PROJECT SUMMARY To devise novel treatments for craniofacial birth defects, disease, and injuries, more research is needed to understand developmental mechanisms that control jaw length. The jaws often display a range of size-related anomalies including mandibular hypoplasia, retrognathia, asymmetry, and clefting. Our study will provide critical data to address this unmet need by focusing on how jaw length is regulated during the deposition and resorption of bone. We employ a unique quail-duck chimeric system that we created for manipulating neural crest mesenchyme (NCM), which are the precursor cells that form the jaw skeleton. Quail have much shorter jaws than do duck. Transplanting NCM between quail and duck embryos generates chimeras with the jaw length of the donor species, but the molecular and cellular mechanisms through which NCM accomplishes this complex task remain largely unknown. In our published and preliminary studies, we uncover a direct correlation between jaw length and the NCM-mediated expression of ligands, receptors, and effectors of the Transforming Growth Factor-Beta (Tgfβ) pathway, which is known to promote bone deposition and resorption. We observe that Tgfβ receptor 1 (Tgfβr1), and targets including an osteogenic factor (Runx2) as well as a bone-resorbing collagenase (Mmp13) are highly upregulated in quail compared to duck. If we over-express Runx2 or Mmp13, we shorten the jaw whereas inhibiting Mmp13 lengthens the jaw. We also find species-specific variation in Runx2 isoforms and in the promoters of Tgfβr1 and Mmp13. Thus, we hypothesize that multiple hierarchical levels of gene regulation in the TgfFβ pathway generate species-specific differences in sensitivity to TGFβ signaling, modulate bone deposition and resorption, and enable NCM to control jaw length. To test our hypothesis, we propose three complementary Specific Aims. In Aim 1, we will determine the extent to which NCM utilizes differential expression of Tgfβr1 to control Tgfβ pathway activation, bone deposition and resorption, and jaw length. We will identify species-specific regulatory elements and assay for cell-autonomous versus context-dependent mechanisms that affect Tgfβr1 expression. In Aim 2, we will determine the extent to which NCM deploys combinatorial, spatial, temporal, and/or species-specific expression of Runx2 isoforms to regulate targets, bone deposition and resorption, and jaw length. We will identify developmental contexts in which Runx2 is alternatively spliced and use gain- and loss-of-function approaches to interrogate the ability of Runx2 isoforms to control bone growth. In Aim 3, we will determine the extent to which NCM leverages species-specific differences in Mmp13 regulation to modulate jaw length. We will manipulate upstream transcriptional inputs and endogenous promoter elements of Mmp13 and use gain- and loss-of-function strategies to test if regulatory changes to Mmp13 provide spatial and temporal control over local resorption and...