Individuals with mutations inCHD8present with gastrointestinal complaints, yet the underlying mechanisms are understudied. Here, using a stable constitutivechd8mutant zebrafish model, we found that the loss ofchd8leads to a reduced number of vagal neural crest cells (NCCs), enteric neural and glial progenitors, emigrating from the neural tube, and that their early migration capability was altered. At later stages, although the intestinal colonization by NCCs was complete, we found the decreased numbers of both serotonin-producing enterochromaffin cells and NCC-derived serotonergic neurons, suggesting an intestinal hyposerotonemia in the absence ofchd8. Furthermore, transcriptomic analyses revealed an altered expression of key receptors and enzymes in serotonin and acetylcholine signaling pathways. The tissue examination ofchd8mutants revealed a thinner intestinal epithelium accompanied by an accumulation of neutrophils and the decreased numbers of goblet cells and eosinophils. Last, single-cell sequencing of whole intestines showed a global disruption of the immune balance with a perturbed expression of inflammatory interleukins and changes in immune cell clusters. Our findings propose a causal developmental link betweenchd8, NCC development, intestinal homeostasis, and autism-associated gastrointestinal complaints.

Gastrointestinal complaints in autism are common and impact the quality of life of affected individuals, yet the underlying mechanisms are understudied. We have found that individuals with mutations in CHD8 present with gastrointestinal disturbances. We have shown that loss of chd8, the sole ortholog of CHD8 in zebrafish, leads to reduced number of enteric neurons and decreased intestinal mobility. However, it remains unclear how chd8 acts during the development of the enteric nervous system and whether CHD8-associated gastrointestinal complaints are solely due to impaired neuronal function in the intestine. Here, utilizing a stable chd8 mutant zebrafish model, we found that the loss of chd8 leads to reduced number of vagal neural crest cells (NCCs), enteric neural progenitors, emigrating from the neural tube and their early migration capability was altered. At later stages, although the intestinal colonization by the NCCs was complete, we found decreased numbers of both NCC-derived serotonergic neurons and serotonin-producing enterochromaffin cells, suggesting an intestinal hyposerotonemia in absence of chd8. Moreover, transcriptomic analyses revealed altered expression of key receptors and enzymes in serotonin and acetylcholine signaling pathways. Next, tissue examination of chd8 mutants revealed thinner intestinal epithelium accompanied by accumulation of neutrophils and decreased numbers of goblet cells and eosinophils. Last, single-cell sequencing of whole mid- and posterior intestines showed a global disruption of the immune balance with perturbed expression of inflammatory interleukins and changes in immune cell clusters. Our findings propose a causal developmental link between chd8, serotonergic pathway, intestinal homeostasis, and autism-associated gastrointestinal complaints.