Decoding regulatory nodes controlling growth and proportion of the skull

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PROJECT SUMMARY The skull is comprised of bones of different embryonic origins that articulate to encase the brain, to protect the sense organs, and to enable mastication. Well-orchestrated signaling and morphological events generate the seamless morphology of the craniofacial complex. Of central importance are the cranial neural crest cells (CNCCs) that migrate from the anterior neural folds to populate the oropharyngeal arches. CNCCs acquire axial identity from midbrain and hindbrain segmentation. Modifications to gene expression of CNCCs, their precursors, their derivatives, or even interacting tissues may underlie both normal variation and common craniofacial malformations. Although the gene regulatory networks that govern early specification of CNCCs are well known, we still lack detailed knowledge of later developmental events involving CNCC derivatives and how this relates to fundamental mechanisms of disease. The experiments outlined in this proposal will tease apart the morphological consequences of genotype. We expect that loci modifying the relative proportions of the skull and face will have commonalities amongst jawed vertebrates, as the head is an ancestral trait. Our prior phylogenomic comparisons identified fixed loci correlated with differential developmental prognathism. One of the regions identified encompasses a locus containing a large, cis-regulatory region highly conserved in all jawed vertebrates. This locus rests in an intron of agap1, and has retained directional synteny with the nearest neighbor, homeobox gene gbx2, over all of vertebrate evolution. This 343 base pair (bp) region is defined as having over 90% identity among vertebrates. A core of over 190bp is retained with 100% identity among primates, suggesting deep conservation preserved by strong selective pressure and a potential role in human development. Preliminary analyses suggest the region may participate in a broader regulatory hub that modulates expression of gbx2. As gbx2 is essential for patterning CNCCs, and is expressed in the oropharyngeal arches, my hypothesis is that the conserved non-coding region we identified acts as a specific enhancer for gbx2, mediating patterning of the forming arches and leading to proportional changes in outgrowth of the jaws. I will test this hypothesis by determining the function of components of the regulatory hub and their contributions to proper growth and form of the jaws. I will analyze necessity and function of orthologous sequences from zebrafish, chimp, and human, as well as determine the role of gbx2 in craniofacial morphology. Outcome measures include long-term assessment of CNCC migration and differentiation in vivo, and evaluating changes to spatiotemporal expression of gbx2 and related homeobox dlx genes in CNCCs and their derivatives. Findings from these experiments will lead to improved clinical strategies addressing disorders with disruptions to growth and form of the jaws as well as shed light on the contribution of coding and non-coding elements to craniofacial development and malformations.