Divergent temporal control of deltoid tuberosity and limb tendon development by an evolutionarily conserved Scleraxis enhancer

Scleraxis (Scx) is a basic helix-loop-helix transcription factor predominantly expressed in tendons, ligaments, and their attachment sites, where it regulates maturation. Here, we report a novel Scx enhancer that drives tissue-specific expression. In transgenic reporter mice, a 5.3 kb downstream Scx enhancer (dSE) conferred robust, stable, and faithful reporter activity reproducing the endogenous Scx expression pattern. Within the dSE, we identified a 343 bp Scx enhancer (CSE), conserved from lobe-finned fishes to tetrapods, that recapitulates Scx expression in limbs. In developing limbs, CSE-deficient mice exhibited a marked reduction of endogenous Scx expression and a complete loss of the deltoid tuberosity (DT) supporting forelimb leverage, accompanied by attenuated BMP and TGF-b signaling, as in Scx-deficient mice. Precise enhancer activation within a critical developmental window was indispensable for DT formation. In contrast, tendon/ligament maturation retained prolonged plasticity, with Scx expression partially recovering later, likely through additional elements within the dSE. These findings uncover an essential role of the CSE in DT morphogenesis and reveal distinct temporal enhancer requirements for DT versus tendon/ligament formation, with additional enhancer dependence confined to tendons/ligaments.