The Epigenetic Factor Landscape of Developing Neocortex Is Regulated by Transcription Factors Pax6→ Tbr2→ Tbr1

Epigenetic factors (EFs) regulate multiple aspects of cerebral cortex development, including proliferation, differentiation, laminar fate, and regional identity. The same neurodevelopmental processes are also regulated by transcription factors (TFs), notably the Pax6→ Tbr2→ Tbr1 cascade expressed sequentially in radial glial progenitors (RGPs), intermediate progenitors, and postmitotic projection neurons, respectively. Here, we studied the EF landscape and its regulation in embryonic mouse neocortex. Microarray and in situ hybridization assays revealed that many EF genes are expressed in specific cortical cell types, such as intermediate progenitors, or in rostrocaudal gradients. Furthermore, many EF genes are directly bound and transcriptionally regulated by Pax6, Tbr2, or Tbr1, as determined by chromatin immunoprecipitation-sequencing and gene expression analysis of TF mutant cortices. Our analysis demonstrated that Pax6, Tbr2, and Tbr1 form a direct feedforward genetic cascade, with direct feedback repression. Results also revealed that each TF regulates multiple EF genes that control DNA methylation, histone marks, chromatin remodeling, and non-coding RNA. For example, Tbr1 activates Rybp and Auts2 to promote the formation of non-canonical Polycomb repressive complex 1 (PRC1). Also, Pax6, Tbr2, and Tbr1 collectively drive massive changes in the subunit isoform composition of BAF chromatin remodeling complexes during differentiation: for example, a novel switch from Bcl7c (Baf40c) to Bcl7a (Baf40a), the latter directly activated by Tbr2. Of 11 subunits predominantly in neuronal BAF, 7 were transcriptionally activated by Pax6, Tbr2, or Tbr1. Using EFs, Pax6→ Tbr2→ Tbr1 effect persistent changes of gene expression in cell lineages, to propagate features such as regional and laminar identity from progenitors to neurons.


INTRODUCTION
Development of the embryonic cerebral cortex is regulated by intrinsic genetic programs and signaling interactions that ultimately give rise to diverse cortical areas, layers, and neuron subtypes with distinct gene expression profiles (Sun and Hevner, 2014;Silbereis et al., 2016). In each cell type, the gene expression profile is determined by a combination of transcription factors (TFs) that bind specific DNA sequences to activate or repress transcription, and epigenetic factors (EFs) that control chromatin structure and accessibility for transcription (Bernstein et al., 2007;Allis and Jenuwein, 2016). Transcriptional activity thus depends on the epigenetic status of the chromatin, as well as the presence or absence of specific TFs that bind promoters, enhancers, and other cis-acting regulatory elements in the genome (Nord et al., 2015;Shibata et al., 2015).
Previous studies have demonstrated physical and genetic interactions between EFs and TFs during neurogenesis. In adult subependymal zone progenitors, Pax6 forms a complex with BAF, a large, multi-subunit ATPase-dependent chromatin remodeler, to activate neurogenic genes such as Sox11 (Ninkovic et al., 2013). In developing neocortex, Tbr2 interacts with Jmjd3 (Gene: Kdm6b), a histone lysine demethylase that removes repressive trimethylation marks on histone H3 lysine 27 (H3K27me3) placed by Polycomb repressive complex 2 (PRC2), to thereby derepress transcription (Sessa et al., 2017). Such interactions illustrate that TFs sometimes function by physically recruiting and targeting EFs to specific genes.
Examples where TFs and EFs regulate each other at the transcriptional level are also known. In developing forebrain, Jarid1b (Kdm5b), a histone lysine demethylase that removes activating epigenetic marks (H3K4me2/3) placed by Trithorax-Group (TrxG) complexes, is required to deactivate and thus limit Pax6 expression (Albert et al., 2013). Similarly, Af9 (Mllt3), a YEATS domain protein that binds acetylated lysine residues, negatively modulates transcription of Tbr1 during genesis of upper cortical layers (Büttner et al., 2010).
Conversely, Pax6, Tbr2, and Tbr1 also regulate the expression of some EF genes, although in many cases it remains unclear whether such regulation is direct. For example, Dnmt3a (a DNA methyltransferase) is upregulated in Pax6 null embryonic cortex (Holm et al., 2007), but it is unknown if Pax6 regulates Dnmt3a directly or indirectly (Ypsilanti and Rubenstein, 2016). A few EF genes are known targets of Tbr2, such as Gadd45g, important in DNA demethylation (Sessa et al., 2017). Tbr1 is known to activate Auts2 (Bedogni et al., 2010a), a Polycomb repressive complex 1 (PRC1) non-canonical subunit (Gao et al., 2014); and Arid1b, an important BAF subunit (Notwell et al., 2016). Building on these few examples, one goal of the present study was to comprehensively identity EF genes that are directly bound and regulated by Pax6,Tbr2,and Tbr1. In addition to studying regulation of EF genes, we also wished to characterize EF genes associated with cortical differentiation, comprising the "EF landscape." In embryonic neocortex, histological zones are correlated with cell identity and differentiation (Bystron et al., 2008), while rostrocaudal and mediolateral gradients of gene expression presage arealization (O'Leary et al., 2007). Indeed, zonal expression patterns can be used to infer specificity of gene expression in RGPs, apical IPs, basal IPs, and neurons (Kawaguchi et al., 2008). In the present study, by combining microarray analysis of RGP and IP transcriptomes  with in situ hybridization (ISH) to define gene expression patterns, we find that dozens of EF genes exhibit celltype or region-specific expression, and together constitute a rich EF landscape involving all categories of epigenetic mechanisms.
Our analysis depicts a new, comprehensive view of the EF landscape in developing neocortex, and its regulation by Pax6,Tbr2,and Tbr1. In addition, this approach yields an updated portrayal of the Pax6→ Tbr2→ Tbr1 cascade, including feedforward and feedback regulation. Importantly, the data indicate that Pax6 is not a specific marker of RGPs, but is also expressed in many Tbr2+ IPs, as we have noted (Englund et al., 2005). Other TFs, such as Sox9, are more specific RGP markers. Together, our results show how a cortical TF network implements cortical differentiation by controlling diverse EFs.

Data Sources
To study gene expression and regulation in the context of cortical neurogenesis, we analyzed data from experiments using embryonic mouse cortex, in the age range from embryonic day (E) 13.5 to E15.5. For microarray and chromatin immunoprecipitation-sequencing (ChIP-seq) experiments, data were reanalyzed from previous studies, and from a new microarray dataset (Supplementary Table S1). For in situ hybridization (ISH), data were sourced from Genepaint (http:// genepaint.org); the Allen Brain Atlas Developing Mouse Brain (http://developingmouse.brain-map.org/); the Brain Gene Expression Map (BGEM), hosted at Gensat (http://gensat.org); and previous literature.

