processes additional to those regulated by dichaete and to test this we expressed dn constructs in the midline of dichaete null mutants.
whereas the wild type sox proteins rescue the cns phenotypes, expression of dn constructs produced phenotypes more severe than those of dichaete mutants , supporting the view that the dn constructs, in addition to disrupting normal dichaete function, are likely to have additional effects and thus act as neomorphic alleles.
.
since previous studies indicate that dichaete acts to repress achaete expression in the neuroectoderm, we used the dn constructs to investigate the effects on achaete expression in the early neuroectoderm using a prosgal <dig> driver  <cit> .
in the stage  <dig> embryo, ac is expressed in a single medial column neuroblast in each hemisegment  <cit> .
consistent with previous reports  <cit> , in dichaete mutants we see a variable increase in the number of ac positive cells in the medial column and the appearance of cells in the intermediate column, with up to  <dig> cells observed in a single hemisegment as well as a more general diffuse staining indicative of low level derepression in the neuroectoderm .
driving the dichaete or msox <dig> Δhmg constructs with prosgal <dig> results in a similar increase in ac positive cells, with both fly and mouse proteins behaving similarly .
in contrast, when constructs carrying the enrep fusions are expressed under the same conditions we see a loss of ac expression in the medial column and no ectopic expression in the intermediate column .
in addition, we found that expressing Δhmg in the early embryo resulted in precocious expression of ac, with 50-60% of dichaeteΔhmg or sox <dig> Δhmg expressing embryos  showing weak ac expression at stage 6/ <dig> in contrast to wild type expression at stage  <dig>  these observations support the view that dichaete directly regulates ac and further confirm that the dn constructs behave as expected.
specifically, we note that expression of the enrep construct results in a loss of ac expression consistent with its expected activity repressing targets genes.
in contrast, the Δhmg construct results in ectopic expression of ac in both medial and intermediate columns, consistent with the idea that it interferes with wild type dichaete function and supports the idea that dichaete normally acts as a repressor of gene expression in some circumstances.
.
intermediate = fraction of hemisegments expressing ac in the intermediate column.
medial down = fraction of hemisegments showing loss of ac expression in the medial column.
medial up/extra = fraction of hemisegments showing stronger ac expression or extra ac expressing cells in the medial column.
in all cases n >  <dig> embryos.
nd = not done.
dichaete regulates comm in the midline
to date only two direct dichaete target genes, slit and ac, have been identified and to learn more about how dichaete regulates gene expression in the developing cns we sought to identify additional direct dichaete targets.
to do this we examined gene expression data, dna binding data and the literature to identify candidates genes for further study, focusing on genes known to be expressed in the midline or early neuroectoderm.
dichaete acts directly in the midline, and in particular is known to function in midline glia  <cit> .
the commisureless gene is expressed in midline glia  <cit>  and in dichaete mutants we observe a loss of comm expression in the midline of stage  <dig> embryos .
significantly, lateral epidermal stripes of comm expression as well as earlier blastoderm and neuroectodermal expression  are not affected in the dichaete mutants, suggesting dichaete does not generally regulate comm expression in the embryo.
.
the hypothesis that comm is a direct target of dichaete in the cns was supported by experiments using the dn constructs: expressing Δhmg or enrep proteins in the midline results in the loss of comm expression along the length of the midline .
further to this, strong and ubiquitous expression of msox2enrep via a maternal gal4-vp <dig> driver results in a loss of comm expression in the midline but has little effect on the lateral and early expression of comm .
this suggests that dichaete interacts with a specific regulatory element of the comm gene that control midline expression and does not act as a general activator of comm expression.
our observations also further emphasise the functional conservation between dichaete and sox <dig>
whereas comm expression is lost from the early midline in dichaete mutant and dn expressing conditions, by stage  <dig>  when high levels of comm are detected in midline glia and commissural axons of wild type embryos , the effects of loss of dichaete function are less pronounced.
while 75-80%  of the dichaete-Δhmg embryos show reduced comm expression, primarily in midline cells and to a much lesser extent in commissural axons, effects are far less pronounced in dichaete null mutants or when enrep constructs are expressed with sim-gal <dig> .
providing enrep constructs early in development via the maternal gal <dig> driver results in a loss of comm in the midline, and again the effects are much less pronounced in axons.
since axonal comm expression arises from lateral commissural neurons extending processes to the midline, this expression is likely to be regulated by enhancers distinct from those controlling midline expression.
we expect that the reduction in axonal comm expression we observed at the midline is therefore most likely due to a general disruption of the midline by, for example, the loss of slit.
together our observations suggest that most dichaete mutant embryos are able to establish glial expression after stage  <dig> in the absence of dichaete and suggests that a different, dichaete independent, regulatory element may control the early and late aspects of comm expression.
taken together with the observation on lateral and early comm expression, our experiments emphasise the modularity of regulatory sequence organisation and suggest that there is little crosstalk between the enhancers controlling specific facets of comm expression.
dichaete-mediated regulation of ase in neuroblasts
the expression of the achaete-scute complex  genes ac, l’sc and sc in dichaete and soxn mutants has already been described  <cit> .
however, the effects on the fourth as-c gene asense  have only been described in soxn mutants  <cit> , despite also being a potential dichaete target gene in the developing neuroectoderm.
we therefore examined the effects of dichaete mutations and dn constructs on its expression.
in the wild type embryo, ase is initially expressed in cns neuroblasts in three well-defined columns at late stage  <dig>  <cit> .
in dichaete null mutant embryos , the expression of ase is detected earlier and is stronger at stages  <dig> and  <dig> than in wild type.
after stage  <dig>  the intensity of ase expression shows no apparent difference between wild type and dichaete mutants.
at later stages, the ase expression pattern becomes disrupted, reflecting, at least in part, the segmentation and cns disruptions characteristic of dichaete mutants.
thus, as with ac in the intermediate column, dichaete appears to act as a repressor of ase.
however, in contrast to the results with ac, the effects on ase expression differ between dichaete mutants and the dn-sox constructs.
with dichaeteΔhmg driven by pros-gal <dig>  we observe a general loss of the ase expressing cells in neuroectoderm in 10-15% of embryos, particularly obvious in the medial and intermediate columns after stage  <dig> .
embryos expressing the other sox dn constructs show much less severe phenotypes.
