INTRODUCTION The wings of Drosophila are derived from relatively simple epithelial bilayers that have proven useful for studying many aspects of development (Blair, 1994; Couso et al., 1994; Diaz- Benjumea and Hafen, 1994; Garcia-Bellido and De Celis, 1992; Schubiger and Palka, 1987; Willliams et al., 1993). Non- neural wing tissue is organized into a patterned distribution of two cell types, intervein (90%) and vein (10% of the wing surface). Each non-neural cell forms an apical cuticular hair so hair density is an indicator of cell surface area (Dobzhansky, 1929). Intervein regions consist of large cells (low hair density) whose major function is to provide an aerodynamic surface. As the fly ecloses intervein cells die leaving a double layer of transparent, hair-studded cuticle (Johnson and Milner, 1987). A reproducible pattern of five longitudinal veins (designated L1 to L5 from anterior to posterior) and two cross veins traverses the cuticular sheet. Veins consist of narrow channels lined by small living cells that provide structural support for the wing and passage for neurons and trachea. During wing development intervein cells are responsible for connecting and holding the two surfaces of the wing together (Fristrom and Fristrom, 1993; Waddington, 1941). To that end, intervein cells differentiate a highly specialized system of cytoskeletal supports, the transalar array (Tucker et al., 1986), anchored in integrin-mediated basal adhesions (Fristrom et al., 1993). Vein cells, in contrast, are relatively unspecialized; they do not form connections with the opposite surface, do not express integrin and do not differentiate transalar arrays. We show here that vein cell differentiation involves an apical con- striction leading to the characteristic small cell size and the acquisition of a laminin-containing basal matrix. Numerous mutations that affect the pattern of wing veins have been isolated (Lindsley and Zimm, 1992). Many of these mutants have been classified according to their loss-of-function phenotypes (Diaz-Benjumea and Garcia-Bellido, 1990; 2661 Development 120, 2661-2671 (1994) Printed in Great Britain © The Company of Biologists Limited 1994 We have characterized the blistered (bs) locus phenotypi- cally, genetically and developmentally using a set of new bs alleles. Mutant defects range from wings with ectopic veins and intervein blisters to completely ballooned wings where the distinction between vein and intervein is lost. Mosaic analyses show that severe bs alleles behave largely autonomously; homozygous patches having vein-like prop- erties. Developmental analyses were undertaken using light and electron microscopy of wild-type and bs wings as well as confocal microscopy of phalloidin- and laminin-stained preparations. bs defects were first seen early in the prepupal period with the failure of apposition of dorsal and ventral wing epithelia. Correspondingly, during definitive vein/intervein differentiation in the pupal period (18-36 hours after puparium formation), the extent of dorsal/ventral reapposition is reduced in bs wings. Regions of the wing that fail to become apposed differentiate prop- erties of vein cells; i.e. become constricted apically and acquire a laminin-containing matrix basally. To further understand bs function, we examined genetic interactions between various bs alleles and mutants of two genes whose products have known functions in wing devel- opment. (i) rhomboid, a component of the EGF-R signalling pathway, is expressed in vein cells and is required for spec- ification of vein cell fate. rho ve mutations (lacking rhomboid in wings) suppress the excess vein formation and associated with bs. Conversely, rho expression in prepupal and pupal bs wings is expanded in the regions of increased vein formation. (ii) The integrin genes, inflated and myospher- oid, are expressed in intervein cells and are required for adhesion between the dorsal and ventral wing surfaces. Loss of integrin function results in intervein blisters. Integrin mutants interact with bs mutants to increase the frequency of intervein blisters but do not typically enhance vein defects. Both developmental and genetic analyses suggest that the bs product is required during metamor- phosis for the initiation of intervein development and the concomitant inhibition of vein development. Key words: laminin, rhomboid, integrin, veinlet, myospheroid, inflated, wing morphogenesis, Drosophila SUMMARY blistered: a gene required for vein/intervein formation in wings of Drosophila Dianne Fristrom 1, *, Philip Gotwals 1,† , Suzanne Eaton 2,‡ , Thomas B. Kornberg 2 , Mark Sturtevant 3 , Ethan Bier 3 and James W. Fristrom 1 1 Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA 2 Department of Biochemistry and Biophysics, University of California, San Francisco, CA 94143, USA 3 Department of Biology, University of California, La Jolla, CA 92093, USA *Author for correspondence † Present address: HHMI, MIT, Cambridge, MA 02139, USA ‡ Present address: EMBL, Meyerhoff Strasse I, 6900 Heidelberg, Germany