Cell lineage and transcriptional analysis of the vertebrate neural plate border

神经嵴祖细胞和外胚层基板细胞都起源于神经板缘（NPB）。 神经嵴形成颅面骨骼，基板形成透镜、耳和嗅觉系统; 两者都对颅感觉神经节有贡献。然而，与其固定在神经嵴或基板上， 我们的结果显示，神经板边缘的细胞似乎共表达转录因子， 多个谱系，从神经嵴到神经基板。此外，我们发现， 在表达多能性因子包括Sall 4、Nanog、Oct 4、Klf 4和cMyc的背神经管内 这在我们的单细胞RNA-seq数据中得到了证实。因此，是什么维持了干细胞的潜力， 形成神经嵴，基板和神经管衍生物在神经板边缘保持开放。我们的初步 数据表明，Sall 4可能与其他多能性基因形成反馈环，包括Pou 5 f3/Oct 4和 Sall1.此外，Sall 4的过表达阻止神经嵴特化并上调Oct 4。测试 假设这些多能性因子维持神经板边缘的多能性， 它们的下调对于神经嵴和/或基板特化是必要的，我们将探讨它们的影响。 它们的功能获得和丧失，鉴定介导它们表达的增强子，并检查动态变化 在神经板边缘成熟过程中的基因表达。为此，将实现以下目标。 目的1：多能性因子异位维持或丢失对神经嵴和基板的影响 发展我们将在体内测试多能性因子Sall 4、Oct 4、Nanog和Klf 4在神经板中的作用 使用功能获得和丧失方法结合单细胞RNA-seq进行边界开发。特别是， 我们将检验这些多能性因子维持神经板边缘多能性的假设 并且它们的下调是完成神经嵴特化所必需的。我们还将研究其他 转录因子，以测试它们在驱动神经板边缘的谱系特化中的作用。 目的2：鉴定和顺式调节分析介导表达的推定增强子 多能性因子和神经板边界转录调节因子。详细的顺式调控分析 允许识别活性增强子及其直接输入，阳性和阴性，从而告知 基因调控网络连接。我们建议使用单细胞ATAC-seq来鉴定推定的调控基因。 在形成神经板边缘有活性的元件，特别是那些介导多能性因子表达的元件 和转录调节因子。将测试推定的增强子的输入及其驱动表达的能力。 目的3：动态分析增强子介导的神经板边缘成熟过程中的表达。 通过将实时成像与增强子驱动的报告基因表达相结合，我们建议测试基因表达的动态变化。 通过多能性基因和转录因子介导的表达。我们将首先关注增强剂， 介导Pax 7、Sox 2和Six 1的表达，分别是神经嵴、神经和基板的特征。

Both neural crest progenitors and ectodermal placode cells arise from the neural plate border (NPB). While the neural crest gives rise to the craniofacial skeleton, the placodes form the lens, ear and olfactory system; both contribute to cranial sensory ganglia. However, rather than being fixed to a neural crest or placodal fate, our results show that cells in the neural plate border appear to coexpress transcription factors characteristic of multiple lineages, ranging from neural crest to neural to placodal. Moreover, we find there is a stem cell niche within the dorsal neural tube that expresses pluripotency factors including Sall4, Nanog, Oct4, Klf4, and cMyc as confirmed in our single cell RNA-seq data. Thus, the question of what maintains stem cells with the potential to form neural crest, placode and neural tube derivatives at the neural plate border remains open. Our preliminary data suggest that Sall4 may form a feed-back loop with other multipotency genes, including Pou5f3/Oct4 and Sall1. Moreover, overexpression of Sall4 prevents neural crest specification and upregulates Oct4. To test the hypothesis that these pluripotency factors maintain multipotency of the neural plate border and that their downregulation is necessary for neural crest and/or placode specification, we will explore the effects of their gain and loss of function, identify enhancers that mediate their expression and examine dynamic changes in their gene expression during neural plate border maturation. To this end, the following aims will be performed. Aim 1: Effects of ectopic maintenance or loss of pluripotency factors on neural crest and placode development. We will test the role of pluripotency factors Sall4, Oct4, Nanog and Klf4 in vivo in neural plate border development using gain and loss of function approaches coupled with single cell RNA-seq. In particular, we will test the hypothesis that these pluripotency factors maintain the multipotency of the neural plate border and their downregulation is necessary for completion of neural crest specification. We will also examine other transcription factors to test their role in driving lineage specification at the neural plate border. Aim 2: Identification and cis-regulatory analysis of putative enhancers mediating expression of pluripotency factors and neural plate border transcriptional regulators. Detailed cis-regulatory analysis allows identification of active enhancers and their direct inputs, both positive and negative, thus informing upon gene regulatory network connections. We propose to use single cell ATAC-seq to identify putative regulatory elements active at the forming neural plate border, particularly those mediating expression of pluripotency factors and transcriptional regulators. Putative enhancers will be tested for inputs and their ability to drive expression. Aim 3: Dynamic analysis of enhancer-mediated expression during maturation of the neural plate border. By coupling live imaging with enhancer driven reporter expression, we propose to test dynamic changes in gene expression mediated by pluripotency genes and transcription factors. We will initially focus on enhancers that mediate expression of Pax7, Sox2 and Six1, characteristic of neural crest, neural and placodes, respectively.