Functional analysis of proteases in tracheal development of Drosophila

Formation and remodeling of extracellular matrix (ECM) structures is critical for epithelial development and organ morphogenesis. The apical ECM of epithelial tissues provides a scaffold for cell movements, contributes to intercellular signaling and acts as a protective barrier against pathogens and environmental factors in both, vertebrates and invertebrates. The type II transmembrane serine protease matriptase and the serine protease prostasin are key factors in a proteolytic cascade that regulates epithelial ECM differentiation during development in vertebrates. While apical ECM formation and remodeling was shown to be essential for Drosophila melanogaster organogenesis, mechanisms and pathways that regulate apical ECM dynamics largely remain elusive. Here, I identify the D. melanogaster proteases Notopleural (Np) and Tracheal- prostasin (Tpr) as functional homologs of vertebrate matriptase and prostasin, respectively. Np mediates morphogenesis and remodeling of apical ECM during embryonic tracheal system development and is essential for maintenance of the transepithelial barrier function. Together with the protease Lumens interrupted (Lint), Np and Tpr mediate degradation of the zona pellucida domain (ZPD) protein Dumpy, which forms elastic fibers in the tracheal apical ECM via the conserved ZPD polymerization module. In addition, Np, Lint and Tpr regulate the formation of the mature tracheal apical ECM cuticle. Furthermore, I demonstrate that Tpr zymogen activation is mediated by Np while zymogen activation of Np occurs in an autocatalytic manner, suggesting Np as an initiator of a proteolytic cascade in the tracheal system. Functional homology of Np and matriptase is based on a conserved substrate specificity, since both proteases cleave Tpr at its zymogen activation site, cleave the D. melanogaster ZPD protein Pio within its ZPD, and mediate cleavage of the human ZPD containing TGF-β receptor type III. The data therefore indicate that cleavage of ZPD proteins by the matriptase-prostasin proteolytic cascade is an evolutionary conserved mechanism