Multipotential progenitors of the mammalian enteric nervous system capable of colonising aganglionic bowel in organ culture

Abstract

The enteric nervous system of vertebrates is derived from neural crest cells that invade the gut wall and generate a highly organised network of enteric ganglia. Among the genes that play an important role in ENS development is c-Ret, mutations of which result in failure of formation of enteric ganglia (intestinal aganglionosis). To further understand the development of the mammalian ENS in general and the mechanism of action of the RET RTK in particular, we have developed and used an organotypic culture system of mouse fetal gut. At the stage of culture initiation, the gut is partially populated by undifferentiated ENS progenitors, but culture for several days results in extensive neuronal and glial differentiation. Using this organ culture system, we have compared the development of the ENS in wild-type and RET-deficient gut and showed that the aganglionic phenotype observed in vivo is consistently reproduced under the in vitro culture conditions. Microinjection of RET+ cells isolated from E11.5 mouse bowel into wild-type or RET-deficient aganglionic gut in organ culture, results in extensive repopulation of their wall by exogenously derived neurons and glia. Finally, using a similar approach, we demonstrate that single RET+ cells introduced into the wall of wild-type gut generate both cell lineages of the ENS, i.e. neurons and glia. Our data show the NC-derived RET+ population of fetal gut in mammalian embryos consists of multipotential progenitors capable of colonising efficiently both wild-type and RET-deficient aganglionic bowel in organ culture.

Sox10 haploinsufficiency affects maintenance of progenitor cells in a mouse model of Hirschsprung disease

“…PGP9.5 and c-Ret, which we show to be preferentially expressed by Sox10-heterozygous mutant cells, are detectable at early stages of normal ENS development and are not only marking committed or even differentiated neuronal cells (33,36,37,42). Although the migratory capacity of c-Ret/ PGP9.5-positive cells has not been directly compared with that of p75/Sox10-double positive and c-Ret-negative ENS-derived neural crest cells, c-Ret-positive cells are able to colonize both wild-type and aganglionic gut (42). This might suggest that it is not a decreased migratory capacity of mutant cells per se that would result in an aganglionic terminal bowel in Hirschsprung disease.…”

“…In both cases, the hindgut becomes aganglionic because it is the last part of the gut to be colonized by neural crest-derived cells (41). PGP9.5 and c-Ret, which we show to be preferentially expressed by Sox10-heterozygous mutant cells, are detectable at early stages of normal ENS development and are not only marking committed or even differentiated neuronal cells (33,36,37,42). Although the migratory capacity of c-Ret/ PGP9.5-positive cells has not been directly compared with that of p75/Sox10-double positive and c-Ret-negative ENS-derived neural crest cells, c-Ret-positive cells are able to colonize both wild-type and aganglionic gut (42).…”

A functional network of highly pure enteric neurons in a dish

“…Third, the use of the antimitotic agent AraC, which is known to inhibit cell proliferation, contributed to the elimination of muscle and enteric glial cells. Most current culture models with enteric neurons are primary cultures of dissociated cells derived from embryonic or adult intestines (Saffrey et al, 1991;Hanani et al, 1994;Heuckeroth et al, 1998;Fex Svenningsen et al, 2003;Chevalier et al, 2008;Gomes et al, 2009;Schonkeren et al, 2022) and organotypic cultures of gut explants (Jessen et al, 1983;Natarajan et al, 1999), in which enteric neurons are in the minority among EGCs, smooth muscle cells, fibroblasts, and epithelial cells. This cellular diversity, which recapitulates, in part, the intestinal cellular microenvironment, makes it extremely difficult to study the intrinsic properties of enteric neurons and their ability to directly respond to various stimuli, including gut cell-derived factors.…”

The <i>RET</i> gene encodes RET protein, which triggers intracellular signaling pathways for enteric neurogenesis, and <i>RET</i> mutation results in Hirschsprung's disease

“…The RET protein has three different isoforms (RET51, RET43, and RET9), which differ in the C-terminal amino acids [27] . Two isoforms, RET9 and RET51, also differ in their intracellular domains [28] , [29] . RET is comprised of 1114 amino acids [30] and has three domains.…”