Development 127, 393-402 (2000) 393 Printed in Great Britain © The Company of Biologists Limited 2000 DEV1479

Glia dictate pioneer trajectories in the Drosophila embryonic CNS

Alicia Hidalgo* and Gwendolen E. Booth Neurodevelopment Group, Department of Genetics, University of Cambridge, UK *Author for correspondence (e-mail: [email protected])

Accepted 19 November; published on WWW 20 December 1999

SUMMARY

Whereas considerable progress has been made in extending growth cones is rich in neuronal cell bodies and understanding the molecular mechanisms of , and also in long processes from both these cell types. across the midline, it is still unclear how the axonal Interactions between , glia and their long processes trajectories of longitudinal pioneer neurons, which never orient extending growth cones. Secondly, glia direct the cross the midline, are established. Here we show that fasciculation and defasciculation of , which pattern longitudinal glia of the embryonic Drosophila CNS direct the pioneer pathways. Together these events are essential formation of pathways. By ablation and for the selective fasciculation of follower axons along the analysis of glial cells missing mutants, we demonstrate that longitudinal pathways. glia are required for two kinds of processes. Firstly, glia are required for guidance, although this requirement is not absolute. We show that the route of Key words: Glia, Axon guidance, Ablation, gcm, CNS, Drosophila

INTRODUCTION Over recent years, most work on guidance has focused on understanding the control of midline crossing by growth cones Axons extend to form intricate and stereotyped trajectories. (Tessier-Lavigne and Goodman, 1996; Thomas, 1998; Tear, Local and long-range cues are thought to aid pathfinding by 1999). In Drosophila, the ventral cord of the embryonic the first axons to trace a pathway (pioneer axons; Bate, 1976). CNS consists of longitudinal connectives, with two As pioneer growth cones navigate they ‘decide’ whether to commissures across the midline linking the connectives in each follow along or move away from a given direction. Such segment (Goodman and Doe, 1992). Most cross decisions are made at stereotyped choice points and may reflect the midline once and fasciculate with pioneer axons to grow a combination of local cues and signals from the target. Once along the longitudinal pathways up to the brain. Both attractive the primary axonal trajectories are established, follower and repulsive signals are secreted by midline cells to control neurons project growth cones, which fasciculate with pioneer midline crossing by axons (Dickson, 1998; Tessier-Lavigne axons. When a pathway is shared by neurons with ultimately and Goodman, 1996; Thomas, 1998; Tear, 1999). These long- different trajectories, follower axons must ‘decide’ which range signals are evolutionarily conserved, implying that route to take, consequently defasciculating from sister axons. midline cells play a fundamental role in controlling axon It is believed that each is ‘able’ to read cues with crossing. Longitudinal pioneer axons, however, are such precision as to execute multiple fasciculation and characterised by their lack of midline crossing. The role of glia defasciculation decisions in an environment heavily dense in in the establishment of these longitudinal pathways remains axons, to finally make correct contacts with its target unclear. (Goodman et al., 1984; Goodman and Shatz, 1993; Tessier- Thus far, the evidence does not favour a role for glia in the Lavigne and Goodman, 1996). guidance of longitudinal axons. CNS glia in some ways It has long been believed that glia preform the pathways that resembling , called here longitudinal glia, axons will follow (Silver et al., 1982; Singer et al., 1979) and enwrap the longitudinal axons (interface glia in Ito et al., there is evidence from vertebrates and grasshopper suggesting 1995). The longitudinal glia originate from a lateral glioblast that glia can guide