Embryogenesis of the peristaltic reflex

Abstract

Key pointsr Neurogenic gut movements start after longitudinal smooth muscle differentiation in three species (mouse, zebrafish, chicken), and at E16 in the chicken embryo.r The first activity of the chicken enteric nervous system is dominated by inhibitory neurons. r 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.r 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. r 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 Nicolas R. Chevalier, a native of Bailly (France) and Vienna (Austria), is a researcher at Laboratoire Matière Systèmes Complexes, CNRS/Université Paris Diderot. He holds an MSc. degree in physics from Ecole Polytechnique Fédérale de Lausanne (Switzerland), and a PhD degree from Université Pierre et Marie Curie (France) in the field of crystal growth. His current investigations pertain to embryonic gut development. He is particularly interested in neural crest cell migration, enteric nervous system development and intestinal motility, and has elucidated the crucial effects of mechanical forces in driving embryonic gut elongation. N. R. Chevalier and others J Physiol 597.10 reflex response to mechanical stimulation after neuronal activity sets in at E16. This asymmetric response is the elementary mechanism responsible for ...

Development of the intrinsic innervation of the small bowel mucosa and villi

“…Previously, immunohistochemical studies have reported innervation of the mouse intestinal mucosa at E16 (10) and E18.5 (57). Recently, one study in the developing chick gut has also revealed neurites projecting to the mucosa at E16.5 (13). Our study shows that the first neurites to innervate the mucosa are present as early as E13.5, preceding the formation of the submucous plexus.…”

How Smooth Muscle Contractions Shape the Developing Enteric Nervous System

“…The decreased density is also clearly seen on whole-mount labeling of postnatal mouse ( Breau et al, 2009 ; Hatch and Mukouyama, 2015 ) and human ( Wester et al, 1999 ) myenteric plexus. As from ∼E10, thin neurites extending circumferentially from the ganglia grow to innervate the circular smooth muscle layer ( Chevalier et al, 2019 ).…”

“…Neurons are already present and differentiated throughout the gut as from E8 but staining with 4-Di-2-Asp yielded a uniform, non-specific staining of the gut tissue for guts younger than E12. This difference of staining at different stages may reflect the level of electrical and mitochondrial activity of the neurons; we have indeed previously shown that the first signs of neuromuscular activity in the embryonic chicken gut only appear at E14-E16 (Chevalier et al, 2019). Stained guts were mounted on thin glass rods inserted in the lumen and we made sure the bottom of the gut did not contact the dish (see section "Materials and Methods" and insert Figure 2A).…”

“…These contractions start as early as embryonic day 6 (E6) in the chicken embryo (Chevalier et al, 2017), and E13.5 in the mouse (Roberts et al, 2010), they take on the form of waves that propagate longitudinally along the gut at a frequency of 1-5 cycles per minute and with increasing amplitude as the intestine matures. This early muscular activity is independent of neural input (Roberts et al, 2010;Chevalier et al, 2019). The goal of this report is to investigate the effects of this smooth-muscle generated mechanical stress on the morphogenesis of the ENS in chicken and mice.…”

“…Each plexus is comprised of a mesh network of glial, neural and progenitor neural crest cells condensed in ganglia, that are interconnected by inter-ganglionic, axonal nerve fibers. The myenteric plexus is sandwiched between the longitudinal and the circular smooth layer, and projects neurites to smooth muscle and is interconnected with the submucosal plexus ( Chevalier et al, 2019 ); the submucosal plexus is located right above the mucosa and projects to the epithelium. Unlike the central nervous system which is protected by a skull or a spine, the ENS is embedded in a soft, deformable tissue and is constantly subject to mechanical stress, both during development and in homeostasis in the adult.…”