Sunday, May 11, 2025
5/11/2025
Modular Control of Cranial Skeletal Connectivity through Joint-Specific Enhancers - PROJECT SUMMARY/ABSTRACT Temporomandibular Joint Disorder (TMJ) and precocious ossification of cranial synchondroses are just two examples of defects caused by disruptions to cranial joints. Proper form and function of joints are required for connectivity and flexibility of the vertebrate skeleton. While the cranium has a vast number and subtype of joints, most joint biology studies have focused on the limbs, leaving a gap in our understanding of how the cranial joints, which are unique in their cranial neural crest cell contribution, develop. Our lab has recently generated single - cell transcriptome and chromatin accessibility data for cranial neural crest-derived cells across 7 timepoints (embryo to adult) in zebrafish. I have been able to extract preliminary global information about the transcription factors and cis-regulatory elements, or enhancers, which separate cranial joints from other types of skeletal tissues. While prior models have proposed joint cartilage is simply immature cartilage, my preliminary data shows several enhancers drive expression in only cranial joints. Additionally, other enhancers drive expression in only replacement cartilage. These data suggest the existence of two completely separate populations, with joints being specified distinctly from replacement cartilage. In this proposal, I use the powerful genetics of the zebrafish model to investigate what enhancers and transcription factors differentiate cranial joints from replacement cartilage. My preliminary bioinformatic analyses suggest that Ap-1 transcription factors (Jun/Fos) work with the master cartilage transcription factor Sox9 to generally specify joint cartilage. Interestingly, mutations in several transcription factors can independently cause defects to only specific cranial joints, suggesting localized transcription factors may specialize joints. The aims outlined in this proposal investigate the neural crest-derived cells in developing cranial joints (Aim 1), how they are uniquely patterned separately from replacement cartilage (Aim 2), and how region-specific transcription factors are responsible for specializing cranial joints in different parts of the head and face (Aim 3). I plan to utilize techniques such as snATAC-seq and CUT&Tag to determine if enhancers are uniquely opened or activated in joints. By combining these large datasets with transgenic assays to confirm if transcription factor motifs are necessary and sufficient for joint activity and identity, I will significantly enhance our understanding of cranial joint development. This project and activity plan for fellowship period are designed to lay the groundwork for my long-term goal of obtaining a position as a tenure-track Professor at a top-tier academic research institution. Furthermore, the data generated in this project will prepare me to generate a competitive K99 application. I will receive mentorship from Dr. Gage Crump, a leading scientist in zebrafish craniofacial development. The experiments in this proposal will take place on the Health Sciences Campus of the University of Southern California, which hosts one of the most experienced communities of craniofacial and skeletal biologists in the country.
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