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

骶神经嵴对脐后胃肠道周围神经系统的贡献

基本信息

批准号：
10644256
负责人：
Marianne Bronner
金额：
$ 55.64万
依托单位：
CALIFORNIA INSTITUTE OF TECHNOLOGY
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-07-01 至 2027-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10644256
关键词：
Antibodies Birds Brain Burn injury CRISPR/Cas technology Cell Differentiation process Cells Central Nervous System Chick Colon Compensation Congenital Abnormality Congenital Megacolon Data Defect Development Disease Embryo Enteral Enteric Nervous System Environment Gastrointestinal Hormones Gastrointestinal Motility Gastrointestinal tract structure Gene Expression Profile Genetic Transcription Genome Genomic approach Genus Alpharetrovirus Hindgut Hormone secretion Human In Situ Individual Interspecific Recombination Intestines Knowledge Label Length Life Mediating Modernization Motor Neurons Neck Nervous System control Neural Crest Neural Crest Cell Neural tube Neuroglia Neuronal Differentiation Neurons Patients Peripheral Nervous System Plasmids Population Primitive foregut structure Process Proliferating Publishing Quail Regulator Genes Retroviridae Role SOX21 gene SOX4 gene Sampling Somites Sorting Tail Techniques Testing Time Tyrosine 3-Monooxygenase Umbilicus cell motility cholinergic enteric neuropathy experimental study fluorophore gut colonization hatching healing hindbrain insight loss of function migration neural novel programs public health relevance single-cell RNA sequencing time use transcription factor

项目摘要

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.

肠神经系统（ENS）是周围神经系统的最大部分，来源于神经嵴群称为“迷走神经”和“骶神经”，分别来自颈部和尾部区域。然而，关于骶神经嵴的文献报道却比迷走神经嵴少得多。为了弥补这一知识差距，我们建议：描述骶骨神经细胞迁移和分化的时间顺序后肠嵴细胞分化为神经元亚型; 2.转录分析骶骨神经嵴，多个时间点与迷走神经嵴比较; 3.转录因子功能检测可能驱动骶骨神经嵴细胞的命运选择。结果将使我们能够测试迷走神经和骶神经神经嵴细胞产生类似或不同类型的肠神经元，并阐明了神经嵴细胞对肠神经元的影响。肠道环境对其分化的影响。我们将描述小鸡骶神经嵴使用一种新的复制无能禽（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瞄准并分离骶骨神经嵴衍生群体，以实现以下目标：特定目的1：鸡骶骨与迷走神经的逆转录病毒介导谱系分析 crest：我们将用RIA逆转录病毒标记第27体节尾侧的神经管，以遵循它们的长期命运。初步结果表明，骶神经嵴来源的细胞填充后脐肠并分化成胆碱能运动神经元以及酪氨酸羟化酶阳性细胞将骶神经嵴与迷走神经嵴以及两者之间的相互作用进行比较人口调查。最后，骶嵴细胞之间的克隆关系将被表征。具体目标2：脐后肠中骶骨神经嵴衍生细胞的单细胞RNA-seq。到阐明控制骶骨神经嵴细胞向神经元进行性分化的基因调控程序和神经胶质细胞，我们建议用流式细胞仪来表征骶嵴衍生细胞的转录谱从后脐肠中分选，并在胚胎日（E）2.5，E6，E8， 10、15、21（孵化前）;这些将与可比较阶段的迷走嵴衍生细胞进行比较。初步 E10的scRNA-seq数据表明，骶神经与迷走神经产生的神经元亚型存在差异，冠。scRNA-seq将使我们能够对不同的神经元亚型进行采样并推断发育轨迹。具体目标3：转录调节因子在骶神经嵴分化为神经元中的作用和神经胶质亚型。关注存在于肠前体簇中的转录因子（例如， Nfatc 1，Foxn 2，Sox 4，Sox 21，Elf 2，Znf 536），我们将测试是否骶神经嵴富集转录因子对于介导增殖、肠内迁移和/或细胞命运决定至关重要。为此我们将使用单质粒CRISPR-Cas9策略在骶神经嵴中进行靶向功能丧失，检查对骶骨神经嵴衍生细胞的分布和/或分化的后续影响。