INTRODUCTION Organization of epithelial cells into higher order structures requires a complex series of morphogenetic events (Rodriquez- Boulan and Nelson, 1989; Gumbiner, 1992). Three possible modes of morphogenetic signal transmission have been postu- lated (Grobstein, 1956; Saxén et al., 1976): cell-extracellular matrix (ECM) interactions (Ekblom, 1981; Saxén, 1987; Laurie et al., 1989; Wang et al., 1990a,b), cell-cell interactions (Takeichi, 1991; Hirai et al., 1992) and diffusion of soluble factors (Grobstein, 1956; Saxén et al., 1976). In recent years, a fibroblast-derived soluble factor, identified as hepatocyte growth factor/scatter factor (HGF/SF), has been shown to induce tubulogenesis by Madin-Darby canine kidney (MDCK) epithelial cells (Montesano et al., 1991a,b) providing important molecular data on the role of diffusable factors in epithelial morphogenesis (Birchmeier and Birchmeier, 1993). Also, additional studies indicated that the MDCK morphogenetic model is relevant to kidney development. Coculturing MDCK cells, seeded in collagen type I, with the mouse embryonic kidney induced branching morphogenesis (Santos et al., 1994). Furthermore, antibodies directed against HGF/SF markedly inhibited embryonic kidney induced morphogenesis. Growing evidence has indicated that, in addition to soluble cytokines, ECM components such as collagen and laminin mediate, at least in part, the organization of epithelial cells into three-dimensional structures (Bennett, 1980; Hall et al., 1982; Montesano et al., 1983; Montesano and Orci, 1985; Barcellos- Hoff et al., 1987). Studies with kidney epithelial cells revealed a potential role for laminin and laminin receptors in establish- ing epithelial polarity and tubulogenesis (Klein et al., 1988; Ekblom et al., 1990; Sorokin et al., 1990). Although a previous study showed that the MDCK kidney cell line exhibits cell surface collagen binding activity (Salas et al., 1987), no putative collagen receptors were identified. More recent studies of integrin expression on MDCK cells revealed three integrins, α1β1, α2β1 and α3β1, that may function as collagen or collagen/laminin receptors (Schoenen- berger et al., 1994; Ojakian and Schwimmer, 1994; Spong and Garrod, 1994). However, the molecular basis of collagen- 3531 Journal of Cell Science 108, 3531-3540 (1995) Printed in Great Britain © The Company of Biologists Limited 1995 JCS8987 Cellular interactions with collagen in a model of kidney tubulogenesis were investigated using Madin-Darby canine kidney (MDCK) cells in an in vitro morphogenetic system. MDCK cells adhered to collagen types I and IV in a Mg 2+ - dependent manner, typical of the α2β1 integrin. Collagen- Sepharose affinity chromatography and immunoblotting demonstrated the presence and collagen binding activity of the α2β1 integrin on MDCK cells. To assess the function of α2β1 integrin, MDCK cells were transfected with a plasmid pRSVα2′ which allowed the expression of α2-integrin subunit antisense RNA. Three G418-resistant clones showing reduced adhesion to collagen, stable genomic inte- gration of the antisense construct, decreased α2-integrin subunit mRNA and decreased α2-integrin subunit protein expression were selected for analysis in morphogenetic experiments. MDCK cells and plasmid-only control trans- fectants, cultured in three-dimensional collagen type I gels, showed normal cyst formation, whereas the antisense RNA transfectants showed increased apoptosis and formed small rudimentary cysts. Stimulation with hepatocyte growth factor/scatter factor-containing 3T3 fibroblast-conditioned medium or recombinant hepatocyte growth factor/scatter factor resulted in extensive branching of the preformed control cysts whereas the surviving small cysts formed by antisense expressing cells increased in size but failed to elongate and branch upon stimulation. We conclude that α2β1 integrin collagen interactions play a crucial role in the hepatocyte growth factor/scatter factor-induced tubu- logenesis and branching morphogenesis of MDCK cells in collagen gels as well as an important role in cell survival. Key words: morphogenesis, integrin, antisense RNA, hepatocyte growth factor/scatter factor, apoptosis SUMMARY Loss of MDCK cell α2β1 integrin expression results in reduced cyst formation, failure of hepatocyte growth factor/scatter factor-induced branching morphogenesis, and increased apoptosis Edwin U. M. Saelman, Patricia J. Keely and Samuel A. Santoro* Department of Pathology, Box 8118, Washington University School of Medicine, 660 South Euclid Avenue, St Louis, MO 63110- 1093, USA *Author for correspondence