Testing the Ret and Sema3d genetic interaction in mouse enteric nervous system development

For most multigenic disorders, clinical manifestation (penetrance) and presentation (expressivity) are likely to be an outcome of genetic interaction between multiple susceptibility genes. Here, using gene knockouts in mice, we evaluated genetic interaction between loss of Ret and loss of Sema3d, two Hirschsprung disease susceptibility genes. We intercrossed Ret and Sema3d double null heterozygotes to generate mice with the nine possible genotypes and assessed survival by counting various genotypes, myenteric plexus presence by acetylcholinesterase staining and embryonic day 12.5 (E12.5) intestine transcriptome by RNA-sequencing. Survival rates of Ret wild-type, null heterozygote and null homozygote mice at E12.5, birth and weaning were not influenced by the genotypes at Sema3d locus and vice versa. Loss of myenteric plexus was observed only in all Ret null homozygotes, irrespective of the genotypes at Sema3d locus, and Sema3d null heterozygote and homozygote mice had normal intestinal innervation. As compared with wild-type mice intestinal gene expression, loss of Ret in null homozygotes led to differential expression of 300 genes, whereas loss of Sema3d in null homozygotes had no major consequence and there was no evidence supporting major interaction between the two genes influencing intestine transcriptome. Overall, given the null alleles and phenotypic assays used, we did not find evidence for genetic interaction between Ret and Sema3d affecting survival, presence of myenteric plexus or intestine transcriptome.