.
dichaete binding at target genes
to strengthen the view that dichaete directly regulates comm and ase, we examined the genome-wide chip-array binding profiles generated by bdtnp  <cit>  and the modencode project  <cit>  and found binding intervals identified at 1% false discovery rate  cut-offs.
the bdtnp data were generated using chromatin from 2– <dig> hr  embryos and the modencode data from 0– <dig> hr  embryos.
together these data may not entirely reflect aspects of late dichaete binding.
we therefore performed specific chip-pcr assays around the ase and comm genes as well as the known target sli using chromatin extracted from 0– <dig> hr embryos .
in the case of sli, the bdtnp data shows  <dig> high confidence binding intervals distributed across various introns and we assayed two of these regions with a series of chip-pcr assays .
using a series of  <dig> primer pairs encompassing a region around the known midline enhancer  <cit>  and a region spanning the 5’ end of the gene, we find evidence for extensive dichaete binding that is as broad, if not broader, than the binding regions identified using chip-array.
encouragingly, the strongest enrichment was observed with primers covering the known midline enhancer .
in the case of ase, the bdtnp data identified four regions, two 5’ and two 3’ to the transcription unit.
we found the strongest chip-pcr signals from primers encompassing the immediate 3’ region  but also found weak signals across the body of the genes.
in the case of comm, we found the strongest chip-pcr signals at the immediate 5’ end of the gene  and also found evidence for dichaete binding further 5’, partially overlapping a region identified in the bdtnp and modencode data .
while there is agreement between the chip-array and pcr-based assays, there are clearly some differences.
the bdtnp data from the blastoderm stage appears to identify much broader binding than modencode, and we note that we see evidence for considerable low level amplification in the pcr assays, with many fragments producing weak but detectable products in comparison to the pre-immune control.
these differences may simply reflect the different embryonic stages used in each study or reflect differences in crosslinking conditions.
while higher resolution chip-seq data will be required to adequately define the extent of dichaete binding at particular regulatory elements, our experiments confirm that dichaete is directly associated with the potential targets genes.
taken together with the evidence from immunohistochemistry experiments on the potential targets in dichaete mutants and dn conditions, these data strongly suggest that ase and comm are direct dichaete targets in the embryonic cns.
interestingly, the ascl <dig> and slit <dig> genes in mouse, which are orthologous to ase and sli respectively, are associated with sox <dig> binding  <cit> , hinting that, along with the functional conservation displayed by dichaete and sox <dig>  <cit> , some of their genetic networks in neural precursors may also be conserved.
tissue-specific expression profiling
while candidate gene approaches are useful for identifying a small number of target genes, genomics methods can offer a more global perspective.
to this end, we used the dominant negative constructs to perform genome-wide gene expression studies to identify a broader range of potential dichaete target genes in specific subsets of the cns.
microarray gene expression analysis was performed with rna extracted from whole stage  <dig> embryos expressing the dichaete dn constructs in the ventral midline or in neuroblasts; the wild-type construct was also included to identify any effects of dichaete overexpression in the tissues in question.
each construct was expressed in the midline with sim-gal <dig> and in neuroblasts with pros-gal <dig>  and four biological replicates were performed for each experiment.
a uas-gfp construct was crossed to both gal <dig> drivers in parallel with the dichaete lines to provide control embryos.
using our standard microarray analysis, a number of differentially expressed genes were detected .
the thresholds used to identify differentially expressed genes were average m-value of < − <dig>  or >  <dig> , p-value <  <dig> .
across the six microarray studies, we found a total of  <dig> genes with a significant change in expression in at least one of the experiments.
focusing first on the ventral midline,  <dig> genes in total changed expression in at least one of the experiments driving a dichaete construct with sim-gal <dig> , with the Δhmg construct showing a stronger effect  than the enrep  or wild type  constructs.
a total of  <dig> genes are common to all three experiments, and  <dig> are common to both dn expressing conditions , including bancal, photorepair and cyp4p <dig>  which are downregulated.
the gene bancal was flagged as a target in all  <dig> gene expression studies, the bdtnp chip data shows dichaete binding around it, and it is annotated with gene ontology  terms “transcription factor binding” and “mrna binding”, as well as “cell proliferation” and “cell fate commitment”, making it an interesting potential target of dichaete.
myosin61f, ras-related protein  and cap-h <dig> are upregulated, suggesting they may be indirect targets.
while we did not identify a significant change in sli expression, we did find  <dig> genes identified in a screen for midline expressed genes, including kek <dig>  kr-h <dig>  sema-5c and nemy <cit> .
the comparatively small number of gene expression changes we identified is likely to be a reflection of the fact that the ventral midline is a very small component of the embryo and thus only very pronounced changes in gene expression are expected to be detected by the microarray analysis.
.
in the neuroblast experiment, driving the dn constructs with pros-gal <dig>  we identified a total of  <dig> genes that changed expression in at least one of the experiments .
it was not unexpected that this experiment affects a larger number of genes since the tissue targeted is larger than the midline and consequently it is easier to pick up more subtle changes in gene expression.
again the Δhmg construct showed the strongest effect  with a smaller number of changes detected when expressing for the enrep  or wild type .
while we might have expected the enrep construct to consistently downregulate genes, we were surprised to find that 30-40% of misregulated genes showed significant upregulation .
focusing on the genes changing expression in both the dn conditions, we identified  <dig> genes and of these  <dig> were downregulated in the presence of the enrep construct.
we found that  <dig> of these have go annotations associated with proteolysis and while we considered that this could be a stress or cellular damage response, we note that  <dig> of these genes were downregulated by the expression of the Δhmg construct, suggesting they require dichaete function for their activation.
we identified  <dig> genes that changed expression in both the midline and the neuroblast experiments, suggesting they are good candidate target genes.
while we did not identify any significantly enriched gene ontology terms associated with this set of genes, we did find  <dig> terms linked to development with corrected p-values lower than  <dig>  when we examined the overall list of list of  <dig> differentially regulated genes .
to find further evidence supporting direct dichaete regulation, we compared the set of  <dig> genes with published genome-wide chip datasets  <cit>  and found that  <dig>  of these have evidence for dichaete binding, suggesting that a substantial fraction of the genes we identify may be genuine dichaete targets .
significantly, when we examined the list of  <dig> genes showing expression changes and evidence of dichaete binding in the chip study,  <dig> development related ontology terms were found with much lower p values , including ontology terms associated with nervous system development  and generation of neurons .
of these  <dig> dichaete bound genes,  <dig> have in situ based gene expression data available in the flyexpress database  <cit> .
creating a gene expression map from these genes shows an enrichment in the ventral neuroectoderm and midline early in embryogenesis, and very strong cns enrichment at stage 9– <dig>  when the gene expression profiling was performed .
this suggests that the genes showing expression changes and dichaete binding are likely to constitute bona fide dichaete targets.
we submitted the list of dichaete bound genes to i-cistarget  <cit>  to search for enriched transcription factor binding site matrices and identified a dichaete-like aacaa motif  associated with 45% of the genes .
interestingly, we also found enrichment for motifs associated with tfs identified as potential dichaete targets , e =  <dig> ), hinting at potential regulatory interactions.
these new dichaete target genes include a number of transcriptional regulators  as well over  <dig> genes annotated with the go term signalling, including several components of the notch , egfr , wnt  and jak-stat  pathways.
we noticed that  <dig> of the new target genes are annotated as being involved in lateral inhibition, further implicating dichaete in the early events in the neuroectoderm.
if notch signalling is compromised in the early neuroectoderm, lateral inhibition breaks down and excess neural cells are produced at the expense of epidermis  <cit> .
in our analysis of ac expression we found extra ac-positive cells in the medial column consistent with a reduction in lateral inhibition .
in particular, we noticed that while expression of enrep constructs generally results in loss of ac expression, we did observe hemisegments with increased numbers of ac-positive cells .
as well as these early events in neural development, we found  <dig> genes involved in neuron differentiation , suggesting that dichaete acts at the level of terminal differentiation genes.
focusing on aspects of neural development, we found that 18%  of the bound and mis-regulated genes have cns development go annotations .
