The craniofacial skeleton of vertebrates develops under the guidance of highly conserved gene regulatory networks active in cranial neural crest cells. Alterations to these networks can lead to numerous human disorders such as cleft palate, premature closing of the skull, and reduced teeth size. Laboratory studies of traditional animal models have contributed to our understanding of the functional role of the core gene networks but have largely involved significant gene modifications through induced mutations. As such, these systems may be less fruitful for discovering how craniofacial gene networks adapt to gene loss and unique morphologies because the abrogation of gene function can have a significant systemic effect. A complementary approach is the study of evolutionary mutant models with adaptations that recapitulate human diseases with similar altered morphology and/or gene changes. Variation that is detrimental in humans may be neutral or even beneficial in evolutionary mutant models, therefore allowing the study of gene regulatory networks in the context of normal organismal function. Previously, limited genetic tools necessitated examining craniofacial models in select animal models. Recent advances in sequencing (e.g. single cell) and functional (e.g. CRISPR) technologies enable fruitful studies in less traditional species. By integrating single cell analysis, whole genome comparisons, and functional assays, gene regulatory networks can be successfully compared across species. My project will evaluate craniofacial gene network conservation and malleability through cross species comparisons and studies of syngnathid fishes (pipefish, seahorses, and seadragons). These amazing fish have elongated ethmoid bones, altered hyoids, and a complete loss of teeth. In addition, we recently discovered that syngnathids have lost key craniofacial developmental genes (fgf3 and fgf4) that we hypothesize has led to rewiring of craniofacial gene regulatory networks. First, I will complete single cell sequencing to capture the RNA and chromatin accessibility of cells in zebrafish and stickleback (fish models with ‘normal’ craniofacial features), and pipefish (evolutionary mutant model). Second, I will build whole genome alignments of these fish and 13 other vertebrates to identify regulatory elements. Third, I will functionally test five identified regulatory elements using zebrafish. These three approaches will reveal how well conserved craniofacial gene expression patterns and sequences are across numerous species. In addition, genes and sequences unique to syngnathids may play a role in adaptation to gene loss and produce altered faces, and may identify novel genes and regulatory factors that can lead to human therapies.