Overlapping Targets of Shh and Gata3 during Craniofacial Development

Background Congenital birth defects are among the leading causes of infant mortality. Phenotypic variation in these defects results from a combination of genetic and environmental factors. One example of this is the modulation of palate phenotypes caused by mutation of the transcription factor Gata3 via the Sonic hedgehog (Shh) pathway. We exposed zebrafish to a subteratogenic dose of the Shh antagonist cyclopamine, and another group to both cyclopamine and gata3 knockdown. We performed RNA-seq on these zebrafish to characterize the overlap of Shh and Gata3 targets. Results We examined those genes whose expression patterns mirrored the biological effect of exacerbated misregulation. These genes were not significantly misregulated by the subteratogenic dose of ethanol but more misregulated by combinatorial disruption of Shh and Gata3 relative to Gata3 alone. Using gene-disease association discovery, we were able to refine this gene list to 11 that have published links to clinical outcomes similar to the gata3 phenotype or with craniofacial malformations. We also used weighted gene co-expression network analysis to identify a module of genes that correlate strongly with co-regulation by Shh and Gata3. This module is enriched in genes related to Wnt signaling. Conclusions We found numerous differentially expressed genes in response to cyclopamine treatment, and even more from double treatment. Most notably, we found a group of genes whose expression profile mirrored the biological effect of Shh/Gata3 interaction. Pathway analysis implicated the importance of Wnt signaling in Gata3/Shh interactions in palate development.