PROJECT SUMMARY Growth of the skeleton occurs at specialized sites called growth plates and sutures. Where these growth sites are located and how long they persist postnatally determine the final shape and size of the skeleton. The prevalence of skeletal dysplasias affecting bone or cartilage growth in humans (estimated to impact approximately one in 4,000-5,000 births) has motivated a vast body of research into the pathways regulating growth plate and suture development and function. However, the field still lacks a basic understanding of the mechanisms that determine where these critical sites form within the skeleton. Growth plates in particular have been typically assumed to form passively between centers of endochondral ossification. However, a scattering of cell polarity data from zebrafish, mice, and chick indicate that growth plates may be prefigured in the cartilage template well before ossification begins, suggesting an active patterning process. This application presents a novel hypothesis for growth plate origins, to be tested in zebrafish using an innovative fate-mapping strategy newly applied to the skeletal system. Growth plates are present in endochondral bones of the craniofacial skeleton as well as the long bones of the limbs. Prior to the onset of bone or cartilage differentiation in the head, skeletal progenitors for the face express distinct cohorts of genes depending on their position on the dorsal-ventral (DV) axis. These domains are not entirely mutually exclusive: a line of cells co-expressing markers characteristic of the neighboring domains can typically be detected at each boundary. Importantly, the positions of these boundaries appear to correlate with the eventual distribution of growth plates and cartilaginous joints in the adult skull. This observation prompted the hypothesis that cartilaginous growth zones in the skull are destined to form at these molecular boundaries. Testing this hypothesis requires following the fate of embryonic boundary cells to adulthood. To this end, an underutilized but powerful intersectional split-Intein-Cre lineage-tracing method will be used to specifically label cells co-expressing markers of adjacent DV domains. The pilot study outlined here will provide the first test of this original hypothesis, validate the methodology, and create reagents for future extensions of this work, all prerequisites for future R01-level support. This proposal directly aligns with the NIDCR's call to investigate mechanisms of development, maintenance, and remodeling of craniofacial tissues. Though the scope is currently limited to the skull, the principles under study may also apply to many other parts of the endochondral skeleton and possibly also to cranial sutures. Results from this work could stimulate a major advance in understanding the logic governing skeletal patterning, with clear relevance for human craniofacial malformations and other skeletal dysplasias.