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, neuronal 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, 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. The results 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 noncoding RNA. Tbr1 knockout (KO), Tbr2 conditional knockout (cKO), and Tbr1/2 double KO/cKO (dKO) mouse embryos were produced as described (Bedogni et al., 2010a; Elsen et al., 2013). The Tbr1/2 double mutants were generated by breeding to combine the necessary alleles (Tbr1/;Tbr22F/2F;Nes11Cre). On E14.5, embryos were harvested, and neocortex was immediately dissected and frozen. Genotypes were determined by PCR 216 of tail DNA. Controls were wild type (+/+) for Tbr1 and non-recombined for Tbr2. Each embryonic neocortex was an independent biological replicate. For each genotype, n = 4 samples of neocortex were analyzed by microarray (Affymetrix ST 1.0). The microarray results were analyzed statistically as described (Elsen et al., 2013). In the current paper, we also analyzed previous microarray data from Tbr1 KO (Bedogni et al., 2010a) and Tbr2 cKO (Elsen et al., 2013) neocortices, designated microarray 1 (MA1); the new microarray data were designated microarray 2 (MA2). Tbr1/2 dKO neocortex was analyzed only in MA2.表观遗传因子（Epigenetic factors, EFs）调控大脑皮层发育的多个维度，涵盖细胞增殖、神经元分化、皮层层命运以及区域特性。同类神经发育过程同时受转录因子（transcription factors, TFs）调控，其中尤为关键的是分别在放射状胶质祖细胞、中间祖细胞与有丝分裂后投射神经元中依次表达的Pax6→Tbr2→Tbr1级联反应。本研究针对胚胎小鼠新皮层内的表观遗传因子调控网络及其调控机制展开系统探究。通过微阵列（microarray）与原位杂交实验发现，众多表观遗传因子基因可在特定皮层细胞类型（如中间祖细胞）中特异性表达，或呈现吻尾轴的梯度表达模式。进一步结合染色质免疫沉淀测序（chromatin immunoprecipitation-sequencing, ChIP-seq）与转录因子突变皮层的基因表达分析证实，诸多表观遗传因子基因可被Pax6、Tbr2或Tbr1直接结合并实现转录调控。研究结果表明，Pax6、Tbr2与Tbr1构成直接的前馈遗传级联反应，且存在直接的反馈抑制作用。此外，各转录因子均可调控多个参与DNA甲基化、组蛋白修饰、染色质重塑以及非编码RNA调控的表观遗传因子基因。 按照已有报道（Bedogni等人，2010a；Elsen等人，2013）的方法，本研究构建了Tbr1敲除（knockout, KO）、Tbr2条件性敲除（conditional knockout, cKO）以及Tbr1/2双敲除/条件性双敲除（double KO/cKO, dKO）的小鼠胚胎。Tbr1/2双突变体通过杂交携带必要等位基因的小鼠获得：Tbr1^-/-;Tbr2^flox/flox;Nes11Cre⁺。在胚胎发育第14.5天（E14.5），收集胚胎并立即分离新皮层组织进行冷冻保存。通过PCR对尾端DNA进行基因型鉴定。对照组为Tbr1野生型（+/+）且Tbr2未发生重组的小鼠。每个胚胎的新皮层均作为独立生物学重复。针对每种基因型，选取4份新皮层样本通过Affymetrix ST 1.0微阵列进行分析。微阵列数据的统计分析参照已有方法（Elsen等人，2013）完成。本研究同时整合了此前发表的Tbr1 KO（Bedogni等人，2010a）与Tbr2 cKO（Elsen等人，2013）的新皮层微阵列数据，记为微阵列数据集1（microarray 1, MA1）；本次新增的微阵列数据记为微阵列数据集2（microarray 2, MA2）。Tbr1/2双敲除新皮层的数据仅纳入MA2分析。