BACKGROUND
the neocortex is a six-layered structure composed of myriad excitatory and inhibitory neuron types.
developmental programs, controlled by multiple cell-intrinsic and –extrinsic cues, establish unique patterns of connectivity for the diverse neuronal subtypes located in different layers.
while the granular neurons of layer iv form networks of local connections within cerebral hemispheres, projection neurons of layers ii/iii, v and vi mediate long distance communications both within and outside the cortex.
developmentally, excitatory neurons are born from dividing progenitors located in distinct germinal domains situated at ventricular and subventricular zones of the dorsal telencephalon.
once born, they migrate to superficial cortical layers, with each wave of migrating neurons surpassing predecessors, resulting in six distinct cortical layers generated in an inside-out manner  <cit> .
consequently, birthdate of a neuron determines its final layer of residence and confers its layer-specific properties.
during and following migration, cortical projection neurons initiate expression of specific sets of axon guidance molecules that allows pathfinding to correct targets  <cit> .
the end product of this highly complex and regulated migration and axon guidance process is a neocortex organized into six layers, with neurons residing in each layer exhibiting unique physiological properties and patterns of connectivity  <cit> .
almost all aspects of neurogenesis, migration and neuronal differentiation are orchestrated by hierarchical and combinatorial functioning of transcription factor  networks  <cit> .
within the embryonic germinal zones, expression of neurog <dig> and neurog <dig> proneural basic helix loop helix  transcription factors commit neural progenitors to cortical excitatory neuron identity  <cit> .
neurog <dig> is both necessary and sufficient for generation of these neurons  <cit>  and neurog <dig> initiated specification of projection neuron identity involves a complex transcription factor network composed of effector transcription factors, such as tbr <dig> , neurod <dig>  and neurod <dig>  .
despite our relatively thorough understanding of the tfs required for specifying a cortical excitatory neuron identity, little is known about how differentiation programs are executed by effector tfs and the nature of the downstream target genes required for corticogenesis.
for example, neurod <dig> is expressed within a wide temporal window  outside of the proliferative zones and constitutes a potential regulator of critical aspects of differentiation and/or maintenance of different types of cortical excitatory neurons  <cit> .
indeed, several studies investigating the consequences of neurod <dig> loss-of-function in mice have demonstrated that neurod <dig> is required for commissural axon pathfinding of layer ii/iii callosal projection neurons, formation of cortical somatosensory maps within layer iv granular neurons and maturation of dendrites and synapses in the hippocampus .
furthermore, a gain-of-function study has demonstrated that misexpression of neurod <dig> in ventral telencephalon progenitors is sufficient to prevent their normal gabaergic differentiation  <cit> .
although during development neurod <dig> controls the execution of a wide range of functions, its target genes during cortex development are largely unknown.
in this study we identified genome-wide targets of neurod <dig> during mid-embryogenesis.
we demonstrate that neurod <dig> binds to a large number of target genes with prominent roles in radial migration, layer-specific differentiation and axon pathfinding of cortical projection neurons.
moreover, we find that neurod <dig> is positioned to control cortical radial migration by regulating members of the reelin signaling pathway.
importantly, we demonstrate that expression of cdk5r <dig>  the regulatory subunit of cyclin-dependent kinase  <dig>  is critically dependent on neurod <dig>  our identification of neurod <dig> targets offers mechanistic insight into known and potentially novel functions of this effector transcription factor and points to the importance of carrying out chip-seq analysis at developmental timepoints within relevant tissue.
RESULTS
identification of neurod <dig> binding sites in embryonic cortex
in mice, the bulk of cortical excitatory neurogenesis takes place during the last ten days of gestation, a period which also coincides with high levels of neurod <dig> expression  <cit> .
therefore, in order to determine the genome-wide targets of neurod <dig> during cortical development, we carried out chromatin-immunoprecipitation followed by high-throughput sequencing  .
first, we identified three antibodies that could successfully immunoprecipitate neurod <dig> following overexpression in neuro2a cell line .
after isolating neurod2-associated chromatin from mouse embryonic day  <dig>   cortex, we confirmed that the promoter region of the nhlh <dig> gene, a previously identified neurod <dig> target  <cit> , was amplified by pcr using chip template dna precipitated by all three neurod <dig> antibodies, but not from chip dna prepared by an unrelated antibody against gfp .
after massively parallel sequencing of all three neurod <dig> chip dna and two separate gfp chip dna samples, we mapped reads using bowtie short read aligner  <cit> .
neurod <dig> peaks were identified by macs   <cit>  and gfp peak alignments were used as input control dataset for signal normalization .
from the three experimental samples a total of approximately  <dig> , <dig> unique reads were identified, which mapped onto  <dig>  peaks .fig.
<dig> chip-seq reveals neurod <dig> target genes during cortical development.
a chromatin dna was immunoprecipitated with three different antibodies against neurod <dig>  or with a gfp antibody which was used as a negative control.
pcr was performed using immunoprecipitated dna as template.
specific primers amplified the promoter region of a previously identified neurod <dig> target gene nhlh <dig>  <cit> .
an enrichment for the positive control nhlh <dig> gene was demonstrated in neurod <dig> chip-pcr samples as compared to gfp chip-pcr.
b the developing cortex  was dissected from e <dig>  mouse brain and used for neurod <dig> chip-seq.
<dig>  peak regions and  <dig>  target genes were identified
the quality of our data was validated by the high degree of correlation among the three neurod <dig> antibodies .
in contrast, comparison of neurod <dig> peaks generated by the three individual antibodies to those of another transcription factor expressed in the mouse embryonic brain, ctcf, resulted in much lower r <dig> values .
in an effort to reduce false-positive binding sites due to non-specific interactions of the particular antibody used, we decided to discard peaks that are identified in only one of the three chip-seq datasets acquired using the three separate antibodies.
as a result, out of a total of  <dig>  peaks from three separate datasets,  <dig>  peaks were found to be located in regions containing overlapping peaks from at least two antibodies.
next, we collapsed all the overlapping peaks to obtain the union of peak coordinates and as a result identified  <dig>  neurod <dig> binding locations.
following analyses were carried out using these  <dig>  neurod <dig> binding sites .
neuronal bhlh transcription factors like neurod <dig> bind to an e-box motif, which is loosely defined as canntg .
therefore, we asked whether or not the neurod <dig> peak sequences were enriched in e-box elements.
by using the meme-chip suite  <cit> , a tool designed to identify enriched sequence elements in large datasets, we discovered that an extended e-box motif, catg, was significantly associated with neurod <dig> peak sequences .fig.
