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，源自神经rest人群分别称为“迷走神经”和“ s骨”，分别由颈部和尾部区域引起。但是，关于sacral的发表少于迷走神经rest。为了纠正这一知识差距，我们提出：1。特征术的临时迁移和分化术的神经元序列在后肠中的cr峰细胞成神经元亚型； 2。转录介绍了s骨神经元冠多个时间点，并与迷走神经rest相比； 3。转录因子的测试功能这可能会驱动s骨神经rest细胞命运。结果将使我们能够测试迷走神经和s骨神经rest细胞产生相似或不同类型的肠神经元，并阐明肠道环境在分化方面。我们将使用小说来表征雏鸡的神经rest 复制无能力的鸟类（RIA）逆转录病毒的谱系标记技术，使我们能够具体 FACS的靶标和分离的s骨神经衍生的人群执行以下目标：特定目的1：与迷走神经中性相比，逆转录病毒介导的雏形谱系分析波峰：我们将用RIA逆转录病毒将神经元管尾尾标记为27 波峰细胞以遵循其长期命运。初步结果表明s骨神经rest衍生的细胞填充后肠道并分化为胆碱能运动神经元以及酪氨酸羟化酶阳性细胞。 s骨神经rest将与迷走神经rest和两者之间的相互作用进行比较人口检查。最后，将表征s骨冠细胞之间的克隆关系。具体目标2：骨骼后肠道中s骨神经rest衍生细胞的单细胞RNA-seq。到阐明控制s骨神经元细胞逐步分化为神经元的基因调节程序和胶质在后肠中，我们建议表征s骨衍生的细胞FACS的转录曲线从后肠道中分类，并在胚胎天（E）2.5，E6，E8，通过单细胞（SC）RNA-Seq处理 10、15、21（匹配）；这些将与迷走神经衍生的细胞在可比的阶段进行比较。初步的 E10上的SCRNA-SEQ数据表明，s骨与迷走神经产生的神经元亚型存在差异波峰。 SCRNA-SEQ将使我们能够采样不同的神经元亚型和推断发育轨迹。特定目的3：转录调节剂在分化s骨神经元为神经元中的作用和神经胶质亚型。专注于Enter前体簇中存在的转录因子（例如 NFATC1，FOXN2，SOX4，SOX21，ELF2，ZNF536），我们将测试是否含有s骨神经含有富含的转录因子对于介导增殖，肠道内的迁移和/或细胞脂肪决策至关重要。为此，我们将使用单质粒CRISPR-CAS9策略和检查随后对萨克拉神经rest衍生细胞的分布和/或分化的影响。