Genetic Approaches to Study Tissue Morphogenesis in Drosophila

Abstract

Morphogenesis is defined as the change of body shape over time, the understanding of which is one of the central questions of developmental biology. To achieve proper overall organ and body shape, morphogenetic movements have to be precisely controlled during development. I have used both genetic and cell biological approaches to study control mechanisms of morphogenesis in the model organism Drosophila melanogaster. Nonmuscle myosin II (myosin hereafter) has well-established roles in generating contractile force on actin filaments during morphogenetic processes. Myosin activation is regulated by phosphorylation of the myosin regulatory light chain (MRCL, encoded by spaghetti squash or sqh in Drosophila) first on Ser-21 and subsequently on Thr-20. These phosphorylation events are positively controlled by a variety of kinases including myosin light chain kinase, Rho kinase, citron kinase, and AMP kinase and are negatively regulated by myosin phosphatase. The activation of myosin is thus highly regulated and is likely developmentally controlled. Therefore in order to monitor the activity of myosin during development, we have generated antibodies against the monophosphorylated (Sqh1P) and diphosphorylated (Sqh2P) forms of Sqh. We first show that the antibodies are highly specific for each phosphorylated form of the protein. We next used these antibodies on wild type Drosophila tissues. Interestingly, Sqh1P predominantly localizes in the adherens junction in imaginal disc cells, whereas Sqh2P locates to the apical domain. Sqh1P and Sqh2P also show distinct patterns of expression in embryos. Sqh1P is expressed nearly ubiquitously and outlines cells consistent with a junctional localization, whereas Sqh2P is strongly expressed in the ventral furrow, the invaginating fore- and hindgut, the invaginating tracheal system, head segments during head involution, and the dorsal most row of epidermal (DME) cells during dorsal closure. These represent tissues that are undergoing extensive cell shape change or cell rearrangements. Sqh2P is localized very apically in these cells, and is noticeably enriched in filopodia emanating from the DME cells during dorsal closure. These antibodies will thus be very useful in monitoring myosin activation for functional studies of morphogenesis. The tracheal system of Drosophila melanogaster has proven to be an excellent model system for studying the development of branched tubular organs. Mechanisms regulating the patterning and initial maturation of the tracheal system have been largely worked out, yet important questions remain regarding how the mature tubes inflate with air at the end of embryogenesis, and how the tracheal system grows in response to the oxygen needs of a developing larva that increases nearly 1000-fold in volume over a four day period. In chapter 2, I describe the cloning and characterization of uninflatable (uif), a gene that encodes a large transmembrane protein containing carbohydrate binding and cell signaling motifs in its extracellular domain. Uif is highly conserved in insect species, but does not appear to have a true ortholog in vertebrate species. uif uif is expressed zygotically beginning in stage 5 embryos, and Uif protein localizes to the apical plasma membrane in all ectodermally derived epithelia, most notably in the tracheal system. uif mutant animals show defects in tracheal inflation at the end of embryogenesis, and die primarily as larvae. Tracheal tubes in mutant larvae are often crushed or twisted, although tracheal patterning and maturation appear normal during embryogenesis. uif mutant larvae also show defects in tracheal growth and molting of their tracheal cuticle. RNAi-induced knockdown of uif in the leg and wing imaginal discs attenuates Notch signaling and produces Notch loss-of-function phenotypes in corresponding adult tissues, indicating that Uif might be a positive regulator of Notch signaling during leg and wing imaginal disc development.