CURRENT RESEARCH
Multiplex HCR in Slide, enabling whole mount SGA. Protocol from Moreno-Campos et al., 2025
Enteric Nervous System (ENS) Development and Differentiation
The ENS is a vast branch of the peripheral nervous system resident within the entire gut. It is also known as the gut brain and an essential portion of the gut-brain-axis. The main constituents of the ENS are hundreds of thousands of neurons and glia embedded within the walls of the gut. The ENS exhibits diverse enteric neuron subtypes and glial cells, which together regulates gut peristalsis, water balance and hormone secretions. While we know that the ENS is largely derived from the neural crest and enteric neural progenitor cells, we still know very little about its vast construction in the context of the live gut. When ENS development goes wrong, it can lead to severe congenital disorders such as Hirschsprung disease, as well as broader functional gut–neurological conditions.
Zebrafish offer a simplified vertebrate model to study ENS development. Using the zebrafish model, we investigate how:
The ENS is a vast branch of the peripheral nervous system resident within the entire gut. It is also known as the gut brain and an essential portion of the gut-brain-axis. The main constituents of the ENS are hundreds of thousands of neurons and glia embedded within the walls of the gut. The ENS exhibits diverse enteric neuron subtypes and glial cells, which together regulates gut peristalsis, water balance and hormone secretions. While we know that the ENS is largely derived from the neural crest and enteric neural progenitor cells, we still know very little about its vast construction in the context of the live gut. When ENS development goes wrong, it can lead to severe congenital disorders such as Hirschsprung disease, as well as broader functional gut–neurological conditions.
Zebrafish offer a simplified vertebrate model to study ENS development. Using the zebrafish model, we investigate how:
- enteric neural progenitor cells migrate, colonize the gut, and differentiate, (Baker et al., 2022, Howard et al., 2022)
- distinct neuron types emerge and assemble into 3D gut-wide networks, (Baker et al., 2022, Moreno-Campos et al., 2025)
- transcriptional programs and spatial cues regulate ENS patterning, (Howard et al., 2022, Baker et al., 2022, Moore et al., 2025, Moreno-Campos et al., 2025)
- disease-associated genes disrupt ENS formation and function, (Howard et al., 2022, Baker et al., 2022, Moore et al., 2025, Moreno-Campos et al., 2024)
- whole-gut spatial genomics and 3D imaging
- single-cell and spatial transcriptomics–guided discovery
- CRISPR F0 functional genetic screening
- custom pipelines for high-content semi-automated confocal microscopy
From Moreno-Campos et al., 2024. Illustrates how an F0-CRISPR screen in zebrafish targeting candidate genes prioritized from single-cell RNA-seq ENS data sets (Howard et al., 2021) can uncover novel regulators of ENS formation.
From Moreno-Campos et al., 2025. Illustrates how sequential, multiplexed HCR technology can be used in whole intact zebrafish larvae, revealing the 3D spatial expression patterns of dozens of mRNAs in the same specimen and revealing novel 3D gene expression data long the entire gut tube.
Neural crest development. From Howard and Uribe, 2022
Neural Crest Cell Diversification
Neural crest cells are stem cells that migrate to various locations and give rise to diverse and fundamental cell types in the vertebrate body–including craniofacial tissues, cardiac cells and peripheral nervous system. Neural crest stem cells originate from the dorsal neural tube, a transient structure during development that will eventually give rise to the central nervous system. Some of the neural crest cells, called "vagal" neural crest cells, eventually can give rise to cells of the outflow tract of the heart, enteric peripheral ganglia, sympathetic ganglia, thymic connective tissue, as well as pigment cells of the skin.
What dictates whether neural crest will give rise to ganglia or other derivatives, such as pigment cells or connective tissues, remains elusive. To address this challenge, we study what regulates neural crest cell delineation, in the zebrafish embryo, a robust vertebrate model.
Towards that end, and begin shedding light on neural crest differentiation, we have undertaken studies on neural crest diversification in zebrafish using single-cell transcriptomics (Howard, Baker et al., 2021), and discovered dozens of transcriptionally-distinct neural crest-derived cellular subpopulations, greatly expanding the field's basic understanding of neural crest cell development. From these populations, we have uncovered previously unappreciated signatures of various genes, including those encoding transcription factors, implicating them in diversification of the vagal neural crest derivatives, including the ENS. Ongoing studies are building upon our recent single-cell discoveries in the lab right now.