<dig> neurod <dig> binds to e-box elements proximal to tsss in vivo.
a neurod <dig> consensus sequence was predicted using  <dig>  peak regions as input for the meme-chip suite  <cit> .
b among the  <dig>  neurod <dig> peaks, approximately 45 % mapped onto ensembl annotated genes   <cit> .
among them approximately 83 % mapped onto introns, and 17 % to either 5′utr, cds or 3′utr exons.
c the binding preference of neurod <dig> to introns, 5′utr exons, 3′utr exons and cds exons was calculated.
the number of neurod <dig> binding sites observed on introns , 5′utr , 3′utr  and cds  exons were normalized to the length of each of these gene segments and compared to a random distribution profile .
the data suggests approximately an 8-fold enrichment in neurod <dig> binding to the first 5′utr exon, a 5-fold enrichment in binding to the first cds exon and approximately 2-fold enrichment in binding to the first and second 3′utr exons.
***p-value ~  <dig>  *p-value <  <dig> .
d a binding map of neurod <dig> relative to closest tsss, revealed that neurod <dig> has a binding preference within +/− 1000 bps of tsss.
peaks that mapped upstream  and downstream  of tsss are color-coded.
e the distribution profiles of closestgene scores  <cit>  calculated by using neurod <dig> peaks  were very significantly different from those calculated using randomly distributed peaks  on the genome .
f distribution of normalized scores for individual genes were generated by subtracting the closestgene score calculated using neurod <dig> peaks from that of random peaks.
dark green represents genes whose scores were greater than one standard deviation  above the mean and were used for further analyses
in order to determine neurod <dig> binding preference along the genome and therefore gain insight into its mechanism of action, we examined peak positions relative to annotated transcriptional units.
analysis of all  <dig>  sites revealed that approximately half of neurod <dig> binding locations  mapped onto annotated ensembl transcripts   <cit>  and the remainder mapped onto intergenic regions.
within a gene, 83 % of intragenic sites were associated with intronic regions and 17 % were associated with 5′utr, coding , or 3′utr exons .
however, on average introns are ~25– <dig> times longer than exons  <cit> , therefore in order to normalize for this length bias we calculated the probability of binding along a gene compared to a random distribution profile.
our calculations revealed that neurod <dig> binding was most likely to be embedded in the first 5′utr exon with an approximately 8-fold enrichment in probability compared to random distribution  .
next, we generated a neurod <dig> binding map relative to annotated transcription start sites  and observed a major accumulation of neurod <dig> peaks within 1000 bps from a tss .
therefore, in vivo neurod <dig> preferentially binds to tss proximal sequences located within or upstream of 5′utr exons.
a previous study used a chip-seq approach to investigate genome-wide targets of neurod <dig> after its viral transduction into the p <dig> cell line  <cit> .
however, since this study was conducted in cell culture, it may not provide an accurate reflection of the in vivo targets of neurod <dig> during cortex development.
in fact, recent studies propose that transcription factor-chromatin associations are context dependent and can vary between developmental stages and tissue types .
therefore, within the context of cortical development, with the exception of a small number of candidate genes studied so far, neurod2’s genome-wide targets and the kinds of biological processes regulated are largely unknown .
since our data demonstrate that neurod <dig> binding was not exclusive to promoter regions or tss-proximal sequences, we decided to employ a method for assigning individual peaks to genes which would also account for tss-distant peaks, as opposed to methods which set fixed windows around tsss for peak capturing.
for this reason, we decided to use the closestgene method which assigns scores to individual genes based on the number of associated peaks and their proximity to respective transcription start sites  <cit> .
we calculated two scores for each gene in the mouse genome by using either the  <dig>  neurod <dig> peaks or the same number of randomly distributed peaks as the input dataset.
plotting the distribution profiles of the scores calculated by neurod <dig> peaks or random peaks revealed a very significant difference between these two datasets  .
next, we calculated a normalized closestgene score for each gene by simply subtracting the random score from the neurod <dig> score .
we then selected putative target genes, with varying scores, for confirmation by neurod <dig> chip followed by quantitative pcr  using the immunoprecipitated dna as template.
we selected genes from the top  <dig> , representing genes whose scores were above one standard deviation  of the mean and were able to confirm  <dig> out of  <dig> .
in addition, as negative controls we tested  <dig> genes  which were not identified as neurod <dig> targets .
these five genes are expressed in interneurons and not in neurod2-expressing cortical projection neurons.
we did not observe any significant enrichment in neurod <dig> binding to 4/ <dig> of the non-target genes and only a slight enrichment in gsx <dig>  a finding worthy of future investigation .
in sum, in light of our high confirmation rate of neurod <dig> binding to putative target genes we decided to use the top  <dig>  in all further analyses.
neurod <dig> target genes are associated with radial migration of cortical neurons
to further gain broad understanding into the biological processes regulated by neurod <dig> we carried out gene ontology analysis.
we identified significantly over-represented biological processes in the top  <dig>  targets against all of the genes in the mouse genome.
ranking based on fold enrichment of biological processes above a significance threshold , revealed that the highly ranked neurod <dig> controlled activities broadly converged onto two major developmental processes; cortical radial migration and axon guidance/fasciculation .table  <dig> gene ontology analysis of neurod <dig> targets
biological process categories above a cutoff p-value  are ranked by fold enrichment.
fold enrichment is calculated as log2
to gain further understanding of the signaling pathways that were regulated by neurod <dig>  we carried out pathway analysis with the neurod <dig> target genes against all pathways present in the biocarta database .
interestingly, we identified reelin signaling as the most significantly targeted pathway  .
the reelin protein is secreted by the cajal-retzius cells, located on the surface of the developing cortex.
by binding to receptors on migrating cortical neurons, reelin promotes reorganization of the cytoskeleton and regulates neuronal migration   <cit> .
our analysis revealed that neurod <dig> targets included several key members of this pathway, such as fyn, cdk5r <dig>  dab <dig> and lrp <dig>  genes .fig.
<dig> neurod <dig> targets cdk5r <dig> and lrp <dig> genes during corticogenesis.
a diagrammatic representation of the reelin signaling pathway that is required for cortical radial migration.
genes that are targets of neurod <dig> are marked with a red star.
b neurod <dig> western blotting confirms knockdown in primary cortical neuronal culture transfected with neurod <dig> shrna as compared to a non-targeting shrna.
egfp is used to verify transfection efficiency and beta-actin is used as a loading control.
c, d and e mrna expression data is obtained from allen developing mouse brain atlas  for neurod <dig>  cdk5r <dig> and lrp <dig> genes in e <dig>  mouse brain.
f and i neurod <dig> peaks associated with cdk5r <dig> and lrp <dig> are plotted relative to enhancer  and promoter  marks.
h3k4me <dig> and h3k4me <dig> chip-seq data is from   <cit> .
peaks are plotted using the genome browser at genome.ucsc.edu.
g and j neurod <dig> chip followed by qpcr confirms neurod <dig> binding to the promoter regions of cdk5r <dig> and lrp <dig> relative to a negative control .
data is normalized to the amount of input dna as described in the methods section .
bars represent standard error of mean.