analysis of this set of  <dig> genes with the string interaction database  <cit>  reveals a highly connected network centred on the wnt and egfr pathways, further emphasising the dichaete role in regulating multiple signalling pathways during nervous system development .
taken together, our expression analysis combined with published dichaete binding data identify a large number of likely direct targets.
clearly these genes will need further validation, however the lists provide a platform for the further analysis of group b sox activity in subsets of the cns.
our experiments also emphasise that ideally data from multiple experiments is needed when interpreting results generated by experiments expressing dominant negative proteins due to the lack of sensitivity and existence of off-target effects.
discussion
in this study we have investigated the use of dominant negative alleles of the sox domain transcription factor dichaete to identify tissue-specific functions and potential target genes.
we show that expression of dichaete alleles with a deleted dna binding domain or fused to a strong transcriptional repressor in subsets of the embryonic cns produce dichaete-like phenotypes.
analysis of predicted target genes in the cns midline  or neuroectoderm  suggest that dichaete directly regulates these genes as judged by molecular phenotypes observed both in dichaete mutants and when exposed to the dn constructs.
expression profiling embryos expressing dn constructs in the cns midline or in neuroblasts identifies approximately  <dig> genes showing altered expression.
while some of the identified gene expression changes will be attributable to off-target effects of the dn constructs, we were encouraged to find that half of misexpressed genes are associated with in vivo dichaete binding, indicating that we have identified a substantial repertoire of new dichaete targets.
we validated the activity of the dn constructs on the established dichaete midline target gene slit, showing that expression of both proteins in the midline led to loss of slit expression and dichaete-like midline phenotypes.
similarly, in the neuroectoderm we confirmed previous work showing that ac is a direct dichaete target  <cit>  with the Δhmg construct leading to derepression of ac in the medial column of the neuroectoderm, expression of the enrep fusion leading to ac repression and chip-pcr assays with dichaete antiserum enriching regulatory sequences in the slit gene.
we went on to identify two new dichaete targets in the cns, comm in the midline and ase in the neuroectoderm.
in midline glia, a cell type where dichaete is known to control slit expression, the comm gene is also expressed in the midline although its function here is not understood  <cit> .
targeting dn expression to the midline, we found a loss of comm expression.
significantly, we also found that when enrep fusions were expressed throughout the embryo via a maternal gal <dig> driver, only midline comm expression was affected while lateral domains were unaffected.
these observations indicate that the action of dn dichaete constructs on comm is not a general effect, but instead targets specific regulatory elements.
again we found support for direct dichaete action by analysing published dichaete chip-array data and performing specific chip-pcr assays.
these observations suggest that dichaete is only involved in the initial establishment of comm expression in the midline, since later in development comm becomes dichaete independent.
this is analogous to our previous finding that dichaete is required for the establishment of primary pair-rule gene expression at the blastoderm stage, but that after cellularisation these genes are expressed independently of dichaete  <cit> .
this difference between expression establishment and maintenance has previously been described for the runt repressor and may be a more widespread feature of transcription factor biology  <cit> .
however, arguing against sox-independent late comm expression, we note that stage  <dig>  when comm expression recovers in dichaete mutants, is when the soxn gene first becomes active in the midline  <cit> , suggesting loss of dichaete may rescued by soxn expression.
since we find that late comm expression is affected in Δhmg expressing embryos and when the enrep fusions are supplied ubiquitously, it is possible that dichaete and soxn use the same co-factors to regulate comm.
in the neuroectoderm we found that ase, a member of the as-c that acts as a neural precursor gene  <cit> , is derepressed in dichaete mutants.
we found earlier than normal expression of ase in the early neuroectoderm along with evidence for dichaete binding in chip assays.
thus, like the proneural as-c complex member ac, dichaete appears to act as a repressor.
whether this is also in conjunction with the ind and vnd homeodomain proteins remains to be determined.
interestingly, in dichaete mutants we observe derepression of ase in all three columns of the neuroectoderm, whereas dichaete expression is restricted to the medial and intermediate columns after gastrulation.
this observation suggests that earlier dichaete expression at the blastoderm stage may influence prepatterning throughout the neuroectoderm.
we expect that expression of the enrep constructs would have little effect at stage 9– <dig> since ase expression is already very low in the wild type at this stage.
it was also observed that expression of Δhmg constructs has little effect during early stages of development.
there are a number of possible reasons for this: most likely dichaete mediated ase repression occurs earlier in development than the activation of the pros-gal <dig> driver, alternatively the Δhmg constructs might be unable to sequester the relevant co-factors.
it is also possible that ase may be an indirect target, however our other evidence suggests this is not the case.
we found severe disruption of ase expression when all of the dn constructs are expressed via a maternal gal <dig> driver , however it is likely that this is due to pleiotropic effects of inhibiting dichaete function.
our analysis uncovered a number of potential off-target effects resulting from the expression of both versions of dn dichaete.
in particular we note that phenotypes tended to be stronger than those observed in dichaete null embryos and we also saw stronger phenotypes when expression of dn constructs was performed in a dichaete null background.
we do not attribute these effects to misexpression per se, since expression of wild type dichaete under the same conditions resulted in very mild phenotypes and also rescues dichaete null phenotypes.
in addition, expression of wild type dichaete resulted in a small number of significant gene expression changes, presumably reflecting some dosage-dependent effects of dichaete.
unexpectedly from the phenotypic analysis, we found that both in the midline and in neuroblasts the Δhmg construct affected the expression of twice as many genes as the enrep construct.
expression of Δhmg mostly led to down regulation of transcript levels, with 70-90% of significantly changed genes repressed compared to controls.
we imagine that the Δhmg protein acts by sequestering dna binding proteins or other cofactors that normally interact with dichaete but that this will also result in these cofactors being unavailable for interaction with other tfs and thus affect other, dichaete-independent, processes.
for example, we know that dichaete interacts with vvl and sim in the midline  <cit> , however, both these proteins play much more extensive roles in embryonic development and, in particular, show stronger midline phenotypes than dichaete mutants  <cit> .
similarly, dichaete is known to interact with ind  <cit>  in the neuroectoderm, but only in a subset of this transcription factor’s expression domain.
therefore at least some of the effects we see from expression of the Δhmg will be attributable to direct action on dichaete targets.
in the case of the enrep fusion, we anticipated that binding of this protein to dichaete targets in the genome would result in the repression of these genes  <cit> , a view reinforced by preliminary data from expression profiling dichaete null mutant embryos indicating that dichaete mainly acts as a transcriptional activator .
we were therefore surprised to find as much as 40% of misregulated genes identified in the enrep screens showed increased expression compared to controls.
these observations may indicate a substantial level of off-target effect, where upregulated genes reflect secondary changes in expression due to repression of a gene encoding a repressor.
alternatively, the upregulation may result from unanticipated interactions between the regulatory proteins binding together at cis-regulatory elements such that the effects of the enrep domain are masked.
finally, it is possible that in the case of some highly expressed genes the engrailed repressor domain may not function and the enrep construct behaves like a dichaete overexpression construct.
while our focused analysis identified two likely dichaete targets, the published chip data and our preliminary dichaete mutant expression profiling suggest that dichaete may regulate thousands of targets in the drosophila genome, and the expression profiling experiments presented here support this view.
