Contribution of the sacral neural crest to the peripheral nervous system of the post-umbilical gastrointestinal tract

肠神经系统(ENS)是周围神经系统的最大部分，起源于 神经脊群被称为“迷走神经”和“骶骨”，分别起源于颈部和尾部。 然而，有关骶骨的文献比迷走神经脊要少得多。为了弥补这一知识鸿沟， 我们建议：1.描述神经移行和分化的时间序列。 在后肠中将脊细胞转化为神经元亚型；2.在转录水平上描述骶骨神经脊。 多个时间点，并与迷走神经峰进行比较；3.转录因子功能检测 这可能会驱动神经脊细胞命运的选择。结果将使我们能够测试迷走神经和骶骨 神经脊细胞产生相似或不同类型的肠神经元，并阐明 肠道环境对其分化的影响。我们将使用一种新的方法来描述鸡的骶骨神经脊。 复制不能(RIA)逆转录病毒的谱系标记技术使我们能够特异性地 通过流式细胞仪检测和分离来自神经脊区的群体，以达到以下目的： 具体目标1：逆转录病毒介导的鸡骶骨和迷走神经的谱系分析比较 CREST：我们将用RIA逆转录病毒对神经管尾部至躯体27进行标记，这些逆转录病毒可以永久地标记骶神经 为了追随它们的长期命运。初步研究结果表明，由骶骨神经沟衍生的细胞 填充脐带后的肠道并分化为胆碱能运动神经元和酪氨酸羟基酶 阳性细胞。并将其与迷走神经脊线进行比较，以及两者之间的相互作用 被检查的人口。最后，我们将描述这些细胞之间的克隆关系。 特定目的2：脐带后肠道中的单细胞RNA序列。至 阐明控制骶骨神经脊细胞向神经元渐进分化的基因调控程序 和后肠中的神经胶质细胞，我们建议用来描述Socage Crest来源的细胞的转录图谱。 从脐带后肠道分离并在胚胎2.5天、E6天、E8天、 10、15、21(孵化前)；这些细胞将与类似时期的迷走神经脊源性细胞进行比较。初步 E10上的scrna-seq数据表明，由坐骨神经和迷走神经产生的神经元亚型存在差异。 克雷斯特。ScRNA-seq将使我们能够采样不同的神经元亚型并推断发育轨迹。 具体目标3：转录调控因子在骶神经脊向神经元分化中的作用 和神经胶质亚型。关注存在于肠道前体簇中的转录因子(例如 NFATc1，Foxn2，Sox4，Sox21，elF2，Znf536)，我们将检测是否有丰富的骶神经脊转录因子 对于调节肠道内的增殖、迁移和/或细胞命运的决定至关重要。为此，我们将 使用单质粒CRISPR-CAS9策略在骶神经脊进行靶向性功能丧失 观察对骶骨神经脊源性细胞分布和/或分化的后续影响。

The enteric nervous system (ENS), the largest portion of the peripheral nervous system, is derived from neural crest populations referred to as “vagal” and “sacral”, arising from the neck and tail regions, respectively. However, much less has been published about sacral than vagal neural crest. To rectify this knowledge gap, we propose to: 1. characterize the temporal sequence of migration and differentiation of sacral neural crest cells into neuronal subtypes in the hindgut; 2. transcriptionally profile the sacral neural crest as at multiple time points and compare with that of vagal neural crest; 3. test function of transcription factors that may drive sacral neural crest cell fate choice. The results will enable us to test whether vagal and sacral neural crest cells give rise to similar or distinct types of enteric neurons and elucidate the influence of the intestinal environment on their differentiation. We will characterize the chick sacral neural crest using a novel lineage labeling technique of Replication Incompetent Avian (RIA) retroviruses that enables us to specifically target and isolate by FACS the sacral neural crest-derived population to perform the following aims: Specific Aim 1: Retrovirus-mediated lineage analysis of the chick sacral compared with vagal neural crest: We will label the neural tube caudal to somite 27 with RIA retroviruses that permanently label sacral neural crest cells in order to follow their long term fate. Preliminary results suggest that sacral neural crest-derived cells populate the post-umbilical gut and differentiate into cholinergic motor neurons as well as tyrosine hydroxylase positive cells. Sacral neural crest will be compared with vagal neural crest and interactions between the two populations examined. Finally, clonal relationships between sacral crest cells will be characterized. Specific Aim 2: Single cell RNA-seq of sacral neural crest-derived cells in the post-umbilical gut. To elucidate gene regulatory programs controlling progressive differentiation of sacral neural crest cells into neurons and glia in the hindgut, we propose to characterize the transcriptional profile of sacral crest-derived cells FACS sorted from the post-umbilical gut and processed by single cell (sc) RNA-seq at embryonic days (E) 2.5, E6, E8, 10, 15, 21 (prehatching); these will be compared with vagal crest-derived cells at comparable stages. Preliminary scRNA-seq data on E10 suggest that there are differences in neuronal subtypes produced by sacral vs vagal crest. scRNA-seq will enable us to sample different neuronal subtypes and infer developmental trajectories. Specific Aim 3: Role of transcriptional regulators into differentiation of sacral neural crest into neuronal and glial subtypes. Focusing on transcription factors that are present in the enteric precursor cluster (e.g. Nfatc1, Foxn2, Sox4, Sox21, Elf2, Znf536), we will test whether sacral neural crest-enriched transcription factors are critical for mediating proliferation, migration within the gut, and/or cell fate decisions. To this end, we will perform targeted loss of function in the sacral neural crest using a single-plasmid CRISPR-Cas9 strategy and examine subsequent effects on distribution and/or differentiation of sacral neural crest-derived cells.