data represents two biological and five technical replicates.
h and k reverse transcription and qpcr analysis of neurons transfected with neurod <dig> shrna or non-targeting shrna reveals a significant reduction in cdk5r <dig> mrna levels  and a trend of reduction in lrp <dig> levels  after neurod <dig> knockdown.
all rt-qpcr results represented data from  <dig> biological samples each analyzed in technical triplicates.
bars represent standard error of mean
we next focused our analysis on two important regulators of cortical radial migration, cdk5r <dig> and lrp <dig> genes.
initially, examination of expression patterns at mid-embryogenesis  revealed that neurod <dig> expression largely overlapped with zones of lrp <dig> and cdk5r <dig> expression .
next, we examined whether neurod <dig> binding along cdk5r <dig> and lrp <dig> genes were associated with enhancer or promoter regions.
we acquired chip-seq data from e <dig>  whole brain for h3k4me <dig> histone modification as a marker for enhancer regions and h3k4me <dig> modification as a marker for promoter regions  <cit>  .
overlaying of these datasets with neurod <dig> peaks revealed that neurod <dig> binding was associated with both promoter and enhancer specific histone marks on these two genes .
we further confirmed neurod <dig> association with the promoter regions of lrp <dig> and cdk5r <dig> genes by neurod <dig> chip followed by qpcr .
finally, we asked whether neurod <dig> controls cdk5r <dig> and lrp <dig> transcription.
we knocked down neurod <dig> expression almost completely in primary cortical neurons by high-efficiency transfection of small hairpin rna .
remarkably, our rt-qpcr results demonstrated a significant, five-fold reduction in cdk5r <dig> mrna levels after neurod <dig> knockdown, as compared to a control sample transfected with a non-targeting shrna .
lrp <dig> mrna also exhibited a trend in reduction in neurod <dig> lacking neurons, however with lower significance .
in sum, our results strongly support neurod <dig> control of radial migration and the reelin signaling pathway through direct regulation of key genes such as cdk5r <dig> and lrp <dig>
next, we asked whether neurod <dig> was expressed in neurons of the intermediate zone , which is a developmentally transient cortical region through which post-mitotic neurons born in subventricular  and ventricular  zones migrate in order to reach their final destination within the cortical plate   <cit> .
to mark neurons that have completed their migration and are localized to the cortical plate, we performed co-staining with antibodies against the tbr <dig> transcription factor and neurod <dig>  as expected, we identified neurod2+/tbr1+ neurons localized to the cp .
in addition, a large fraction of neurod2+ neurons were localized within the iz, below the layer of tbr1+ cp, most likely constituting the migratory population.
taken together, our results are consistent with a model in which neurod <dig> expression is initiated in post-mitotic neurons right before the onset of migration and regulates a set of genes, that includes critical components of the reelin pathway.fig.
<dig> neurod <dig> target genes are enriched in the subventricular-intermediate zone of the developing cortex.
a immunofluorescent staining against neurod <dig> and tbr <dig>  and dapi staining on coronal sections prepared from e <dig>  mouse brain.
neurod <dig> positive neurons are observed in the svz-iz region of the developing cortex.
scale bar, 62 μm.
b the percentage of overlap between neurod <dig> target genes and differentially expressed genes  in the cortical plate , subventricular zone/intermediate zone , ventricular zone , and those that are preferentially expressed in two regions  are plotted.
neurod <dig> target genes  display an enrichment in degs of the different cortical regions  as compared to randomly selected genes  from the mouse genome
next, in an unbiased approach to identify probable gene candidates regulating migration through the iz, we asked whether the expression of a particular set of neurod <dig> targets were enriched in the neurons of this zone.
towards this goal, we took advantage of a study describing differentially expressed transcripts by laser capture microscopy followed by mrna-seq within three major regions of the e <dig> cortex; cortical plate , subventricular-intermediate zones  and ventricular zone   <cit> .
in this study, each differentially expressed gene  was categorized as being strictly specific to one zone , or as being expressed in two of the three zones .
a comparison of neurod <dig> targets with the expression profiles of degs revealed that approximately 35 % of all svz-iz specific genes  were also targets of neurod <dig>  .
this comparison revealed lower percentages for the other four categories.
for example, in the vz where post-mitotic neurons are excluded and cell division of progenitor neurons takes place, only 6 % of degs were also targets of neurod <dig>  the overlap between degs in any category and equivalent numbers of randomly selected genes from the mouse genome was approximately 3 % .
consistent with known functions of previously identified genes that are required for radial migration, the functions of the  <dig> svz-iz specific neurod <dig> targets converged on axon guidance, regulation of neuronal polarity and actin cytoskeleton   <cit> .
taken together, our combined data strongly support a role for neurod <dig> in radial migration of cortical neurons.
neurod <dig> is positioned to control differentiation and axonal guidance of distinct cortical projection neuron lineages
neurons that are migrating through the iz during mid-embryogenesis differentiate into different types of cortical projection neurons  <cit> .
the genetic programs that specify the identities of distinct cortical projection neuron lineages are only partially understood.
loss-of-function studies of a set of layer-specific transcription factors have unveiled their requirement for distinct axon guidance programs and establishment of lamina-specific connectivity .
interestingly, we noticed the presence of such a group of transcription factors that control the differentiation of layer-specific properties of cortical projection neurons in our neurod <dig> target gene dataset.
we identified that both deep layer markers, such as fezf <dig> and bcl11b  and upper layer markers such as cux <dig> and satb <dig> scored highly in our neurod <dig> target gene list .
analysis of neurod <dig> peaks that mapped onto the genetic loci of fezf <dig>  bcl11b, cux <dig> and satb <dig> uncovered association at several distinct points which overlapped with enhancer  and promoter  specific histone marks .
examination of expression patterns at mid-embryogenesis revealed that neurod <dig> expression overlapped with the regions expressing fezf <dig>  bcl11b, cux <dig> and satb <dig> .fig.
