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 defects, 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 in model systems is often obtainable from naturally occurring systems that
display analogous divergent phenotypes. These natural systems are now feasible for genomic
and transgenic approaches and provide an opportunity for ‘evolutionary’ forward genetics.
Here I propose to leverage my lab’s extensive experience developing a new vertebrate
system from the ground up: highly divergent craniofacial morphology in Caribbean pupfishes.
Our preliminary genome-wide divergence scans and association mapping have identified both
well-known craniofacial candidate genes and ten new candidate genes associated with jaw
length variation. Divergent genomic regions are often restricted to a single gene with only one
or a few candidate variants in upstream regulatory or intronic regions. Our preliminary
quantitative trait locus mapping also indicates that some of these regions explain up to 15% of
jaw length variation in lab-reared F2 intercrosses. Thus, I hypothesize that fixed mutations in
these species control spatiotemporal expression of both known and novel craniofacial
genes underlying highly divergent craniofacial features 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 assembly, quantitative genetics,
transcriptomics, in situ hybridization, and CRISPR-Cas9 genome editing.
Our initial success in Caribbean pupfishes demonstrates the power of our approach and
potential for expansion. Pupfish exhibit novel craniofacial traits; ongoing gene flow and strong
selection provide an ideal natural ‘experiment’ for fine-mapping candidate variants associated
with these traits. We are also pioneering in situ hybridization and CRISPR-Cas9 approaches in
pupfishes. By integrating candidate gene and variant discovery in a natural system exhibiting
diverse craniofacial features with powerful functional assays, 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.
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