SOX1 Determines the Regional Identity of Neural Progenitors Differentiated from Human Embryonic Stem Cells

During human embryogenesis, primitive neural cells start to be generated at the time of gastrulation and gradually acquire regional identities, which is a process called neural patterning. But how intrinsic factors respond to exogenous patterning signals remains poorly understood. Human Embryonic Stem Cells (hESCs) provide a great model to recapitulate this process. Through exogenous manipulation of canonical WNT signaling activation during neural differentiation, dose-dependent specification of regionally defined neural progenitors ranging from the telencephalic forebrain to posterior hindbrain could be rapidly and efficiently induced. Unexpectedly, we find that SOX1, generally referred as a pan-neural gene, displays a regional specific distribution in the human neural patterning process. To investigate the expression and function of SOX1 efficiently, we have generated the SOX1-EGFP reporter and SOX1-knockout (KO) hESC lines using the CRISPR/Cas9 system. SOX1 is initially expressed across the mes–met border and peaked in the metencephalon region at the early regional specification stage. Its depletion leads to the posterior shift of the mes–met border. Therefore, SOX1 is required to define the border at a correct region. In-depth analysis of SOX1 ChIP-sequencing and transcriptome data will provide more insights into how SOX1 determines the mes-met border formation and identify the downstream targets of SOX1. This study identifies SOX1 as one of the intrinsic factors key for the prepattern establishment in the developing central nervous system, particularly for defining the isthmus position. Overall design: To analysis the function of SOX1 in early neural differentiation, we compared the gene expression profiles between wild type and SOX1-/- cells at 3 time points (Day 0, 4, 8) during neural differentiation by mRNA sequencing.

在人类胚胎发生过程中，原始神经细胞于原肠胚形成阶段开始产生，并逐步获得区域身份特征，这一过程被称为神经模式形成（neural patterning）。然而，内在因子如何响应外在模式形成信号，目前仍不甚明晰。人类胚胎干细胞（human embryonic stem cells, hESCs）是重现这一过程的优秀模型。在神经分化过程中，通过对外源调控经典WNT信号通路的激活，可快速且高效地诱导出剂量依赖性的区域特异性神经前体细胞，其范围涵盖端脑前脑至后脑后部。令人意外的是，我们发现通常被称为泛神经基因的SOX1，在人类神经模式形成过程中呈现出区域特异性分布。为高效探究SOX1的表达与功能，我们利用CRISPR/Cas9系统构建了SOX1-EGFP报告基因及SOX1敲除（knockout, KO）人类胚胎干细胞系。SOX1最初在中脑-后脑边界处表达，并在早期区域特化阶段于菱脑区域达到表达峰值。敲除SOX1会导致中脑-后脑边界向后移位。因此，SOX1对于在正确区域界定该边界是必需的。对SOX1染色质免疫沉淀测序（ChIP-sequencing, ChIP-seq）和转录组数据的深入分析，将进一步揭示SOX1如何调控中脑-后脑边界的形成，并鉴定其下游靶基因。本研究证实SOX1是发育中的中枢神经系统中预模式建立的关键内在因子之一，尤其对于界定峡部位置至关重要。实验整体设计：为探究SOX1在早期神经分化中的功能，我们通过mRNA测序，比较了神经分化过程中第0、4、8三个时间点下野生型与SOX1基因敲除（SOX1-/-）细胞的基因表达谱。