Ret loss-of-function decreases neural crest progenitor proliferation and restricts developmental fate potential during enteric nervous system development

Abstract

The receptor tyrosine kinase gene RET plays a critical role in the fate specification of enteric neural crest cells (ENCCs) during enteric nervous system (ENS) development. Ret loss of function (LoF) alleles are associated with Hirschsprung disease (HSCR), which is marked by aganglionosis of the gastrointestinal (GI) tract. ENCCs invade the developing GI tract, proliferate, migrate caudally, and differentiate into all of the major ENS cell types. Although the major phenotypic consequences, and the underlying transcriptional changes from Ret LoF in the developing ENS have been described, its cell type and state-specific effects are unknown. Consequently, we performed single-cell RNA sequencing (scRNA-seq) on an enriched population of ENCCs isolated from the developing GI tract of Ret null heterozygous and homozygous mouse embryos at embryonic day (E)12.5 and E14.5. We demonstrate four significant findings: (1) Ret-expressing ENCCs are a heterogeneous population composed of ENS progenitors as well as glial and neuronal committed cells; (2) neurons committed to a predominantly inhibitory motor neuron developmental trajectory are not produced under Ret LoF, leaving behind a mostly excitatory motor neuron developmental program; (3) HSCR-associated and Ret gene regulatory network genes exhibit distinct expression patterns across Ret-expressing ENCC cells with their expression impacted by Ret LoF; and (4) Ret deficiency leads to precocious differentiation and reduction in the number of proliferating ENS precursors. Our results support a model in which Ret contributes to multiple distinct cellular phenotypes and that Ret LoF contributes to GI aganglionosis in multiple independent ways.

RET enhancer haplotype-dependent remodeling of the human fetal gut development program

“…We hypothesize that quantitative decreases in RET ENCDC numbers, thereby reducing expression of ENCDC-expressed genes, while consequently leading to a relative increase in the proportion of non-ENCDC cells (increasing expression of genes in this cell population). This hypothesis is supported by our single cell expression study on purified Ret expressing ENCDC cells in the developing mouse gastrointestinal tract (Vincent, Chatterjee et al 2021). Gene set enrichment analysis of differentially expressed genes between wildtype and Ret deficient cells identified several cell cycle-associated gene sets including mitotic cell cycle processes, cell division, DNA replication, regulation of cell cycle as top scoring Gene Ontology Biological Processes.…”

“…The magnitude and diversity of genetic changes arising from RET deficiency suggests to us a parsimonious hypothesis for these observations since RET plays a direct role in the proliferation of the ENCDCs prior to enteric neuron differentiation (Vincent, Chatterjee et al 2021). We hypothesize that quantitative decreases in RET ENCDC numbers, thereby reducing expression of ENCDC-expressed genes, while consequently leading to a relative increase in the proportion of non-ENCDC cells (increasing expression of genes in this cell population).…”

“…Gene set enrichment analysis of differentially expressed genes between wildtype and Ret deficient cells identified several cell cycle-associated gene sets including mitotic cell cycle processes, cell division, DNA replication, regulation of cell cycle as top scoring Gene Ontology Biological Processes. Furthermore , immune-fluorescence assays on the mitotic marker phosphohistone H3 (pH3) demonstrated that there is overall reduction of pH3-marked Ret null cells indicating that a smaller fraction of these ENCDC cells are actively engaging the cell cycle in the absence of Ret leading to an overall reduction of these cells in the developing mouse gut (Vincent, Chatterjee et al 2021).…”

Ret deficiency decreases neural crest progenitor proliferation and restricts fate potential during enteric nervous system development

“…Raw and processed sequencing reads data have been deposited in NCBI Gene Expression Omnibus (GEO) ( GSE192676 ) ( 88 ).…”