BACKGROUND
during development of the mammalian nervous system, neural progenitors within the neuroepithelium give rise sequentially to neuronal cells and glia.
to achieve these well-orchestrated waves of differentiation, neuroepithelial progenitors are progressively constrained via specific extrinsic and intrinsic signals.
by directly promoting the neuronal fate in neural progenitors, the proneural transcription factors of the bhlh family are essential regulators of neurogenesis from invertebrates to mammals  <cit> .
three proneural genes have been characterized to date in the embryonic mouse telencephalon: neurogenin <dig> and neurogenin <dig> are expressed in neuronal progenitors in dorsal telencephalon  <cit> , which gives rise to the cerebral cortex, while mash <dig> determines the fate of neuronal progenitors in the ventral telencephalon  <cit> , giving rise to the basal ganglia.
in addition to their role in the selection of neuronal progenitors within the neuroepithelium, vertebrate proneural genes have also been shown to specify neuronal subtype identities.
indeed, ngn <dig> is necessary for the proper differentiation of excitatory glutamatergic projection neurons in the cerebral cortex, while mash <dig> promotes the fate of gaba-ergic inhibitory interneurons in the basal ganglia  <cit> .
proneural transcription factors are thus critical regulators for both the initiation of neuronal differentiation and the specification of neurons into distinct regional subtypes.
in order to understand how ngn <dig> and mash <dig> regulate these two different aspects of neurogenesis we have developed a genomic approach incorporating the generation of microarray datasets of mice either lacking or overexpressing proneural genes, phylogenetic footprinting, and analysis using bayesian statistical regression.
the utility of bayesian-based network analyses to determine predictive gene regulatory network structures has been demonstrated in in vitro and invertebrate models  <cit> , but few attempts have been made using datasets derived from mammalian species due to the dependence of these techniques on a relatively large sample size  <cit> .
however, this limitation may be eased through incorporation of data from other sources, such as prior information from the literature and sequence-based transcription factor binding site  information  <cit> .
here, we initially identify a gene regulatory network  from a compilation of the current literature on proneural bhlh protein regulation of telencephalic development during neurogenesis.
next, microarray data from gain-of-function  analysis obtained after electroporation of either ngn <dig> or mash <dig> in the developing mouse dorsal or ventral telencephalon, respectively, were fused with previous and new microarray results from proneural loss-of-function  experiments  <cit> .
this allowed us to identify novel targets of ngn <dig> and mash <dig>  and to formally test literature-based grn hypotheses through bayesian statistical analysis of global gene expression patterns.
a total of nine predicted proneural targets were confirmed by in situ rna hybridization on brain slices from embryos lacking or overexpressing mash <dig> or ngn <dig>  including several members of notch signaling pathway  as well as the rna binding protein elavl <dig> .
members of the wnt signaling pathway are predicted as targets of ngn <dig> in the cerebral cortex.
we then took advantage of the identification of putative downstream effectors of proneural genes to identify conserved binding sites for mash <dig> and ngn <dig>  then scanned the sequence surrounding them for other conserved tfbss.
from this analysis, we predict creb, tcf/lef, pou-domain containing transcription factors, sox <dig>  and mef2a as novel co-factors  and co-regulators  of proneural proteins.
finally, a novel bayesian-based algorithm was developed to compile the microarray data, the tfbs analysis, and the literature-based network, generating a proneural grn for the developing mouse telencephalon.
RESULTS
literature-based network structure
a grn that describes the current understanding of proneural bhlh interactions in the developing telencephalon was developed through a review of the literature .
the reader is referred additional file  <dig> and several important reviews <cit>  for more detailed descriptions of the experimental research underlying this literature-based grn.
gof experiments
to further identify genes regulated by the proneural factors ngn <dig> and mash <dig> in the embryonic telencephalon and complement existing lof data  <cit> , we developed a gof approach using electroporation in a whole embryo culture.
the feasibility of the gof study was tested by injection of a ngn2-expressing or mash1-expressing vector in the telencephalic vesicles of e <dig>  mouse embryos.
the time course of ngn <dig> and mash <dig> overexpression was followed with dll <dig> promoter-lacz reporter transgenes monitoring ngn <dig> and mash <dig> activities, respectively ; see also  <cit> ).
lacz reporter activity was detected  <dig> h after electroporation of ngn <dig> or mash <dig> in the dorsal and ventral sides of telencephalic vesicles, respectively, and reached the highest level  <dig> h after proneural gene electroporation ).
in contrast, activation by endogenous proneural proteins of the two reporters in embryos electroporated with an empty expression vector remained low.
the efficiency of electroporation was similar in the control and ngn2- and mash1-electroporated cortices, as assessed by expression of a co-electroporated gfp plasmid before lacz staining .
large-scale electroporation experiments of ngn <dig> in the dorsal telencephalon and mash <dig> in the ventral telencephalon of e <dig>  embryos were thus performed and,  <dig> h later, the electroporated tissue was processed for rna probe preparation .
identification of candidate ngn <dig> and mash <dig> target genes
we reasoned that by comparing the transcriptome of embryos overexpressing proneural genes with expression data obtained from current mash <dig> lof datasets and previously published ngn <dig> lof datasets  <cit> , we would identify novel downstream effectors of mash <dig> and/or ngn <dig> in the developing telencephalon.
for the study of ngn <dig> targets, data from the cortex of mash1-/-;ngn2-/- double mutant mice was utilized rather than ngn2-/- single mutants in order to avoid the compensation due to de-repression of mash <dig> in the cortex of the later mutants  <cit> .
analysis of microarray data allowed us to split potential proneural targets into three groups  and 2):  genes regulated by both mash <dig> and ngn2;  genes regulated by mash <dig> only; and  genes regulated by ngn <dig> only.
a full list of predicted targets categorized by gene ontology  is presented in additional file  <dig>
briefly, among the  <dig> common targets were  <dig> genes involved in notch signaling  and the rna binding protein elavl <dig> expressed in maturing neurons  <cit> .
