Molecular and cellular pathways driving competency for human vagal neural crest specification

摘要/摘要 体外培养的人多能干细胞(HPSCs)可分化为神经峰样细胞(NCCs)。作为回应 对于维甲酸(RA)，神经干细胞可以基于基因表达和基因表达而向迷走神经峰方向分化。 根据谱系分化能力。来自实验室的令人兴奋的初步数据，通过使用 CellTag血统追踪技术表明，迷走身份的获取过程实际上始于 非常早期的分化阶段，在RA暴露之前。我们的数据表明迷走神经的自发出现- 在这些早期分化时间点上，有能力的种群与非有能力的种群相比。此外，我们 观察到有能力的种群呈现出与原始细胞相匹配的基因表达模式 成为后脑的区域中的外胚层。理解这些早期迷走者的过程- 建立有能力的前体，以及维持和执行迷走神经能力的机制 具有广泛的含义。对于NCC规范，改进了对颅骨空间模式的理解 区域可以极大地增强我们产生迷走神经趋化细胞的能力，用于治疗以下疾病 先天性巨结肠。除了NCC图案化，我们的研究可能揭示轴向图案化的一般机制 这影响了包括中枢神经系统在内的许多其他发育中的胚胎组织。 在目标1中，我们建议使用scRNAseq来完全描述在这一点上表现出迷走神经能力的人群 在识别使用双报告器hPSC线路为这些细胞选择性地富集化的信号之前的系统。在AIM 2我们建议用(1)平行SCATAC的雄心勃勃的组合来剖析能力的机制 以及多组学方法中的scRNA测序，以揭示差异可及性区域，这些区域解释 不同的RA反应机制，以及(2)应用CRISPR-a和CRISPR-I来测试候选人的能力 基因分别编程或破坏具有不同空间特性的种群的能力。

Abstract/Summary In vitro neural crest-like cells (NCCs) can be generated from human pluripotent stem cells (hPSCs). In response to retinoic acid (RA), NCCs can be patterned towards vagal neural crest identity based on gene expression and based on lineage differentiation capacity. Exciting preliminary data from the lab, acquired through the use of CellTag lineage tracing technology, indicate that the process of vagal identity acquisition actually begins at a very early differentiation stage, prior to RA exposure. Our data suggest the spontaneous appearance of vagal- competent versus non-competent populations during those early differentiation time points. Furthermore, we observed that the competent population exhibits a gene expression pattern that matches cells of the primordial ectoderm in the region that becomes the hindbrain. Understanding the process by which these early vagal- competent precursors are established, as well as the mechanism that maintains and executes vagal competence has broad implications. For NCC specification, an improved understanding of spatial patterning in the cranial region may greatly enhance our ability to generate vagal NCCs for the treatment of diseases such as Hirschsprung’s Disease. Beyond NCC patterning, our study may reveal general mechanisms of axial patterning that impact many other developing embryonic tissues including the CNS. In Aim 1 we propose to use scRNAseq to fully characterize the population that exhibits vagal competence in this system before identifying signals that selectively enrich for these cells using a double reporter hPSC line. In Aim 2 we propose to dissect the mechanism of competence with the ambitious combination of (1) parallel scATAC and scRNA sequencing in a multiomics approach to uncover regions of differential accessibility that account for distinct RA response mechanisms, and (2) application of CRISPR-a and CRISPR-i to test the ability of candidate genes to respectively program or disrupt competence in populations with distinct spatial identities.