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The Role of Pioneer Neurons in Guidance and Fasciculation in the CNS of Drosophila

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The Role of Pioneer Neurons in Guidance and Fasciculation in the CNS of Drosophila Development 124, 3253-3262 (1997) 3253 Printed in Great Britain © The Company of Biologists Limited 1997 DEV1201 Targeted neuronal ablation: the role of pioneer neurons in guidance and fasciculation in the CNS of Drosophila A. Hidalgo† and A. H. Brand* The Wellcome/CRC Institute, and Department of Genetics, Cambridge University, Tennis Court Road, Cambridge, CB2 1QR, UK *Author for correspondence †Present address: Department of Genetics, Cambridge University, Downing Street, Cambridge CB2 3EH, UK SUMMARY Although pioneer neurons are the first to delineate the axon formation, (2) the interaction between two pioneers is pathways, it is uncertain whether they have unique necessary for the establishment of each fascicle and (3) pathfinding abilities. As a first step in defining the role of pioneer neurons function synergistically to establish the pioneer neurons in the Drosophila embryonic CNS, we longitudinal axon tracts, to guide the fasciculation of describe the temporal profile and trajectory of the axons of follower neurons along specific fascicles and to prevent four pioneer neurons and show that they differ from pre- axons from crossing the midline. viously published reports. We show, by targeted ablation of one, two, three or four pioneer neurons at a time, that (1) Key words: pioneer neurons, cell ablation, CNS, Drosophila, axon no single pioneer neuron is essential for axon tract pathway, neuron INTRODUCTION P pioneer neurons, or that the timing of axon outgrowth is crucial. For example, the A and P neurons may follow cues on The first neurons to extend their axons, the ‘pioneer’ neurons glial cells that the G neuron cannot recognise, or that are not (Bate, 1976), navigate in an environment devoid of other present at the time the G axon grows out. axons. Subsequently, the axons from later differentiating It is not clear whether an individual neuron can act as a neurons, the ‘follower’ neurons, contact the axons of the pioneer neuron (Bastiani et al., 1986), or whether pathways are pioneers and fasciculate with them to form the mature axon pioneered by a group of cells acting in concert at a given time bundles. Pioneer neurons may have intrinsic pathfinding (Raper et al., 1984). In the grasshopper embryo, the A/P pathway abilities that distinguish them from follower neurons. Alterna- is pioneered by several neurons rather than by an individual tively, they may simply be the first neurons to extend their pioneer (Raper et al., 1984). Only when all of the P pioneer axons and, in their absence, follower neurons might be able to neurons are ablated does the follower G axon stall (Raper et al., establish the axonal pathways independently. A functional 1984). This indicates that there is not a functional hierarchy study of the cellular roles of pioneer neurons would aid in the among the P neurons, but that they act together as a group of further molecular analysis of axon guidance. For instance, if pioneer cells required for the formation of the A/P fascicle. pioneer neurons are unique cell types, they might express Similarly, in the grasshopper antenna and the limb bud, axon specific adhesion, signalling or receptor molecules that are not pathways are pioneered by pairs of neurons (Bate, 1976). found in the follower neurons. Experiments to ablate the pioneer neurons of the longitudi- Cell ablation has been carried out in several different nal axon pathway have been carried out both in grasshopper organisms to address the question of whether pioneer neurons (Bastiani et al., 1986) and in Drosophila (Lin et al., 1995). The behave as a unique cell type. There is evidence from vertebrates MP1/dMP2 pathway in the grasshopper embryo is the earliest and invertebrates supporting the notion that pioneer neurons are longitudinal pathway to be formed, and is pioneered by the unique cells, and hence essential for axon guidance (du Lac et descending axons of MP1 and dMP2, along which the al., 1986; Eisen et al., 1986; Ghosh et al., 1990; Gong and ascending axon of pCC extends (Bate and Grunewald, 1981; Shipley, 1995; Klose and Bentley, 1989; Raper et al., 1984). For Jacobs and Goodman, 1989). The MP1 and dMP2 neurons in example, cell ablation in the grasshopper central nervous grasshopper embryos were ablated with a laser and the effects system (CNS) has revealed a hierarchy in neuronal pathfinding on the axon of pCC were analysed. Bastiani et al. (1986) ability: follower neurons cannot navigate in the absence of showed that pCC requires the other pioneers in order to extend pioneers (Raper et al., 1984). The A/P longitudinal pathway is its axon anteriorly. In their absence, the pCC growth cone either pioneered by two A and three P neurons. When these cells are stalls