Screen to Identify Cell-Type and Region-Specific Gene Expression
Previously, transcriptome profiling and unbiased cluster analysis of single cells indicated that the ventricular zone (VZ) and subventricular zone (SVZ) of E14.5 mouse neocortex contain four cell types: RGPs, apical IPs (aIPs), basal IPs (bIPs), and postmitotic projection neurons (PNs) (Kawaguchi et al., 2008). Each cell type occupies characteristic histological zones in developing neocortex: RGPs in VZ; aIPs in VZ; bIPs in SVZ; and PNs in SVZ, intermediate zone (IZ), and cortical plate (CP). Using this information, we screened the top 300 differentially expressed genes (up-and downregulated) from a previous microarray experiment comparing RGP and IP transcriptomes . For the selected genes, we assessed histological expression patterns as revealed by ISH or microdissection (Ayoub et al., 2011). The primary goal was to identify RGP and IP genes, but as it happened, PN-specific genes were also enriched in Tbr2-GFP+ sorted cells, reflecting perdurance of GFP in daughter neurons of IPs . Conversely, non-PN lineages (e.g., meninges) were highly enriched in Tbr2-GFP − sorted cells.
Cell-type specificity was determined using the following criteria. RGP genes were enriched in Tbr2-GFP − cells on microarray (log 2 FC < 0; p < 0.05), and expressed mainly in VZ; aIP genes were enriched in Tbr2-GFP+ cells (log 2 FC > 0; p < 0.05), and expressed mainly in VZ; bIP genes were enriched in Tbr2-GFP+ cells, and expressed mainly in SVZ; PN genes were enriched in Tbr2-GFP+ cells, and expressed in IZ/CP. Some neuronal differentiation genes were expressed by not only neurons, but also progenitor cells undergoing neuronal differentiation. Also, some neuronal genes were widely expressed in forebrain neurons, while others were restricted to cortical PNs. Thus, neuron-specific genes were further classified according to initial zone of expression (VZ earliest, CP latest), and specificity for cortical or general neurons. If different microarray probes for the same gene showed enrichment in Tbr2-GFP+ and Tbr2-GFP − cells ("conflicted" probes), the gene was not considered specific for cell type. Genes with rostrocaudal expression gradients were identified, and classified according to zone of expression, as previously described (Bedogni et al., 2010a;Elsen et al., 2013;Alfano et al., 2014). Further details of our approach, including analysis of gene expression in other cell types (such as GABAergic neurons), will be presented in a separate manuscript (in preparation).
By this approach, 52 EF genes with cell-type-specific expression in developing neocortex were ascertained (Supplementary Table S2), as were 11 EF genes with rostrocaudal gradients; 4 genes exhibited both cell-type and region-specific expression (Supplementary Table S3).

Ethics Statement
This study was carried out in accordance with the recommendations of Guide for the Care and Use of Laboratory Animals, National Research Council. The protocol was approved by the Institutional Animal Care and Use Committee of Seattle Children's Research Institute.

Analysis of ChIP-Seq and Other TF Binding Data
Previous ChIP-seq raw data were obtained and reanalyzed for Pax6 (Pattabiraman et al., 2014), Tbr2 (Sessa et al., 2017), and Tbr1 (Notwell et al., 2016). TF binding sites (peaks) were determined from BED files using the Bioconductor ChIPpeakAnno package (Zhu et al., 2010), as well as the TxDb.Mmusculus.UCSC.mm9.knownGene package, which is simply a re-packaging of the UCSC known gene table for the mm9 genome build (Rosenbloom et al., 2015). Peaks were annotated to the closest gene within 50 kilobases (kb) of the binding site. In the present analysis, TF binding was considered "positive" if the binding site was located anywhere in the transcribed sequence, or within 50 kb upstream or downstream.
The ChIP-seq data listed in Supplementary Table S1 were our main sources, but TF binding was also evaluated by reference to previous literature. For Pax6, previous studies included genome-wide ChIP analyses of Pax6 binding in E12.5 neocortex (Sansom et al., 2009) and forebrain (Sun et al., 2015); as well as computational analysis and prediction of Pax6 binding sites (Coutinho et al., 2011). Results of all TF binding analyses for selected EF genes are included in Supplementary Table S3.

Defining Direct Target Genes Regulated by Transcription Factors
Genes were defined as direct targets of Pax6, Tbr2, or Tbr1 regulation if the gene showed both TF binding by ChIP-seq, and differential expression (p < 0.05) in TF mutant neocortex compared to control on microarray. For analysis of Tbr1 and Tbr2 direct target genes, differential expression (p < 0.05) on either MA1 or MA2 was accepted as evidence of regulation. Genes regulated synergistically by Tbr1 and Tbr2 were identified by the presence of both Tbr1 and Tbr2 binding sites, and significant differential expression (p < 0.05) in Tbr1/2 dKO cortex, but not in Tbr1 KO or Tbr2 cKO cortex independently.
By this approach, 36 EF genes were identified as direct targets of transcriptional regulation by Pax6, Tbr2, and/or Tbr1; direct regulation was also assessed for the key TFs Pax6, Insm1, Tbr2, and Tbr1 (Supplementary Table S4).

RESULTS AND DISCUSSION
Cell-Type Specific Expression of Pax6, Tbr2, and Tbr1 Using the methods described above to evaluate cell-type-specific gene expression, we began by evaluating the expression of Pax6, Tbr2, Tbr1, and other selected TFs. As expected, Tbr2 and Tbr1 were highly enriched in the Tbr2-GFP+ lineage, and showed zonal expression patterns on ISH consistent with IPs (aIPs and bIPs) and PNs, respectively ( Figure 1A). However, Pax6 expression was not cell-type-specific: different probes for Pax6 on the Tbr2-GFP microarray were enriched in different cell groups (conflicted probes), while ISH showed Pax6 in both VZ and SVZ ( Figure 1A). These results accord with our previous observations that Pax6 protein is expressed not only in RGPs, but also in some IPs (Englund et al., 2005). However, other TFs were identified as specific markers of RGPs, such as Sox9 ( Figure 1A). Immunohistochemistry and genetic lineage tracing have confirmed that Sox9 is specifically expressed in RGPs (Kaplan et al., 2017).