growth cones and can also prompt which divides while migrating towards the midline (Jacobs et fasciculation and defasciculation of axons at choice points al., 1989; Schmidt et al., 1997). EM studies had suggested that (Auld, 1999; Pfrieger and Barres, 1995). In grasshopper, glia form a prepattern of for the growth cones ablation of the segment boundary cell prevents exit of the aCC to follow (Jacobs and Goodman, 1989a). However, the axon from the CNS (Bastiani and Goodman, 1986). However, progression of pioneer growth cone extension relative to glial in the CNS it is still uncertain whether glia aid pathfinding or migration patterns remains unknown. Mutations and ablation not (Auld, 1999; Pfrieger and Barres, 1995). of glia have been used to study the role of glia in guidance. 394 A. Hidalgo and G. E. Booth

Glia were ablated by means of the GAL4 system with glia- MATERIALS AND METHODS specific lines available at the time (Hidalgo et al., 1995). However, in the only case where glia were ablated prior to Fly stocks growth cone extension, the MP2 and SP1 neurons were also (1) Wild type: Canton-S; (2) glial cells missing mutants: ablated, obscuring any involvement of glia in guidance. In the gcm∆P1/CyOlacZ (Jones et al., 1995); (3) synthetic glial GAL4 driver: remaining cases, the glia were ablated at a time following w; s-gcmGAL4 15.1, insertion on the X (Booth et al., 2000); (4) w; growth cone extension, so the question of pioneer growth cone UAS-RicinA/CyOen11 lacZ (Hidalgo et al., 1995): CyOen11lacZ guidance could not be addressed. The effects of several drives lacZ expression in stripes in the embryo, in the expression mutations on longitudinal tract formation have been analysed pattern of the wingless gene; (5) double GAL4 line driving expression in glia and MP2: w; s-gcmGAL4 211/CyOlacZ; 15J2/15J2 (for a but, because these genes are involved in midline or neuronal description of 15J2, see Hidalgo and Brand, 1997). development, their effects are not direct (Auld, 1999; Jacobs, 1993). In the more specific repo glial mutants, however, Ablations longitudinal tracts form (Halter et al., 1995). Mutants for the Ablation of glia was carried out with the GAL4 system (Brand and gene glial cells missing (gcm) lack all glia, which are Perrimon, 1993). The line s-gcmGAL4 151 was engineered by fusing transformed into neurons (Hosoya et al., 1995; Jones et al., a synthetic enhancer with 11 repeats of the consensus binding 1995; Pfrieger and Barres, 1995; Vincent et al., 1996). sequence for Gcm upstream of GAL4 (see Booth et al., 2000). Line Embryos lacking gcm can lack all longitudinal tracts. However, s-gcm GAL4 151 drives expression in the glioblast, progenitor of the longitudinal axon tracts can also form, leading to the longitudinal glia, and its progeny, and in other glial classes in a mosaic conclusion that glia play no essential role in guidance (see fashion (Booth et al., 2000). This line also drives sporadic expression in some macrophages and in a reduced number of neurons, mainly references above). However, the transformation to neuronal from stage 16. These neurons are not the pioneer neurons. The fate is incomplete, since in the PNS chordotonal organs only combined stock s-gcmGAL4 211/CyOlacZ; 15J2/15J2 drives GAL4 15-30% of hemisegments have all glia transformed to neurons expression both in the longitudinal glia (in 1-3 hemisegments per (Hosoya et al., 1995; Jones et al., 1995). Furthermore, the embryo) and the dMP2 and vMP2 neurons (in most segments). Only transformed cells may retain some glial features, since they embryos in which ablation had taken place were analysed. Embryos migrate, divide and reach the neuropile as normal glia do in which ablation had not taken place were identified by the (Hosoya et al., 1995; Vincent et al., 1996). Remarkably, the expression of lacZ from the reporter balancer chromosomes, which number of β-gal-positive cells in gcmPlacZ mutants is the same was visualised with anti-β-gal antibodies. Hemisegments