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

该项目旨在确定肠道神经系统(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。 控制胃肠道(GI)，并控制所有基本的肠道功能，包括运动。ENS中的赤字 神经元的丰富与以胃肠道功能障碍为特征的一系列疾病有关，使人虚弱 症状，并降低生活质量。到目前为止，ENS障碍只能有症状地或通过 手术切除受影响的区域。治疗丢失的ENS细胞的一个有希望的方法是刺激局部干细胞 以再生缺失的ENS神经元。然而，关于这些信号的知识有很大的差距 以及成功再生ENS所需的细胞谱系。为了解决这一知识鸿沟，我们需要 建立一个实验上易于驯化的动物模型系统，显示出强大的神经再生能力，包括 肠道功能恢复。在哺乳动物中，神经元只在损伤后部分地重新神经支配和恢复神经元。 与哺乳动物不同，斑马鱼ENS在局部消融少量ENS后再生ENS损伤 神经元。然而，斑马鱼能否修复肠道各部位广泛的ENS损伤，以及修复的程度 再生后的功能恢复尚不清楚。此外，我们对分子所知甚少。 作为神经再生基础的线索、细胞生物学过程和细胞谱系组成。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 引导斑马鱼再生。建立健壮的神经再生动物模型系统 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 足够成功再生ENS。这一提议检验了斑马鱼肠道的中心假设 环境允许肠道干细胞激活产生丢失的ENS神经元有两个目的：(1)建立 斑马鱼胚胎再生能力的研究；(2)细胞群体鉴定、时空基因表达 动力学，以及推动细胞消融后神经元再生的细胞谱系。AIM 1利用了一种基因- 用于精确时空控制细胞丢失和高分辨率全肠道成像的化学消融系统 分析功能恢复情况。AIM 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图谱。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度和卓越的 ScRNA-seq的细胞和分子分辨建立健壮的ENS动物模型系统 从而为神经系统再生的研究开辟了新的天地。这项工作是 意义重大，因为它将为理解哪些分子线索和细胞 响应促进神经再生，以及如何应用这些因素来增强人类神经 再生治疗肠道神经疾病。

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.