specific targets of ngn <dig> include genes involved in signal transduction , transcription factors  such as the cortical differentiation factors nhlh <dig> and bhlhb <dig>  <cit> , proteins with kinase/phosphatase activity  such as the serine/threonine kinase dcx, already shown to be directly regulated by ngn <dig> in the cortex  <cit>  and other cortical genes such as the cell adhesion molecule ephrin a <dig>  <cit> .
interestingly, several components of the wnt pathway are predicted targets of ngn <dig> including wnt7b  <cit> , dix domain containing  <dig>  <cit> , fzd <dig>  fzd <dig>  and tax1bp <dig>  it is noteworthy that neurod, a gene considered as directly regulated by ngn proteins  <cit> , was decreased in the ngn2-/- embryos, but was not significantly upregulated after ngn <dig> electroporation in microarray experiments, suggesting that the window for analysis of electroporated embryos was not suitable.
indeed, in situ hybridization analysis showed that electroporation of ngn <dig> does not induce neurod expression in the telencephalon at  <dig> h, but only at  <dig> h after electroporation .
a similar delay has been reported for induction of neurod by the related gene ngn <dig> in the intestine  <cit> .
this suggests either that neurod is not a direct target of ngn <dig>  or that its expression also requires another factor that is not present at the time of electroporation.
involvement of other genes in the regulation of neurod is supported by the lack of a dramatic change in neurod expression in ngn <dig> null mutant telencephalon  <cit> .
finally, putative targets of mash <dig> include transcription factors  such as the lim homeobox transcription factor isl <dig>  which is potentially involved in the specification of gabaergic projection neurons in the striatum  <cit> , and factors involved in signal transduction such as the gaba vesicular transporter slc32a1/viaat  <cit>  and the gene gp38/podoplanin  <cit> , the notch regulated protein nrarp shown to destabilize the notch intracellular domain   <cit> , and lfng, a modulator of notch signaling regulated directly by mash <dig> in the telencephalon  <cit> .
these putative target genes form the basis for subsequent bioinformatics and network analyses to predict novel co-factors, co-regulators, and grn connectivity.
validation of predicted target genes
to confirm the microarray data, several of the predicted novel targets of ngn <dig> and mash <dig> were validated through rna in situ hybridization analysis of wild type and mutant embryos , and of embryos overexpressing ngn <dig> or mash <dig> .
the predicted ngn <dig> targets nhlh <dig>  mfng, and elavl <dig> are mostly expressed at e <dig>  in the mantle zone of the dorsal telencephalon, while rbdh <dig> and zfp <dig> are also expressed in the ventricular zone.
all five genes are downregulated in the medial part of the dorsal telencephalon of ngn <dig> mutant embryos, indicating that ngn <dig> is indeed required for normal expression of these genes ).
we also show that ngn <dig> can induce expression of the same predicted targets when overexpressed by electroporation in the telencephalon of e <dig>  embryos.
at this stage, their expression is barely detectable in neurons that begin to accumulate above the ventricular zone of the dorsal telencephalon ).
expression of nhlh <dig>  mfng, zfp <dig>  rbdh <dig>  and elavl <dig> is strongly upregulated after electroporation of a ngn2-expressing vector in the dorsal telencephalon , marked by *).
thus, both lof and gof analysis confirm that five predicted targets are indeed regulated by ngn <dig> in the developing mouse telencephalon.
the predicted mash <dig> targets mfng and pdpn are predominantly expressed in the ventricular zone of the ventral telencephalon at e <dig> , while gadd45g is predominantly expressed in the subventricular zone and elavl <dig> in the mantle zone .
all four genes are downregulated in the telencephalon of mash <dig> mutant embryos particularly in the medial part of the ventral telencephalon, which is most severely affected in mash <dig> mutant embryos  <cit>  ).
ectopic expression of mash <dig> in the dorsal telencephalon of e <dig>  embryos strongly induces expression of the four genes ).
mash <dig> also rapidly induces expression of gadd45g and mfng in the embryonal carcinoma cell line p <dig>  as detected by quantitative reverse transcription polymerase chain reaction .
these data suggest that many of the genes identified by microarray analysis of mash <dig> and ngn <dig> gof and lof experiments are indeed regulated by mash <dig> and ngn <dig>
bayesian analysis of literature-based grn
connectivity in the literature-based network structure was quantified based on the gof and lof microarray gene expression datasets.
this bayesian-based method removes the need to rely on fold change cut-offs by examining the strength of the predicted relationships based on concurrently evaluating variability in gene expression patterns of several genes of interest across control and perturbation experiments.
significant connections based on this analysis  are highlighted in red in figure  <dig>  our analysis predicts 86%  of the connections as significant based on the microarray dataset; however, four are considered inhibitory interactions instead of activations, suggesting possible feedback loops between pax <dig> and ngn <dig>  between satb <dig>  ngn <dig> and nhlh <dig>  as well as between neurod and etv <dig> .
specificity of our method was estimated through the application of  <dig> randomly generated datasets by permutation of gene labels using the entire microarray dataset.
this analysis results in an estimated false positive rate of  <dig> %, therefore the chances of seeing  <dig> significant connections out of  <dig> is very low .
two of the connections originating from ngn <dig>  are not considered significant based on the current analysis.
as the algorithm relies on linear relationships between genes, its inability to detect these two connections may be a result of the non-linear variability between ngn <dig> and its targets that is created by the large increase in ngn <dig> transcript levels after electroporation.
in addition, the compensatory action of mash <dig> in the dorsal telencephalon of the ngn2-/- mutants further complicates the algorithm's ability to detect connectivity to common targets such as dll <dig>
bioinformatics prediction of ngn <dig> and mash <dig> co-factors
the identification of potential downstream effectors of mash <dig> and ngn <dig> allowed us to examine the occurrence of defined tfbss in the conserved sequence surrounding those genes.
the e-box recognized by bhlh proteins is a degenerate  <dig> bp  motif .
however, recent evidence suggests that mash <dig> and ngn <dig> have different preferences for the two central residues and that their consensus binding sequences extend even outside of the e-box motif .
based on these and other data, we defined different consensus binding sequences for mash <dig>  and ngn <dig>  .