where glial as that of glia in wild type, indicating that the glioblast lineage ablation was verified by staining with anti-Repo were analysed. In has not been altered (Hosoya et al., 1995). Furthermore, ablations with sgcmGAL4 151, neighbouring or adjacent non-ablated hemisegments in the same embryos were used as controls for normal because lacZ-expressing transformed cells are found along the trajectories at the same stage. axonal pathways of the CNS (Hosoya et al., 1995; Vincent et al., 1996), it is also conceivable that they might still provide Immunocytochemistry novel cues that axons are also able to follow (Pfrieger and Antibody stainings were carried out following standard procedures, Barres, 1995). Consequently, gcm mutations do not simply using the Vectastain Elite kit from Vector Labs, and NiCl was used correspond to lack of glia but to a novel composition of the for colour intensification when necessary. Anti-Repo was used at ventral nerve cord. 1:300 (gift of Travers); anti-Heartless at 1:1000 (gift of Hosono); fasII Longitudinal pathways are pioneered by pCC, MP1, dMP2 at 1:5 (gift of Goodman); 22c10 at 1:10 (gift of Patel). For rhodamine- and vMP2, which extend in pairs in opposite directions (Bate phalloidin staining, embryos were fixed in 80% ethanol, incubated and Grunewald, 1981; Bastiani et al., 1986; Jacobs and first with 22c10 and subsequently with rhodamine-phalloidin (gift of Martin-Bermudo) for 40 minutes together with FITC anti-mouse. Goodman, 1989b; Lin et al., 1994; Hidalgo and Brand, 1997). pCC and vMP2 extend together anteriorly, whereas MP1 and dMP2 extend together posteriorly. All four contact half way to establish the first, single longitudinal fascicle. Subsequently, RESULTS pioneer axons undergo a series of defasciculation and refasciculation events to establish, by the end of Glial and neuronal cues during pathfinding embryogenesis, two primary fascicles at each side of the At the beginning of axonogenesis (stage 12.3), the growth midline: pCC fasciculates with dMP2 along the first fascicle, cones of pioneer neurons pCC and vMP2 extend together closest to the midline, vMP2 runs also along this pathway, but anteriorly, as the growth cones of MP1 and dMP2 extend in a more ventral plane defasciculated from pCC/dMP2 and posteriorly (Bastiani et al., 1986; Jacobs and Goodman, 1989b; MP1 runs along the second, central fascicle (Hidalgo and Hidalgo and Brand, 1997; Lin et al., 1994). Antibody 22c10 Brand, 1997). A third, outer fascicle is visualised by fasII, but recognises the axons of dMP2 and vMP2, whereas fasII its pioneer neurons remain undiscovered. It is not known what recognises the axon of pCC. governs the defasciculation and refasciculation events that Glia migrate ahead of extending growth cones. When the build the final trajectories of pioneer axons. progeny of the glioblast reach the cell bodies of the pioneer Here we have analysed the consequences of interfering with neurons, they stop migrating in the dorsoventral direction and glial function on axonal trajectories, using ablation and gcm cluster around the neurons. At stage 12.3, the growth cones of mutations. We have ablated the longitudinal glia at the glioblast dMP2 and vMP2 extend towards the glia (Fig. 1A). Glia do not stage, prior to the time of axon extension. We show that glia form a prepattern for the axonal trajectories, as most of the are responsible for orienting growth cones and for the segment at this stage is free of glia (Fig. 1B). Glia migrate fasciculation and defasciculation events that trace the pathways posteriorly together with pioneer growth cone extension and, of longitudinal pioneer axons. in particular, the axon of dMP2 follows the glia (Fig. 1B) Glia dictate pioneer axon trajectories 395 sending projections towards them (Fig. 1D). The growth cone used drives expression in the longitudinal glioblast in a mosaic of vMP2/pCC appears to extend further than the most anterior fashion, so that in most embryos only