while some of the gene expression changes we identify reflect secondary consequences downstream of direct dichaete targets, we were reassured to find evidence for dichaete binding associated with over half of the misregulated genes.
significantly, these bound and regulated genes are enriched for nervous system expression and also show significant over-representation of gene ontology terms associated with development and specifically cns development.
a number of genes encoding transcription factors associated with cns development were identified in the screen , indicating that dichaete may play diverse roles in controlling aspects of the regulatory networks underpinning cns development.
we also identified enriched binding motifs for hb and lola associated with these bound and regulated genes, hinting at potential regulatory interactions involving dichaete.
of particular interest is hb, which is known to be involved in the temporal sequence of tfs that specify neuroblast fates  <cit> .
dichaete has been shown to participate in a similar process during larval cns development  <cit>  and our observations suggest that, as well as being expressed together in neuroblasts, there may be a regulatory loop between dichaete and hb in the embryo.
other potentially interesting dichaete targets include nejire , encoding a histone acetyltransferase involved in both segmentation and neuron differentiation, bancal, encoding a splicing regulator, and several components of the notch signalling pathway including the genes encoding the delta ligand and the transcriptional co-repressor hairless.
as we note above, the presence of extra ac expressing cells observed when expressing dn constructs is consistent with a loss of lateral inhibition and suggests that dichaete regulates multiple steps in the neural specification pathway.
group b sox proteins have previously been shown to regulate notch signalling in vertebrates by directly regulating the hes class bhlh genes  <cit>  and we identified the notch pathway repressor encoded by hairless as well as the bearded-family member em <dig> as putative direct dichaete targets.
we also identified several components of the wnt signalling pathway as target genes.
previously both dichaete and soxn have been shown to negatively regulate the wnt pathway in the embryonic epidermis  <cit>  and our studies suggest this extends to other tissues.
interestingly, in the epidermis a key target of soxn/dichaete is shavenbaby, which is regulated by both wnt and egfr signalling.
our screen also identified sprouty and cbl, encoding two global inhibitors of the egfr pathway  <cit> , suggesting a more general sox-mediated interaction between egfr and wnt pathways.
sox factors have been proposed to interact with wnt signalling pathways during a variety of developmental processes  <cit>  and our data suggests that this may occur at multiple levels.
CONCLUSIONS
taken together our study acts as a starting point for further analysis of the role dichaete plays in the gene regulatory networks governing the development of the embryonic cns in drosophila.
we validate new dichaete target genes in the midline and neuroectoderm as well as identifying a set of over  <dig> dichaete bound and regulated genes.
given the functional conservation between dichaete and vertebrate sox <dig>  as well as the wider association between group b sox proteins and cns development, we suggest these data may be more widely applicable to understanding the role sox proteins play in critical developmental processes.

 during the process of development, precise changes in gene expression are critical for correctly timed differentiation and body plan development. tissue-specific gene expression is often highly conserved across distantly related species and changes in gene expression are thought to contribute to speciation and the evolution of phenotypic diversity. however, the search for direct targets of the transcription factors regulating developmental processes and the identification of regulatory networks remains challenging. in particular, it is difficult to differentiate between downstream effects, functional compensation by related genes and genuine direct targets. in the context of conserved families of transcriptional regulators, research in model organisms utilizing a number of different genetic tools can be helpful in elucidating the precise regulatory mechanisms at play in specific tissues with the hope that such insights are generalisable to a broader range of species. group b sox domain factors represent such a family of conserved transcription factors with roles in a number of important developmental processes, including the regulation of stem cell pluripotency, eye development, and drosophila segmentation among others. in the context of the central nervous system, early expression of group b sox genes in the neuroectoderm is detected in all metazoans examined to date, and in at least some cases it is known that this expression is functionally important. however, a degree of functional redundancy between different sox genes expressed in the same tissues makes this a difficult gene family to study and means that comparatively few direct regulatory targets are known despite the importance of this class of transcription factor. in drosophila there are four group b genes: soxneuro, dichaete, sox21a and sox21b, the last three organised in a genomic cluster that is conserved in insects. soxn and dichaete encode group b proteins corresponding to mammalian sox and sox respectively. we have previously shown that the mouse sox gene is able to effectively rescue dichaete mutant phenotypes, highlighting the considerable functional conservation exhibited by sox proteins. dichaete has an early role in embryonic segmentation, where it acts to regulate the expression of primary pair rule genes, and is then active in the anlage of the central nervous system. in the early stages of drosophila cns specification, dichaete and soxn are required for the correct expression of the proneural gene achaete and in this role they cooperate with the dv-patterning homeodomain proteins intermediate neuroblasts defective and ventral neuroblasts defective. after gastrulation, dichaete is dynamically expressed in many segregated neuroblasts and their progeny. in the developing cns, dichaete has a defined phenotype in the ventral midline where it is a uniquely expressed sox gene early in embryogenesis. it is required for the correct specification and development of midline glial cells, directly regulating the slit gene via interaction with the pou domain protein ventral veins lacking and the pas domain protein encoded by single minded. interestingly, a sox-pou interaction is also a critical part of early mouse development, where sox partners with oct. while the precise role dichaete plays during the later stages of embryonic cns development has not yet been clarified, a recent study suggests a role in maintaining the stem cell-like neuroblasts in a self-renewing state, with downregulation of dichaete leading to exit from the cell cycle and premature differentiation, again showing similarity to the function of sox in mammals. dichaete and soxn are both known to be active in the early neuroectoderm and, similarly to mammalian group b genes, display a degree of functional redundancy or compensation. mutations in either of these genes exhibit relatively weak phenotypes in cells where they are co-expressed but double mutants show severe neural hypoplasia, suggesting that at least one group b gene is necessary for the correct specification or differentiation of early neural progenitors. however, while it is clear the fly group b sox proteins can functionally compensate in regions of the neuroectoderm, a careful analysis of the phenotypes suggests that each protein has a unique role, even in cells that they are co-expressed in. this is most apparent in the intermediate column of the neuroectoderm where dichaete acts to repress the expression of the proneural gene achaete whereas soxn appears to have activating functions. to date, the number of characterised direct targets of dichaete in the developing cns is comparatively small, including the proneural gene ac in the neuroectoderm and slit in the midline. the early embryo genome-wide binding profiles published by the berkeley drosophila transcription network project and the modencode consortium identify dichaete binding at thousands of genomic locations, suggesting a considerable number of direct target genes. however, since the binding profiles were obtained using whole embryos, they do not give insight into tissue-specific aspects of dichaete function. similarly, due to functional compensation and pleiotropy it is also difficult to tease apart tissue-specific aspects of dichaete function from the genomic analysis of loss-of-function mutants. in this study, we probed tissue-specific dichaete function in vivo, using two newly developed dominant negative dichaete alleles. we focused on specific neural tissues in the early embryo: the neuroectoderm and the ventral midline. we first characterised the constructs using phenotypic analysis to determine whether their overall effect matches that of dichaete mutants. we then used a mixture of phenotypic and binding analysis to characterise two new putative dichaete direct target genes, commisureless in the midline and asense in the neuroectoderm. we showed that dichaete directly binds to regulatory elements associated with these genes. finally, we expressed the dominant negatives in specific neural tissues and performed whole transcriptome expression profiling, comparing these data with results from existing genome-wide binding studies to identify a set of potential dichaete targets. using these data, we also assess the appropriateness of dominant negatives as a tool for unravelling tissue-specific transcription factor function.  