Smooth muscle contractility causes the gut to grow anisotropically.

The intestine is the most anisotropically shaped organ, but, when grown in culture, embryonic intestinal stem cells form star- or sphere-shaped organoids. Here, we present evidence that spontaneous tonic and phasic contractions of the circular smooth muscle of the embryonic gut cause short-timescale elongation of the organ by a purely mechanical, self-squeezing effect. We present an innovative culture set-up to achieve embryonic gut growth in culture and demonstrate by three different methods (embryological, pharmacological and microsurgical) that gut elongational growth is compromised when smooth muscle contractions are inhibited. We conclude that the cumulated short-term mechanical deformations induced by circular smooth muscle lead to long-term anisotropic growth of the gut, thus demonstrating a self-consistent way by which the function of this organ (peristalsis) directs its shape (morphogenesis). Our model correctly predicts that longitudinal smooth muscle differentiation later in embryogenesis slows down elongation, and that several mice models with defective gut smooth muscle contractility also exhibit gut growth defects. We lay out a comprehensive scheme of forces acting on the gut during embryogenesis and of their role in the morphogenesis of this organ. This knowledge will help design efficient in vitro organ growth protocols and handle gut growth pathologies such as short bowel syndrome.

Embryogenesis of the peristaltic reflex.

KEY POINTS: Neurogenic gut movements start after longitudinal smooth muscle differentiation in three species (mouse, zebrafish, chicken), and at E16 in the chicken embryo. The first activity of the chicken enteric nervous system is dominated by inhibitory neurons. The embryonic enteric nervous system electromechanically couples circular and longitudinal spontaneous myogenic contractions, thereby producing a new, rostro-caudally directed bolus transport pattern: the migrating motor complex. The response of the embryonic gut to mechanical stimulation evolves from a symmetric, myogenic response at E12, to a neurally mediated, polarized, descending inhibitory, 'law of the intestine'-like response at E16. High resolution, whole-mount 3D reconstructions are presented of the enteric nervous system of the chicken embryo at the neural-control stage E16 with the iDISCO+ tissue clarification technique. ABSTRACT: Gut motility is a complex transport phenomenon involving smooth muscle, enteric neurons, glia and interstitial cells of Cajal. Because these different cells differentiate and become active at different times during embryo development, studying the ontogenesis of motility offers a unique opportunity to 'time-reverse-engineer' the peristaltic reflex. Working on chicken embryo intestinal explants in vitro, we found by spatio-temporal mapping and signal processing of diameter and position changes that motility follows a characteristic sequence of increasing complexity: (1) myogenic circular smooth muscle contractions from E6 to E12 that propagate as waves along the intestine, (2) overlapping and independent, myogenic, low-frequency, bulk longitudinal smooth muscle contractions around E14, and (3) tetrodotoxin-sensitive coupling of longitudinal and circular contractions by the enteric nervous system as from E16. Inhibition of nitric oxide synthase neurons shows that the coupling consists in nitric oxide-mediated relaxation of circular smooth muscle when the longitudinal muscle layer is contracted. This mechanosensitive coupling gives rise to a directional, cyclical, propagating bolus transport pattern: the migrating motor complex. We further reveal a transition to a polarized, descending, inhibitory reflex response to mechanical stimulation after neuronal activity sets in at E16. This asymmetric response is the elementary mechanism responsible for peristaltic transport. We finally present unique high-resolution 3D reconstructions of the chicken enteric nervous system at the neural-control stage based on confocal imaging of iDISCO+ clarified tissues. Our study shows that the enteric nervous system gives rise to new peristaltic transport patterns during development by coupling spontaneous circular and longitudinal smooth muscle contraction waves.