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

使用具有时空控制的细胞消融系统研究斑马鱼肠神经系统再生

基本信息

批准号：
10525953
负责人：
Julia Ganz
金额：
$ 43.94万
依托单位：
MICHIGAN STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-08-01 至 2024-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10525953
关键词：
Ablation Address Affect Animal Model Area Automobile Driving Biological Assay Biological Models Biological Process Bromodeoxyuridine Cell Lineage Cell physiology Cells Clinical Congenital Megacolon Cues Development Digestion Digestive System Disorders Disease Disease model Enteral Enteric Nervous System Environment Excision Expenditure Foundations Functional disorder Future Gastrointestinal tract structure Gene Expression Generations Genes Goals Human Image Immune response Immunohistochemistry In Situ Hybridization Individual Injury Investigation Knowledge Lead Mammals Mission Molecular Molecular Genetics Molecular Profiling Natural regeneration Nerve Regeneration Nervous System Trauma Nervous System control Nervous system structure Neuraxis Neuroglia Neurons Peripheral Peripheral Nervous System Population Process Public Health Quality of life RNA Recovery Recovery of Function Regenerative capacity Regenerative response Regulatory Element Research Resolution Signal Transduction Societies System Testing TimeLine Translating United States National Institutes of Health Work Zebrafish autism spectrum disorder base cell motility cell type central nervous system injury chemical genetics disabling symptom gastrointestinal gene regulatory network gut health gut homeostasis high resolution imaging human disease in vivo innovation microbiota nerve supply nervous system disorder neuron regeneration novel therapeutic intervention regenerative regenerative approach reinnervation repaired response single-cell RNA sequencing spatiotemporal stem cells

项目摘要

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.

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 提供内在神经支配胃肠道 (GI) 的控制并控制所有重要的肠道功能，包括蠕动。 ENS 赤字神经元丰度与多种以胃肠道功能障碍、衰弱为特征的疾病有关症状，并降低生活质量。迄今为止，ENS 疾病只能对症治疗或通过手术切除受影响的区域。治疗丢失的 ENS 细胞的一个有前途的途径是刺激局部干细胞再生缺失的 ENS 神经元。然而，关于信号的知识存在很大差距以及 ENS 成功再生所必需的细胞谱系。为了弥补这一知识差距，我们需要建立一个实验上易于处理的动物模型系统，该系统显示出强大的 ENS 再生能力，包括肠道功能的恢复。在哺乳动物中，ENS 仅在损伤后部分重新支配和恢复神经元。与哺乳动物不同，斑马鱼 ENS 局部消融后可再生 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，我们对分子的了解还知之甚少。 ENS 再生的线索、细胞生物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策指导斑马鱼 ENS 再生。建立强大的 ENS 再生动物模型系统将为我们的长期目标铺平道路，即确定必要的基因和基因调控网络足以成功进行 ENS 再生。该提案检验了斑马鱼肠道的中心假设环境允许肠干细胞激活以产生丢失的 ENS 神经元，目的有两个：(1) 建立斑马鱼ENS的再生能力； (2) 识别细胞群体、时空基因表达动力学，以及细胞消融后驱动神经元再生的细胞谱系。目标 1 利用遗传- 化学消融系统，用于精确时空控制细胞损失和高分辨率全肠成像分析功能恢复情况。目标 2 使用单细胞 RNA 序列 (scRNA-seq) 来识别细胞和驱动再生反应的细胞类型和谱系的分子特征。这个提议是创新，因为它将利用遗传化学细胞消融系统的精度和卓越的 scRNA-seq 的细胞和分子分辨率，建立稳健的 ENS 动物模型系统再生，从而为神经系统再生的研究开辟了新的视野。这部作品是意义重大，因为它将为理解哪些分子线索和细胞提供必要的基础反应促进 ENS 再生以及如何应用这些因素来增强人类 ENS 再生来治疗肠道神经系统疾病。