Extracellular matrix deposition precedes muscle-tendon integration during murine forelimb morphogenesis

The development of a functional vertebrate musculoskeletal system requires the combination of contractile muscle and extracellular matrix (ECM)-rich tendons that transmit muscle-generated force to bone. Despite the different embryologic origins, muscle and tendon integrate at the myotendinous junction (MTJ) to seamlessly connect cells and ECM across this interface. While the cell-cell signaling factors that direct development have received considerable attention, how and when the ECM linking these tissues is deposited remains unknown. To address this gap, we analyzed the 3D distribution of different ECM and the influence of skeletal muscle in forelimbs from wildype (WT) and muscle-less Pax3Cre/Cre mice. At E11.5, prior to MTJ integration, an aligned ECM was present at the presumptive insertion of the long triceps into the WT ulna. Mechanically robust tendon-like and muscle compartmentalization structures, positive for type I collagen, type V collagen, and fibrillin-2, still formed when muscle was knocked out. However, MTJ-specific ECM was not observed when muscle was absent. Our results show that an ECM-based template forms independent of muscle, but muscle is needed for the proper assembly of ECM at the MTJ.Summary statementAn aligned ECM template connects tendon and muscle during limb development, independent of muscle progenitor migration into the limb; however, the assembly of MTJ-specific ECM requires the presence of muscle.