Gene expression pattern Gap junctions in Drosophila: developmental expression of the entire innexin gene family Lucy A. Stebbings a , Martin G. Todman b , Rose Phillips c , Claire E. Greer c , Jennifer Tam d , Pauline Phelan e , Kirsten Jacobs c , Jonathan P. Bacon c , Jane A. Davies c, * a Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA, UK b Laboratory of Neuroendocrinology, The Babraham Institute, Cambridge CB2 4AT, UK c Sussex Centre for Neuroscience, School of Biological Sciences, University of Sussex, Brighton BN1 9QG, UK d Division of Medical and Molecular Genetics, GKT School of Medicine, King’s College London, 8th Floor Guy’s Tower, Guy’s Hospital, London SE1 9RT, UK e Department of Biosciences, University of Kent, Canterbury CT2 7NJ, UK Received 7 January 2002; received in revised form 15 January 2002; accepted 16 January 2002 Abstract Invertebrate gap junctions are composed of proteins called innexins and eight innexin encoding loci have been identified in the now complete genome sequence of Drosophila melanogaster. The intercellular channels formed by these proteins are multimeric and previous studies have shown that, in a heterologous expression system, homo- and hetero-oligomeric channels can form, each combination possessing different gating characteristics. Here we demonstrate that the innexins exhibit complex overlapping expression patterns during oogenesis, embryogenesis, imaginal wing disc development and central nervous system development and show that only certain combinations of innexin oligomerization are possible in vivo. This work forms an essential basis for future studies of innexin interactions in Drosophila and outlines the potential extent of gap-junction involvement in development. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Cell–cell communication; Connexin; Gap junction; Innexin; In situ hybridization; Intercellular channel; zpg 1. Results and discussion Gap junctions are clusters of intercellular channels that allow the direct passage of small ions and molecules from one cell to a neighbouring cell in a regulated fashion. They are present in all multicellular organisms and are involved in a range of processes that include electrical coupling, maintenance of homeostasis and the exchange of small ions and signalling molecules between cells (reviewed in Bruzzone et al., 1996). Gap-junction channels are multi- meric and each half channel (hemi-channel) is contributed by a different cell. A hemi-channel consists of a multimeric ring of proteins (six in vertebrates), each with four trans- membrane domains and intracellular N- and C-termini. When two hemi-channels dock across the intercellular gap, a regulated pore linking the two cells’ cytoplasms is created (Unger et al., 1999). The channels are regulated by voltage and pH and can also be gated by phosphorylation (reviewed in Bruzzone et al., 1996). In vertebrates, they are composed of members of the connexin family, 15 of which have been identified so far in rodents (Manthey et al., 1999). In invertebrates, the components have been more recently identified as a family of proteins known as the innexins (Phelan et al., 1998a,b). In paired Xenopus oocytes the invertebrate innexins can, like vertebrate connexins, form either homomeric channels (entirely composed of one protein type), heteromeric chan- nels (the hemi-channel contains more than one protein) or heterotypic channels (each hemi-channel has a different composition) (Phelan et al., 1998b; Landesman et al., 1999; Stebbings et al., 2000a; Todman et al., submitted). Unlike vertebrate connexins, however, only a minority of the innexins studied so far form homomeric homotypic gap- junction channels. Most innexins, therefore, appear to parti- cipate in channels of mixed composition (reviewed in Phelan, 2000; Phelan and Starich, 2001). The completion of the Drosophila sequencing project (Adams et al., 2000) has enabled us to identify the eight innexin genes in this model organism. These genes encode at least 10 proteins, although the number may increase as alternative splice forms and their products are discovered. Here we have performed a comprehensive mRNA expres- Mechanisms of Development 113 (2002) 197–205 0925-4773/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S0925-4773(02)00025-4 www.elsevier.com/locate/modo * Corresponding author. Tel.: 1 44-1273-877436; fax: 1 44-1273- 678535. E-mail address: j.a.davies@sussex.ac.uk (J.A. Davies).