Intestinal dysmotility in a zebrafish (Danio rerio) shank3a;shank3b mutant model of autism

Autism spectrum disorder (ASD) is currently estimated to affect more than 1% of the world population. For people with ASD, gastrointestinal (GI) distress is a commonly reported but a poorly understood co-occurring symptom. Here, we investigate the physiological basis for GI distress in ASD by studying gut function in a zebrafish model of Phelan-McDermid syndrome (PMS), a condition caused by mutations in the SHANK3 gene. To generate a zebrafish model of PMS, we used CRISPR/Cas9 to introduce clinically related C-terminal frameshift mutations in shank3a and shank3b zebrafish paralogues ( shank3abΔC ). Because PMS is caused by SHANK3 haploinsufficiency, we assessed the digestive tract (DT) structure and function in zebrafish shank3abΔC +/− heterozygotes. Human SHANK3 mRNA was then used to rescue DT phenotypes in larval zebrafish. Significantly slower rates of DT peristaltic contractions ( p < 0.001) with correspondingly prolonged passage time ( p < 0.004) occurred in shank3abΔC +/− mutants. Rescue injections of mRNA encoding the longest human SHANK3 isoform into shank3abΔC +/− mutants produced larvae with intestinal bulb emptying similar to wild type (WT), but still deficits in posterior intestinal motility. Serotonin-positive enteroendocrine cells (EECs) were significantly reduced in both shank3abΔC +/− and shank3abΔC −/− mutants ( p < 0.05) while enteric neuron counts and overall structure of the DT epithelium, including goblet cell number, were unaffected in shank3abΔC +/− larvae. Our data and rescue experiments support mutations in SHANK3 as causal for GI transit and motility abnormalities. Reductions in serotonin-positive EECs and serotonin-filled ENS boutons suggest an endocrine/neural component to this dysmotility. This is the first study to date demonstrating DT dysmotility in a zebrafish single gene mutant model of ASD.