to identify potential co-factors for ngn <dig> or mash <dig>  we employed phylogenetic footprinting and tfbs search algorithms on a subset of the predicted targets based on the microarray dataset ).
first,  <dig> conserved  putative ngn <dig> binding sites surrounding  <dig> of the predicted ngn <dig> target genes and  <dig> conserved putative mash <dig> binding sites surrounding  <dig> of the predicted mash <dig> target genes and  <dig> of the common target genes were identified .
subsequently, we identified tfbs from the transfac library co-occurring specifically with the putative ngn <dig> binding sites in ngn <dig> targets or with the putative mash <dig> binding sites in mash <dig> and mash1/ngn <dig> common target genes using fisher's exact two-sided test for significance ).
no specific co-factors for mash <dig> were identified in this analysis, whereas camp response element ), yy <dig>  and nkx binding sites were significantly enriched near ngn <dig> binding sites when compared with the sequence around mash <dig> binding sites ).
several of the predicted co-factors have dorsal/ventral restricted expression patterns in the embryonic telencephalon, consistent with a role in neuronal specification ).
bioinformatics prediction of ngn <dig> and mash <dig> co-regulators
next we sought to predict potential co-regulators that may bind at regulatory modules independent from those that bind ngn <dig> or mash <dig>  in particular, we were interested in identifying transcription factors that may confer regulation at modules closer to the tss .
in order to efficiently search for co-regulators, we identified evolutionary conserved tfbs  using the confac algorithm  <cit>  to analyze a shorter sequence  in the 5' flanking region of  <dig> predicted mash <dig>  ngn <dig>  and common targets .
to predict putative common co-regulators, we tested for enrichment of the identified tfbs in ngn <dig> target genes and mash1/common target genes compared with a set of  <dig> randomly selected genes using the mann-whitney statistical test.
we identified  <dig> potential ngn2- and mash1-specific co-regulators by testing for tfbs enrichment in ngn <dig> targets versus mash <dig> targets and vice versa.
we identified  <dig> putative co-regulators from these analyses after eliminating those transcription factors that are not expressed in either the dorsal or ventral developing telencephalon based on microarray transcript levels .
predicted common co-regulators include e2f <dig>  tef, nfy, egr <dig>  hes <dig>  and pou-domain containing factors.
predicted co-regulators were specifically enriched in ngn <dig> targets when compared with mash <dig> and common targets include sox <dig>  creb, tcf <dig>  lef <dig>  mef2a, and yy <dig>  no transcription factors were significantly enriched when mash <dig> target genes were compared against ngn <dig> target genes.
this approach identified all five of the predicted ngn <dig> co-factors found in the previous analysis, as well as identifying several transcription factors that may co-regulate the putative ngn <dig> and mash <dig> targets at independent regulatory modules including e2f <dig>  egr <dig>  hes <dig>  and nfy in both ngn <dig> and mash <dig> targets and sox <dig> and mef2a in ngn <dig> target genes.
through interrogation of online databases of in situ hybridization in serial brain sections across development  <cit> , we confirmed the expression of several predicted co-factors/co-regulators in the developing dorsal and ventral telencephalon .
interestingly, several of the predicted co-factors/co-regulators, including sox <dig>  crebbp, creb <dig>  tcf <dig>  lef <dig>  pou6f <dig>  pou2f <dig>  pou3f <dig>  tef, hes <dig>  and e2f <dig>  show appreciable expression in the ventricular zone of dorsal and/or ventral telencephalon, where proneural bhlh proteins are expressed.
furthermore, direct protein-protein interactions between proneural bhlh proteins and crebbp, tcf <dig>  and mef2a are reported in the human protein reference database  <cit> .
bayesian network analysis with an informative prior structure
to provide an integrated view of the network regulated by proneural genes, information obtained from the literature, novel expression data, and phylogenetic footprinting analyses were quantitatively linked through application of the bayesian-based tao-gen algorithm  <cit>  with the addition of an informative prior structure .
nodes represented in the network include several candidate mash <dig> and ngn <dig> target genes from the fusion and sorting of the gof/lof microarray datasets, as well as candidate co-factors and co-regulators from the phylogenetic footprinting analyses.
an informative prior structure considered all significant literature-based connections as required  and used the tfbs information from the phylogenetic footprinting analyses to weight connections in which tfbs information was found.
for example, a conserved sox <dig> tfbs was identified in the sequence surrounding lfng, therefore a preference is given to sox <dig> being a parent to lfng, which results in a slightly higher probability that a linear relationship between these two genes will be significant in the posterior distribution.
we have confirmed the reliability of the tao-gen algorithm as well as the utility of the informative prior structure through direct comparisons with results obtained from another bayesian network algorithm  <cit> , as well as with results obtained using tao-gen without an informative prior .
the resulting bayesian-based network structure predicts  <dig> linkages  between the set of  <dig> genes.
sox <dig>  mef2a, elavl <dig>  and pou6f <dig> are predicted as the most prolific co-regulators of the target genes, with  <dig>   <dig>   <dig>  and  <dig> children, respectively.
furthermore, our analysis predicts creb <dig>  crebbp, and yy <dig> as the most likely candidates for dorsally expressed ngn <dig> co-factors, and supports a synergistic interaction between pou-domain containing transcription factors and bhlh proneural proteins in the regulation of common target genes, which is consistent with previous studies  <cit> .
we also identified several transcription factors as candidate co-regulators of ngn <dig> target genes, including hes <dig>  egr <dig>  nfy, mef2a, tef, and sox <dig>  whereas pou6f <dig> is a predicted co-regulator of mash <dig> target genes.
interestingly, these different transcription factors have been previously implicated in regulatory processes in other developmental contexts  <cit> , consistent with a potential role in regulating neurogenesis during telencephalon development.
discussion
as the amount of experimental data grows and becomes more complex, the power of computational models to identify mechanisms of biological processes by integrating diverse sets of experimental data is being realized, particularly in organisms where high throughput perturbation analyses have been developed, including saccharomyces cerevisiae, caenorhabditis elegans, and strongylocentrotus purpuratus  <cit> .
however, finding and quantifying mammalian networks has been challenging due to the data requirements of current methods.
