Le Parco, I. | bioRxiv Science

bioRxiv2026

Physiological caffeine concentrations do not affect enteric neural crest cell migration in a mouse model

The defect in enteric neural crest cell (ENCCs) migration that causes Hirschsprung disease (HD) in newborns is linked to genetic and likely also environmental factors. Among these, maternal coffee consumption has been associated with increased HD risk, yet the role of caffeine on ENCC migration has remained unexplored. Here, we show that although mouse ENCCs express caffeine-sensitive ADORA2A receptors, physiological doses of caffeine do not perturb endogenous ENCC calcium activity. We find that caffeine can modulate ENCC outgrowth in a 2D culture model but that it does not affect migration in a 3D collagen gel and in the ex-vivo embryonic gut. We conclude that physiological doses of caffeine do not hamper ENCC migration in the ex-vivo embryonic mouse intestine, thereby discarding a potential direct environmental mechanism of caffeine in HD.

developmental biology↗

bioRxiv2025

Endothelin 3 and T-type Ca2+ channels drive enteric neural crest cell calcium activity, contractility and migration

Enteric neural crest cells (ENCCs) colonize the gut during embryogenesis and migration defects give rise to Hirschsprung disease (HD). Mutations in GDNF/RET and EDN3/EDNRB are known to be causal in HD. Here, we show that migrating ENCCs in mice exhibit endogenous EDN3/EDNRB-gated calcium activity, mediated by chloride channels, T-type Ca2+ channels and inositol trisphosphate-sensitive intracellular-store release. We find that inhibiting Ca2+ activity results in ENCC migration defects, while exciting it promotes migration by increasing ENCC contractility and traction force to the extracellular matrix. Our study demonstrates that embryonic endothelin-mediated neural crest migration and adult endothelin-mediated vasoconstriction is one and the same phenomenon, taking place in different cell types. Our results suggest a functional link between rare mutations of CACNA1H (the gene encoding CaV3.2) and HD, and pave the way for understanding neurocristopathies in terms of neural crest cell bioelectric activity deficits.

developmental biology↗