one or two glioblasts are glia, as visualised with the nuclear marker Repo (Figs 1B, 2A; killed (Booth et al., 2000). This line is also expressed in other for Repo see Campbell et al., 1994; Halter et al., 1995; Xiong glial types; however, expression is also mosaic in these glia so et al., 1994). However Heartless-positive glial cytoplasm that secondary damage to the ventral nerve cord is minimised. (Shishido et al., 1997) abuts the pCC axon (Fig. 2B) and glial Only the catalytic subunit (A) of Ricin is expressed, such that projections extend ahead of the pCC/vMP2 growth cone (Figs the toxin cannot exit the expressing cells. We have shown in 1G,H, 2C,D). dMP2/MP1 and pCC/vMP2 meet over a glial cell previous work that targeted ablation with Ricin-A is cell to form the first longitudinal pathway, which by stage 13 is autonomous, since cells adjacent to ablated cells are unaffected covered in glia (Figs 1C, 3F, 4D). (Hidalgo et al., 1995). The distance travelled by the vMP2 and dMP2 growth cones At the beginning of axonogenesis (stage 12.3), dMP2 is rich in cell membranes, revealed with rhodamin-phalloidin extends posteriorly, fasciculating with MP1 and aCC and (Fig. 1E). Glial projections contribute to this membrane mesh. surrounded by glia for a short distance (Fig. 3A). The growth Longitudinal glia coexpress the nuclear protein Repo and the cone branches at the location of a glial cell (choice point 1; membrane protein Heartless (Fig. 1F). At the time of pioneer Fig. 3B,G): one branch carries on towards the muscle (the aCC growth cone extension and prior to their fasciculation, axon), and the other branch (dMP2) heads posteriorly to meet Heartless-positive glial projections reach across adjacent the vMP2/pCC growth cone (choice point 2; Fig. 3E,F,H). segments and make glia-to-glia contact (Fig. 1F-H). When the glioblast is ablated, the dMP2 growth cone extends Neuronal filopodia also cover the distance to be travelled by more slowly, as the axon is shorter than that in a normal the pioneer growth cones. The pCC growth cone extends long filopodia that contact glia from the adjacent anterior segment prior to fasciculation with the dMP2/MP1 growth cone (Fig. 2C). While the vMP2 and dMP2 growth cones are still far apart, the pCC/vMP2 growth cone also makes contact with intermediate neurons, which can be visualised with fasII (Fig. 2D) and 22c10 (Fig. 2E). One of these intermediate neurons is SP1 (Fig. 2E; see also Lin et al., 1994). We did not detect coexpression of Repo and fasII at an intermediate cell, as implied in previous work by Lin et al. (1994). Glia migrate along the cell bodies of fasII-positive cells located ahead along their trajectory (Fig. 2A). It appears that the same intermediate neurons are contacted by both growth cones and migrating glia. The fact that glia precede extending growth cones during axonogenesis and that growth cones send filopodia towards glia suggests that glia attract pioneer growth cones. However, along the trajectory of migrating glia and extending growth cones, there are intermediate neurons that are contacted by both cell types. This suggests Fig. 1. Glia migrate and send projections ahead of pioneer growth cones. Longitudinal glia migration and projections relative to extension of pioneer growth cones in wild-type that bidirectional contact between neurons embryos. (A-D) Glia visualised with antibodies to the nuclear marker Repo (black) and and glia occurs during the establishment of the axons of vMP2 and dMP2 with 22c10 antibodies (brown). (A) The growth cones of the first longitudinal trajectory. vMP2 and dMP2 extend towards the glia (stage 12.3). (B) Slightly later, glia start migrating posteriorly and do not yet cover the extent of the segment. vMP2 extends Glia are required at turning choice anteriorly apparently devoid of glia; dMP2 extends following the glia (stage 12.2). points of growth cones (C) dMP2 extends long processes over a longitudinal glial cell to fasciculate with vMP2 The role