Feedforward and Feedback Regulation in the Pax6→ Tbr2→ Tbr1 Cascade
Using an intersectional approach to identify genes that were both bound and regulated by each TF (details in section Materials and Methods), we first examined whether Pax6, Tbr2, and Tbr1 transcriptionally regulate each other and/or themselves.
Previous studies have found that Pax6 directly represses its own transcription (Manuel et al., 2007), and directly activates Tbr2 expression (Sansom et al., 2009). Our analysis confirmed FIGURE 1 | Cell types, TF expression, and histological zones in E14.5 mouse neocortex. (A) Neurogenesis and cell-type-specific TF expression. Histological zones and cell types (left) are aligned with TF gene ISH (right; white ISH signal, blue nuclear counterstain). Arrows indicate common (but not exclusive) pathways of neurogenesis. Numbers above ISH panels indicate log 2 FC on Tbr2-GFP microarray (all p < 0.05). Abbreviations: see text. ISH: Allen Brain Atlas Developing Mouse Brain, E15.5 (colors inverted for figure). Scale bar: 50 µm. (B) The Pax6→ Tbr2→ Tbr1 cascade involves direct feedforward activation (arrows) and feedback repression (bars). The effect of Tbr2 binding at the Tbr2 locus could not be determined from available data (see text), but could be feedback repression.
Previous studies have also suggested that Tbr2 directly binds and activates Tbr1 (Sessa et al., 2017). This was confirmed in the present analysis. Moreover, we found that Tbr2 binds and represses Pax6: in Tbr2 cKO neocortex, Pax6 was significantly upregulated (log 2 FC = +0.36, p = 10 −3 on MA1; log 2 FC = +0.49, p = 10 −3 on MA2). In contrast, Tbr1 was downregulated in Tbr2 cKO cortex. We also noted Tbr2 binding to its own gene (Tbr2), although the functional effects were uncertain: Tbr2 mRNA expression is reduced due to Tbr2 cKO (Elsen et al., 2013), so the effects of Tbr2 on its own transcription could not be evaluated. We speculate that, like Pax6, Tbr2 may repress its own transcription as a feedback mechanism ( Figure 1B).
ChIP-seq analysis of Tbr1 showed that Tbr1 binds to the Tbr2 locus, but not to Pax6 or Tbr1. On microarray, however, Tbr2 expression was not significantly changed in Tbr1 null mice (S3). Thus, Tbr1 does not appear to directly regulate Tbr2, Pax6, or Tbr1.
Together, these data indicate that the Pax6→ Tbr2→ Tbr1 cascade operates as a positive feedforward cascade, but also self-regulates by direct negative feedback effects ( Figure 1B).
Since Pax6, Tbr2, and Tbr1 are expressed in different cell types (differentiation stages in the same lineage)-except for overlapping expression of Pax6 and Tbr2 in some IPs (Englund et al., 2005)-feedforward activation may involve epigenetic mechanisms. For example, Tbr2 and Tbr1 exhibit virtually no overlap of protein expression in developing neocortex, yet Tbr2 expression in IPs is essential for high levels of Tbr1 expression in postmitotic PNs (S4). One explanation is that Tbr2 may drive epigenetic changes at the Tbr1 locus that persist in postmitotic neurons. For example, removal of repressive histone marks by Jmjd3, an interacting protein of Tbr2, may create a permissive chromatin environment for Tbr1 transcription (Sessa et al., 2017).

Identification of EFs With Cellular, Regional, or TF-Regulated Expression
To identify genes with cell-type or region-specific expression in E14.5 mouse neocortex, we screened differentially expressed genes from a previous microarray experiment comparing RGP and IP transcriptomes . We used ISH to characterize expression patterns in developing neocortex (Supplementary Figure S1; Section Materials and Methods). To identify EF genes regulated by Pax6, Tbr2, and Tbr1, we selected EF genes that were both bound by the TF per ChIP-seq, and significantly regulated in TF null neocortex per microarray. All EF genes that were evaluated are listed in Supplementary Table S3, which also includes results from microarrays, ISH, and ChIP-seq; annotations of cell-type and regional identity; and previous literature citations.
Of more than 350 EF genes evaluated, 52 exhibited celltype-specific expression: 14 in RGPs, 2 in aIPs, 6 in bIPs, 9 in aIPs and bIPs, 18 in general neurons or precursors, and 3 in PNs or precursors (Supplementary Table S2). In addition, 11 EF genes exhibited rostrocaudal gradients: 4 high rostral, 7 high caudal (Supplementary Table S3). Furthermore, 36 EF genes were bound and regulated by Pax6, Tbr2, and/or Tbr1 (Supplementary Table S4). Of these, 9 were regulated by two TFs independently, but always in the same direction; and 2 EF genes were regulated only synergistically by Tbr2 and Tbr1. The effects of TFs on target gene expression were mixed: Pax6 activated 5 EF genes, and repressed 5; Tbr2 activated 8, and repressed 10; Tbr1 activated 13, and repressed 2; Tbr1 and Tbr2 (Tbr1/2) coordinately activated 2 EF genes. In sum, 73 EF genes showed cell-type or regional specificity, or were directly regulated by at least one of the TFs (Pax6,Tbr2,and Tbr1).
Results for each category of EFs are presented and discussed in the following sections. Neurodevelopmental implications are discussed in the final sections.
In the present analysis, all three Dnmt genes (Dnmt1/3a/3b) were specifically enriched in RGPs (Figure 2). In addition, Mbd2 and Uhrf1 were enriched in Tbr2-GFP − cells, but they were not detected on ISH, and could not be assigned RGP identity with confidence. Downregulation of DNA methylation activity in IPs was directed in part by Tbr2, which directly repressed Dnmt3a. Also, Mbd2 was directly repressed by Tbr2, consistent with the possibility that Mbd2 is RGP-specific, and actively repressed upon IP differentiation.
Among DNA demethylation genes, Gadd45g was regionally enriched with a high caudal gradient in VZ/SVZ, and was directly repressed by Pax6 and Tbr2 ( Figure 2F). Gadd45a, although not detected by ISH, was also directly repressed by Tbr2 ( Figure 2E). Tet1 was significantly enriched in Tbr2-GFP+ cells (although not detected on ISH), and was directly activated by Tbr1.
Mecp2, a methyl-cytosine reader linked to Rett syndrome (Qiu, 2017), was enriched in Tbr2-GFP+ cells (log 2 FC = +0.72), but not in any specific cell type, as ISH showed high levels in multiple zones. During embryonic neurogenesis, Mecp2 is  Table S2). Gadd45g (D), part of a pathway for DNA demethylation, was expressed in a high caudal gradient in the VZ, but was not significantly enriched in RGPs or IPs on microarray. (Significant log 2 FC values are indicated by bold text, in red or green). Sagittal sections, rostral left, ventral down (see also Supplementary Figure S1). ISH: Genepaint (A,C,D) and BGEM (B; darkfield). Scale bar: 100 µm. (E) Cell-type-specific gene expression and regulation by TFs. Arrows, direct transcriptional activation; bars, direct repression. (F) Pax6 and Tbr2 may shape the Gadd45g gradient by direct repression.
Frontiers in Neuroscience | www.frontiersin.org necessary to limit Pax6 expression in Tbr2+ IPs, and to modulate the pace of PN maturation (Cobolli Gigli et al., 2018).
These results indicate that DNA methylation activity is mainly enriched in RGPs, and that PN differentiation is associated with reduced DNA methylation, and increased DNA demethylation. Also, the high caudal gradient of Gadd45g in progenitor zones implicates DNA demethylation in cortical regionalization. Pax6, Tbr2, and Tbr1 regulate this system by repressing and activating key genes, including repression of the caudal marker (Gadd45g) by Pax6 and Tbr2 ( Figure 2F). Thus, DNA methylation and demethylation may regulate not only neuron differentiation (Sharma et al., 2016) and astrogenesis (Fan et al., 2005), but also cortical regionalization under the control of Pax6 and Tbr2.