<dig> neurod <dig> target genes encode transcription factors that control the differentiation of distinct populations of cortical layer specific projection neurons.
mrna expression data is obtained from allen developing mouse brain atlas  for fezf <dig> , bcl11b , cux <dig>  and satb <dig>  for e <dig>  mouse brain.
neurod <dig> expression overlaps with the domains expressing each of the cortical layer specific transcription factors.
neurod <dig> peaks are plotted relative to enhancer  and promoter  marks.
h3k4me <dig> and h3k4me <dig> chip-seq data is from   <cit> .
peaks are plotted using the genome browser at genome.ucsc.edu.
neurod <dig> chip followed by qpcr confirms neurod <dig> binding to the promoter regions of all four layer markers relative to a negative control  .
data is normalized to the amount of input dna as described in the methods section.
bars represent standard error of mean.
data represents two biological and five technical replicates.
mrna levels of fezf <dig>  bcl11b, cux <dig> and satb <dig> were quantified by rt-qpcr in neurons transfected with either a non-targeting or a neurod <dig> shrna.
a significant reduction in cux <dig> mrna levels was observed in the absence of neurod <dig>  each experiment represents  <dig> biological samples each analyzed in technical triplicates.
data is presented as fold change of control sample .
p-values for changes in expression of fezf <dig>  bcl11b and satb <dig> were between  <dig>  and  <dig> .
a non-targeting shrna was used as control.
bars are standard error of the mean
next, we confirmed neurod <dig> association with the promoter regions of these genes, by neurod <dig> chip coupled with quantitative pcr .
our analysis revealed that neurod <dig> associated strongly with the promoter regions of fezf <dig>  bcl11b, cux <dig> and satb <dig> genes.
in order to determine whether neurod <dig> is required for the expression of cortical layer-specific transcription factors, we quantified their mrna levels by rt-qpcr after neurod <dig> knockdown in primary cortical neurons .
interestingly, we observed a general trend in which all four transcription factor levels were reduced, albeit at varying levels of significance.
in particular, the expression of cux <dig> mrna was very significantly decreased by approximately 60 %.
together, our results argue that neurod <dig> is a principal regulator of a group of transcription factors that confer layer-specific identities to cortical projection neurons.
given that a major function of layer-specific transcription factors during cortical development is to express the correct set of axon guidance receptors and establish proper connectivity, next we focused on how neurod <dig> might be involved in this process.
evidence supporting this possibility comes from a study demonstrating that neurod <dig> genetically interacts with another bhlh neuronal differentiation factor, neurod <dig>  to establish the corpus callosum and the anterior commissure fiber tract  <cit> .
these axon guidance defects have been correlated with dysregulation of the cell adhesion protein cntn <dig> and the axonal receptor robo <dig> in neurod2/ <dig> double knockout mice  <cit> .
to our knowledge no other study has investigated a role for neurod <dig> in fasciculation and guidance of cortical projection neuron axons.
consequently, the full extent of the neurod <dig> regulated axon guidance pathways is unknown.
our previous pathway analysis using the biocarta database identified the reelin signaling pathway as a primary target of neurod <dig>  however did not reveal any suggestions of neurod <dig> regulation of cortical neuronal connectivity.
since different pathway databases have the potential to illuminate different aspects of a given gene set, to further explore neurod <dig> controlled pathways we compared our set of target genes against the kegg  pathway database using david bioinformatics resources  <cit> .
interestingly, our pathway analysis revealed axon guidance as the most significantly targeted pathway  .
among the  <dig> genes which construct different axon guidance pathways in the kegg database,  <dig> were targets of neurod <dig> .
in particular, we observed an enrichment for genes that constitute the ephrin/eph receptor, semaphorin/plexin receptor and slit/robo receptor signaling pathways.
our results are consistent with a role for neurod <dig> in callosal axon guidance and in formation of commissural fiber tracts via direct expression of receptors or ligands mediating axon repulsion and attraction during the mid-embryogenic period.table  <dig> top ten axon guidance genes targeted by neurod2
in this study, we have identified the in vivo targets of neurod <dig>  an effector transcription factor during the peak of cortical excitatory neurogenesis in mice.
we have generated our neurod <dig> target gene dataset by carrying out a chip-seq analysis on the cortex region of e <dig>  stage mouse embryos.
we discovered an enrichment of neurod <dig> binding locations at tsss.
however, we also observed that many binding sites were distant to tsss and overlapped with enhancer regions.
our observations were in agreement with recent evidence suggesting that tss-distal transcription factor binding sites are critical regulators of cell-type specific gene expression programs .
therefore, to assign binding locations to individual genes, we utilized the closestgene method in order to capture the tss-proximal and –distal peaks.
following our computational analysis we predicted  <dig>  neurod <dig> target genes and verified a selected subset at a high rate of confirmation .
we found that neurod <dig> gene targets include critical regulators of radial migration of cortical neurons from the ventricular and subventricular zones to the cortical plate.
a precisely regulated migration process in the developing cortex is essential for the establishment of a six-layered cortex.
molecularly, cortical radial migration requires reelin, a glycosylated extracellular protein that is secreted by the cajal-retzius cells located in the most superficial marginal zone of the developing cortex.
reelin binds to lrp <dig>  and vldlr receptors that are expressed by migrating cortical neurons and by doing so activates downstream signaling pathways that eventually reorganize the cytoskeleton and promote migration  <cit> .
our data revealed that neurod <dig> binds to lrp <dig> gene at regions overlapping with promoter and enhancer specific histone markers and also possibly regulates its expression.
other essential regulators of cortical radial migration are the serine-threonine kinase cdk <dig>  and its regulatory subunit cdk5r <dig> .
loss-of-function mutations of both cdk <dig> and cdk5r <dig> result in impaired radial migration and abnormal cortical lamination  <cit> .
intriguingly, we demonstrated that neurod <dig> binds to and regulates the expression of cdk5r <dig> gene in cortical neurons.
to date, a direct functional role for neurod <dig> in cortical migration has not been tested.
however, consistent with our findings, a recent study reports abnormal laminar positioning of cortical projection neurons in neurod <dig> knockout mice  <cit> .
looking forward we are greatly interested in directly testing a role for neurod <dig> in the cortical migration process.
consistent with our gene ontology results we showed that neurod <dig> was expressed in the neurons of the svz and iz, which are zones of migrating neurons.
further, overlaying our dataset with a study describing differentially expressed genes in functionally distinct zones of the embryonic cortex revealed that neurod <dig> is associated with approximately 35 % of all genes enriched in the svz and iz  <cit> .
many of these genes have not been investigated with regard to their functions in radial migration, and are highly likely to have essential roles in this process as they encode proteins that organize the neuronal cytoskeleton and control cellular adhesion.
we also observed a number of genes that regulate axonal pathfinding within this overlapping dataset, including epha <dig>  plxna <dig> and robo <dig>  which might indicate additional roles for these receptors in migration, as it has been recently suggested .
in sum, our study is in agreement with a model in which neurod <dig> expression is initiated in post-mitotic cortical neurons and regulates their migration through the transcription of a set of genes controlling actin and microtubule dynamics and cell surface molecules with roles in cellular adhesion.
finally, our data suggested that in addition to regulating the migration of cortical projection neurons, neurod <dig> also targets transcription factors that control the layer-specific identities and connectivity patterns of neurons located in different layers.
in particular, we provided evidence that neurod <dig> controls both deep-layer markers, such as fezf <dig> and bcl11b , as well as upper-layer markers such as satb <dig> and cux <dig>  in summary, our dataset offers an exciting list of genes and pathways with putative roles in migration, layer-specific differentiation and axonal migration.