in the present analysis, we have demonstrated the utility of bioinformatics approaches for elucidation of a grn describing proneural bhlh  transcription factor regulation of murine telencephalon specification.
to develop a robust grn, we first measured global gene expression patterns in gof and lof analyses to identify novel putative target genes for ngn <dig> and mash <dig>  several of which were validated by in situ hybridization in slice preparations from mutant and electroporated mouse embryos.
using bayesian network analysis, we have corroborated  <dig> literature-based grn hypotheses by quantifying connections based on the compiled microarray datasets.
as highly conserved long-range enhancers are known to be particularly important for developmentally regulated genes  <cit> , we utilized phylogenetic footprinting analyses to identify putative long-range enhancers in several predicted ngn <dig> and mash <dig> target genes.
this approach suggests that no putative co-factor binding sites are specifically associated with conserved mash <dig> binding sites in both mash <dig> and common target genes, consistent with an instructive and relatively context-independent role for mash <dig> in ventral cell fate determination  <cit> .
in addition, the analysis identifies several conserved tfbss in close proximity to ngn <dig> binding sites surrounding the ngn <dig> target genes, consistent with previous findings supporting a more cell context-dependent role for ngn <dig> in dorsal telencephalic fate specification  <cit> .
in addition, we hypothesized that important regulatory modules that bind transcription factors other than ngn <dig> and mash <dig> may be present surrounding the identified target genes.
therefore, our second comparative genomics analysis was designed to search for conserved tfbs enriched in target genes, but not necessarily in close proximity to mash <dig> or ngn <dig> binding sites.
using a novel bayesian network analysis approach with an informative prior structure, we were able to synthesize the knowledge gained from each of the above experimental and computational approaches to predict connectivity between the novel target genes, co-factors, and co-regulators.
our resultant grn predicts that creb <dig> and crebbp are the most likely candidates for a dorsally expressed ngn <dig> co-factor regulating cortical targets such as neurod <dig>  eomes and neurod <dig>  interestingly, previous analyses have indicated interactions between bhlh transcription factors, creb, and the creb binding protein  in the differentiation of several cell types  <cit> , as well as in neurotrophin-mediated expression of vgf  <cit> .
most notably, a conserved neurod <dig> promoter has recently been shown to contain both creb and e-box binding sites and is activated via camp exposure  <cit> .
yy <dig> is also a potential ngn <dig> co-factor, yet our microarray data suggest that yy <dig> mrna expression is confined to the ventral telencephalon.
this finding suggests an inhibitory role for yy <dig> in ngn2-regulated transcription, which is supported by evidence showing yy <dig> inhibition of both bmp induced cell differentiation  <cit>  and notch transcriptional activity  <cit> .
our grn also supports a synergistic interaction between pou-domain containing transcription factors and bhlh proneural proteins in the activation of several target genes.
interestingly, both pou6f <dig> and mash <dig> have been identified as important mediators of oligodendrocyte development  <cit> , and pou6f <dig> is thought to synergistically interact with members of the sox family  <cit> .
moreover, other members of the pou domain, class  <dig> family of transcription factors, brn <dig> or brn <dig>  which exhibit similar affinity with pou6f <dig> for certain binding sites  <cit>  have been experimentally shown to co-regulate several mash <dig> targets in the ventral telencephalon  <cit> .
finally, our analysis predicts connectivity between sox <dig>  fzd <dig>  and wnt7b, which is consistent with a recent report suggesting sox <dig> regulation through wnt signaling  <cit> .
sox <dig> may serve as an inhibitory factor for several ngn <dig> target genes including gng <dig>  npdc <dig>  eomes, fzd <dig>  and coro2b, consistent with the opposing roles of sox <dig> and ngn <dig> in the specification of glial fate  <cit> .
we also show that mef2a, which has been shown previously to have complex regulatory functions in neuronal differentiation and plasticity  <cit> , may co-activate several of the predicted ngn <dig> targets, such as neurod, ngn <dig>  wnt7b, mgst <dig>  gng <dig>  acpl <dig>  and dusp <dig>  in addition, our findings suggest that transcriptional regulators downstream of wnt signaling  may bind to regulatory modules that also bind ngn <dig>  which is consistent with the role of wnt signaling in the specification of the dorsal forebrain  <cit> , and offers a hypothesis in which coordinated wnt activation and ngn <dig> expression act in concert to transcriptionally activate target genes.
interestingly, a recent report suggests that wnt pathway-initiated neural differentiation, but not proliferation, requires specific interactions between tcf/β-catenin and crebbp  <cit> .
another intriguing aspect of our analysis is the prediction of elavl <dig> as an important regulator of ngn <dig> targets, including bhlhb <dig>  robo <dig>  nhlh <dig>  coro2b, sox <dig>  and dll <dig>  possibly through stabilization of mrna  <cit> .
it is of note that two related mrna stabilization genes, elavl <dig> and elavl <dig>  are also predicted targets of ngn <dig>
our current research identifies important research steps for further refinement of the grn.
the cyclic nature of delayed negative feedback in the notch pathway is thought to act as a molecular clock regulating the timing of several developmental processes  <cit> .
future production of robust time-series datasets will allow for application of bayesian methods that are not limited to the discovery of acyclic networks and linear relationships  <cit> .
several recent reports have suggested connectivity between notch and wnt pathways  <cit> .
for example, one way in which the notch pathway may regulate wnt signaling is through lef protein stabilization by nrarp activation  <cit> .
however, because no data currently exist that would allow quantitative prediction via direct protein-protein interactions, we are unable to predict this relationship in the current network analysis.
nonetheless, our network analyses of transcriptional regulation offers other hypotheses such as a tcf/lef regulation of lfng and mfng, which is consistent with evidence during somitogenesis suggesting a wnt mediated regulation of lfng expression  <cit> .
one important application of the network analysis is the prediction of the most useful perturbation or chromatin immunoprecipitation experiments for resolving the overall network structure  <cit> .
this is particularly relevant to studies in mammalian species, in which perturbation analyses are much more time and resource intensive.
our network analysis would suggest centg <dig> and elavl <dig> are important candidates for perturbation and subsequent global gene expression analysis for further network resolution, as they are both highly connected nodes and little is known about their function in the developing telencephalon.
interestingly, centg <dig> may protect against neurodegeneration in polyglutamine diseases  <cit> .
the central role of elavl <dig>  which regulates through mrna stabilization  <cit> , highlights the importance of moving beyond cis regulatory binding to elucidate network relationships.
