Gene regulatory programs underlying diversification of facial ligaments and tendons in zebrafish
The musculoskeletal complex is held together by ligaments connecting skeletal elements and tendons bridging the musculature with the skeleton. How connective tissues of the right type and function are specified in distinct regions of the developing body remains understudied. To understand the gene regulatory programs specifying connective tissue subtypes, we generated single-cell datasets of RNA expression and chromatin accessibility for scxa:mCherry+ connective tissue cells of the developing zebrafish face. We identified cell clusters corresponding to tendon, ligament, periligament, perichondrium, and other connective tissue types, which we validated by in situ hybridization. We also uncovered tendon and ligament cells sharing an osteogenic signature, which may explain the remodeling of ligament-bone interfaces and the formation of sesamoid bone from tendons. This osteogenic potential of tendon and ligament cells is further reflected by co-enrichment of transcription factor-binding motifs for Scx and the osteogenic factor Runx2 in their enhancers. Moreover, several enhancers drive spatially-restricted transgenic activity in distinct ligaments, periligament tissue, perichondrium, and other jaw mesenchyme types, which we used to express a photoconvertible nlsEOS protein and reveal posterior-directed growth of the major jaw-stabilizing ligament. At the anterior end of this same ligament, we show that Nkx3.2 is required for its attachment to the jaw joint. Our study reveals gene regulatory programs for jaw connective tissue diversification and provides a mechanism underlying the propensity of tendons and ligaments to ossify in normal and pathological contexts. Overall design: Tg(scxa:mCherry)+ single cells or nuclei were extracted and sorted from 5 and 14dpf zebrafish embryos. ScRNA, snRNA, and snATAC libraries were prepared using the 10X Genomics platform.