CONCLUSIONS
significant progress in recent years has identified critical transcription factors which specify progenitors to the cortical projection neuron fate.
however, little is known about the effector programs that further control the differentiation of this important neuronal subtype  <cit> .
in this study, we report genome-wide targets of an effector transcription factor, neurod <dig>  using a chip-seq approach.
our results demonstrate that in mice at mid-embryogenesis , a time point which overlaps with the peak of cortical excitatory neurogenesis, neurod <dig> binds to a set of genes whose functions coalesce into two essential processes; radial migration and axon guidance.
prominently, we verify that expression of cdk5r <dig>  a critical regulator of radial migration, and cux <dig>  a transcription factor which controls the differentiation of upper layer  projection neurons  is dependent on neurod <dig>  our study highlights the value of chip-seq experiments conducted within relevant tissue and during a developmental period  <cit>  and offers the potential of unveiling novel genes and pathways regulating cortical projection neuron differentiation.
neurod <dig> controls the differentiation and physiology of a variety of different types of neurons such as those located in the hippocampus, amygdala and cerebellum.
looking forward, it is of interest to identify neurod <dig> targets in the context of different spatiotemporal settings within the developing nervous system.

 the neocortex is a six-layered structure composed of myriad excitatory and inhibitory neuron types. developmental programs, controlled by multiple cell-intrinsic and –extrinsic cues, establish unique patterns of connectivity for the diverse neuronal subtypes located in different layers. while the granular neurons of layer iv form networks of local connections within cerebral hemispheres, projection neurons of layers ii/iii, v and vi mediate long distance communications both within and outside the cortex. developmentally, excitatory neurons are born from dividing progenitors located in distinct germinal domains situated at ventricular and subventricular zones of the dorsal telencephalon. once born, they migrate to superficial cortical layers, with each wave of migrating neurons surpassing predecessors, resulting in six distinct cortical layers generated in an inside-out manner. consequently, birthdate of a neuron determines its final layer of residence and confers its layer-specific properties. during and following migration, cortical projection neurons initiate expression of specific sets of axon guidance molecules that allows pathfinding to correct targets. the end product of this highly complex and regulated migration and axon guidance process is a neocortex organized into six layers, with neurons residing in each layer exhibiting unique physiological properties and patterns of connectivity. almost all aspects of neurogenesis, migration and neuronal differentiation are orchestrated by hierarchical and combinatorial functioning of transcription factor networks. within the embryonic germinal zones, expression of neurog and neurog proneural basic helix loop helix transcription factors commit neural progenitors to cortical excitatory neuron identity. neurog is both necessary and sufficient for generation of these neurons and neurog initiated specification of projection neuron identity involves a complex transcription factor network composed of effector transcription factors, such as tbr, neurod and neurod. despite our relatively thorough understanding of the tfs required for specifying a cortical excitatory neuron identity, little is known about how differentiation programs are executed by effector tfs and the nature of the downstream target genes required for corticogenesis. for example, neurod is expressed within a wide temporal window outside of the proliferative zones and constitutes a potential regulator of critical aspects of differentiation and/or maintenance of different types of cortical excitatory neurons. indeed, several studies investigating the consequences of neurod loss-of-function in mice have demonstrated that neurod is required for commissural axon pathfinding of layer ii/iii callosal projection neurons, formation of cortical somatosensory maps within layer iv granular neurons and maturation of dendrites and synapses in the hippocampus. furthermore, a gain-of-function study has demonstrated that misexpression of neurod in ventral telencephalon progenitors is sufficient to prevent their normal gabaergic differentiation. although during development neurod controls the execution of a wide range of functions, its target genes during cortex development are largely unknown. in this study we identified genome-wide targets of neurod during mid-embryogenesis. we demonstrate that neurod binds to a large number of target genes with prominent roles in radial migration, layer-specific differentiation and axon pathfinding of cortical projection neurons. moreover, we find that neurod is positioned to control cortical radial migration by regulating members of the reelin signaling pathway. importantly, we demonstrate that expression of cdk5r the regulatory subunit of cyclin-dependent kinase is critically dependent on neurod our identification of neurod targets offers mechanistic insight into known and potentially novel functions of this effector transcription factor and points to the importance of carrying out chip-seq analysis at developmental timepoints within relevant tissue.  identification of neurod binding sites in embryonic cortex in mice, the bulk of cortical excitatory neurogenesis takes place during the last ten days of gestation, a period which also coincides with high levels of neurod expression. therefore, in order to determine the genome-wide targets of neurod during cortical development, we carried out chromatin-immunoprecipitation followed by high-throughput sequencing. first, we identified three antibodies that could successfully immunoprecipitate neurod following overexpression in neuro2a cell line. after isolating neurod2-associated chromatin from mouse embryonic day cortex, we confirmed that the promoter region of the nhlh gene, a previously identified neurod target, was amplified by pcr using chip template dna precipitated by all three neurod antibodies, but not from chip dna prepared by an unrelated antibody against gfp. after massively parallel sequencing of all three neurod chip dna and two separate gfp chip dna samples, we mapped reads using bowtie short read aligner. neurod peaks were identified by macs and gfp peak alignments were used as input control dataset for signal normalization. from the three experimental samples a total of approximately, unique reads were identified, which mapped onto peaks.fig. chip-seq reveals neurod target genes during cortical development. a chromatin dna was immunoprecipitated with three different antibodies against neurod or with a gfp antibody which was used as a negative control. pcr was performed using immunoprecipitated dna as template. specific primers amplified the promoter region of a previously identified neurod target gene nhlh. an enrichment for the positive control nhlh gene was demonstrated in neurod chip-pcr samples as compared to gfp chip-pcr. b the developing cortex was dissected from e mouse brain and used for neurod chip-seq. peak regions and target genes were identified the quality of our data was validated by the high degree of correlation among the three neurod antibodies. in contrast, comparison of neurod peaks generated by the three individual antibodies to those of another transcription factor expressed in the mouse embryonic brain, ctcf, resulted in much lower r values. in an effort to reduce false-positive binding sites due to non-specific interactions of the particular antibody used, we decided to discard peaks that are identified in only one of the three chip-seq datasets acquired using the three separate antibodies. as a result, out of a total of peaks from three separate datasets, peaks were found to be located in regions containing overlapping peaks from at least two antibodies. next, we collapsed all the overlapping peaks to obtain the union of peak coordinates and as a result identified neurod binding locations. following analyses were carried out using these neurod binding sites. neuronal bhlh transcription factors like neurod bind to an e-box motif, which is loosely defined as canntg. therefore, we asked whether or not the neurod peak sequences were enriched in e-box elements. by using the meme-chip suite, a tool designed to identify enriched sequence elements in large datasets, we discovered that an extended e-box motif, catg, was significantly associated with neurod peak sequences.fig. neurod binds to e-box elements proximal to tsss in vivo. a neurod consensus sequence was predicted using peak regions as input for the meme-chip suite. b among the neurod