PROJECT SUMMARY The craniofacial skeleton is composed of multiple tissue types that must be precisely integrated during development to ensure normal structure and function. While the mechanisms orchestrating this process are key to revealing the etiology of congenital disorders with craniofacial differences, our current understanding of the craniofacial skeleton is largely based on studies that focus on bone and cartilage. Conversely, the mechanisms that regulate differentiation and patterning of diverse soft connective tissues, inter-tissue crosstalk, and the formation of the connections between different tissue types in the head remain largely unknown. The scope of the proposed study is comprehensive and forward-thinking, as it investigates the craniofacial skeleton as a multi-tissue system, addressing critical gaps in our understanding of craniofacial connective tissue development and maintenance in four complementary research programs. These programs leverage Dr. Amy Merrill’s expertise craniofacial development to decode the molecular identities of connective tissue cells, reveal their role in joint development and maintenance, identify the signals that bridge connective tissue and bone, and establish novel associations between connective tissue development and human disease. Utilizing both disease-first and gene-first approaches from this distinct perspective, alongside an expert team of human geneticists, this research aims to identify new genes variants that cause human craniofacial disorders. Cutting-edge techniques in spatial transcriptomics and mouse genetics will be used to identify genes and pathways regulating assembly of the craniofacial skeleton, engineer new tools that target and track understudied skeletal tissue types, and establish powerful models of human congenital disorders. The increased flexibility and duration of the R35 grant will facilitate these technological advances, breaking new ground to uncover fundamental programs that regulate skeletal connectivity in craniofacial development and disease. Dr. Merrill is uniquely poised to successfully lead this study at the University of Southern California in the Center for Craniofacial Molecular Biology, a rapidly growing research center with exceptional resources directed by Dr. Yang Chai. She has an outstanding record of research productivity and impact in the field of craniofacial biology and currently holds two R01’s from NIDCR. Dr. Merrill has shown excellence in mentoring, which is reflected by the success of her trainees, recognition with the prestigious USC Mentoring Award in 2022, and role as Co-Investigator on USC’s NIDCR T90 Training Grant for Craniofacial Biology. She has also shown sustained commitment to professional service within the research community, serving in various leadership roles, including President-elect of the Society of Craniofacial Genetics and Developmental Biology. Dr. Merrill’s contributions to research, mentoring, and service position her on a...