Discovery and functional analysis of novel candidate genes and variants underlying craniofacial diversification in Cyprinodon pupfishes

Project Summary: Craniofacial abnormalities are the most common form of human birth defect, but their molecular basis remains poorly understood. Highly conserved craniofacial developmental pathways shared across diverse vertebrate species have been shaped by adaptive evolution to produce a tremendous diversity of adaptive craniofacial phenotypes. Fundamental investigation of the genetic basis of these phenotypes will lead to better diagnosis, prevention, and treatment of human birth defects. Indeed, complementary or new information on the genetic basis of many human pathologies can be obtained from naturally occurring organisms, particularly non-model vertebrates, that display analogous divergent phenotypes, known as ‘evolutionary mutant’ models. These natural systems are becoming increasingly tractable for genomic and transgenic approaches and provide an opportunity for ‘evolutionary’ forward genetics. Here I propose to build on my lab’s demonstrated success developing a new vertebrate system studying the genetic basis of highly divergent craniofacial morphology in Caribbean pupfishes. Pupfish exhibit novel craniofacial features not found in other non-model fish systems and are highly tractable for laboratory studies with life histories and eggs comparable to zebrafish. Ongoing gene flow and strong selection for divergent craniofacial features provide an ideal natural ‘experiment’ for fine-mapping candidate variants associated with these traits. Our initial success confirming three new craniofacial genes in Caribbean pupfishes and identifying 27 candidate craniofacial genes found in other vertebrates demonstrates the power and potential of our approach. I hypothesize that fixed mutations between these species control spatiotemporal expression of both known and novel craniofacial genes underlying the highly divergent craniofacial features observed in pupfishes. I propose to investigate the genetic basis of novel adaptive phenotypes in this non-model system using a combination of population genomics, de novo genome assemblies, phenomics, quantitative genetics, transcriptomics, in situ hybridization, gene overexpression and chemical inhibition, and CRISPR-Cas9 genome editing. By integrating candidate gene and variant discovery with functional genetics in a natural system exhibiting diverse craniofacial features the proposed research will demonstrate the feasibility and power of new non-model systems to gain novel insights into the developmental genetics of human diseases.