peaks, approximately 45 % mapped onto ensembl annotated genes. among them approximately 83 % mapped onto introns, and 17 % to either 5′utr, cds or 3′utr exons. c the binding preference of neurod to introns, 5′utr exons, 3′utr exons and cds exons was calculated. the number of neurod binding sites observed on introns, 5′utr, 3′utr and cds exons were normalized to the length of each of these gene segments and compared to a random distribution profile. the data suggests approximately an 8-fold enrichment in neurod binding to the first 5′utr exon, a 5-fold enrichment in binding to the first cds exon and approximately 2-fold enrichment in binding to the first and second 3′utr exons. ***p-value ~  *p-value < . d a binding map of neurod relative to closest tsss, revealed that neurod has a binding preference within +/− 1000 bps of tsss. peaks that mapped upstream and downstream of tsss are color-coded. e the distribution profiles of closestgene scores calculated by using neurod peaks were very significantly different from those calculated using randomly distributed peaks on the genome. f distribution of normalized scores for individual genes were generated by subtracting the closestgene score calculated using neurod peaks from that of random peaks. dark green represents genes whose scores were greater than one standard deviation above the mean and were used for further analyses in order to determine neurod binding preference along the genome and therefore gain insight into its mechanism of action, we examined peak positions relative to annotated transcriptional units. analysis of all sites revealed that approximately half of neurod binding locations mapped onto annotated ensembl transcripts and the remainder mapped onto intergenic regions. within a gene, 83 % of intragenic sites were associated with intronic regions and 17 % were associated with 5′utr, coding, or 3′utr exons. however, on average introns are ~25– times longer than exons, therefore in order to normalize for this length bias we calculated the probability of binding along a gene compared to a random distribution profile. our calculations revealed that neurod binding was most likely to be embedded in the first 5′utr exon with an approximately 8-fold enrichment in probability compared to random distribution. next, we generated a neurod binding map relative to annotated transcription start sites and observed a major accumulation of neurod peaks within 1000 bps from a tss. therefore, in vivo neurod preferentially binds to tss proximal sequences located within or upstream of 5′utr exons. a previous study used a chip-seq approach to investigate genome-wide targets of neurod after its viral transduction into the p cell line. however, since this study was conducted in cell culture, it may not provide an accurate reflection of the in vivo targets of neurod during cortex development. in fact, recent studies propose that transcription factor-chromatin associations are context dependent and can vary between developmental stages and tissue types. therefore, within the context of cortical development, with the exception of a small number of candidate genes studied so far, neurod2’s genome-wide targets and the kinds of biological processes regulated are largely unknown. since our data demonstrate that neurod binding was not exclusive to promoter regions or tss-proximal sequences, we decided to employ a method for assigning individual peaks to genes which would also account for tss-distant peaks, as opposed to methods which set fixed windows around tsss for peak capturing. for this reason, we decided to use the closestgene method which assigns scores to individual genes based on the number of associated peaks and their proximity to respective transcription start sites. we calculated two scores for each gene in the mouse genome by using either the neurod peaks or the same number of randomly distributed peaks as the input dataset. plotting the distribution profiles of the scores calculated by neurod peaks or random peaks revealed a very significant difference between these two datasets. next, we calculated a normalized closestgene score for each gene by simply subtracting the random score from the neurod score. we then selected putative target genes, with varying scores, for confirmation by neurod chip followed by quantitative pcr using the immunoprecipitated dna as template. we selected genes from the top, representing genes whose scores were above one standard deviation of the mean and were able to confirm out of. in addition, as negative controls we tested genes which were not identified as neurod targets. these five genes are expressed in interneurons and not in neurod2-expressing cortical projection neurons. we did not observe any significant enrichment in neurod binding to 4/ of the non-target genes and only a slight enrichment in gsx a finding worthy of future investigation. in sum, in light of our high confirmation rate of neurod binding to putative target genes we decided to use the top in all further analyses. neurod target genes are associated with radial migration of cortical neurons to further gain broad understanding into the biological processes regulated by neurod we carried out gene ontology analysis. we identified significantly over-represented biological processes in the top targets against all of the genes in the mouse genome. ranking based on fold enrichment of biological processes above a significance threshold, revealed that the highly ranked neurod controlled activities broadly converged onto two major developmental processes; cortical radial migration and axon guidance/fasciculation.table gene ontology analysis of neurod targets biological process categories above a cutoff p-value are ranked by fold enrichment. fold enrichment is calculated as log2 to gain further understanding of the signaling pathways that were regulated by neurod we carried out pathway analysis with the neurod target genes against all pathways present in the biocarta database. interestingly, we identified reelin signaling as the most significantly targeted pathway. the reelin protein is secreted by the cajal-retzius cells, located on the surface of the developing cortex. by binding to receptors on migrating cortical neurons, reelin promotes reorganization of the cytoskeleton and regulates neuronal migration. our analysis revealed that neurod targets included several key members of this pathway, such as fyn, cdk5r dab and lrp genes.fig. neurod targets cdk5r and lrp genes during corticogenesis. a diagrammatic representation of the reelin signaling pathway that is required for cortical radial migration. genes that are targets of neurod are marked with a red star. b neurod western blotting confirms knockdown in primary cortical neuronal culture transfected with neurod shrna as compared to a non-targeting shrna. egfp is used to verify transfection efficiency and beta-actin is used as a loading control. c, d and e mrna expression data is obtained from allen developing mouse brain atlas for neurod cdk5r and lrp genes in e mouse brain. f and i neurod peaks associated with cdk5r and lrp are plotted relative to enhancer and promoter marks. h3k4me and h3k4me chip-seq data is from. peaks are plotted using the genome browser at genome.ucsc.edu. g and j neurod chip followed by qpcr confirms neurod binding to the promoter regions of cdk5r and lrp relative to a negative control. data is normalized to the amount of input dna as described in the methods section. bars represent standard error of mean. data represents two biological and five technical replicates. h and k reverse transcription and qpcr analysis of neurons transfected with neurod shrna or non-targeting shrna reveals a significant reduction in cdk5r mrna levels and a trend of reduction in lrp levels  after neurod knockdown. all rt-qpcr results represented data from biological samples each analyzed in technical triplicates. bars represent standard error of mean we next focused our analysis on two important regulators of cortical radial migration, cdk5r and lrp genes. initially, examination of expression patterns at mid-embryogenesis revealed that neurod expression largely overlapped with zones of lrp and cdk5r expression. next, we examined whether neurod binding along cdk5r and lrp genes were associated with enhancer or promoter regions. we acquired chip-seq data from e whole brain for h3k4me histone modification as a marker for enhancer regions and h3k4me modification as a marker for promoter regions. overlaying of these datasets with neurod peaks revealed that neurod binding was associated with both promoter and enhancer specific histone marks on these two genes. we further confirmed neurod association with the promoter regions of lrp and cdk5r genes by neurod chip followed by qpcr. finally, we asked whether neurod controls cdk5r and lrp transcription. we knocked down neurod expression almost completely in primary cortical neurons by high-efficiency