Histone Marks
Histone marks are covalent modifications associated with regulation of chromatin structure and transcriptional activity (Allis and Jenuwein, 2016;Gates et al., 2017). Histone marks include acetylation, methylation, ubiquitylation, sumoylation, phosphorylation, and crotonylation. Generally, histone marks are placed by multisubunit enzyme complexes, are recognized by reader proteins, and are reversible by other enzyme complexes. Many EFs that place or remove histone marks have multiple subunit isoforms encoded by different genes, expressed in specific tissues or differentiation stages.
Another HDAC, Hdac5, was specifically expressed by PNs in IZ/CP. Recent studies suggest that Hdac5 limits the expression of Mef2c target genes, thus restraining neurite outgrowth (Gu et al., 2018). In turn, Hdac5 has been identified as a target of miR-124 and miR-9 (Gu et al., 2018), elements of the ncRNA system in developing neocortex (described below). This is noteworthy because both Tbr1 and Tbr2 directly repress Mir9-2 (host gene of miR-9), and thus indirectly potentiate Hdac5 expression. Hdac3 was moderately enriched in Tbr2-GFP+ cells, and widely expressed on ISH.
Of the class I HDACs, Hdac2 was enriched in Tbr2-GFP+ cells, and was expressed predominantly by differentiating neurons in the IZ/CP of cortex ( Figure 3F), and other forebrain regions (not shown). Thus, Hdac2 was classified as a marker of general neuronal differentiation starting in the IZ (N-iz; Supplementary Table S2). In contrast, Hdac1 showed no lineage bias on Tbr2-GFP microarray, and was widely expressed with highest levels in the VZ (see the section on Rest/CoRest complexes, below). In sum, Hdac1 and Hdac2 showed complementary enrichment in progenitors and neurons, respectively.
Among related factors in histone acetylation, Uhrf1, which recruits Dnmt1 and HATs to chromatin during proliferation (Murao et al., 2014), was RGP-specific, as noted ( Figure 2E). Ankrd11, a scaffolding molecule that potentiates Hdac3 signaling (Gallagher et al., 2015), was significantly enriched in the neuronal lineage, and was activated by Tbr1.
Together, these results reveal an important genetic circuit in IPs that regulates layer 5 differentiation. Also, Hdac9 and Hdac5 seem to play similar roles limiting Mef2-and activity-driven gene expression in mature cells, but their expression and regulation in IPs and new PNs suggest they may possibly have distinct functions during neurogenesis. During the IP-PN transition, both Tbr2 and Tbr1 promote the shift from Hdac9 to Hdac5 expression. Tbr2 directly represses Hdac9, while Tbr2 and Tbr1 indirectly potentiate Hdac5 expression, by directly repressing MiR9-2 and thus limiting targeted degradation of Hdac5 by miR-9 ( Figure 3H). These findings support our view that Tbr2 drives the transition from IP to PN, while Tbr1 drives PN differentiation (Mihalas et al., 2016;Mihalas and Hevner, 2017).

