Most investigations of craniosynostosis focus on the dermatocranium, the second cranial skeleton to form during embryogenesis that comprises the dermal bones of the cranial vault and facial skeleton. A completely separate cranial skeleton, the chondrocranium, develops before the dermatocranium to support the embryonic brain and other sense organs. Historically, the chondrocranium has been studied across the vertebrates and is recognized as fundamental to craniofacial development, but it is not well known to craniofacial biologists and has never been studied in the laboratory mouse until now. The chondrocranium is formed of cartilage and though parts of it ossify endochondrally, other portions begin to degenerate by about embryonic day 15-16 in the mouse. By careful analysis of whole mount and histological specimens, we have documented the synchronized deterioration of select chondrocranial elements with the appearance and superimposition of particular dermal bones of the growing dermatocranium. These observations signal the existence of a mechanism for the coordinated, localized expansion (dermal bones) and resorption (cartilage) of two developmentally and evolutionarily separate skeletal systems. Our project, supported by strong preliminary data of the mouse chondrocranium, is designed to test a central hypothesis: that the chondrocranium serves as a structural and functional scaffold for the later development of dermatocranial elements including the formation of cranial vault sutures. Based on the common finding that boundaries between different cell populations often serve as tissue organizers, we recognize the establishment and maintenance of stable boundaries that restrict the mixing of different cell populations as critical to proper development, and propose a research design that interrogates the chondrocranial/dermatocranial boundary as significant to the coordinated development of the skull. We will interrogate cells at specific sites to determine the processes that function to maintain the boundaries. Then using the Fgfr2c+/C342Y mouse model for craniosynostosis, we will investigate relevant chondrocranial/dermatocranial boundaries operative in the development of two craniosynostosis phenotypes: premature closure of the coronal suture and abnormal growth of the midface. That the chondrocranium is composed of irregularly shaped cartilages, many of which are short-lived, requires that we conceive new tools for analysis. We will complete development of an innovative system to dissect and reconstruct the chondrocranium in silico from micro computed tomography images with tight temporal control, precisely delineate chondrocranial anatomy in 3D over embryonic time, and establish the role of the chondrocranium in development of the dermatocranium. Achieving our goals will enrich textbook knowledge of craniofacial development by defining the role of the chondrocranium in the production of dermatocranial phenotypes, provide information relative ...