transfection of small hairpin rna. remarkably, our rt-qpcr results demonstrated a significant, five-fold reduction in cdk5r mrna levels after neurod knockdown, as compared to a control sample transfected with a non-targeting shrna. lrp mrna also exhibited a trend in reduction in neurod lacking neurons, however with lower significance. in sum, our results strongly support neurod control of radial migration and the reelin signaling pathway through direct regulation of key genes such as cdk5r and lrp next, we asked whether neurod was expressed in neurons of the intermediate zone, which is a developmentally transient cortical region through which post-mitotic neurons born in subventricular and ventricular zones migrate in order to reach their final destination within the cortical plate. to mark neurons that have completed their migration and are localized to the cortical plate, we performed co-staining with antibodies against the tbr transcription factor and neurod as expected, we identified neurod2+/tbr1+ neurons localized to the cp. in addition, a large fraction of neurod2+ neurons were localized within the iz, below the layer of tbr1+ cp, most likely constituting the migratory population. taken together, our results are consistent with a model in which neurod expression is initiated in post-mitotic neurons right before the onset of migration and regulates a set of genes, that includes critical components of the reelin pathway.fig. neurod target genes are enriched in the subventricular-intermediate zone of the developing cortex. a immunofluorescent staining against neurod and tbr and dapi staining on coronal sections prepared from e mouse brain. neurod positive neurons are observed in the svz-iz region of the developing cortex. scale bar, 62 μm. b the percentage of overlap between neurod target genes and differentially expressed genes in the cortical plate, subventricular zone/intermediate zone, ventricular zone, and those that are preferentially expressed in two regions are plotted. neurod target genes display an enrichment in degs of the different cortical regions as compared to randomly selected genes from the mouse genome next, in an unbiased approach to identify probable gene candidates regulating migration through the iz, we asked whether the expression of a particular set of neurod targets were enriched in the neurons of this zone. towards this goal, we took advantage of a study describing differentially expressed transcripts by laser capture microscopy followed by mrna-seq within three major regions of the e cortex; cortical plate, subventricular-intermediate zones and ventricular zone. in this study, each differentially expressed gene was categorized as being strictly specific to one zone, or as being expressed in two of the three zones. a comparison of neurod targets with the expression profiles of degs revealed that approximately 35 % of all svz-iz specific genes were also targets of neurod. this comparison revealed lower percentages for the other four categories. for example, in the vz where post-mitotic neurons are excluded and cell division of progenitor neurons takes place, only 6 % of degs were also targets of neurod the overlap between degs in any category and equivalent numbers of randomly selected genes from the mouse genome was approximately 3 %. consistent with known functions of previously identified genes that are required for radial migration, the functions of the svz-iz specific neurod targets converged on axon guidance, regulation of neuronal polarity and actin cytoskeleton. taken together, our combined data strongly support a role for neurod in radial migration of cortical neurons. neurod is positioned to control differentiation and axonal guidance of distinct cortical projection neuron lineages neurons that are migrating through the iz during mid-embryogenesis differentiate into different types of cortical projection neurons. the genetic programs that specify the identities of distinct cortical projection neuron lineages are only partially understood. loss-of-function studies of a set of layer-specific transcription factors have unveiled their requirement for distinct axon guidance programs and establishment of lamina-specific connectivity. interestingly, we noticed the presence of such a group of transcription factors that control the differentiation of layer-specific properties of cortical projection neurons in our neurod target gene dataset. we identified that both deep layer markers, such as fezf and bcl11b and upper layer markers such as cux and satb scored highly in our neurod target gene list. analysis of neurod peaks that mapped onto the genetic loci of fezf bcl11b, cux and satb uncovered association at several distinct points which overlapped with enhancer and promoter specific histone marks. examination of expression patterns at mid-embryogenesis revealed that neurod expression overlapped with the regions expressing fezf bcl11b, cux and satb.fig. neurod target genes encode transcription factors that control the differentiation of distinct populations of cortical layer specific projection neurons. mrna expression data is obtained from allen developing mouse brain atlas for fezf, bcl11b, cux and satb for e mouse brain. neurod expression overlaps with the domains expressing each of the cortical layer specific transcription factors. neurod peaks are plotted relative to enhancer and promoter marks. h3k4me and h3k4me chip-seq data is from. peaks are plotted using the genome browser at genome.ucsc.edu. neurod chip followed by qpcr confirms neurod binding to the promoter regions of all four layer markers relative to a negative control. data is normalized to the amount of input dna as described in the methods section. bars represent standard error of mean. data represents two biological and five technical replicates. mrna levels of fezf bcl11b, cux and satb were quantified by rt-qpcr in neurons transfected with either a non-targeting or a neurod shrna. a significant reduction in cux mrna levels was observed in the absence of neurod each experiment represents biological samples each analyzed in technical triplicates. data is presented as fold change of control sample. p-values for changes in expression of fezf bcl11b and satb were between and. a non-targeting shrna was used as control. bars are standard error of the mean next, we confirmed neurod association with the promoter regions of these genes, by neurod chip coupled with quantitative pcr. our analysis revealed that neurod associated strongly with the promoter regions of fezf bcl11b, cux and satb genes. in order to determine whether neurod is required for the expression of cortical layer-specific transcription factors, we quantified their mrna levels by rt-qpcr after neurod knockdown in primary cortical neurons. interestingly, we observed a general trend in which all four transcription factor levels were reduced, albeit at varying levels of significance. in particular, the expression of cux mrna was very significantly decreased by approximately 60 %. together, our results argue that neurod is a principal regulator of a group of transcription factors that confer layer-specific identities to cortical projection neurons. given that a major function of layer-specific transcription factors during cortical development is to express the correct set of axon guidance receptors and establish proper connectivity, next we focused on how neurod might be involved in this process. evidence supporting this possibility comes from a study demonstrating that neurod genetically interacts with another bhlh neuronal differentiation factor, neurod to establish the corpus callosum and the anterior commissure fiber tract. these axon guidance defects have been correlated with dysregulation of the cell adhesion protein cntn and the axonal receptor robo in neurod2/ double knockout mice. to our knowledge no other study has investigated a role for neurod in fasciculation and guidance of cortical projection neuron axons. consequently, the full extent of the neurod regulated axon guidance pathways is unknown. our previous pathway analysis using the biocarta database identified the reelin signaling pathway as a primary target of neurod however did not reveal any suggestions of neurod regulation of cortical neuronal connectivity. since different pathway databases have the potential to illuminate different aspects of a given gene set, to further explore neurod controlled pathways we compared our set of target genes against the kegg pathway database using david bioinformatics resources. interestingly, our pathway analysis revealed axon guidance as the most significantly targeted pathway. among the genes which construct different axon guidance pathways in the kegg database, were targets of neurod. in particular, we observed an enrichment for genes that constitute the ephrin/eph receptor, semaphorin/plexin receptor and slit/robo receptor signaling pathways. our results