Trithorax/COMPASS Activating Complexes
Another important category of histone marks consists of lysine methylation (mono-, di-, and trimethylation) and demethylation. The best-known epigenetic systems using these marks are Trithorax/COMPASS complexes, which place H3K4 trimethyl (H3K4me3) and other marks at active promoters; and PRC2, which places repressive H3K27me3 marks that silence chromatin. The PRC2 system is furthermore connected to PRC1, which places another silencing histone mark-monoubiquitylation of H2A on K119 (H2AK119u1)-and functions synergistically with PRC2. In Drosophila, TrxG and Polycomb group (PcG) systems are considered antagonistic; genes marked with both H3K4me3 (activating) and H3K27me3 (repressive) are considered to be in a "bivalent" state, poised for long-term repression or activation. In mammals, the Trithorax and Polycomb systems have become more complex and diverse, with many tissue-specific isoforms and non-canonical subunits. While TrxG genes (as defined by PcG antagonism) also encompass other classes of molecules, such as chromatin remodelers (Schuettengruber et al., 2011;Moccia and Martin, 2018), those other molecules are classified separately for purposes of this article.
Kdm5a (Jarid1a), another H3K4me3 demethylase, was expressed in a regional gradient (high caudal) in the VZ/SVZ ( Figure 4E). On microarray, different Kdm5a probes were enriched in Tbr2-GFP+ and GFP − cells (conflicted), so expression of Kdm5a could not be specifically assigned to RGPs or IPs.
Ash1l, an H3K36 methylase that may activate or repress transcription in different contexts (Schuettengruber et al., 2011;Zhu et al., 2016), was highly enriched in aIPs and bIPs ( Figure 4B; Supplementary Table S2), but was not regulated by Pax6,Tbr2,or Tbr1. These results indicate that deposition and removal of TrxG marks are actively regulated by Tbr2 and Tbr1 during neuronal differentiation ( Figure 4F). Also, cortical regionalization may be influenced by Jarid1a (Kdm5a), without direct regulation by Pax6, Tbr2, or Tbr1 ( Figure 4G).
Previously, the PRC2 system has been shown to regulate the timing of neurogenesis in developing neocortex. RGPs lacking Ezh2 undergo accelerated differentiation to produce IPs and neurons, followed by precocious gliogenesis (Pereira et al., 2010). Moreover, Tbr2 and other key IP-genic or neurogenic genes are marked by high levels of H3K27me3 in RGPs, but these repressive PRC2 marks are removed during IP or neuron differentiation (Albert et al., 2017). PRC2 also regulates rostrocaudal patterning of cortex, as Suz12 heterozygous null mice have reduced occipital cortex (Miró et al., 2009).
In the present study (Figure 5), analysis of core PRC2 subunits showed that Ezh2 was widely expressed in developing neocortex, with slight enrichment in Tbr2-GFP+ cells; while Ezh1 was not detectable. In contrast to the widespread expression of Ezh2, the other core PRC2 subunits Suz12 and Eed were expressed almost exclusively in VZ/SVZ, although neither was specifically enriched in Tbr2-GFP+ or GFP-cells. Moreover, both Suz12 (Miró et al., 2009) and Eed ( Figure 5G) exhibited high caudal to low rostral gradients within VZ/SVZ.
The gradient of Suz12 expression has previously been linked to cortical regionalization. In Suz12 heterozygous null mice, occipital cortex was greatly reduced, suggesting that high PRC2 activity instructs occipital identity (Miró et al., 2009). With parallel gradients of core Suz12 and Eed subunit genes, overall PRC2 activity may be steeply graded within the VZ/SVZ. Also, the low levels of Suz12 and Eed expression outside progenitor compartments suggest that PRC2 activity may be essentially limited to the VZ and SVZ.
Other canonical and non-canonical subunits of PRC2 also displayed cell-type-specific or regional expression patterns. Rbbp7 was specifically expressed in RGPs (Figure 5B), while Rbbp4 was enriched in Tbr2-GFP+ cells. Aebp2, encoding a protein that enhances PRC2 activity on PRC1-marked chromatin, was also specifically expressed in RGPs ( Figure 5A). In contrast, Jarid2 (jumonji), a non-canonical PRC2 subunit that may inhibit PRC2 activity (Shen et al., 2009), was specifically enriched in bIPs (Figure 5C), and was directly activated by Tbr2 (Supplementary Table S4). Mtf2 (PCL2) was highly enriched in the neuronal lineage (Figure 5D), and was directly activated by Tbr1. Phf19 (PCL3), which targets PRC2 to H3K36me3-marked chromatin, was expressed in a high rostral gradient in VZ/SVZ (counter to Suz12 and Eed). The Phf19 (PCL3) countergradient suggests that not only the abundance of PRC2 complexes, but also the formation of non-canonical PRC2 complexes, are regionally modulated within VZ/SVZ.
Overall, differentiation of IPs and neurons was associated with upregulation of Kdm6a (Utx) and Kdm6b (Jmjd3), which "unlock" chromatin by remove the H3K27me3 marks placed by PRC2. For regionalization, high canonical PRC2 activity is necessary in caudal VZ/SVZ for occipital cortex identity (Miró et al., 2009), but non-canonical PRC2 is also implicated in regionalization, by the high rostral gradient of Phf19 (PCL3). Despite the important role of PRC2 in regionalization, the subunits with graded expression are not directly regulated by Pax6, Tbr2, or Tbr1 ( Figure 5J).
In developing neocortex, PRC1 is thought to regulate the tempo of differentiation, and the balance of neuron subtypes. In Ring1b (Rnf2)-deficient RGPs, neurogenesis is prolonged (Hirabayashi et al., 2009), and Ctip2+ layer 5 neurons are increased at the expense of upper layer neurons due to impaired repression of Fezf2 (Morimoto-Suzki et al., 2014). Non-canonical PRC1-Auts2 complexes are implicated in mouse behavioral development (Gao et al., 2014). In humans, AUTS2 is an important intellectual disability and autism gene (Beunders et al., 2016).
In the present analysis, Rnf2 (Ring1b) appeared to be the predominant E3 ligase in developing neocortex. Rnf2 was enriched in Tbr2-GFP+ cells, and was seen in all zones by ISH, though highest in the VZ (Figure 6A). In contrast, Ring1 (Ring1a) was barely detectable on microarrays and ISH.
Canonical PRC1 subunits were, for the most part, widely expressed and little regulated by Pax6, Tbr2, or Tbr1. Bmi1 (Pcgf4; Figure 6B) and Pcgf2 were both detected in all zones of neocortex, but highest in VZ. Also, Bmi1 (Pcgf4) was moderately enriched in Tbr2-GFP+ cells, and more highly expressed than Pcgf2. Multiple Cbx genes were expressed in developing neocortex, but none exhibited cell-type specificity. However, Cbx4 was directly activated by Pax6. Since Cbx4 promotes sumoylation of Dnmt3a (Li et al., 2007), the upregulation of Cbx4 by Pax6 may suppress de novo DNA methylation during IP genesis. Cbx2 was expressed in a high caudal gradient in VZ/SVZ (Figures 6F,I). Phc1-3 were enriched in Tbr2-GFP+ cells, but none showed cell-type specificity by ISH. Overall, these findings are consistent with previous studies of PRC1 gene expression in embryonic mouse cortex (Vogel et al., 2006).
These data suggest that canonical PRC1 complexes are present in all types of cortical cells (although most abundant in progenitors), and are minimally regulated by Pax6→ Tbr2→ Tbr1. In contrast, non-canonical PRC1 complexes exhibit differentiation-related changes, such as upregulation of Rybp in IPs and new PNs. Notably, Tbr1 directly activated two noncanonical PRC1 subunits (Rybp, Auts2) implicated in brain development (Gao et al., 2014).
Mllt3 (Af9), a histone H3K9ac reader, was enriched in neurons of the IZ and CP ( Figure 7C). Previously, Af9 has been reported to inhibit deep layer identity by repressing Tbr1 transcription (Büttner et al., 2010). In the present study, we found that Pax6 directly activated Mllt3 (Supplementary Table S4). Since previous studies have also found that Pax6 drives upper layer identity (Schuurmans et al., 2004), it seems plausible that Pax6 indirectly represses Tbr1 by activating high expression of Mllt3 in precursors of upper layer neurons. Thus, Pax6 indirectly activates Tbr1 via Tbr2, and indirectly represses Tbr1 via Mllt3 ( Figure 7D).

