Functionally specialized paralogues of Mmp11 and Timp4 interact during myotome boundary development in zebrafish
University of New Brunswick
Myotome boundary maturation occurs in zebrafish embryos between 24 and 48 hours post fertilization, and is fundamentally similar to the maturation of the myotendinous junction (MTJ) in tetrapods. During this process extracellular matrix is remodelled both mechanically and biochemically, changing shape and composition from being a rectangular fibronectin-dominated structure to a chevron-shaped laminin-dominated structure. Suggestively, both matrix metalloproteinase 11 alpha and beta (Mmp11α + β), as well as tissue inhibitor of matrix metalloproteinases 4 (Timp4) accumulate at the myotome boundary during this process. The MMPs are zinc-dependent proteinases responsible for much of the matrix remodelling that takes places during development, as well as during physiological and pathological processes like wound healing and tumour metastasis. MMP activity is regulated largely post-translationally, with TIMPs playing central roles both in the inhibition of MMP activity, and in coordinating the proteolytic activation of proMMPs. To understand better how MMP activity is regulated during maturation of MTJs, I have investigated if, when, where and how the various zebrafish paralogues of Mmp11 and Timp4 interact at the MTJ during this process. Proximity ligation assays demonstrate that Mmp11α and β interact with Timp4, and that Mmp11β interacts with fibronectin, but neither MMP appears to interact with laminin during the maturation of the myotome boundary. Depending on the domains interacting, the MMP-TIMP complex can either be inhibitory or modulatory. Using yeast two hybrid assays, I show that the interactions between Timp4β and both Mmp11 paralogues are likely of the modulatory type, but that Timp4α likely acts to inhibit Mmp11β. Taken together, I conclude that Mmp11 and Timp4 paralogues are important effectors of matrix remodelling at the MTJ, and that functional specialization of these paralogues has likely occurred.