evaluation of dominant negative constructs since dichaete is widely and dynamically expressed during drosophila development and null mutations have pleiotropic effects, its function in specific tissues is difficult to study in detail. to facilitate tissue-specific analysis of dichaete action, we generated dominant negative dichaete proteins and expressed them in specific tissues via the gal4-uas system. we produced three types of construct: two dominant negative proteins, either lacking the dichaete hmg dna-binding domain or a fusion between the full length dichaete and the strong repressor domain from the engrailed protein, we also produced a full length wild-type dichaete control. both types of dominant negative protein have been previously reported in work with vertebrate group b sox proteins: the enrep in xenopus, chick and zebrafish, and the Δhmg in xenopus. enrep fusions are reported to turn transcription factors into strong repressors, effectively shutting down the expression of target genes. the way that Δhmg proteins act is less clear but it is likely they function by sequestering essential co-factors. thus our expectations when using the dn constructs was that the enrep fusion would reveal sets of genes where dichaete function is required for activation but would not detect genes where dichaete normally acts as a repressor, whereas the Δhmg construct should uncover genes repressed or activated by dichaete via interactions with a cofactor. since we have previously shown that sox effectively rescues dichaete mutant phenotypes, we also generated a similar set of dominant negative constructs using mouse sox to test the generality of our approach. to assess the effectiveness of the dn constructs we expressed each of them in the midline of wild type flies using a sim-gal driver and in neuroblasts using a pros-gal driver. in the case of sim-gal we used a driver line heterozygous for sim-gal on the 2nd and homozygous for sim-gal on the 3rd chromosome crossed to homozygous uas lines. for pros-gal a 2nd chromosome homozygous driver line was crossed with homozygous uas lines. in both cases, all progeny express the transgene. we have previously shown that expressing wild type dichaete or msox with a midline driver rescues dichaete null mutant phenotypes: both dichaete Δhmg and enrep constructs produced dichaete-like phenotypes in the cns when expressed with sim-gal bp staining of dn-dichaete expressing embryos showed collapsed commissures, reduced separation between the longitudinal tracts, gaps in longitudinal connectives, misrouting and inappropriate midline crossing by longitudinal axons. similar phenotypes were observed with the msox constructs and in all cases 50-70% of embryos exhibited some degree of cns disruption. in agreement with our previous observations, expression of the wild type sox proteins under the same conditions produced very mild phenotypes. focusing on the longitudinal connectives as revealed by fasii staining, we observed strong cns phenotypes with both the fly and mouse dn constructs. since slit is a dichaete target gene in the midline, we examined slit in dn expressing conditions and found, as expected, a reduction in expression. this observation indicates that, at the level of an individual target gene, the dn constructs mimic dichaete loss of function. however, with both fasii and bp staining, the phenotypes we observed were more severe than those exhibited by dichaete null mutants. this suggests that the dn constructs affect processes additional to those regulated by dichaete and to test this we expressed dn constructs in the midline of dichaete null mutants. whereas the wild type sox proteins rescue the cns phenotypes, expression of dn constructs produced phenotypes more severe than those of dichaete mutants, supporting the view that the dn constructs, in addition to disrupting normal dichaete function, are likely to have additional effects and thus act as neomorphic alleles.. since previous studies indicate that dichaete acts to repress achaete expression in the neuroectoderm, we used the dn constructs to investigate the effects on achaete expression in the early neuroectoderm using a prosgal driver. in the stage embryo, ac is expressed in a single medial column neuroblast in each hemisegment. consistent with previous reports, in dichaete mutants we see a variable increase in the number of ac positive cells in the medial column and the appearance of cells in the intermediate column, with up to cells observed in a single hemisegment as well as a more general diffuse staining indicative of low level derepression in the neuroectoderm. driving the dichaete or msox Δhmg constructs with prosgal results in a similar increase in ac positive cells, with both fly and mouse proteins behaving similarly. in contrast, when constructs carrying the enrep fusions are expressed under the same conditions we see a loss of ac expression in the medial column and no ectopic expression in the intermediate column. in addition, we found that expressing Δhmg in the early embryo resulted in precocious expression of ac, with 50-60% of dichaeteΔhmg or sox Δhmg expressing embryos showing weak ac expression at stage 6/ in contrast to wild type expression at stage these observations support the view that dichaete directly regulates ac and further confirm that the dn constructs behave as expected. specifically, we note that expression of the enrep construct results in a loss of ac expression consistent with its expected activity repressing targets genes. in contrast, the Δhmg construct results in ectopic expression of ac in both medial and intermediate columns, consistent with the idea that it interferes with wild type dichaete function and supports the idea that dichaete normally acts as a repressor of gene expression in some circumstances.. intermediate = fraction of hemisegments expressing ac in the intermediate column. medial down = fraction of hemisegments showing loss of ac expression in the medial column. medial up/extra = fraction of hemisegments showing stronger ac expression or extra ac expressing cells in the medial column. in all cases n >  embryos. nd = not done. dichaete regulates comm in the midline to date only two direct dichaete target genes, slit and ac, have been identified and to learn more about how dichaete regulates gene expression in the developing cns we sought to identify additional direct dichaete targets. to do this we examined gene expression data, dna binding data and the literature to identify candidates genes for further study, focusing on genes known to be expressed in the midline or early neuroectoderm. dichaete acts directly in the midline, and in particular is known to function in midline glia. the commisureless gene is expressed in midline glia and in dichaete mutants we observe a loss of comm expression in the midline of stage embryos. significantly, lateral epidermal stripes of comm expression as well as earlier blastoderm and neuroectodermal expression are not affected in the dichaete mutants, suggesting dichaete does not generally regulate comm expression in the embryo.. the hypothesis that comm is a direct target of dichaete in the cns was supported by experiments using the dn constructs: expressing Δhmg or enrep proteins in the midline results in the loss of comm expression along the length of the midline. further to this, strong and ubiquitous expression of msox2enrep via a maternal gal4-vp driver results in a loss of comm expression in the midline but has little effect on the lateral and early expression of comm. this suggests that dichaete interacts with a specific regulatory element of the comm gene that control midline expression and does not act as a general activator of comm expression. our observations also further emphasise the functional conservation between dichaete and sox whereas comm expression is lost from the early midline in dichaete mutant and dn expressing conditions, by stage when high levels of comm are detected in midline glia and commissural axons of wild type embryos, the effects of loss of dichaete function are less pronounced. while 75-80% of the dichaete-Δhmg embryos show reduced comm expression, primarily in midline cells and to a much lesser extent in commissural axons, effects are far less pronounced in dichaete null mutants or when enrep constructs are expressed with sim-gal. providing enrep constructs early in development via the maternal gal driver results in a loss of comm in the midline, and again the effects are much less pronounced in axons. since axonal comm expression arises from lateral commissural neurons extending processes to the midline, this expression is likely to be regulated by enhancers distinct from those controlling midline expression. we expect that the reduction in axonal comm expression we observed at the midline is therefore most likely due to a general disruption of the midline by, for example, the loss of slit. together our observations suggest that most dichaete mutant embryos are able to establish glial expression after stage in the absence of dichaete and suggests that a different, dichaete independent, regulatory