PROJECT SUMMARY Cartilage is a specialized connective tissue with structural roles in many parts of the vertebrate body. Depending on location and developmental stages, cartilaginous tissues in the body exist in a range of phenotypic forms with different physiological properties. In mammals, this diversity is most striking in the head, where hyaline cartilages of the embryonic skull (transient) and jaw joints (permanent) coexist with histologically distinct elastic cartilage, a permanent subtype found in the external ear and larynx. Yet nearly all studies of cartilage development focus on hyaline cartilage. This has led to a poor understanding of how elastic cartilage is specified, and more generally of regulatory networks that differentiate permanent and transient cartilages. Single-cell RNA sequencing (scRNAseq) of cranial neural crest (CNCC) derivatives in zebrafish reveals unexpected diversity of cartilages in the larval and adult head. Through in situ validation, I find a cartilage subtype highly distinct from hyaline cartilage that localizes to the gills and resembles mammalian elastic cartilage. Through single-cell analysis of chromatin accessibility, we have identified putative enhancers specifically accessible in hyaline or elastic cartilage. Motif analysis of these regions reveals a strong co-enrichment of Gata and Sox9 motifs specifically along the elastic cartilage trajectory, with Gata3 in particular showing selective expression in elastic cartilage and associated mesenchyme. I propose a model in which Gata3 primes enhancers specific to gill elastic cartilage for binding and activation by Sox9, leading to divergence of elastic and hyaline subtypes during cartilage development. In my first aim, I plan to validate elastic cartilage-specific cis-regulatory networks in vivo by testing putative enhancers in zebrafish. I then test whether validated enhancers can drive specific expression in mouse elastic cartilage, further establishing homology between fish gill and mammalian elastic cartilage. Using a combination of cutting-edge genetic, transgenic, and genomics tools, I will test that Gata3 modifies Sox9 enhancer binding and activation in elastic cartilage, leading to divergence from hyaline cartilage. My research will interrogate regulatory networks underlying cartilage subtype divergence during development, with particular relevance to developing therapies for specific repair of elastic cartilage defects of the external ear (microtia/anotia) that occur in many craniofacial syndromes. More broadly, my project will pave the way for understanding the divergence between transient and permanent cartilage subtypes. I plan to carry out this zebrafish-focused project under the mentorship of Dr. Gage Crump at USC, with additional collaborators allowing me to gain complementary expertise in mouse genetics. My training plan is tailored to provide me focused training in bioinformatics and expose me to the clinical correlates of my basic research. ...