Snakeskin, a membrane protein associated with smooth septate junctions, is required for intestinal barrier function in Drosophila Yuichi Yanagihashi 1,2 , Tadao Usui 3 , Yasushi Izumi 1 , Shigenobu Yonemura 4 , Motoyuki Sumida 5 , Shoichiro Tsukita 2, *, Tadashi Uemura 3 and Mikio Furuse 1,` 1 Division of Cell Biology, Department of Physiology and Cell Biology, Graduate School of Medicine, Kobe University, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan 2 Department of Cell Biology, Faculty of Medicine, Kyoto University, Yoshida Konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan 3 Graduate School of Biostudy, Kyoto University, Yoshida Konoe-cho, Sakyo-ku, Kyoto 606-8507, Japan 4 Electron Microscopy Unit, RIKEN Center for Developmental Biology, 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe 650-0047, Japan 5 Division of Insect Resources, Center for Bioscience Field Science, Kyoto Institute of Technology, Saga, Ukyo-ku, Kyoto 616-8354, Japan *Deceased ` Author for correspondence (furuse@med.kobe-u.ac.jp) Accepted 12 December 2011 Journal of Cell Science 125, 1980–1990 ß 2012. Published by The Company of Biologists Ltd doi: 10.1242/jcs.096800 Summary Septate junctions (SJs) are the membrane specializations observed between epithelial cells in invertebrates. SJs play a crucial role in epithelial barrier function by restricting the free diffusion of solutes through the intercellular space. In arthropod species, two morphologically different types of SJs have been described: pleated septate junctions (pSJs) and smooth septate junctions (sSJs), which are specific to ectodermal and endodermal epithelia, respectively. In contrast to the recent identification of pSJ-related proteins, the molecular constituents of sSJs are mostly unknown. Here, we report the discovery of a new sSJ-specific membrane protein, designated ‘Snakeskin’ (Ssk). Ssk is highly concentrated in sSJs in the Drosophila midgut and Malpighian tubules. Lack of Ssk expression is embryonically lethal in Drosophila and results in defective sSJ formation accompanied by abnormal morphology of midgut epithelial cells. We also show that the barrier function of the midgut to a fluorescent tracer is impaired in ssk-knockdown larvae. These results suggest that Ssk is required for the intestinal barrier function in Drosophila. Key words: Drosophila, Septate junctions, Cell–cell junction, Endoderm, Snakeskin, CG6981 Introduction The epithelium isolates the body from the outer environment and separates distinct fluid compartments within the body. To accomplish these functions as barriers, epithelial cells have specialized intercellular junctions, termed occluding junctions, that restrict the free diffusion of solutes across the cellular sheets through the paracellular pathway (Furuse and Tsukita, 2006). In vertebrates and tunicates, tight junctions (TJs) create a diffusion barrier within the intercellular space of epithelial and endothelial cells (Lane et al., 1994). Membrane proteins of the claudin family, which have four membrane-spanning domains, are the major players in the core structure of TJs and determine the barrier and channel property of TJs (Angelow et al., 2008; Anderson and Van Itallie, 2009; Furuse, 2010). In contrast to vertebrate species, epithelial cells in invertebrates generally lack TJs, although a few exceptions have been reported (Lane and Chandler, 1980). Instead, they possess another membrane specialization, called septate junctions (SJs), as their occluding junctions (Lane et al., 1994; Tepass et al., 2001). In ultra- thin section electron microscopy, SJs are visualized as parallel plasma membranes of adjacent cells with an obvious intercellular gap containing ladder-like septa. Morphological variations of SJs exist across invertebrate phyla. Some animals, including coelenterates, arthropods, echinoderms and hemichordates, possess multiple types of SJs depending on the ectodermal or endodermal origin of epithelial cells (Green and Bergquist, 1982; Lane et al., 1994). However, it is largely unknown whether the different SJs are evolutionary variations from a common origin because of a lack of information on their molecular composition. In light of the concept that the paracellular diffusion barrier is a fundamental cellular mechanism in metazoans, the variation in SJs is of great interest in terms of cell biology, comparative physiology and the evolution of animal species. In arthropods, SJs are divided into two types, pleated SJs (pSJs) and smooth SJs (sSJs) (Lane et al., 1994; Tepass and Hartenstein, 1994). Although both SJs have ladder-like septa, those along the plasma membranes in tracer-infiltrated specimens show zigzagging and smooth lines in pSJs and sSJs, respectively (Lane et al., 1994). In freeze-fracture replicas, rows of intramembranous particles, separated from one another, are observed in pSJs, whereas rows of particles fused into ridges are prominent in sSJs (Lane and Swales, 1982). In Drosophila, pSJs are found in ectodermal epithelia, such as the epidermis, foregut, hindgut and tracheae, and in glia, whereas sSJs are observed in endodermal epithelia, such as the midgut (Tepass and Hartenstein, 1994). The outer epithelial layer of the proventriculus and the Malpighian tubules also have sSJs, although these cells are originally derived from ectoderm during development. 1980 Research Article Journal of Cell Science