element may control the early and late aspects of comm expression. taken together with the observation on lateral and early comm expression, our experiments emphasise the modularity of regulatory sequence organisation and suggest that there is little crosstalk between the enhancers controlling specific facets of comm expression. dichaete-mediated regulation of ase in neuroblasts the expression of the achaete-scute complex genes ac, l’sc and sc in dichaete and soxn mutants has already been described. however, the effects on the fourth as-c gene asense have only been described in soxn mutants, despite also being a potential dichaete target gene in the developing neuroectoderm. we therefore examined the effects of dichaete mutations and dn constructs on its expression. in the wild type embryo, ase is initially expressed in cns neuroblasts in three well-defined columns at late stage. in dichaete null mutant embryos, the expression of ase is detected earlier and is stronger at stages and than in wild type. after stage the intensity of ase expression shows no apparent difference between wild type and dichaete mutants. at later stages, the ase expression pattern becomes disrupted, reflecting, at least in part, the segmentation and cns disruptions characteristic of dichaete mutants. thus, as with ac in the intermediate column, dichaete appears to act as a repressor of ase. however, in contrast to the results with ac, the effects on ase expression differ between dichaete mutants and the dn-sox constructs. with dichaeteΔhmg driven by pros-gal we observe a general loss of the ase expressing cells in neuroectoderm in 10-15% of embryos, particularly obvious in the medial and intermediate columns after stage. embryos expressing the other sox dn constructs show much less severe phenotypes.. dichaete binding at target genes to strengthen the view that dichaete directly regulates comm and ase, we examined the genome-wide chip-array binding profiles generated by bdtnp and the modencode project and found binding intervals identified at 1% false discovery rate cut-offs. the bdtnp data were generated using chromatin from 2– hr embryos and the modencode data from 0– hr embryos. together these data may not entirely reflect aspects of late dichaete binding. we therefore performed specific chip-pcr assays around the ase and comm genes as well as the known target sli using chromatin extracted from 0– hr embryos. in the case of sli, the bdtnp data shows high confidence binding intervals distributed across various introns and we assayed two of these regions with a series of chip-pcr assays. using a series of primer pairs encompassing a region around the known midline enhancer and a region spanning the 5’ end of the gene, we find evidence for extensive dichaete binding that is as broad, if not broader, than the binding regions identified using chip-array. encouragingly, the strongest enrichment was observed with primers covering the known midline enhancer. in the case of ase, the bdtnp data identified four regions, two 5’ and two 3’ to the transcription unit. we found the strongest chip-pcr signals from primers encompassing the immediate 3’ region but also found weak signals across the body of the genes. in the case of comm, we found the strongest chip-pcr signals at the immediate 5’ end of the gene and also found evidence for dichaete binding further 5’, partially overlapping a region identified in the bdtnp and modencode data. while there is agreement between the chip-array and pcr-based assays, there are clearly some differences. the bdtnp data from the blastoderm stage appears to identify much broader binding than modencode, and we note that we see evidence for considerable low level amplification in the pcr assays, with many fragments producing weak but detectable products in comparison to the pre-immune control. these differences may simply reflect the different embryonic stages used in each study or reflect differences in crosslinking conditions. while higher resolution chip-seq data will be required to adequately define the extent of dichaete binding at particular regulatory elements, our experiments confirm that dichaete is directly associated with the potential targets genes. taken together with the evidence from immunohistochemistry experiments on the potential targets in dichaete mutants and dn conditions, these data strongly suggest that ase and comm are direct dichaete targets in the embryonic cns. interestingly, the ascl and slit genes in mouse, which are orthologous to ase and sli respectively, are associated with sox binding, hinting that, along with the functional conservation displayed by dichaete and sox, some of their genetic networks in neural precursors may also be conserved. tissue-specific expression profiling while candidate gene approaches are useful for identifying a small number of target genes, genomics methods can offer a more global perspective. to this end, we used the dominant negative constructs to perform genome-wide gene expression studies to identify a broader range of potential dichaete target genes in specific subsets of the cns. microarray gene expression analysis was performed with rna extracted from whole stage embryos expressing the dichaete dn constructs in the ventral midline or in neuroblasts; the wild-type construct was also included to identify any effects of dichaete overexpression in the tissues in question. each construct was expressed in the midline with sim-gal and in neuroblasts with pros-gal and four biological replicates were performed for each experiment. a uas-gfp construct was crossed to both gal drivers in parallel with the dichaete lines to provide control embryos. using our standard microarray analysis, a number of differentially expressed genes were detected. the thresholds used to identify differentially expressed genes were average m-value of < − or > , p-value < . across the six microarray studies, we found a total of genes with a significant change in expression in at least one of the experiments. focusing first on the ventral midline, genes in total changed expression in at least one of the experiments driving a dichaete construct with sim-gal, with the Δhmg construct showing a stronger effect than the enrep or wild type constructs. a total of genes are common to all three experiments, and are common to both dn expressing conditions, including bancal, photorepair and cyp4p which are downregulated. the gene bancal was flagged as a target in all gene expression studies, the bdtnp chip data shows dichaete binding around it, and it is annotated with gene ontology terms “transcription factor binding” and “mrna binding”, as well as “cell proliferation” and “cell fate commitment”, making it an interesting potential target of dichaete. myosin61f, ras-related protein and cap-h are upregulated, suggesting they may be indirect targets. while we did not identify a significant change in sli expression, we did find genes identified in a screen for midline expressed genes, including kek kr-h sema-5c and nemy. the comparatively small number of gene expression changes we identified is likely to be a reflection of the fact that the ventral midline is a very small component of the embryo and thus only very pronounced changes in gene expression are expected to be detected by the microarray analysis.. in the neuroblast experiment, driving the dn constructs with pros-gal we identified a total of genes that changed expression in at least one of the experiments. it was not unexpected that this experiment affects a larger number of genes since the tissue targeted is larger than the midline and consequently it is easier to pick up more subtle changes in gene expression. again the Δhmg construct showed the strongest effect with a smaller number of changes detected when expressing for the enrep or wild type. while we might have expected the enrep construct to consistently downregulate genes, we were surprised to find that 30-40% of misregulated genes showed significant upregulation. focusing on the genes changing expression in both the dn conditions, we identified genes and of these were downregulated in the presence of the enrep construct. we found that of these have go annotations associated with proteolysis and while we considered that this could be a stress or cellular damage response, we note that of these genes were downregulated by the expression of the Δhmg construct, suggesting they require dichaete function for their activation. we identified genes that changed expression in both the midline and the neuroblast experiments, suggesting they are good candidate target genes. while we did not identify any significantly enriched gene ontology terms associated with this set of genes, we did find terms linked to development with corrected p-values lower than when we examined the overall list of list of differentially regulated genes. to find further evidence supporting direct dichaete regulation, we compared the set of genes with published genome-wide chip datasets and found that of these have evidence for dichaete binding, suggesting that a substantial fraction of the genes we identify may be genuine dichaete targets. significantly, when we examined the list of genes showing expression changes and evidence of dichaete binding in the chip study, development related ontology terms were found with much lower p values, including ontology terms associated with nervous system development and generation of neurons. of these dichaete bound genes, have in situ based gene