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

• 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
• 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

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提供了内在的神经 胃肠道（GI）的大道并控制所有必需的肠道功能，包括运动。 ENS中的缺陷 神经元丰度与特征在于gi功能障碍的多种疾病，使人衰弱 症状和生活质量降低。迄今为止，ENS疾病只能症状或通过 手术去除受影响区域。治疗丢失的ENS细胞的有希望的途径是刺激局部干细胞 再生丢失的ENS神经元。但是，关于信号的知识有很大的差距 成功的ENS再生所需的细胞谱系。为了解决这个知识差距，我们需要 建立一个实验可牵引的动物模型系统，显示可靠的ENS再生 肠道功能的恢复。在哺乳动物中，ENS仅部分加剧并在受伤后恢复神经元。 与哺乳动物不同，斑马鱼ENS会在少量ENS的局部消融后再生ENS损伤 神经元。但是，斑马鱼是否可以在肠道的所有部位修复巨大的ENS伤害，以及 再生后的功能恢复尚不清楚。此外，我们对分子知之甚少 线索，细胞生物学过程和细胞谱系组成，其基于Ens再生。因此，有一个 建立细胞和分子机制以及细胞谱系决策的迫切需要 指南斑马鱼的再生。建立一个强大的ENS再生动物模型系统将 为我们的长期目标铺平道路，以确定必要的基因和基因调节网络 足够成功的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两个目的产生丢失的ENS神经元：（1）建立 斑马鱼的再生能力； （2）识别细胞群，时空基因表达 动力学和细胞谱系在细胞消融后驱动神经元再生。 AIM 1利用遗传 化学消融系统，用于精确的时空控制细胞损失和高分辨率全态成像 分析功能恢复。 AIM 2使用单细胞RNA-SEQ（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谱。该提议是 创新的，因为它将利用遗传化学细胞消融系统的精度和杰出的 SCRNA-SEQ的细胞和分子分辨率建立了鲁棒的动物模型系统 再生，从而为神经系统再生研究开辟了新的视野。这项工作是 显着，因为它将为理解哪些分子提示和细胞提供必要的基础 响应促进ENS再生以及如何应用此类因素来增强人类ENS 再生以治疗肠道神经疾病。