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.