expression data available in the flyexpress database. creating a gene expression map from these genes shows an enrichment in the ventral neuroectoderm and midline early in embryogenesis, and very strong cns enrichment at stage 9– when the gene expression profiling was performed. this suggests that the genes showing expression changes and dichaete binding are likely to constitute bona fide dichaete targets. we submitted the list of dichaete bound genes to i-cistarget to search for enriched transcription factor binding site matrices and identified a dichaete-like aacaa motif associated with 45% of the genes. interestingly, we also found enrichment for motifs associated with tfs identified as potential dichaete targets, e =  ), hinting at potential regulatory interactions. these new dichaete target genes include a number of transcriptional regulators as well over genes annotated with the go term signalling, including several components of the notch, egfr, wnt and jak-stat pathways. we noticed that of the new target genes are annotated as being involved in lateral inhibition, further implicating dichaete in the early events in the neuroectoderm. if notch signalling is compromised in the early neuroectoderm, lateral inhibition breaks down and excess neural cells are produced at the expense of epidermis. in our analysis of ac expression we found extra ac-positive cells in the medial column consistent with a reduction in lateral inhibition. in particular, we noticed that while expression of enrep constructs generally results in loss of ac expression, we did observe hemisegments with increased numbers of ac-positive cells. as well as these early events in neural development, we found genes involved in neuron differentiation, suggesting that dichaete acts at the level of terminal differentiation genes. focusing on aspects of neural development, we found that 18% of the bound and mis-regulated genes have cns development go annotations. analysis of this set of genes with the string interaction database reveals a highly connected network centred on the wnt and egfr pathways, further emphasising the dichaete role in regulating multiple signalling pathways during nervous system development. taken together, our expression analysis combined with published dichaete binding data identify a large number of likely direct targets. clearly these genes will need further validation, however the lists provide a platform for the further analysis of group b sox activity in subsets of the cns. our experiments also emphasise that ideally data from multiple experiments is needed when interpreting results generated by experiments expressing dominant negative proteins due to the lack of sensitivity and existence of off-target effects. discussion in this study we have investigated the use of dominant negative alleles of the sox domain transcription factor dichaete to identify tissue-specific functions and potential target genes. we show that expression of dichaete alleles with a deleted dna binding domain or fused to a strong transcriptional repressor in subsets of the embryonic cns produce dichaete-like phenotypes. analysis of predicted target genes in the cns midline or neuroectoderm suggest that dichaete directly regulates these genes as judged by molecular phenotypes observed both in dichaete mutants and when exposed to the dn constructs. expression profiling embryos expressing dn constructs in the cns midline or in neuroblasts identifies approximately genes showing altered expression. while some of the identified gene expression changes will be attributable to off-target effects of the dn constructs, we were encouraged to find that half of misexpressed genes are associated with in vivo dichaete binding, indicating that we have identified a substantial repertoire of new dichaete targets. we validated the activity of the dn constructs on the established dichaete midline target gene slit, showing that expression of both proteins in the midline led to loss of slit expression and dichaete-like midline phenotypes. similarly, in the neuroectoderm we confirmed previous work showing that ac is a direct dichaete target with the Δhmg construct leading to derepression of ac in the medial column of the neuroectoderm, expression of the enrep fusion leading to ac repression and chip-pcr assays with dichaete antiserum enriching regulatory sequences in the slit gene. we went on to identify two new dichaete targets in the cns, comm in the midline and ase in the neuroectoderm. in midline glia, a cell type where dichaete is known to control slit expression, the comm gene is also expressed in the midline although its function here is not understood. targeting dn expression to the midline, we found a loss of comm expression. significantly, we also found that when enrep fusions were expressed throughout the embryo via a maternal gal driver, only midline comm expression was affected while lateral domains were unaffected. these observations indicate that the action of dn dichaete constructs on comm is not a general effect, but instead targets specific regulatory elements. again we found support for direct dichaete action by analysing published dichaete chip-array data and performing specific chip-pcr assays. these observations suggest that dichaete is only involved in the initial establishment of comm expression in the midline, since later in development comm becomes dichaete independent. this is analogous to our previous finding that dichaete is required for the establishment of primary pair-rule gene expression at the blastoderm stage, but that after cellularisation these genes are expressed independently of dichaete. this difference between expression establishment and maintenance has previously been described for the runt repressor and may be a more widespread feature of transcription factor biology. however, arguing against sox-independent late comm expression, we note that stage when comm expression recovers in dichaete mutants, is when the soxn gene first becomes active in the midline, suggesting loss of dichaete may rescued by soxn expression. since we find that late comm expression is affected in Δhmg expressing embryos and when the enrep fusions are supplied ubiquitously, it is possible that dichaete and soxn use the same co-factors to regulate comm. in the neuroectoderm we found that ase, a member of the as-c that acts as a neural precursor gene, is derepressed in dichaete mutants. we found earlier than normal expression of ase in the early neuroectoderm along with evidence for dichaete binding in chip assays. thus, like the proneural as-c complex member ac, dichaete appears to act as a repressor. whether this is also in conjunction with the ind and vnd homeodomain proteins remains to be determined. interestingly, in dichaete mutants we observe derepression of ase in all three columns of the neuroectoderm, whereas dichaete expression is restricted to the medial and intermediate columns after gastrulation. this observation suggests that earlier dichaete expression at the blastoderm stage may influence prepatterning throughout the neuroectoderm. we expect that expression of the enrep constructs would have little effect at stage 9– since ase expression is already very low in the wild type at this stage. it was also observed that expression of Δhmg constructs has little effect during early stages of development. there are a number of possible reasons for this: most likely dichaete mediated ase repression occurs earlier in development than the activation of the pros-gal driver, alternatively the Δhmg constructs might be unable to sequester the relevant co-factors. it is also possible that ase may be an indirect target, however our other evidence suggests this is not the case. we found severe disruption of ase expression when all of the dn constructs are expressed via a maternal gal driver, however it is likely that this is due to pleiotropic effects of inhibiting dichaete function. our analysis uncovered a number of potential off-target effects resulting from the expression of both versions of dn dichaete. in particular we note that phenotypes tended to be stronger than those observed in dichaete null embryos and we also saw stronger phenotypes when expression of dn constructs was performed in a dichaete null background. we do not attribute these effects to misexpression per se, since expression of wild type dichaete under the same conditions resulted in very mild phenotypes and also rescues dichaete null phenotypes. in addition, expression of wild type dichaete resulted in a small number of significant gene expression changes, presumably reflecting some dosage-dependent effects of dichaete. unexpectedly from the phenotypic analysis, we found that both in the midline and in neuroblasts the Δhmg construct affected the expression of twice as many genes as the enrep construct. expression of Δhmg mostly led to down regulation of transcript levels, with 70-90% of significantly changed genes repressed compared to controls. we imagine that the Δhmg protein acts by sequestering dna binding proteins or other cofactors that normally interact with dichaete but that this will also result in these cofactors being unavailable for interaction with other tfs and thus affect other, dichaete-independent, processes. for example, we know that dichaete interacts with vvl and sim in the midline, however, both these proteins play much more extensive roles in embryonic development and, in particular, show stronger midline phenotypes than dichaete