CONCLUSIONS
in this manuscript, we have combined a number of different approaches to identify regulatory relationships that are important for mammalian telencephalic development, using existing knowledge from the literature, global gene expression data from gof and lof studies, bioinformatics-based sequence analyses, and bayesian network algorithms.
systems biology methodologies that take into account several data sources, such as that presented here, will aid in more rapid identification and quantification of gene regulation which is useful for discovering critical steps in the progression of normal and perturbed human telencephalon development.
eventually, the linkage of mammalian genetic perturbation network analysis with protein interactions and ultimately phenotypic outcome will become possible, as has been initiated in invertebrate models.
the methods developed and applied in this manuscript are a first step towards this broader goal.

 during development of the mammalian nervous system, neural progenitors within the neuroepithelium give rise sequentially to neuronal cells and glia. to achieve these well-orchestrated waves of differentiation, neuroepithelial progenitors are progressively constrained via specific extrinsic and intrinsic signals. by directly promoting the neuronal fate in neural progenitors, the proneural transcription factors of the bhlh family are essential regulators of neurogenesis from invertebrates to mammals. three proneural genes have been characterized to date in the embryonic mouse telencephalon: neurogenin and neurogenin are expressed in neuronal progenitors in dorsal telencephalon, which gives rise to the cerebral cortex, while mash determines the fate of neuronal progenitors in the ventral telencephalon, giving rise to the basal ganglia. in addition to their role in the selection of neuronal progenitors within the neuroepithelium, vertebrate proneural genes have also been shown to specify neuronal subtype identities. indeed, ngn is necessary for the proper differentiation of excitatory glutamatergic projection neurons in the cerebral cortex, while mash promotes the fate of gaba-ergic inhibitory interneurons in the basal ganglia. proneural transcription factors are thus critical regulators for both the initiation of neuronal differentiation and the specification of neurons into distinct regional subtypes. in order to understand how ngn and mash regulate these two different aspects of neurogenesis we have developed a genomic approach incorporating the generation of microarray datasets of mice either lacking or overexpressing proneural genes, phylogenetic footprinting, and analysis using bayesian statistical regression. the utility of bayesian-based network analyses to determine predictive gene regulatory network structures has been demonstrated in in vitro and invertebrate models, but few attempts have been made using datasets derived from mammalian species due to the dependence of these techniques on a relatively large sample size. however, this limitation may be eased through incorporation of data from other sources, such as prior information from the literature and sequence-based transcription factor binding site information. here, we initially identify a gene regulatory network from a compilation of the current literature on proneural bhlh protein regulation of telencephalic development during neurogenesis. next, microarray data from gain-of-function analysis obtained after electroporation of either ngn or mash in the developing mouse dorsal or ventral telencephalon, respectively, were fused with previous and new microarray results from proneural loss-of-function experiments. this allowed us to identify novel targets of ngn and mash and to formally test literature-based grn hypotheses through bayesian statistical analysis of global gene expression patterns. a total of nine predicted proneural targets were confirmed by in situ rna hybridization on brain slices from embryos lacking or overexpressing mash or ngn including several members of notch signaling pathway as well as the rna binding protein elavl. members of the wnt signaling pathway are predicted as targets of ngn in the cerebral cortex. we then took advantage of the identification of putative downstream effectors of proneural genes to identify conserved binding sites for mash and ngn then scanned the sequence surrounding them for other conserved tfbss. from this analysis, we predict creb, tcf/lef, pou-domain containing transcription factors, sox and mef2a as novel co-factors and co-regulators of proneural proteins. finally, a novel bayesian-based algorithm was developed to compile the microarray data, the tfbs analysis, and the literature-based network, generating a proneural grn for the developing mouse telencephalon.  literature-based network structure a grn that describes the current understanding of proneural bhlh interactions in the developing telencephalon was developed through a review of the literature. the reader is referred additional file and several important reviews for more detailed descriptions of the experimental research underlying this literature-based grn. gof experiments to further identify genes regulated by the proneural factors ngn and mash in the embryonic telencephalon and complement existing lof data, we developed a gof approach using electroporation in a whole embryo culture. the feasibility of the gof study was tested by injection of a ngn2-expressing or mash1-expressing vector in the telencephalic vesicles of e mouse embryos. the time course of ngn and mash overexpression was followed with dll promoter-lacz reporter transgenes monitoring ngn and mash activities, respectively; see also ). lacz reporter activity was detected h after electroporation of ngn or mash in the dorsal and ventral sides of telencephalic vesicles, respectively, and reached the highest level h after proneural gene electroporation ). in contrast, activation by endogenous proneural proteins of the two reporters in embryos electroporated with an empty expression vector remained low. the efficiency of electroporation was similar in the control and ngn2- and mash1-electroporated cortices, as assessed by expression of a co-electroporated gfp plasmid before lacz staining. large-scale electroporation experiments of ngn in the dorsal telencephalon and mash in the ventral telencephalon of e embryos were thus performed and, h later, the electroporated tissue was processed for rna probe preparation. identification of candidate ngn and mash target genes we reasoned that by comparing the transcriptome of embryos overexpressing proneural genes with expression data obtained from current mash lof datasets and previously published ngn lof datasets, we would identify novel downstream effectors of mash and/or ngn in the developing telencephalon. for the study of ngn targets, data from the cortex of mash1-/-;ngn2-/- double mutant mice was utilized rather than ngn2-/- single mutants in order to avoid the compensation due to de-repression of mash in the cortex of the later mutants. analysis of microarray data allowed us to split potential proneural targets into three groups and 2): genes regulated by both mash and ngn2; genes regulated by mash only; and genes regulated by ngn only. a full list of predicted targets categorized by gene ontology is presented in additional file briefly, among the common targets were genes involved in notch signaling and the rna binding protein elavl expressed in maturing neurons. specific targets of ngn include genes involved in signal transduction, transcription factors such as the cortical differentiation factors nhlh and bhlhb, proteins with kinase/phosphatase activity such as the serine/threonine kinase dcx, already shown to be directly regulated by ngn in the