INTRODUCTION Many networks of epithelial tubes develop from isolated branching units which interconnect by fusion of tubes from different units. In the development of the vertebrate vascular system, for example, blood islands form and then coalesce to establish the network of major vessels (Risau and Flamme, 1995). Later in development, new tubules sprout from these vessels and grow out and fuse with other vessels to further interconnect the vascular network. Fusion of epithelial tubes also occurs during development of the kidney: the developing nephron fuses with the extending ureteric duct system to establish continuity between these tubes, so that urine can flow from the nephron to the ureters (Bard et al., 1994). Fusion of epithelial tubes requires that growing tubes locate a fusion partner, adhere and establish a patent connection between them. Little is known of the cellular mechanisms or molecular control of such epithelial tube fusion events. The Drosophila tracheal (respiratory) system is a branched tubular epithelial network that transports oxygen throughout the body. The twenty tracheal metameres arise independently from sacs of ~80 tracheal cells that undergo a series of sequen- tial branching events (Manning and Krasnow, 1993; Samakovlis et al., 1996). While most tracheal branches continue forming new branches throughout embryonic and larval life, five branches in each metamere cease branching during embryogenesis and grow towards and fuse to branches from the neighboring hemisegments to interconnect the tracheal network (Fig. 1A-D). Each fusion event is mediated by a single, specialized cell in each of the fusing branches, which expresses a set of fusion markers that were identified in a P[lacZ] transposon enhancer trap screen (Samakovlis et al., 1996; see below). In this paper, we describe the cellular dynamics of a tracheal fusion event and identify a gene regulatory hierarchy that controls it. Each fusion cell and its partner undergo a sophis- ticated morphogenetic program involving cytoplasmic outgrowth, cell adhesion and formation of an intracellular lumen, generating a connecting joint composed of two doughnut-shaped cells. Fusion markers are expressed in a specific sequence that anticipates the cellular events of fusion. One of the two early markers is identified as the escargot gene, which encodes a zinc finger transcription factor that has pre- viously been found to function in the development of imaginal histoblasts (Ashburner et al., 1990; Whiteley et al., 1992; Hayashi et al., 1993; Fuse et al., 1994). escargot is an activator of the fusion program, as well as a repressor of branching, that can drive ectopic tracheal fusion events and repress terminal branching when misexpressed. MATERIALS AND METHODS Drosophila strains The enhancer trap markers 1-eve-1 (Tracheal-1), Fusion-1 to 4, Fusion-7 (Branch-2), and Terminal-1 and 2 have been described 3531 Development 122, 3531-3536 (1996) Printed in Great Britain © The Company of Biologists Limited 1996 DEV7512 During development of tubular networks such as the mammalian vascular system, the kidney and the Drosophila tracheal system, epithelial tubes must fuse to each other to form a continuous network. Little is known of the cellular mechanisms or molecular control of epithelial tube fusion. We describe the cellular dynamics of a tracheal fusion event in Drosophila and identify a gene regulatory hierarchy that controls this extraordinary process. A tracheal cell located at the developing fusion point expresses a sequence of specific markers as it grows out and contacts a similar cell from another tube; the two cells adhere and form an intercellular junction, and they become doughnut-shaped cells with the lumen passing through them. The early fusion marker Fusion-1 is identi- fied as the escargot gene. It lies near the top of the regula- tory hierarchy, activating the expression of later fusion markers and repressing genes that promote branching. Ectopic expression of escargot activates the fusion process and suppresses branching throughout the tracheal system, leading to ectopic tracheal connections that resemble certain arteriovenous malformations in humans. This establishes a simple genetic system to study fusion of epithelial tubes. Key words: tube fusion, epithelial morphogenesis, branching morphogenesis, trachea, escargot, Drosophila SUMMARY Genetic control of epithelial tube fusion during Drosophila tracheal development Christos Samakovlis 1,2 , Gerard Manning 1 , Pär Steneberg 2 , Nir Hacohen 1 , Rafael Cantera 3 and Mark A. Krasnow 1, * 1 Department of Biochemistry, Beckman Center, Stanford University School of Medicine, Stanford, CA 94305, USA 2 Umeå Center for Molecular Pathogenesis, Umeå University, S-90187 Umeå, Sweden 3 Department of Zoology, University of Stockholm, S-10691 Stockholm, Sweden *Author for correspondence (e-mail: krasnow@cmgm.stanford.edu)