are consistent with a role for neurod in callosal axon guidance and in formation of commissural fiber tracts via direct expression of receptors or ligands mediating axon repulsion and attraction during the mid-embryogenic period.table top ten axon guidance genes targeted by neurod2 in this study, we have identified the in vivo targets of neurod an effector transcription factor during the peak of cortical excitatory neurogenesis in mice. we have generated our neurod target gene dataset by carrying out a chip-seq analysis on the cortex region of e stage mouse embryos. we discovered an enrichment of neurod binding locations at tsss. however, we also observed that many binding sites were distant to tsss and overlapped with enhancer regions. our observations were in agreement with recent evidence suggesting that tss-distal transcription factor binding sites are critical regulators of cell-type specific gene expression programs. therefore, to assign binding locations to individual genes, we utilized the closestgene method in order to capture the tss-proximal and –distal peaks. following our computational analysis we predicted neurod target genes and verified a selected subset at a high rate of confirmation. we found that neurod gene targets include critical regulators of radial migration of cortical neurons from the ventricular and subventricular zones to the cortical plate. a precisely regulated migration process in the developing cortex is essential for the establishment of a six-layered cortex. molecularly, cortical radial migration requires reelin, a glycosylated extracellular protein that is secreted by the cajal-retzius cells located in the most superficial marginal zone of the developing cortex. reelin binds to lrp and vldlr receptors that are expressed by migrating cortical neurons and by doing so activates downstream signaling pathways that eventually reorganize the cytoskeleton and promote migration. our data revealed that neurod binds to lrp gene at regions overlapping with promoter and enhancer specific histone markers and also possibly regulates its expression. other essential regulators of cortical radial migration are the serine-threonine kinase cdk and its regulatory subunit cdk5r. loss-of-function mutations of both cdk and cdk5r result in impaired radial migration and abnormal cortical lamination. intriguingly, we demonstrated that neurod binds to and regulates the expression of cdk5r gene in cortical neurons. to date, a direct functional role for neurod in cortical migration has not been tested. however, consistent with our findings, a recent study reports abnormal laminar positioning of cortical projection neurons in neurod knockout mice. looking forward we are greatly interested in directly testing a role for neurod in the cortical migration process. consistent with our gene ontology results we showed that neurod was expressed in the neurons of the svz and iz, which are zones of migrating neurons. further, overlaying our dataset with a study describing differentially expressed genes in functionally distinct zones of the embryonic cortex revealed that neurod is associated with approximately 35 % of all genes enriched in the svz and iz. many of these genes have not been investigated with regard to their functions in radial migration, and are highly likely to have essential roles in this process as they encode proteins that organize the neuronal cytoskeleton and control cellular adhesion. we also observed a number of genes that regulate axonal pathfinding within this overlapping dataset, including epha plxna and robo which might indicate additional roles for these receptors in migration, as it has been recently suggested. in sum, our study is in agreement with a model in which neurod expression is initiated in post-mitotic cortical neurons and regulates their migration through the transcription of a set of genes controlling actin and microtubule dynamics and cell surface molecules with roles in cellular adhesion. finally, our data suggested that in addition to regulating the migration of cortical projection neurons, neurod also targets transcription factors that control the layer-specific identities and connectivity patterns of neurons located in different layers. in particular, we provided evidence that neurod controls both deep-layer markers, such as fezf and bcl11b, as well as upper-layer markers such as satb and cux in summary, our dataset offers an exciting list of genes and pathways with putative roles in migration, layer-specific differentiation and axonal migration.  significant progress in recent years has identified critical transcription factors which specify progenitors to the cortical projection neuron fate. however, little is known about the effector programs that further control the differentiation of this important neuronal subtype. in this study, we report genome-wide targets of an effector transcription factor, neurod using a chip-seq approach. our results demonstrate that in mice at mid-embryogenesis, a time point which overlaps with the peak of cortical excitatory neurogenesis, neurod binds to a set of genes whose functions coalesce into two essential processes; radial migration and axon guidance. prominently, we verify that expression of cdk5r a critical regulator of radial migration, and cux a transcription factor which controls the differentiation of upper layer projection neurons is dependent on neurod our study highlights the value of chip-seq experiments conducted within relevant tissue and during a developmental period and offers the potential of unveiling novel genes and pathways regulating cortical projection neuron differentiation. neurod controls the differentiation and physiology of a variety of different types of neurons such as those located in the hippocampus, amygdala and cerebellum. looking forward, it is of interest to identify neurod targets in the context of different spatiotemporal settings within the developing nervous system. 
our understanding of effector transcription factors and their downstream transcriptional programs that direct the differentiation process of cortical projection neurons is far from complete. in this study, we carried out a chip-seq analysis of neurod an effector transcription factor expressed in lineages of cortical projection neurons during the peak of cortical excitatory neurogenesis. our analysis of in vivo neurod target genes offers mechanistic insight into signaling pathways that regulate neuronal migration and axon guidance and identifies genes that are likely to be required for proper cortical development. neurod2cortical projection neuroncortical layersradial migrationaxon guidanceissue-copyright-statement© the author 2015 our results suggest that during cortical development neurod targets key genes that are required for reelin signaling, a major pathway that regulates the migration of neurons from germinal zones to their final layers of residence within the cortex. cortical projection neurons constitute the major excitatory neuron population within the cortex and mediate long distance communication between the cortex and other brain regions. we also find that neurod binds to a large set of genes with functions in layer-specific differentiation and in axonal pathfinding of cortical projection neurons. cellular differentiation programs are controlled, to a large extent, by the combinatorial functioning of specific transcription factors.
 cellular differentiation programs are controlled, to a large extent, by the combinatorial functioning of specific transcription factors. cortical projection neurons constitute the major excitatory neuron population within the cortex and mediate long distance communication between the cortex and other brain regions. our understanding of effector transcription factors and their downstream transcriptional programs that direct the differentiation process of cortical projection neurons is far from complete.  in this study, we carried out a chip-seq analysis of neurod an effector transcription factor expressed in lineages of cortical projection neurons during the peak of cortical excitatory neurogenesis. our results suggest that during cortical development neurod targets key genes that are required for reelin signaling, a major pathway that regulates the migration of neurons from germinal zones to their final layers of residence within the cortex. we also find that neurod binds to a large set of genes with functions in layer-specific differentiation and in axonal pathfinding of cortical projection neurons.  our analysis of in vivo neurod target genes offers mechanistic insight into signaling pathways that regulate neuronal migration and axon guidance and identifies genes that are likely to be required for proper cortical development. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords neurod2cortical projection neuroncortical layersradial migrationaxon guidanceissue-copyright-statement© the author 2015 