cortex and other cortical genes such as the cell adhesion molecule ephrin a. interestingly, several components of the wnt pathway are predicted targets of ngn including wnt7b, dix domain containing, fzd fzd and tax1bp it is noteworthy that neurod, a gene considered as directly regulated by ngn proteins, was decreased in the ngn2-/- embryos, but was not significantly upregulated after ngn electroporation in microarray experiments, suggesting that the window for analysis of electroporated embryos was not suitable. indeed, in situ hybridization analysis showed that electroporation of ngn does not induce neurod expression in the telencephalon at h, but only at h after electroporation. a similar delay has been reported for induction of neurod by the related gene ngn in the intestine. this suggests either that neurod is not a direct target of ngn or that its expression also requires another factor that is not present at the time of electroporation. involvement of other genes in the regulation of neurod is supported by the lack of a dramatic change in neurod expression in ngn null mutant telencephalon. finally, putative targets of mash include transcription factors such as the lim homeobox transcription factor isl which is potentially involved in the specification of gabaergic projection neurons in the striatum, and factors involved in signal transduction such as the gaba vesicular transporter slc32a1/viaat and the gene gp38/podoplanin, the notch regulated protein nrarp shown to destabilize the notch intracellular domain, and lfng, a modulator of notch signaling regulated directly by mash in the telencephalon. these putative target genes form the basis for subsequent bioinformatics and network analyses to predict novel co-factors, co-regulators, and grn connectivity. validation of predicted target genes to confirm the microarray data, several of the predicted novel targets of ngn and mash were validated through rna in situ hybridization analysis of wild type and mutant embryos, and of embryos overexpressing ngn or mash. the predicted ngn targets nhlh mfng, and elavl are mostly expressed at e in the mantle zone of the dorsal telencephalon, while rbdh and zfp are also expressed in the ventricular zone. all five genes are downregulated in the medial part of the dorsal telencephalon of ngn mutant embryos, indicating that ngn is indeed required for normal expression of these genes ). we also show that ngn can induce expression of the same predicted targets when overexpressed by electroporation in the telencephalon of e embryos. at this stage, their expression is barely detectable in neurons that begin to accumulate above the ventricular zone of the dorsal telencephalon ). expression of nhlh mfng, zfp rbdh and elavl is strongly upregulated after electroporation of a ngn2-expressing vector in the dorsal telencephalon, marked by *). thus, both lof and gof analysis confirm that five predicted targets are indeed regulated by ngn in the developing mouse telencephalon. the predicted mash targets mfng and pdpn are predominantly expressed in the ventricular zone of the ventral telencephalon at e, while gadd45g is predominantly expressed in the subventricular zone and elavl in the mantle zone. all four genes are downregulated in the telencephalon of mash mutant embryos particularly in the medial part of the ventral telencephalon, which is most severely affected in mash mutant embryos ). ectopic expression of mash in the dorsal telencephalon of e embryos strongly induces expression of the four genes ). mash also rapidly induces expression of gadd45g and mfng in the embryonal carcinoma cell line p as detected by quantitative reverse transcription polymerase chain reaction. these data suggest that many of the genes identified by microarray analysis of mash and ngn gof and lof experiments are indeed regulated by mash and ngn bayesian analysis of literature-based grn connectivity in the literature-based network structure was quantified based on the gof and lof microarray gene expression datasets. this bayesian-based method removes the need to rely on fold change cut-offs by examining the strength of the predicted relationships based on concurrently evaluating variability in gene expression patterns of several genes of interest across control and perturbation experiments. significant connections based on this analysis are highlighted in red in figure our analysis predicts 86% of the connections as significant based on the microarray dataset; however, four are considered inhibitory interactions instead of activations, suggesting possible feedback loops between pax and ngn between satb ngn and nhlh as well as between neurod and etv. specificity of our method was estimated through the application of randomly generated datasets by permutation of gene labels using the entire microarray dataset. this analysis results in an estimated false positive rate of %, therefore the chances of seeing significant connections out of is very low. two of the connections originating from ngn are not considered significant based on the current analysis. as the algorithm relies on linear relationships between genes, its inability to detect these two connections may be a result of the non-linear variability between ngn and its targets that is created by the large increase in ngn transcript levels after electroporation. in addition, the compensatory action of mash in the dorsal telencephalon of the ngn2-/- mutants further complicates the algorithm's ability to detect connectivity to common targets such as dll bioinformatics prediction of ngn and mash co-factors the identification of potential downstream effectors of mash and ngn allowed us to examine the occurrence of defined tfbss in the conserved sequence surrounding those genes. the e-box recognized by bhlh proteins is a degenerate bp motif. however, recent evidence suggests that mash and ngn have different preferences for the two central residues and that their consensus binding sequences extend even outside of the e-box motif. based on these and other data, we defined different consensus binding sequences for mash and ngn. to identify potential co-factors for ngn or mash we employed phylogenetic footprinting and tfbs search algorithms on a subset of the predicted targets based on the microarray dataset ). first, conserved putative ngn binding sites surrounding of the predicted ngn target genes and conserved putative mash binding sites surrounding of the predicted mash target genes and of the common target genes were identified. subsequently, we identified tfbs from the transfac library co-occurring specifically with the putative ngn binding sites in ngn targets or with the putative mash binding sites in mash and mash1/ngn common target genes using fisher's exact two-sided test for significance ). no specific co-factors for mash were identified in this analysis, whereas camp response element ), yy and nkx binding sites were significantly enriched near ngn binding sites when compared with the sequence around mash binding sites ). several of the predicted co-factors have dorsal/ventral restricted expression patterns in the embryonic telencephalon, consistent with a role in neuronal specification ). bioinformatics prediction of ngn and mash co-regulators next we sought to predict potential co-regulators that may bind at regulatory modules independent from those that bind ngn or mash in particular, we were interested in identifying transcription factors that may confer regulation at modules closer to the tss. in order to efficiently search for co-regulators, we identified evolutionary conserved tfbs using the confac algorithm to analyze a shorter sequence in the 5' flanking region of predicted mash ngn and common targets. to predict putative common co-regulators, we