Neural crest cells are stem cells that migrate to various locations and give rise to diverse and fundamental cell types in the vertebrate body–including craniofacial tissues, cardiac cells and peripheral nervous system. Neural crest stem cells originate from the dorsal neural tube, a transient structure during development that will eventually give rise to the central nervous system. Some of the neural crest cells, called "vagal" neural crest cells, eventually can give rise to cells of the outflow tract of the heart, enteric peripheral ganglia, sympathetic ganglia, thymic connective tissue, as well as pigment cells of the skin.
What dictates whether neural crest will give rise to ganglia or other derivatives, such as pigment cells or connective tissues, remains elusive. To address this challenge, we study what regulates neural crest cell delineation, in the zebrafish embryo, a robust vertebrate model.
Towards that end, and begin shedding light on neural crest differentiation, we have undertaken studies on neural crest diversification in zebrafish using single-cell transcriptomics (Howard, Baker et al., 2021), and discovered dozens of transcriptionally-distinct neural crest-derived cellular subpopulations, greatly expanding the field's basic understanding of neural crest cell development. From these populations, we have uncovered previously unappreciated signatures of various genes, including those encoding transcription factors, implicating them in diversification of the vagal neural crest derivatives, including the ENS. Ongoing studies are building upon our recent single-cell discoveries in the lab right now.
Neural crest to neuron progression as seen in single-cell RNA-seq. From Howard, Baker et al., 2021
Relevance to Human Health and the Nervous System
Understanding the genetic programs and cellular interactions that drive stem cells to form the enteric nervous system is of crucial concern. From a large view, having knowledge about how neural crest differentiate into neural tissue is essential for understanding how cells make fundamental decisions in their native tissue context, and significantly, for also informing targeted designs for neural therapeutics.
Because improper neural crest development leads to developmental anomalies such as Hirschsprung disease (colonic aganglionosis), and neural crest-derived cancers, such as Melanoma and Neuroblastoma, there has been great interest in understanding the migration and differentiation of neural crest cells. It is important to study development of the ENS so that we can understand not only how it forms and functions, but also to help us to understand how things go wrong in various gastrointestinal autonomic neuropathies (Hirschsprung disease, Achalasia), as well as neural crest stem cell defects, such as when neural crest become cancerous.
Hirschsprung disease is characterized by a paucity of ganglia along variable lengths of the gut, with colonic aganglionosis being the most common form, occurring every 1 in 5000 births.. The current treatment for this pediatric developmental defect is surgical resection of the aganglionic intestinal segment--however eventual outcomes of patients varies greatly and most exhibit functional enteric defects throughout life--highlighting the need for alternative treatments and understanding the ontogeny of the disorder.
Melanoma and Neuroblastoma are neural crest-derived cancers affecting adults and children alike throughout the world. It is hypothesized that several genetic mutations in neural crest cell lineages are the basis for formation of neuroblastoma and melanoma cancers, however the signaling landscape conducive to formation of melanoma and neuroblastoma are not entirely known. Other current studies in the lab endeavor to understand how neural crest cell pathways may contribute to oncogenesis.
Understanding the genetic programs and cellular interactions that drive stem cells to form the enteric nervous system is of crucial concern. From a large view, having knowledge about how neural crest differentiate into neural tissue is essential for understanding how cells make fundamental decisions in their native tissue context, and significantly, for also informing targeted designs for neural therapeutics.
Because improper neural crest development leads to developmental anomalies such as Hirschsprung disease (colonic aganglionosis), and neural crest-derived cancers, such as Melanoma and Neuroblastoma, there has been great interest in understanding the migration and differentiation of neural crest cells. It is important to study development of the ENS so that we can understand not only how it forms and functions, but also to help us to understand how things go wrong in various gastrointestinal autonomic neuropathies (Hirschsprung disease, Achalasia), as well as neural crest stem cell defects, such as when neural crest become cancerous.
Hirschsprung disease is characterized by a paucity of ganglia along variable lengths of the gut, with colonic aganglionosis being the most common form, occurring every 1 in 5000 births.. The current treatment for this pediatric developmental defect is surgical resection of the aganglionic intestinal segment--however eventual outcomes of patients varies greatly and most exhibit functional enteric defects throughout life--highlighting the need for alternative treatments and understanding the ontogeny of the disorder.
Melanoma and Neuroblastoma are neural crest-derived cancers affecting adults and children alike throughout the world. It is hypothesized that several genetic mutations in neural crest cell lineages are the basis for formation of neuroblastoma and melanoma cancers, however the signaling landscape conducive to formation of melanoma and neuroblastoma are not entirely known. Other current studies in the lab endeavor to understand how neural crest cell pathways may contribute to oncogenesis.