Whereas ligaments hold skeletal elements together, tendons bridge the musculature with the skeleton. How connective tissues of the right type and function are specified in distinct regions of the developing body remains unclear. Here, we generated single-cell datasets of RNA expression and chromatin accessibility for scxa:mCherry+ connective tissues of the developing zebrafish face. We identified cell clusters corresponding to tendon, ligament, periligament, perichondrium, and other types, as well as tendon and ligament subtypes with an osteogenic signature that may explain the remodeling of ligament-bone interfaces and formation of sesamoid bones. We further identified several enhancers driving spatially-restricted transgenic activity in ligaments, periligament tissue, and other connective tissues. By utilizing a ligament-specific photoconvertible nlsEOS transgenic line, we revealed directional growth of ligaments. In addition, we found that nkx3.2 is expressed within the joint-proximal domain of the major jaw-stabilizing ligament, with this domain being lost in nkx3.2 mutants. Our study reveals distinct 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.
Roberts et al. (2026) studied this question.