tested for enrichment of the identified tfbs in ngn target genes and mash1/common target genes compared with a set of randomly selected genes using the mann-whitney statistical test. we identified potential ngn2- and mash1-specific co-regulators by testing for tfbs enrichment in ngn targets versus mash targets and vice versa. we identified putative co-regulators from these analyses after eliminating those transcription factors that are not expressed in either the dorsal or ventral developing telencephalon based on microarray transcript levels. predicted common co-regulators include e2f tef, nfy, egr hes and pou-domain containing factors. predicted co-regulators were specifically enriched in ngn targets when compared with mash and common targets include sox creb, tcf lef mef2a, and yy no transcription factors were significantly enriched when mash target genes were compared against ngn target genes. this approach identified all five of the predicted ngn co-factors found in the previous analysis, as well as identifying several transcription factors that may co-regulate the putative ngn and mash targets at independent regulatory modules including e2f egr hes and nfy in both ngn and mash targets and sox and mef2a in ngn target genes. through interrogation of online databases of in situ hybridization in serial brain sections across development, we confirmed the expression of several predicted co-factors/co-regulators in the developing dorsal and ventral telencephalon. interestingly, several of the predicted co-factors/co-regulators, including sox crebbp, creb tcf lef pou6f pou2f pou3f tef, hes and e2f show appreciable expression in the ventricular zone of dorsal and/or ventral telencephalon, where proneural bhlh proteins are expressed. furthermore, direct protein-protein interactions between proneural bhlh proteins and crebbp, tcf and mef2a are reported in the human protein reference database. bayesian network analysis with an informative prior structure to provide an integrated view of the network regulated by proneural genes, information obtained from the literature, novel expression data, and phylogenetic footprinting analyses were quantitatively linked through application of the bayesian-based tao-gen algorithm with the addition of an informative prior structure. nodes represented in the network include several candidate mash and ngn target genes from the fusion and sorting of the gof/lof microarray datasets, as well as candidate co-factors and co-regulators from the phylogenetic footprinting analyses. an informative prior structure considered all significant literature-based connections as required and used the tfbs information from the phylogenetic footprinting analyses to weight connections in which tfbs information was found. for example, a conserved sox tfbs was identified in the sequence surrounding lfng, therefore a preference is given to sox being a parent to lfng, which results in a slightly higher probability that a linear relationship between these two genes will be significant in the posterior distribution. we have confirmed the reliability of the tao-gen algorithm as well as the utility of the informative prior structure through direct comparisons with results obtained from another bayesian network algorithm, as well as with results obtained using tao-gen without an informative prior. the resulting bayesian-based network structure predicts linkages between the set of genes. sox mef2a, elavl and pou6f are predicted as the most prolific co-regulators of the target genes, with and children, respectively. furthermore, our analysis predicts creb crebbp, and yy as the most likely candidates for dorsally expressed ngn co-factors, and supports a synergistic interaction between pou-domain containing transcription factors and bhlh proneural proteins in the regulation of common target genes, which is consistent with previous studies. we also identified several transcription factors as candidate co-regulators of ngn target genes, including hes egr nfy, mef2a, tef, and sox whereas pou6f is a predicted co-regulator of mash target genes. interestingly, these different transcription factors have been previously implicated in regulatory processes in other developmental contexts, consistent with a potential role in regulating neurogenesis during telencephalon development. discussion as the amount of experimental data grows and becomes more complex, the power of computational models to identify mechanisms of biological processes by integrating diverse sets of experimental data is being realized, particularly in organisms where high throughput perturbation analyses have been developed, including saccharomyces cerevisiae, caenorhabditis elegans, and strongylocentrotus purpuratus. however, finding and quantifying mammalian networks has been challenging due to the data requirements of current methods. in the present analysis, we have demonstrated the utility of bioinformatics approaches for elucidation of a grn describing proneural bhlh transcription factor regulation of murine telencephalon specification. to develop a robust grn, we first measured global gene expression patterns in gof and lof analyses to identify novel putative target genes for ngn and mash several of which were validated by in situ hybridization in slice preparations from mutant and electroporated mouse embryos. using bayesian network analysis, we have corroborated literature-based grn hypotheses by quantifying connections based on the compiled microarray datasets. as highly conserved long-range enhancers are known to be particularly important for developmentally regulated genes, we utilized phylogenetic footprinting analyses to identify putative long-range enhancers in several predicted ngn and mash target genes. this approach suggests that no putative co-factor binding sites are specifically associated with conserved mash binding sites in both mash and common target genes, consistent with an instructive and relatively context-independent role for mash in ventral cell fate determination. in addition, the analysis identifies several conserved tfbss in close proximity to ngn binding sites surrounding the ngn target genes, consistent with previous findings supporting a more cell context-dependent role for ngn in dorsal telencephalic fate specification. in addition, we hypothesized that important regulatory modules that bind transcription factors other than ngn and mash may be present surrounding the identified target genes. therefore, our second comparative genomics analysis was designed to search for conserved tfbs enriched in target genes, but not necessarily in close proximity to mash or ngn binding sites. using a novel bayesian network analysis approach with an informative prior structure, we were able to synthesize the knowledge gained from each of the above experimental and computational approaches to predict connectivity between the novel target genes, co-factors, and co-regulators. our resultant grn predicts that creb and crebbp are the most likely candidates for a dorsally expressed ngn co-factor regulating cortical targets such as neurod eomes and neurod interestingly, previous analyses have indicated interactions between bhlh transcription factors, creb, and the creb binding protein in the differentiation of several cell types, as well as in neurotrophin-mediated expression of vgf. most notably, a conserved neurod promoter has recently been shown to contain both creb and e-box binding sites and is activated via camp exposure. yy is also a potential ngn co-factor, yet our microarray data suggest that yy mrna expression is confined to the ventral telencephalon. this finding suggests an inhibitory role for yy in ngn2-regulated transcription, which is supported by evidence showing yy inhibition of