INTRODUCTION Flies, like vertebrates, are bilateral symmetrical organisms, in which the two sides are separated by the midline. The fly ventral nerve cord (VNC) of the central nervous system (CNS) extends along the main body axis, like the spinal cord of vertebrates, linking the brain with the sensory and motor systems. Interneurons project axons that cross the midline once and then extend along the longitudinal pathways (Goodman and Doe, 1992). The insect ventral midline and its vertebrate equivalent, the floorplate, are sources of antagonistic repulsive (Slit) and attractive (Netrins) signals that regulate the crossing of axons (Tear, 1999; Tessier-Lavigne and Goodman, 1996; Thomas, 1998). The combination of these molecules instructs axons to cross the midline, to leave the midline once they have reached it and never to cross it again. However, despite our understanding of the control of midline crossing, not enough is known about how longitudinal axons are maintained laterally. Repulsion from the midline is a key mechanism to keep axons along longitudinal pathways (Tear, 1999; Tessier-Lavigne and Goodman, 1996; Thomas, 1998). The repulsive signal Slit (Sli) is produced by the midline glia (Kidd et al., 1999). Interneuron axons express the Sli receptor Roundabout (Robo) and thus remain parallel to the midline from a certain distance (Kidd et al., 1998a). To allow robo-expressing axons to reach the midline prior to extending along the longitudinal pathways, the midline glia also express the Commissureless protein, which is responsible for downregulating robo expression as axons approach the midline (Kidd et al., 1998b; Tear et al., 1996). At the end of axonogenesis, all longitudinal axons express robo and remain contralateral, away from the midline. The longitudinal pathways are pioneered by four neurons per hemisegment, pCC, MP1, dMP2 and vMP2, whose axons never cross the midline (Bastiani et al., 1986; Bate and Grunewald, 1981; Hidalgo and Brand, 1997; Jacobs and Goodman, 1989; Lin et al., 1994). These ipsilateral pioneer axons form a scaffold for the later selective fasciculation of follower axons. During the formation of the first longitudinal fascicle, pioneer growth cones also express the Robo receptor, which prevents them from crossing the midline (Kidd et al., 1998a). During their pathfinding, the pioneer axons interact with a class of glial cells, the interface glia (Ito et al., 1995), which at the end of embryogenesis overlie the longitudinal axons (Hidalgo and Booth, 2000). The longitudinal glia are the interface glia derived from the segmentally repeated lateral glioblasts, located at the edge of the neuroectoderm. Longitudinal glia, like the midline glia, are reminiscent of vertebrate oligodendrocytes since they originate from highly migratory and proliferative precursors and enwrap CNS axons (Halter et al., 1995; Jacobs et al., 1989; Schmidt et al., 1997). The longitudinal glioblasts divide and migrate ventrally until they contact the cell bodies of the pioneer neurons, where they halt at a certain distance from the midline. The first longitudinal fascicle is formed as the descending axons of 207 Development 128, 207-216 (2001) Printed in Great Britain © The Company of Biologists Limited 2001 DEV9748 Contrary to our knowledge of the genetic control of midline crossing, the mechanisms that generate and maintain the longitudinal axon pathways of the Drosophila CNS are largely unknown. The longitudinal pathways are formed by ipsilateral pioneer axons and the longitudinal glia. The longitudinal glia dictate these axonal trajectories and provide trophic support to later projecting follower neurons. Follower interneuron axons cross the midline once and join these pathways to form the longitudinal connectives. Once on the contralateral side, longitudinal axons are repelled from recrossing the midline by the midline repulsive signal Slit and its axonal receptor Roundabout. We show that longitudinal glia also transiently express roundabout, which halts their ventral migration short of the midline. Once in contact with axons, glia cease to express roundabout and become dependent on neurons for their survival. Trophic support and cell-cell contact restrict glial movement and axonal trajectories. The significance of this relationship is revealed when neuron-glia interactions are disrupted by neuronal ablation or mutation in the glial cells missing gene, which eliminates glia, when axons and glia cross the midline despite continued midline repellent signalling. Key words: robo, Connectives, Glia, Cell survival, Drosphila melanogaster, CNS SUMMARY Roundabout signalling, cell contact and trophic support confine longitudinal glia and axons in the Drosophila CNS Edward F. V. Kinrade 1 , Tamar Brates 1 , Guy Tear 2 and Alicia Hidalgo 1, * 1 NeuroDevelopment Group, Department of Genetics, University of Cambridge, Downing Street, Cambridge CB2 3EH, UK 2 Molecular Neurobiology Group, New Hunt’s House, Guy’s Hospital Campus, King’s College, London SE1 1UL, UK *Author for correspondence (e-mail: a.hidalgo@gen.cam.ac.uk) Accepted 3 November; published on WWW 21 December 2000