mutants. similarly, dichaete is known to interact with ind in the neuroectoderm, but only in a subset of this transcription factor’s expression domain. therefore at least some of the effects we see from expression of the Δhmg will be attributable to direct action on dichaete targets. in the case of the enrep fusion, we anticipated that binding of this protein to dichaete targets in the genome would result in the repression of these genes, a view reinforced by preliminary data from expression profiling dichaete null mutant embryos indicating that dichaete mainly acts as a transcriptional activator. we were therefore surprised to find as much as 40% of misregulated genes identified in the enrep screens showed increased expression compared to controls. these observations may indicate a substantial level of off-target effect, where upregulated genes reflect secondary changes in expression due to repression of a gene encoding a repressor. alternatively, the upregulation may result from unanticipated interactions between the regulatory proteins binding together at cis-regulatory elements such that the effects of the enrep domain are masked. finally, it is possible that in the case of some highly expressed genes the engrailed repressor domain may not function and the enrep construct behaves like a dichaete overexpression construct. while our focused analysis identified two likely dichaete targets, the published chip data and our preliminary dichaete mutant expression profiling suggest that dichaete may regulate thousands of targets in the drosophila genome, and the expression profiling experiments presented here support this view. while some of the gene expression changes we identify reflect secondary consequences downstream of direct dichaete targets, we were reassured to find evidence for dichaete binding associated with over half of the misregulated genes. significantly, these bound and regulated genes are enriched for nervous system expression and also show significant over-representation of gene ontology terms associated with development and specifically cns development. a number of genes encoding transcription factors associated with cns development were identified in the screen, indicating that dichaete may play diverse roles in controlling aspects of the regulatory networks underpinning cns development. we also identified enriched binding motifs for hb and lola associated with these bound and regulated genes, hinting at potential regulatory interactions involving dichaete. of particular interest is hb, which is known to be involved in the temporal sequence of tfs that specify neuroblast fates. dichaete has been shown to participate in a similar process during larval cns development and our observations suggest that, as well as being expressed together in neuroblasts, there may be a regulatory loop between dichaete and hb in the embryo. other potentially interesting dichaete targets include nejire, encoding a histone acetyltransferase involved in both segmentation and neuron differentiation, bancal, encoding a splicing regulator, and several components of the notch signalling pathway including the genes encoding the delta ligand and the transcriptional co-repressor hairless. as we note above, the presence of extra ac expressing cells observed when expressing dn constructs is consistent with a loss of lateral inhibition and suggests that dichaete regulates multiple steps in the neural specification pathway. group b sox proteins have previously been shown to regulate notch signalling in vertebrates by directly regulating the hes class bhlh genes and we identified the notch pathway repressor encoded by hairless as well as the bearded-family member em as putative direct dichaete targets. we also identified several components of the wnt signalling pathway as target genes. previously both dichaete and soxn have been shown to negatively regulate the wnt pathway in the embryonic epidermis and our studies suggest this extends to other tissues. interestingly, in the epidermis a key target of soxn/dichaete is shavenbaby, which is regulated by both wnt and egfr signalling. our screen also identified sprouty and cbl, encoding two global inhibitors of the egfr pathway, suggesting a more general sox-mediated interaction between egfr and wnt pathways. sox factors have been proposed to interact with wnt signalling pathways during a variety of developmental processes and our data suggests that this may occur at multiple levels.  taken together our study acts as a starting point for further analysis of the role dichaete plays in the gene regulatory networks governing the development of the embryonic cns in drosophila. we validate new dichaete target genes in the midline and neuroectoderm as well as identifying a set of over dichaete bound and regulated genes. given the functional conservation between dichaete and vertebrate sox as well as the wider association between group b sox proteins and cns development, we suggest these data may be more widely applicable to understanding the role sox proteins play in critical developmental processes. 
an analysis of the broader range of candidate dichaete targets indicates that dichaete plays diverse roles in cns development, with the or so dichaete-bound putative targets including a number of transcription factors, signalling pathway components and terminal differentiation genes. our analysis also reveals that dominant negatives cause off-target effects, highlighting the need to use other experimental data for validating findings from dominant negative studies. as well as identifying commisureless as a target, our data indicate that dichaete helps establish its expression during early midline development but has less effect on its established later expression, highlighting dichaete action on tissue specific enhancers. in drosophila melanogaster, the sox gene dichaete is dynamically expressed in the embryonic cns, where it is known to have functional roles in neuroblasts and the ventral midline. we expressed these tissue-specifically in the midline and in neuroblasts using the uas/gal system, validating their use at the phenotypic level and with known target genes. using chip and immunohistochemistry, we identified two new likely direct dichaete target genes, commisureless in the midline and asense in the neuroectoderm. these include a number of genes with known roles in cns development, including several components of the notch, wnt and egfr signalling pathways. they are, however, difficult to study as mutations often have pleiotropic effects and other sox family members can mask phenotypes due to functional compensation. group b sox domain transcription factors play important roles in metazoan central nervous system development. in this study, we use inducible dominant negative proteins in combination with chip, immunohistochemistry and genome-wide expression profiling to further dissect the role of dichaete in these two tissues. in the early neurectoderm we implicate dichaete in the lateral inhibition pathway and show that dichaete acts to repress the proneural gene asense.
 group b sox domain transcription factors play important roles in metazoan central nervous system development. they are, however, difficult to study as mutations often have pleiotropic effects and other sox family members can mask phenotypes due to functional compensation. in drosophila melanogaster, the sox gene dichaete is dynamically expressed in the embryonic cns, where it is known to have functional roles in neuroblasts and the ventral midline. in this study, we use inducible dominant negative proteins in combination with chip, immunohistochemistry and genome-wide expression profiling to further dissect the role of dichaete in these two tissues.  we generated two dominant negative dichaete constructs, one lacking a dna binding domain and the other fused to the engrailed transcriptional repressor domain. we expressed these tissue-specifically in the midline and in neuroblasts using the uas/gal system, validating their use at the phenotypic level and with known target genes. using chip and immunohistochemistry, we identified two new likely direct dichaete target genes, commisureless in the midline and asense in the neuroectoderm. we performed genome-wide expression profiling in stage 8– embryos, identifying almost a thousand potential tissue-specific dichaete targets, with half of these genes showing evidence of dichaete binding in vivo. these include a number of genes with known roles in cns development, including several components of the notch, wnt and egfr signalling pathways.  as well as identifying commisureless as a target, our data indicate that dichaete helps establish its expression during early midline development but has less effect on its established later expression, highlighting dichaete action on tissue specific enhancers. an analysis of the broader range of candidate dichaete targets indicates that dichaete plays diverse roles in cns development, with the or so dichaete-bound putative targets including a number of transcription factors, signalling pathway components and terminal differentiation genes. in the early neurectoderm we implicate dichaete in the lateral inhibition pathway and show that dichaete acts to repress the proneural gene asense. our analysis also reveals that dominant negatives cause off-target effects, highlighting the need to use other experimental data for validating findings from dominant negative studies. soxdichaetedrosophilacnsdominant-negativegenomics 