Investigating the emergence of vertebrate cranial cartilage diversity - 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. A strong research project
combined with career development and interactions within our highly dynamic stem cell center will allow me to
achieve my goal of becoming an independent researcher.