Persistent larval sensory neurones are required for the normal development of the adult sensory afferent projections in Drosophila

Darren W. Williams 0 1 David Shepherd 0 0 School of Biological Sciences, University of Southampton , Southampton, SO16 7PX , UK 1 Present address: Department of Zoology, University of Washington , Seattle, WA 98195 , USA SUMMARY We have tested the hypothesis that larval neurones guide growth of adult sensory axons in Drosophila. We show that ablation of larval sensory neurones causes defects in the central projections of adult sensory neurones. Spiralling axons and ectopic projections indicate failure in axon growth guidance. We show that larval sensory neurones are required for peripheral pathfinding, entry into the CNS and growth guidance within the CNS. Ablation of subsets of neurones shows that larval sensory neurones serve During development, insect sensory neurones arise peripherally in stereotyped locations and grow axons to the CNS to synapse with their targets. Within the CNS, the sensory axons form highly organised sensory arrays, such that sensory neurones that serve different sensory modalities terminate in different layers of the CNS (Merritt and Murphey, 1992; Murphey et al., 1989a; Murphey et al., 1989b). Within these different layers, further order is apparent with many systems exhibiting topographic ordering of sensory afferents (Johnson and Murphey, 1985; Newland, 1991). This rigid anatomical organisation of sensory axons is a fundamental feature of sensory systems in many organisms, including mammals, and is a necessary prerequisite for the formation of ordered patterns of synaptic connectivity (Bacon and Murphey, 1984; Burrows and Newland, 1993; Shepherd et al., 1988). Despite the apparent wealth of knowledge concerning the organisation of insect sensory systems, little is known about the mechanisms that control the developmental assembly of these complex arrays. To address these processes, we have been studying the development of the adult sensory system of Drosophila and have identified a mechanism of axon guidance that provides a simple means for organising complex sensory arrays. As part of its life cycle, Drosophila undergoes a complete metamorphosis which sees the sensory system assembled during embryogenesis replaced by a new adult sensory system. While this transition sees a turnover of the embryonic sensory system, not all embryonic sensory neurones are eliminated during metamorphosis. A defined subset of embryonic sensory neurones persist into adult stages (Shepherd and Smith, 1996; specific guidance roles. Dorsal neurones are required for axon guidance across the midline, whereas lateral neurones are required for posterior growth. We conclude that larval sensory neurones pioneer the assembly of sensory arrays in adults. Tix et al., 1989a; Tix et al., 1989b; Williams and Shepherd, 1999) and retain their central axonal projections within the CNS (Shepherd and Smith, 1996). Furthermore the central projections of these persistent neurones prefigure the central pathways taken by the ingrowing adult sensory axons (Shepherd and Smith, 1996). On the basis of this evidence, it was hypothesised that the axon pathways defined by the persistent neurones might provide a pre-assembled axon scaffold to guide the growth of the adult sensory axons and play an essential role in the development of ordered sensory arrays in the adult CNS. The idea that persistent neurones might guide adult axon growth has been tested a number of times but with mixed results. The first studies found that persistent sensory neurones serve no role in peripheral axon growth guidance (Kunes et al., 1993; Tix et al., 1989a) while more recent work has shown that embryonic multidendritic neurones in the notum guide adult sensory axon growth towards the CNS (Usui-Ishihara et al., 2000). Importantly, however, all these studies focussed on peripheral pathfinding and did not consider whether persistent neurones are required for axon guidance within the CNS. The aim of our work was, therefore, to extend these observations and test the role of persistent neurones in guiding adult sensory axon growth within the CNS. To achieve this, we used laser ablation to kill persistent sensory neurones during larval stages and assayed the organisation of the adult sensory afferents within the CNS. The results show that after ablation of persistent sensory neurones, the central afferent projections of the adult sensory neurones on the notum and wing show defects indicative of errors in axon growth. Studies of single sensory axons show that the larval neurones pioneer the central pathways taken by adult sensory neurones and provide precise guidance cues, which allow neurones to make specific growth decisions within the CNS and ensure that axons innervate the appropriate regions of neuropil. MATERIALS AND METHODS Fly strains and crosses For all ablation experiments, GAL4-C161, an insert on the third chromosome, was used (Shepherd and Smith, 1996). In ablation experiments where morphology of the larval projections were visualised, fly stocks with two copies of UAS-mCD8GFP were established. In experiments where adult projections were visualised, a single copy of UAS-mCD8GFP and a single copy of UAS-nlsGFP were used together. This dual reporter combination provided sufficient GFP signal for identification and ablation of the soma while allowing the fine detail of the adult central afferent projections to be revealed. Laser ablations The cell bodies of individual neurones were ablated in third instar larvae. These animals were anaesthetised with diethyl ether vapour and placed in phosphate-buffered saline (PBS) under a coverslip. The larvae and neurones of interest were visualised using a Zeiss Axioskop FS fitted with Nomarski, epifluorescence and a low light CCD video camera (JVC). Ablations were observed in real time on the video monitor. Laser light from a pulsed dye laser (VSL 33: Laser Sciences, Newton, MA) was delivered through a Micropoint laser system (Photonics Instruments, Arlington Heights, IL) containing Coumarin 440 dye. The intensity and alignment of the laser was established at the beginning of each experiment. Laser energy was attenuated using neutral density filters until just sufficient to crack a coverslip with the 63 lens. Thirty pulses were delivered at a frequency of 2 Hz. Survival to eclosed adult was routinely 90%. Dissections, immunocytochemistry and microscopy Pupal ventral nerve cords were dissected out in saline (Jan and Jan, 1976) on Sylgard coated dishes under a dissecting microscope. Adult flies were collected as virgins and maintained at 25C for 24 hours. Flies were anaesthetised with carbon dioxide, the head and abdomen removed and the intact thorax fixed overnight in 4% paraformaldehyde at 4C on a rotator. Apart from a 30 minute acid permeabilisation step performed with 2N HCl/PBST, immunocytochemistry was as described previously (Smith and Shepherd, 1996). GFP was revealed with an antiGFP monoclonal antibody mix (Boehringer Roche) at 1:250. An antimouse secondary (...truncated)