ATP-Dependent Chromatin Remodeling Complexes
Chromatin remodeling complexes use ATP to modify the positioning, conformation, and isoform composition of histones in nucleosomes-and thereby alter the availability of genes for TF binding (reviewed by López and Wood, 2015;Hota and Bruneau, 2016). These types of complexes contain an Snf2-domain ATPase, along with other proteins that modulate the ATPase activity and confer chromatin target specificity.
Most chromatin remodeling complexes contain multiple subunits: up to 16 in BAF, 4 in ISWI, 7 in CHD (NuRD), and 15 in INO80 complexes (Hota and Bruneau, 2016). Some subunit isoforms exhibit tissue-specific or differentiation-related expression. For example, BAF complex subunits are extensively switched in cortical differentiation (Son and Crabtree, 2014).
Besides these large complexes, other ATP-dependent chromatin remodelers, such as Atrx (a Snf2-type ATPase and histone reader protein that places H3.3 in heterochromatin) are also implicated in epigenetic regulation of neurodevelopment (Iwase et al., 2017).

ISWI Chromatin Remodeling Complexes
At least eight ISWI complexes have been described in mammals (Goodwin and Picketts, 2017). Furthermore, the ATPase core subunits of ISWI complexes (Snf2h/l) have been shown to be important in brain development. Smarca1 (Snf2l) mutant mice exhibit excessive, prolonged proliferation of cortical progenitors, especially IPs (Yip et al., 2012); while Smarca5 (Snf2h) mutant mice exhibit reduced proliferation, at least in cerebellum (Alvarez-Saavedra et al., 2014).
Overall, the present analysis suggests that NuRF complexes are specifically present in RGPs, while NoRC complexes are particularly abundant in IPs (Figure 8F). The direct repression of Baz2b by Tbr2 and Tbr1 suggests that downregulation of some ISWI complexes (possibly a Baz2b-containing NoRC variant) is important for differentiation from IPs to PNs.

INO80 Chromatin Remodeling Complexes
Among ATPase subunit genes, Ino80 was detected primarily in VZ, but was not enriched in Tbr2-GFP − or GFP+ lineages (Supplementary Table S3). Ino80b (Ies2), which activates the ATPase activity of Ino80, was specifically expressed in RGPs (log 2 FC = −0.45), suggesting that Ino80-containing complexes are enriched and activated in RGPs. The INO80 remodelers are important in DNA replication and repair, as well as transcriptional regulation (Poli et al., 2017), so the enrichment of Ino80 activity in RGPs may be related to high proliferative activity in this cell type.
Together, these findings suggest that Ino80-containing complexes are specifically active in RGPs, while p400/Tip60 complexes are most active in postmigratory CP neurons. The functions of INO80 complexes in cortical development are unknown.

CHD Chromatin Remodeling Complexes
Among Chd ATPase genes, only Chd7 exhibited cell-type or region-specific expression-indeed, both. Chd7 was enriched in Tbr2-GFP+ cells (log 2 FC = +0.80) on microarray, and was expressed specifically in VZ on ISH, identifying Chd7 as a specific marker of aIPs. Within VZ, Chd7 exhibited high caudal expression (Figure 9A), suggesting its involvement in regionalization. Consistent with this possibility, we also found that Chd7 was directly bound and repressed by Pax6 and Tbr2 ( Figure 9I; Supplementary Table S4), both of which promote rostral identity. Previous studies suggest that Chd7 binds mainly to enhancers and active transcription start sites, and is essential for activation of neuronal differentiation genes (Moccia and Martin, 2018). Mutations in human CHD7 cause CHARGE syndrome, a complex disorder with significant brain and somatic anomalies (Feng et al., 2017;Moccia and Martin, 2018).
Other Chd genes regulated by TFs included Chd1, repressed by Pax6; and Chd3, jointly activated by Tbr1 and Tbr2. Chd1 was not specifically enriched in Tbr2-GFP+ or GFP-lineages, nor was ISH available, so the topography of Chd1 expression is unknown. Chd1 protein recognizes H3K4me3 marks (active promoters) and globally activates transcription (Guzman-Ayala et al., 2015). Also, Chd1 interacts with FACT complex (Ssrp1 and Supt16) at centromeres to facilitate histone exchange (Okada et al., 2009). Of the FACT subunits, Ssrp1 was RGP-specific ( Figure 9B), while Supt16 was widely expressed. These data suggest that FACT-Chd1 complexes may be abundant in RGPs, but downregulated in IPs, in part by Pax6 repression of Chd1 (Figures 9G,H).
Direct activation of Chd3 by Tbr2 and Tbr1 supports the conclusion that Chd3 expression increases with neuronal differentiation. In the present analysis, Chd4 was not, however, specifically enriched in RGPs as previously suggested (Nitarska et al., 2016). Rather, Chd4 exhibited widespread expression in cortical zones, and Chd4 was (like Chd3) enriched in Tbr2-GFP+ cells on microarray (Figures 9C,D), while Chd5 was essentially undetectable. These data suggest that in RGPs, NuRD complexes contain mainly Chd4, while in neurons, NuRD complexes contain both Chd3 and Chd4 (Figure 9H).
Most other NuRD subunits did not exhibit cell-typespecific expression, but a few did. As noted above, Mbd2 was specifically enriched in Tbr2-GFP − cells (likely RGPs; ISH not informative), and was directly repressed by Tbr2 (Figure 2E; Supplementary Tables S3, S4). In contrast, Mbd3 was widely expressed (Figure 9E). Rbbp7 was specifically expressed in RGPs (Figure 5B), while Rbbp4 was primarily enriched in neuron lineages (see also sections on Rbbp4/7 in PRC2 and NuRF complexes). Hdac1 was expressed in all zones but enriched in VZ/SVZ, while Hdac2 was moderately enriched in neurons ( Figure 3F). Mta1/2 were widely expressed, while Mta3 was essentially undetectable. Gatad2a/b were both enriched in Tbr2-GFP+ cells, and Gatad2a was widely expressed on ISH, but Gatad2b ISH was not available. Ctbp2, a NuRD partner that targets it to active genes that require silencing during differentiation (Kim et al., 2015), was directly activated by Tbr2 and Tbr1 (Figures 9F,G).
Overall, these findings suggest that NuRD subunit composition and silencing activity are modulated during differentiation from RGPs to neurons. These changes are driven in part by Tbr2 and Tbr1 (Figures 9G,H). Also, the graded expression of Chd7, and its repression by Pax6 and Tbr2, implicate Chd7 in cortical regionalization (Figure 9I), although further studies will be necessary to substantiate this role.

