A Conserved Transcription Factor Regulatory Program Promotes Tendon Fate

Tendons, which transmit force from muscles to bones, are highly prone to injury. Understanding the mechanisms driving tendon fate would impact efforts to improve tendon healing, yet this knowledge is limited. To find direct regulators of tendon progenitor emergence, we performed a zebrafish high-throughput chemical screen. We established Forskolin as a tenogenic inducer across vertebrates, functioning through Creb1a, which is required and sufficient for tendon fate. Putative enhancers containing cAMP response elements (CRE) in human, mouse and fish, drove specific expression in zebrafish cranial and fin tendons. Analysis of these genomic regions identified motifs for Ebf/EBF transcription factors. Mutation of CRE or Ebf/EBF motifs significantly disrupted enhancer activity and specificity in tendons. Zebrafish ebf1a/ebf3a mutants displayed defects in tendon formation. Notably, Creb1a/CREB1 and Ebf1a/Ebf3a/EBF1 overexpression facilitated tenogenic induction in zebrafish and human pluripotent stem cells. Together, our work reveals functional conservation of two novel transcription factors in promoting tendon fate. Col2a1-egfp or Prg4-tdtomato cells were isolated from the 3xR mouse ESC line and subjected to bulk RNA sequencing