The first contractile waves in the developing embryonic gut are purely myogenic, they only involve smooth muscle. Here, we provide evidence for a transition from smooth-muscle to Interstitial Cell of Cajal (ICC) driven contractile waves in the developing chicken gut. In-situ hybridization staining for ANO1, a known ICC marker, shows that ICCs are already present throughout the gut as from embryonic day 7 (E7). We devised a protocol to reveal ICC oscillatory and propagative calcium activity in embryonic gut whole-mount and find that the first steady calcium oscillations in ICCs occur at E14. We show that the activation of ICCs leads to an increase in contractile wave frequency, regularity, directionality and velocity between E12 and E14. We finally demonstrate that application of the c-KIT antagonist imatinib mesylate in organ culture specifically depletes the ICC network and inhibits the transition to a regular, rhythmic wave pattern. We compare our findings to existing results in the mouse and predict that a similar transition should take place in the human fetus between 12 and 14 weeks of development. Together, our results point to an abrupt physiological transition from smooth-muscle mesenchyme self-initiating waves to ICC-driven motility in the fetus, and clarify the contribution of ICCs to the contractile wave pattern.

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.