or follows the axon of its sibling neuron aCC, extending ablated, the follower G neuron is unable to extend its axon in towards the periphery. This suggests that pCC behaves in this pathway (Raper et al., 1984), indicating either that the G grasshopper as a follower neuron, and that there is an absolute neuron does not possess the pathfinding abilities of the A and requirement for the MP1 and dMP2 pioneer neurons in axon 3254 A. Hidalgo and A. H. Brand guidance. The consequences of ablating MP1 and dMP2 were that we use is non-invasive, cell-specific and cell-autonomous only studied at the time of pioneer axon extension and the effect (Hidalgo et al., 1995). We have selectively killed each of the on later follower neurons was not reported. pioneer neurons of the longitudinal tracts of the ventral nerve More recently, ablation of the MP1, dMP2, vMP2 and pCC cord of the embryonic CNS. We have also ablated several neurons in Drosophila was reported and the effects on follower pioneer neurons in combination. This has allowed us to address neurons analysed. Lin et al. (1995) reported that follower the following questions. (1) Are the pioneer neurons essential neurons were able to establish the longitudinal pathways in the for the formation of the longitudinal axon pathways? (2) Are absence of the pioneers. This contrasts with the result in pioneer neurons different from each other? (3) Do pioneer grasshopper, but might be explained by the random, mosaic neurons function in isolation or as a group of cells? Interest- nature of the ablation technique used by Lin et al. (1995). For ingly, our results differ dramatically from those that have pre- example, Bastiani et al. (1986) had previously shown that the viously been published (Lin et al., 1995). loss of a pioneer neuron in one segment can be rescued by a pioneer axon extending from a neighbouring segment. MATERIALS AND METHODS Similar discrepancies between grasshopper and Drosophila are found in pioneering the intersegmental nerve (ISN). In Flies were raised on standard Drosophila media at 25°C. Embryonic grasshopper, the ISN, which extends from the CNS to the stages are as described by Campos-Ortega and Hartenstein (1985). periphery, is pioneered by the U neurons followed by aCC (du Lac et al., 1986). Ablation of the U neurons leads to stalling GAL4 expressing lines of the aCC axon (du Lac et al., 1986). In Drosophila, this nerve Lines that express GAL4 in a restricted cell- or tissue-specific pattern is pioneered by aCC and the U neurons extend along its axon were generated by enhancer detection (Brand and Perrimon, 1993). An enhancerless gene encoding the yeast transcriptional activator (Lin et al., 1995). Ablation of aCC causes some early defects GAL4 is inserted randomly into the Drosophila genome where, in pathfinding by the Us, but these are later corrected to form depending upon the site of integration, expression is directed by any a normal nerve (Lin et al., 1995). This suggests that aCC is not one of a diverse array of genomic enhancers. A second gene, con- required for the formation of the ISN in the fly embryo. taining GAL4-binding sites within its promoter, can then be intro- Experiments in different organisms have provided conflict- duced into this background where it will only be transcribed in those ing views on the roles of pioneer neurons (Chitnis and Kuwada, cells where GAL4 is expressed. Transgenic lines that drive GAL4 1991; Eisen, 1991; Ghosh et al., 1990; Gong and Shipley, 1995; expression in the pioneer neurons were identified by activation of a Keshishian and Bentley, 1983; Klose and Bentley, 1989; Pike UAS-tau-lacZ gene fusion (Hidalgo et al., 1995). In a screen of et al., 1992). For instance, there is evidence from zebrafish and approximately 1100 independent GAL4 insertion lines (E. Watkins, grasshopper showing that pioneer neurons are dispensable E. L. Dormand, A. Wade, R. Barbosa, S. Park, N. Keller, N. Sheard, because, after their elimination, follower neurons can find their N. Perrimon and A. H. B., unpublished data), we found two lines that express GAL4 primarily in the pioneer neurons (15J2, C544). The pathways normally (Chitnis and Kuwada, 1991; Eisen, 1991; third pioneer neuron line (MZ465) was a kind gift from J. Urban and Keshishian and Bentley, 1983; Pike et al., 1992). If the neurons G. Technau. The ftzNGAL4 line (Lin et al., 1995) was a kind gift from that pioneer the grasshopper limb bud nerve are ablated, a David Van Vactor. normal axon pathway is formed by follower neurons (Keshishian and Bentley, 1983). It has also been suggested that GAL4 expression in pioneer neurons a different environment (provided by the basal lamina and (1) Line 15J2 expresses GAL4 in both the vMP2 and dMP2 interneu- guidepost neurons and glia) is present at the time of pioneer rons (Fig. 1a,b,g,j). GAL4-driven expression of Tau-β-galactosidase outgrowth and that this, and not cell type, drives axon guidance.
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