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.