Abstract
The extracellular matrix (ECM) of the myotendinous junction (MTJ) undergoes dramatic physical and biochemical remodeling during the first 48 h of development in zebrafish, transforming from a rectangular fibronectin-dominated somite boundary to a chevron-shaped laminin-dominated MTJ. Matrix metalloproteinase 11 (Mmp11, a.k.a. Stromelysin-3) is both necessary and sufficient for the removal of fibronectin at the MTJ, but whether this protease acts directly on fibronectin and how its activity is regulated remain unknown. Using immunofluorescence, we show that both paralogues of Mmp11 accumulate at the MTJ during this time period, but with Mmp11a present early and later replaced by Mmp11b. Moreover, Mmp11a also accumulates intracellularly, associated with the Z-discs of sarcomeres within skeletal muscle cells. Using the epitope-mediated MMP activation (EMMA) assay, we show that despite having a weaker paired basic amino acid motif in its propeptide than Mmp11b, Mmp11a is activated by furin, but may also be activated by other mechanisms intracellularly. One or both paralogues of tissue inhibitors of metalloproteinase-4 (Timp4) are also present at the MTJ throughout this process, and yeast two-hybrid assays reveal distinct and specific interactions between various domains of these proteins. We propose a model in which Mmp11a activity is modulated (but not inhibited) by Timp4 during early MTJ remodeling, followed by a phase in which Mmp11b activity is both inhibited and spatially constrained by Timp4 in order to maintain the structural integrity of the mature MTJ.
Highlights
The development of skeletal muscle is highly conserved in vertebrates, with the bulk of the musculature arising from the somitic mesoderm, which differentiates into segmented myotomes early in embryonic development [1]
We propose a model in which Mmp11a activity is modulated by tissue inhibitors of metalloproteinase-4 (Timp4) during early myotendinous junction (MTJ) remodeling, followed by a phase in which Mmp11b activity is both inhibited and spatially constrained by Timp4 in order to maintain the structural integrity of the mature MTJ
Timp4 is present in the MTJ throughout the developmental period examined, and we find that domains of both paralogues of Timp4 interact with domains of both Mmp11 paralogues, but with distinctly different specificities
Summary
The development of skeletal muscle is highly conserved in vertebrates, with the bulk of the musculature arising from the somitic mesoderm, which differentiates into segmented myotomes early in embryonic development [1]. Like many provisional fibronectin-rich matrices, the ECM between developing somites is replaced by a more complex matrix consisting of laminin-rich basement membranes flanking a core rich in fibrillar collagens as the somites mature into myotomes, and as the rectangular somite boundaries mature into chevron-shaped myotendinous junctions (MTJs) [1,5,6,7]. This biochemical and structural remodeling of the ECM is essential for the normal development of skeletal muscle and is an ideal context in which to study developmentally regulated matrix remodeling in vivo.
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