BAF Chromatin Remodeling Complexes
Among EFs with documented importance in cortical development, the BAF chromatin remodeling complex plays a well-established role in regulating cerebral cortex size and function (Narayanan et al., 2015;Sokpor et al., 2017). Moreover, BAF subunit switching occurs at specific stages of neuronal differentiation (Son and Crabtree, 2014). The BAF complex is important for human brain development, as genetic defects of BAF subunits, such as Baf250b (Arid1b), cause Coffin-Siris syndrome, a microcephaly disorder with intellectual disability (Son and Crabtree, 2014).
Mammalian BAF complexes are sometimes categorized by Baf250 isoform, as Baf250a-(BAF-A) and BAF250b-containing (BAF-B) complexes (Hota and Bruneau, 2016). We observed that Arid1a (Baf250a) was ubiquitously expressed, while Arid1b (Baf250b) was enriched in the CP (Figure 10D), and was directly activated by Tbr1 (Supplementary Table S4). These results suggest that BAF-A predominates in progenitors, while cortical PNs express BAF-A and BAF-B complexes, the latter driven by Tbr1-mediated activation of Arid1b.
The present results indicate that the subunit composition of BAF complexes is highly regulated in cortical PN differentiation; and that the Pax6→ Tbr2→ Tbr1 cascade is responsible for activation of many BAF subunit genes in IPs and neurons, as well as the activation of Smarca2 in a high rostral gradient (Figures 10J-L). Interestingly, Pax6, Tbr2, and Tbr1 did not directly repress any npBAF subunit genes. Recently, BAF complexes were reported to interact with Utx (Kdm6a) and Jmjd3 (Kdm6b), and potentiate their H3K27me3 demethylase activity (Narayanan et al., 2015). Thus, the Pax6→ Tbr2→ Tbr1 cascade drives the formation of two complexes that recruit H3K27me3 demethylases: BAF (Narayanan et al., 2015) and Mll3/COMPASS-like (Schuettengruber et al., 2011).
In the present analysis (Figure 11), Rest was specifically expressed in RGPs (Figure 11A), consistent with its established function of suppressing neuronal differentiation. Of corepressors, Sin3a and Rcor1 were expressed mainly in VZ (and Rcor1 was enriched in Tbr2-GFP+ cells), while Rcor2 was expressed mainly in SVZ/IZ and inner VZ (Figures 11B,D,E). The enrichment of Rcor2 in Tbr2-GFP+ cells (log 2 FC = +1.94), together with its bilaminar expression pattern in VZ and SVZ ( Figure 11E), indicated specific enrichment in aIPs and bIPs (Supplementary Table S2). Of the interacting HDACs, Hdac1 was expressed at highest levels in the VZ (Figure 11C), while Hdac2 was expressed mainly in IZ/CP, and was enriched in Tbr2-GFP+ cells ( Figure 3F). Thus, Rest/CoRest complexes form predominantly in RGPs, where Rest recruits mainly Sin3a and Hdac1, and possibly Rcor1 ( Figure 11H). Interestingly, one function of Rest is to repress miR-9 * and miR-124 (Yoo et al., 2009); as shown below in the section on ncRNA, miR-9 * is also repressed by Tbr1 and Tbr2.
Importantly, Insm1 has previously been implicated in the genesis of IPs: Insm1 null mice have decreased IP abundance, and reduced Tbr2 expression (Farkas et al., 2008). One function of Insm1 is to promote the delamination of cortical progenitors, by directly repressing Plekha7 (Tavano et al., 2018). Since Insm1 is thought to be a transcriptional repressor, and directly represses Rest (Monaghan et al., 2017), it seems unlikely that Insm1 directly activates Tbr2. Nevertheless, Insm1 is an integral component of the TF network regulated by Pax6→ Tbr2→ Tbr1 ( Figure 11I).
Together, these findings indicate that several lncRNAs are specifically expressed at high levels in IPs and new PNs, and that several miR genes are expressed with cellular or regional specificity. The gradient of Mir99ahg, and its possible targeting Fgfr3, suggest a new role for miR in cortical patterning. Finally, their direct regulation by Tbr2 and Tbr1 suggests that lncRNA and miR genes have significant functions in cortical development ( Figure 12G).

Neurodevelopmental Processes Controlled by EFs and Regulated by Pax6, Tbr2, and Tbr1
The major findings from our analysis, summarized in Table 1, indicate that all kinds of EFs exhibit cell type-specific expression, and many EFs are regulated by Pax6, Tbr2, and/or Tbr1. These results implicate EFs in regulating cortical development at every stage of differentiation. Together with available functional information, our findings show that Pax6, Tbr2, and Tbr1 use transcriptional regulation of EF genes to modulate many important processes, notably IP genesis, laminar identity, and rostrocaudal regionalization of neocortex.

Regulation of IP Genesis
Previous studies have found that Pax6, Insm1, and Tbr2 each play distinct roles in IP genesis ( Figure 13A). In Pax6 null embryos, basal progenitors divide in the SVZ but do not express Tbr2, because Pax6 is required for Tbr2 activation (Quinn et al., 2007). Insm1 mutants exhibit severe reduction (∼50%) of basal IPs with proportionately decreased Tbr2 expression (Farkas et al., 2008).
In Tbr2 cKO embryos, conflicting phenotypes have been reported. In studies using Foxg1-Cre recombinase, Tbr2 inactivation caused ∼75% reduction of basal IPs (Sessa et al., 2008). However, Foxg1-Cre heterozygosity itself causes ∼38% IP deficiency (Siegenthaler et al., 2008), making Foxg1-Cre a sensitized, anomalous background. In contrast, Tbr2 cKO mice FIGURE 13 | Cortex-specific neurodevelopmental processes regulated by EFs under the control of Pax6, Tbr2, and Tbr1. (A) IP genesis is reportedly regulated by multiple EFs, as well as TFs such as Pax6 and Insm1. Tbr2 directly represses IP-genic factors. Arrows: (B) Differentiation of all cortical layers is regulated by interacting EFs and TFs. SP: subplate. (C) Rostrocaudal regionalization is extensively regulated by TFs and EFs in an expansive gene regulatory network. Pax6 and Tbr2 regulate several regionally graded EF genes (italic), which are components of several different epigenetic complexes or systems (bold). Abbreviations: dm, demethylation; others as in text. Lines indicate zonal separation of IZ/CP above, and VZ/SVZ below. Arrows indicate direct transcriptional activation; bars, repression. produced with Nes11-Cre have normal or increased numbers of bIPs, which migrate into the IZ and divide ectopically (Mihalas et al., 2016). Importantly, Nes11-Cre is a transgene that does not interfere with cortical development. Thus, the data suggest that Insm1 and Pax6 promote IP genesis and differentiation, respectively; while Tbr2 promotes the transition from IP to PN, in part by repressing IP genes ( Figure 11I).
Previously, many EFs have also been implicated in controlling IP genesis ( Figure 13A). Among these, Kat6b (Morf, querkopf) was directly repressed by Tbr2 (Figure 3H; Supplementary Table S4). Morf (Kat6b) is a MYST family HAT that activates gene expression, and is required for forebrain growth (Thomas et al., 2000). It is unknown if IPs are reduced in Kat6b (Morf) deficient embryos, but deficiency of the MYST coactivator, Brpf1, has been found to reduce IP genesis and cortical growth (You et al., 2015). These findings indicate that Tbr2 is required to repress IP-genic EF (Kat6b) and TF (Pax6, Insm1) genes in IPs ( Figure 13A).
The present analysis found that Pax6 directly activated Mllt3 (Af9), a YEATS domain acetylation reader that directly mediates Tbr1 repression for upper layer identity (Büttner et al., 2010). Thus, Pax6 may promote upper layer identity in part by repressing lower layer identity. Paradoxically, Pax6 activates Tbr1 indirectly (via Tbr2) to promote PN differentiation (Figure 11I), but also represses Tbr1 indirectly (via Mllt3) to control laminar identity ( Figure 13B).
Tbr2 may suppress layer 5 differentiation in part by directly repressing expression of Kat6b (Morf), a MYST family HAT that promotes layer 5 differentiation, as well as cortical growth (Thomas et al., 2000). In Tbr2 cKO cortex, upregulation of Kat6b (log 2 FC = +0.18; p = 0.005) was associated with increased abundance of layer 5 neurons (Mihalas et al., 2016). The involvement of Morf (Kat6b) in layer 5 differentiation is supported by the phenotype of Brpf1 mutant mice: Brpf1 is an activator of Morf (Kat6b), and Brpf1 mutants have prominent layer 5 defects (You et al., 2015).