both bmp induced cell differentiation and notch transcriptional activity. our grn also supports a synergistic interaction between pou-domain containing transcription factors and bhlh proneural proteins in the activation of several target genes. interestingly, both pou6f and mash have been identified as important mediators of oligodendrocyte development, and pou6f is thought to synergistically interact with members of the sox family. moreover, other members of the pou domain, class family of transcription factors, brn or brn which exhibit similar affinity with pou6f for certain binding sites have been experimentally shown to co-regulate several mash targets in the ventral telencephalon. finally, our analysis predicts connectivity between sox fzd and wnt7b, which is consistent with a recent report suggesting sox regulation through wnt signaling. sox may serve as an inhibitory factor for several ngn target genes including gng npdc eomes, fzd and coro2b, consistent with the opposing roles of sox and ngn in the specification of glial fate. we also show that mef2a, which has been shown previously to have complex regulatory functions in neuronal differentiation and plasticity, may co-activate several of the predicted ngn targets, such as neurod, ngn wnt7b, mgst gng acpl and dusp in addition, our findings suggest that transcriptional regulators downstream of wnt signaling may bind to regulatory modules that also bind ngn which is consistent with the role of wnt signaling in the specification of the dorsal forebrain, and offers a hypothesis in which coordinated wnt activation and ngn expression act in concert to transcriptionally activate target genes. interestingly, a recent report suggests that wnt pathway-initiated neural differentiation, but not proliferation, requires specific interactions between tcf/β-catenin and crebbp. another intriguing aspect of our analysis is the prediction of elavl as an important regulator of ngn targets, including bhlhb robo nhlh coro2b, sox and dll possibly through stabilization of mrna. it is of note that two related mrna stabilization genes, elavl and elavl are also predicted targets of ngn our current research identifies important research steps for further refinement of the grn. the cyclic nature of delayed negative feedback in the notch pathway is thought to act as a molecular clock regulating the timing of several developmental processes. future production of robust time-series datasets will allow for application of bayesian methods that are not limited to the discovery of acyclic networks and linear relationships. several recent reports have suggested connectivity between notch and wnt pathways. for example, one way in which the notch pathway may regulate wnt signaling is through lef protein stabilization by nrarp activation. however, because no data currently exist that would allow quantitative prediction via direct protein-protein interactions, we are unable to predict this relationship in the current network analysis. nonetheless, our network analyses of transcriptional regulation offers other hypotheses such as a tcf/lef regulation of lfng and mfng, which is consistent with evidence during somitogenesis suggesting a wnt mediated regulation of lfng expression. one important application of the network analysis is the prediction of the most useful perturbation or chromatin immunoprecipitation experiments for resolving the overall network structure. this is particularly relevant to studies in mammalian species, in which perturbation analyses are much more time and resource intensive. our network analysis would suggest centg and elavl are important candidates for perturbation and subsequent global gene expression analysis for further network resolution, as they are both highly connected nodes and little is known about their function in the developing telencephalon. interestingly, centg may protect against neurodegeneration in polyglutamine diseases. the central role of elavl which regulates through mrna stabilization, highlights the importance of moving beyond cis regulatory binding to elucidate network relationships.  in this manuscript, we have combined a number of different approaches to identify regulatory relationships that are important for mammalian telencephalic development, using existing knowledge from the literature, global gene expression data from gof and lof studies, bioinformatics-based sequence analyses, and bayesian network algorithms. systems biology methodologies that take into account several data sources, such as that presented here, will aid in more rapid identification and quantification of gene regulation which is useful for discovering critical steps in the progression of normal and perturbed human telencephalon development. eventually, the linkage of mammalian genetic perturbation network analysis with protein interactions and ultimately phenotypic outcome will become possible, as has been initiated in invertebrate models. the methods developed and applied in this manuscript are a first step towards this broader goal. 
the proneural proteins mash and ngn are key cell autonomous regulators of neurogenesis in the mammalian central nervous system, yet little is known about the molecular pathways regulated by these transcription factors. next, we searched the non-coding sequence surrounding the predicted proneural downstream effector genes for evolutionarily conserved transcription factor binding sites associated with newly defined consensus binding sites for ngn and mash this allowed us to identify potential novel co-factors and co-regulators for proneural proteins, including creb, tcf/lef, pou-domain containing transcription factors, sox and mef2a. novel targets of ngn and/or mash were identified, such as members of the notch and wnt pathways, and proteins involved in adhesion and signal transduction. finally, a gene regulatory network was delineated using a novel bayesian-based algorithm that can incorporate information from diverse datasets. here we identify the downstream effectors of proneural genes in the telencephalon using a genomic approach to analyze the transcriptome of mice that are either lacking or overexpressing proneural genes. together, these data shed light on the molecular pathways regulated by proneural genes and demonstrate that the integration of experimentation with bioinformatics can guide both hypothesis testing and hypothesis generation.
 the proneural proteins mash and ngn are key cell autonomous regulators of neurogenesis in the mammalian central nervous system, yet little is known about the molecular pathways regulated by these transcription factors.  here we identify the downstream effectors of proneural genes in the telencephalon using a genomic approach to analyze the transcriptome of mice that are either lacking or overexpressing proneural genes. novel targets of ngn and/or mash were identified, such as members of the notch and wnt pathways, and proteins involved in adhesion and signal transduction. next, we searched the non-coding sequence surrounding the predicted proneural downstream effector genes for evolutionarily conserved transcription factor binding sites associated with newly defined consensus binding sites for ngn and mash this allowed us to identify potential novel co-factors and co-regulators for proneural proteins, including creb, tcf/lef, pou-domain containing transcription factors, sox and mef2a. finally, a gene regulatory network was delineated using a novel bayesian-based algorithm that can incorporate information from diverse datasets.  together, these data shed light on the molecular pathways regulated by proneural genes and demonstrate that the integration of experimentation with bioinformatics can guide both hypothesis testing and hypothesis generation. 