A neural crest cell isotropic-to-nematic phase transition in the developing mammalian gut

Abstract

While the colonization of the embryonic gut by neural crest cells has been the subject of intense scrutiny over the past decades, we are only starting to grasp the morphogenetic transformations of the enteric nervous system happening in the fetal stage. Here, we show that enteric neural crest cell transit during fetal development from an isotropic cell network to a square grid comprised of circumferentially-oriented cell bodies and longitudinally-extending interganglionic fibers. We present ex-vivo dynamic time-lapse imaging of this isotropic-to-nematic phase transition and show that it occurs concomitantly with circular smooth muscle differentiation in all regions of the gastrointestinal tract. Using conditional mutant embryos with enteric neural crest cells depleted of β1-integrins, we show that cell-extracellular matrix anchorage is necessary for ganglia to properly reorient. We demonstrate by whole mount second harmonic generation imaging that fibrous, circularly-spun collagen I fibers are in direct contact with neural crest cells during the orientation transition, providing an ideal orientation template. We conclude that smooth-muscle associated extracellular matrix drives a critical reorientation transition of the enteric nervous system in the mammalian fetus.

Regional cytoarchitecture of the adult and developing mouse enteric nervous system

“…In chick, the circular muscle differentiates just before the arrival of NCCs, 27,28 and inhibition of circular muscle contractions changes the anisotropy of the embryonic chick ENS. 29 Both mechanical forces and morphogens influence intestinal smooth muscle orientation during development, 28 and whether these factors affect neuronal stripe development and patterning has yet to be explored. Such mechanistic insights will not only improve our understanding of ENS development in vivo but will also be useful for generating intestinal organoids, examples of which have two ENS plexuses but lack patterning along the longitudinal axis.…”

Physical organogenesis of the gut

“…Organ culture allows for direct control of physical (mechanical, electrical), chemical (pH, O2, metabolism, pharmacology) and biochemical growth conditions. Two to three day cultures recapitulate key developmental events like smooth muscle differentiation (Huycke et al, 2019), ENCC migration (Young et al, 2001), enteric ganglia reorientation (Chevalier et al, 2021a), epithelial structuring (Shyer et al, 2013), elongational growth (Khalipina et al, 2019). Longer culture periods can result in straying from physiological development, and should be examined carefully.…”

“…3a, dashed lines), in either the rostro-caudal or caudo-rostral direction, with a speed in the range 20-500 µm/sec. The frequency of contractions increases with developmental time from 0.5 to 3 cycles per minute (Chevalier et al, 2017;Chevalier et al, 2019;Chevalier et al, 2021a;Roberts et al, 2010) between E5 and E15 in chickens (E13.5 and E18.5 in mice).. The only direct evidence we have of those contractions in human development come from MRI images of fixed embryos (Fig.…”

“…Circumferentially oriented α-SMA fibers characteristic of contractile smooth muscle start appearing in the chicken, mouse and human at respectively E5 (Chevalier et al, 2017), E13.5 (McHugh, 1995 and before 7 weeks (Beaulieu et al, 1993;Romanska et al, 1996). This differentiation proceeds rostro-caudally in the mouse lower digestive tract, with colonic SM differentiating at E14.5 (Chevalier et al, 2021a;Roberts et al, 2010). The two most important biochemical factors involved in SM differentiation are the Sonic hedgehog (Shh) and the BMP pathways (Huycke et al, 2019).…”

“…These material considerations may play a role in the Hirschsprung phenotype of the Holstein mouse (Soret et al, 2015), in which collagen VI is over-abundantly secreted by ENCCs. In humans and mice, differentiation of CSM occurs after ENCC migration in the midgut, and concomitantly with ENCC migration at E14.5 in the hindgut (Chevalier et al, 2021a). CSM is therefore not required for ENCC migration.…”