Muscle explants myenteric neural ganglion WT-like neural ganglia, colon function and microbiota inflammed submucosa disrupted epithelial barrier thicker muscles dysbiosis Untreated Hirschsprung mouse GDNF-treated Hirschsprung mouse Wild type mouse P20 distal colon no enteric nervous system, megacolon, premature death Mouse models of Hirschsprung disease exogenous GDNF P20/P56 enema P4 P8 P0 e n d o g e n o u s G D N F hypertrophic extrinsic nerve with Schwann cells submucosal neural ganglion People with Hirschsprung disease resected aganglionic colon enteric neuron +GDNF ctl extrinsic nerve and Schwann cells +GDNF ctl nervous system regeneration, WT-like colon function, survival

Numerous studies in chordates and arthropods currently indicate that Cdx proteins have a major ancestral role in the organization of posthead tissues. In urochordate embryos, Cdx loss-of-function has been shown to impair axial elongation, neural tube (NT) closure and pigment cell development. Intriguingly, in contrast to axial elongation and NT closure, a Cdx role in neural crest (NC)-derived melanocyte/ pigment cell development has not been reported in any other chordate species. To address this, we generated a new conditional pan-Cdx functional knockdown mouse model that circumvents Cdx functional redundancy as well as the early embryonic lethality of Cdx mutants. Through directed inhibition in the neuroectoderm, we provide in vivo evidence that murine Cdx proteins impact melanocyte and enteric nervous system development by, at least in part, directly controlling the expression of the key early regulators of NC ontogenesis Pax3, Msx1 and Foxd3. Our work thus reveals a novel role for Cdx proteins at the top of the trunk NC gene regulatory network in the mouse, which appears to have been inherited from their ancestral ortholog.

Based on the bilateral relationship between the gut microbiota and formation/function of the enteric nervous system (ENS), we sought to determine whether antibiotics-induced dysbiosis might impact the expressivity of genetically-induced ENS abnormalities. To address this, we took advantage of the TashT mouse model of Hirschsprung disease, in which colonic aganglionosis and hypoganglionosis are both much more severe in males. These defects result into two male-biased colon motility phenotypes: either megacolon that is lethal around weaning age or chronic constipation in adults, the latter being also associated with an increased proportion of nitrergic neurons in the distal ENS. Induction of dysbiosis using a cocktail of broad-spectrum antibiotics specifically impacted the colonic ENS of TashTTg/Tg mice in a stage-dependent manner. It further decreased the neuronal density at post-weaning age and differentially modulated the otherwise increased proportion of nitrergic neurons, which appeared normalized around weaning age and further increased at post-weaning age. These changes delayed the development of megacolon around weaning age but led to premature onset of severe constipation later on. Finally, local inhibition of nitric oxide signaling improved motility and prevented death by megacolon. We thus conclude that exposure to antibiotics can negatively influence the expressivity of a genetically-induced enteric neuropathy.

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