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Transcriptional regulation of neuronal cell lineage decisions in the developing enteric nervous system

Award Number: R01HD105604
ORGANIZATION: NATIONAL INSTITUTE OF CHILD HEALTH AND HUMAN DEVELOPMENT
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)
PERIOD OF PERFORMANCE START DATE: 06/15/2022
PERIOD OF PERFORMANCE END DATE: 04/30/2027

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Transcriptional regulation of neuronal cell lineage decisions in the developing enteric nervous system - The vertebrate enteric nervous system (ENS), the largest portion of the peripheral nervous system, mostly derives from the vagal neural crest which arises in the caudal hindbrain, migrates to the foregut and along the entire length of the gut, differentiating into many different neuronal subtypes. In humans, defects in ENS formation cause Hirschsprung’s Disease, or colonic agangliogenesis. While ENS neurons play critical roles in regulating gastrointestinal motility, surprisingly little is known about how or what controls neuronal lineage specification in the ENS. The recent advent of single-cell technologies promises to help elucidate identification of neuronal cell types and molecular mechanisms underlying enteric neuronal differentiation. Zebrafish offer several advantages for tackling important questions in ENS development due to their simplified enteric nervous system, accessibility to genetic manipulation and facility of imaging. Similar to amniotes, the zebrafish gut contains neural crest-derived neuronal subtypes, ranging from serotonergic, cholinergic and dopaminergic neurons to VIP, Substance P and Nitric Oxide (NO)-containing neurons. Here, we propose to perform single cell RNA-seq of individual enteric precursors and neurons at different developmental stages (2-6 dpf) within the developing ENS. The function of candidate transcription and signaling factors in ENS neuronal specification will be tested by CRISPR-Cas9 perturbation experiments in both zebrafish and chick. Finally, single cell ATAC-seq will be used to identify and then dissect enteric enhancers to build an ENS gene regulatory network. We propose to perform the following aims: Aim 1: Transcriptional profiling of the enteric neural crest-derived cells at individual cell resolution using single cell RNA-seq and multiplex fluorescent in situ hybridization. We will perform single cell RNA-seq on thousands of cells per time point (2-6 days post-fertilization) of enteric precursors and neurons dissected and sorted from the zebrafish embryonic gut. We will validate expression of genes of interest, in particular transcription factors and signaling molecules, using hybridization chain reaction (HCR) and infer developmental trajectories from progenitor to neuronal differentiation. Aim 2: Role of transcription factors in differentiation of ENS neuronal subtypes in zebrafish and chick. We will mine the scRNA-seq to identify transcription factors whose expression correlates with the progenitor state (e.g. hey1a) and various neuronal subtype markers (e.g. ebf1a, etv1, Klf6a, Insm1a) for functional validation using CRISPR-Cas9 mediated knock-out in zebrafish and in chick. Aim 3: Identifying active enhancers associated with neuronal differentiation in the ENS using single cell ATAC-seq. We will use single cell ATAC-seq to identify and test putative regulatory elements functioning in neuronal precursor and differentiating neurons in the developing zebrafish ENS. Putative enhancing regions will be tested for their ability to drive ENS expression in zebrafish, mutated and tested for conservation with amniotes.


Issue Date FY	Funding FY	Legal Entity Name	Legal Entity Address	Legal Entity City	Legal Entity State	Legal Entity Zip Code	Legal Entity COUNTY	Legal Entity COUNTRY	Assistance Listing	Award Code	Budget Year	Action Date	Action Type	Action Amount

Issue Date FY: 2026 ( Subtotal = $521,786 )
2026	2026	CALIFORNIA INSTITUTE OF TECHNOLOGY	1200 E CALIFORNIA BLVD	PASADENA	CA	91125	LOS ANGELES	USA	Child Health and Human Development Extramural Research	000	5	4/7/2026	NON-COMPETING CONTINUATION	$521,786
														Subtotal = $521,786

Issue Date FY: 2025 ( Subtotal = $521,786 )
2025	2025	CALIFORNIA INSTITUTE OF TECHNOLOGY	1200 E CALIFORNIA BLVD	PASADENA	CA	91125	LOS ANGELES	USA	Child Health and Human Development Extramural Research	000	4	4/3/2025	NON-COMPETING CONTINUATION	$521,786
														Subtotal = $521,786

Issue Date FY: 2024 ( Subtotal = $516,568 )
2024	2024	CALIFORNIA INSTITUTE OF TECHNOLOGY	1200 E CALIFORNIA BLVD	PASADENA	CA	91125	LOS ANGELES	USA	Child Health and Human Development Extramural Research	000	3	4/30/2024	NON-COMPETING CONTINUATION	$516,568
														Subtotal = $516,568

Issue Date FY: 2023 ( Subtotal = $521,786 )
2023	2023	CALIFORNIA INSTITUTE OF TECHNOLOGY	1200 E CALIFORNIA BLVD	PASADENA	CA	91125	LOS ANGELES	USA	Child Health and Human Development Extramural Research	000	2	3/30/2023	NON-COMPETING CONTINUATION	$521,786
														Subtotal = $521,786

Issue Date FY: 2022 ( Subtotal = $521,786 )
2022	2022	CALIFORNIA INSTITUTE OF TECHNOLOGY	1200 E CALIFORNIA BLVD	PASADENA	CA	91125	LOS ANGELES	USA	Child Health and Human Development Extramural Research	000	1	6/15/2022	NEW	$521,786
														Subtotal = $521,786

Grand Total All Awards = $2,603,712

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Transcriptional regulation of neuronal cell lineage decisions in the developing enteric nervous system

Award Number: R01HD105604

ORGANIZATION: NATIONAL INSTITUTE OF CHILD HEALTH AND HUMAN DEVELOPMENT

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)

PERIOD OF PERFORMANCE START DATE: 06/15/2022

PERIOD OF PERFORMANCE END DATE: 04/30/2027

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