Rostrocaudal Regionalization
The cerebral cortex is patterned by molecular expression gradients that confer different properties on cortical cells, according to their rostrocaudal and mediolateral coordinates (O'Leary et al., 2007). As part of this system, Pax6, Tbr2, and Tbr1 regulate molecular gradients at each stage of differentiation from RGPs→ IPs→ PNs (Bishop et al., 2000;Bedogni et al., 2010a;Elsen et al., 2013;Mihalas and Hevner, 2017). In the present study, many EFs that are expressed in rostrocaudal gradients were identified, including some that are directly regulated by Pax6 and Tbr2 ( Figure 13C). Both Pax6 and Tbr2 directly repressed two EF genes with high caudal gradients in VZ/SVZ: Gadd45g and Chd7 ( Figure 13C). These findings suggest that Pax6 and Tbr2 shape the Gadd45g and Chd7 gradients. However, the roles of Gadd45g and Chd7 in cortical regionalization remain unknown.
Interestingly, Pax6 directly activated the expression of BAF subunits Smarca2 (Brm) and Bcl11a (Ctip1), in CP and IZ/CP respectively ( Figure 13C). Since Pax6 is not expressed in IZ/CP, its ability to activate Smarca2 and Bcl11a may depend on epigenetic mechanisms, such that Pax6 "unlocks" these genes in neurogenic progenitors, making them available for activation in PNs. The dependence of Bcl11a, a caudal enriched gene, on Pax6, a rostral enriched TF, suggests that while Pax6 may be necessary to unlock Bcl11a, Pax6 probably does not drive the Bcl11a gradient. While Smarca2 has no known role in cortical regionalization, Bcl11a has been implicated in the acquisition of sensory cortex identity .
Although Mir99ahg was not directly regulated by Pax6→ Tbr2→ Tbr1, its high rostral expression gradient in the VZ (Figure 12F) was noteworthy because miR-99 targets Fgfr3 (Jiang et al., 2014), which is expressed in a high caudal gradient and promotes growth of occipitotemporal cortex (Hevner, 2005;Thomson et al., 2009). Also, canonical PRC2 complexes play an important role in promoting occipital identity with high caudal gradients of Suz12 and Eed (Figure 5J), but these PRC2 core genes were, in our analysis, not directly regulated by Pax6→ Tbr2→ Tbr1 (Figure 13C).

Coordinate Regulation of Cortical Development by TFs and EFs
The present study demonstrates that many types of EFs are direct targets of gene activation or repression by Pax6, Tbr2, or Tbr1 (Table 1). In many examples, the regulation of EFs by TFs was robust and affected multiple elements in an epigenetic system or signaling pathway. For example, Pax6, Tbr2, and Tbr1 activated multiple BAF subunit genes, to effect subunit switching and neuronal differentiation (Figure 10). In another example, Tbr1 activated non-canonical PRC1 subunits (Rybp, Auts2) in PNs (Figure 6). Also, many HATs and HDACs were regulated by this TF cascade (Figure 3). Overall, our results indicate that Pax6, Tbr2, and Tbr1 utilize EFs to modulate neurodevelopmental processes such as IP genesis, laminar fate acquisition, and regional identity (Figure 13). The Pax6→ Tbr2→ Tbr1 cascade itself emerges as a complex network with feedforward and feedback regulation ( Figure 1B).
Epigenetic mechanisms appear well-suited to regulation of regional and laminar identity, persistent phenotypes that are initially determined in progenitor cells, then propagated into IPs and finally, new PNs. For example, the cortical "protomap" is initially specified in RGPs, then propagated into IPs and PNs, where regional identity continues to be refined (Bedogni et al., 2010a;Elsen et al., 2013;Alfano et al., 2014).
Besides EFs, other target genes regulated by Pax6, Tbr2, and Tbr1 can be identified using the same approach, and are currently under analysis. Through these studies, it will be possible to comprehensively profile gene expression by RGPs, IPs, and PNs; and to better understand how Pax6, Tbr2, and Tbr1 control the genesis of cortical PNs.

AUTHOR CONTRIBUTIONS
GE designed and conducted experiments, produced new microarray data from Tbr1/2 mutant embryos, analyzed results, and wrote the manuscript. FB and RH produced microarray data. TB and JM analyzed data from microarray and ChIP-seq experiments. SL and JR produced and analyzed Pax6 ChIP-seq data. RH designed experiments, analyzed data, and wrote the manuscript.

FUNDING
Supported by research grants from NINDS to RH (R01 NS092339, R01 NS085081) and to JR (R01 NS34661); and by grants from the Lejeune Foundation, Umberto Veronesi Foundation, and ProRett Italia to FB.