Investigation of Enteric Nervous System Regeneration in Zebrafish Using a Cell Ablation System with Spatio-Temporal Control

该项目旨在确定肠神经系统（ENS）的再生能力，并确定 控制ENS再生的细胞和分子机制。ENS提供内在的神经支配 胃肠道（GI）和控制所有基本的肠道功能，包括运动。ENS赤字 神经元丰度与以GI功能障碍、使人衰弱、 症状和生活质量下降。迄今为止，ENS疾病只能通过外科手术或 手术切除受影响的区域。一个有希望的治疗失去ENS细胞的方法是刺激局部干细胞 再生缺失的ENS神经元然而，在有关信号的知识方面存在重大差距， 和成功ENS再生所必需的细胞谱系。为了弥补这一知识差距，我们需要 建立实验上易于处理的动物模型系统，其显示出稳健的ENS再生，包括 恢复肠道功能。在哺乳动物中，ENS在损伤后仅部分地再神经支配和恢复神经元。 与哺乳动物不同，斑马鱼ENS在局灶性消融少量ENS后再生ENS损伤 神经元然而，斑马鱼是否可以修复肠道所有部位的广泛ENS损伤，以及 再生后的功能恢复是未知的。此外，我们对分子生物学知之甚少。 提示，细胞生物学过程，以及ENS再生的细胞谱系组成。因此，有一个 迫切需要建立细胞和分子机制以及细胞谱系决定， 引导斑马鱼的ENS再生。建立ENS再生的动物模型体系， 为我们的长期目标铺平道路，以确定必要的基因和基因调控网络， 足以成功再生ENS。这项提议验证了中心假设，即斑马鱼肠道 环境允许肠干细胞活化以产生丢失的ENS神经元，这有两个目的：（1）建立肠干细胞的神经元模型。 斑马鱼ENS的再生能力;（2）鉴定细胞群体，时空基因表达 动力学和细胞消融后驱动神经元再生的细胞谱系。目标1利用基因- 用于精确时空控制细胞损失和高分辨率全肠成像的化学消融系统 来分析功能恢复情况目的2使用单细胞RNA-seq（scRNA-seq）来鉴定细胞和 驱动再生反应的细胞类型和谱系的分子概况。这项建议是 创新，因为它将利用遗传化学细胞消融系统的精确性和特殊的 scRNA-seq的细胞和分子解析以建立稳健ENS的动物模型系统 从而为神经系统再生的研究开辟了新的视野。这项工作是 重要的是，因为它将为理解哪些分子线索和细胞信号提供必要的基础。 反应促进ENS再生，以及这些因素如何应用于增强人类ENS 再生来治疗肠道神经疾病。

This project aims to determine the regenerative capacity of the enteric nervous system (ENS) and identify the cellular and molecular mechanisms that control ENS regeneration. The ENS provides the intrinsic innervation of the gastrointestinal (GI) tract and controls all essential gut functions including motility. Deficits in ENS neuron abundance are associated with a wide range of disorders characterized by GI dysfunction, debilitating symptoms, and reduced quality of life. To date, ENS disorders can only be treated symptomatically or by surgical removal of the affected area. A promising avenue to treat lost ENS cells is to stimulate local stem cells to regenerate missing ENS neurons. However, there is a significant gap in knowledge regarding the signals and cell lineages necessary for successful ENS regeneration. To address this knowledge gap, we need to establish an experimentally tractable animal model system that displays robust ENS regeneration including recovery of gut functions. In mammals, the ENS only partially reinnervates and recovers neurons after injury. Unlike mammals, the zebrafish ENS regenerates ENS injury after focal ablation of a small number of ENS neurons. However, whether zebrafish can repair extensive ENS injuries in all parts of the gut, and the extent of functional recovery following regeneration is not known. Furthermore, we know very little about the molecular cues, cell biological processes, and cell lineage composition that underlie ENS regeneration. Thus, there is a critical need to establish the cellular and molecular mechanisms as well as the cell lineage decisions that guide ENS regeneration in zebrafish. Establishing an animal model system of robust ENS regeneration will pave the way for our long-term goal to identify the genes and gene regulatory networks necessary and sufficient for successful ENS regeneration. This proposal tests the central hypothesis that the zebrafish gut environment allows enteric stem cell activation to generate lost ENS neurons in two Aims: (1) establish the regenerative ability of the zebrafish ENS; (2) identify cell populations, spatio-temporal gene expression dynamics, and cell lineages that drive neuronal regeneration after cell ablation. Aim 1 utilizes a genetic- chemical ablation system for precise spatio-temporal control of cell loss and high-resolution whole-gut imaging to analyze functional recovery. Aim 2 uses single-cell RNA-seq (scRNA-seq) to identify the cellular and molecular profiles of the cell types and lineages that drive the regenerative response. This proposal is innovative, as it will capitalize on the precision of the genetic-chemical cell ablation system and the exceptional cellular and molecular resolution of scRNA-seq to establish an animal model system of robust ENS regeneration and thereby open new horizons for the study of nervous system regeneration. This work is significant, as it will provide the necessary foundation for understanding which molecular cues and cellular responses promote ENS regeneration and how such factors can be applied to enhance human ENS regeneration to treat neurological diseases of the gut.