of glia in guidance was studied by and form the first longitudinal fascicle (stage 12.1). (D) Filopodia on the dMP2 growth ablating longitudinal glia with Ricin cone reaching towards longitudinal glia. (E) Rhodamin-phalloidin (red) staining of cell expression driven by the GAL4 system (see membranes at the time of dMP2 and vMP2 (22c10, green) growth cone extension. Materials and Methods; Brand and Perrimon, Before dMP2 contacts vMP2 a thick mesh of cell membranes covers the distance between them. (F) Coexpression of the membrane protein Htl (blue) and nuclear Repo 1993; Hidalgo et al., 1995) and by studying (brown) in longitudinal glia. Glial projections link longitudinal glia from adjacent glial cells missing (gcm) mutations, in which segments (arrows). (G,H) Glial projections (anti-Htl, blue, arrows) from adjacent glia are transformed towards a neuronal fate segments in contact while the growth cones of vMP2 and dMP2 (arrowheads) are still far (Hosoya et al., 1995; Jones et al., 1995; apart. Image B corresponds to one segment, C to two; A,D to one hemisegment, E-H to Vincent et al., 1996). The s-gcmGAL4 line two, midline to the left. Anterior is up. 396 A. Hidalgo and G. E. Booth

Fig. 2. Contact with intermediate neurons and glia prior to longitudinal fascicle establishment in wild type. (A) Longitudinal glia (anti-Repo, black) migrate posteriorly along the cell bodies of fasII-expressing neurons (brown): pCC (black arrowhead, cell body) and other neurons (arrows). The pCC growth cone (white arrowhead) appears to extend in an area devoid of glia. (B) Glial cytoplasm (anti-Htl, blue, arrow) closely abuts the pCC axon along its entire length. (C) The pCC growth cone (white arrowheads) sends a fan-like mesh of filopodia (stained with fasII, brown, arrows) that contact the glia (anti-Htl, blue) of the adjacent anterior segment; (black arrowhead indicates pCC cell body). (D) The pCC growth cone (fasII, brown, white arrowhead) contacts fasII-positive neurons (arrow) prior to fasciculating with dMP2/MP1 (black arrowhead indicates pCC cell body). Glial cytoplasm (white arrows) covers the distance between the two adjacent segments. (E) The growth cone of vMP2 (22c10, brown) contacts a neuron (arrows) prior to reaching the growth cone of dMP2. This intermediate neuron might be SP1. Images represent two hemisegments, midline to the left, anterior is up. adjacent segment (n=5/7 ablated hemisegments; Fig. 3C) or it does not meet the pCC/vMP2 fascicle and it appears thickened and with a large growth cone (n=5/7). However, dMP2 does eventually extend and, slightly later, the growth cone may not branch out: the dMP2/MP1 axon heads towards the muscle fasciculating with aCC (n=2/7; Fig. 3D,I). At stage 13, normally the ascending growth cone of vMP2 and descending dMP2 meet and fasciculate over a glial cell (Figs 1C, 3F) to form the first longitudinal single fascicle (Fig. 4D). Ablation of longitudinal glia can lead to failure of contact and fasciculation between pCC/vMP2 and dMP2/MP1 fascicles (n=5/9) or to loss of the dMP2/MP1 pathway (n=6/18; Fig. 4E,F). In many cases, formation of the first fascicle is not affected by the absence of glia. These data suggest that glia attract the dMP2/MP1 growth cone, provoke axonal defasciculation at the branching choice point and aid fasciculation of all growth cones into a single fascicle (stage 13). Glia are dispensable for the extension of the pCC/vMP2 growth cone, since ablation of Fig. 3. Glia required at growth cone choice points. Pioneer growth cone trajectories in longitudinal glia does not affect the pCC axon normal hemisegments (A,B,E,F) and in hemisegments where glia have been ablated (C,D) between stages 12.3 and 13. Glia are visualised with anti-Repo (blue) and (n=27/27; Fig. 4B,C,E,F). No effect was observed axons with 22c10 (brown); white arrowheads indicate cell bodies, black ones axons. when either the glia from its own segment or from The midline is to the right. (A) vMP2 extends anteriorly, dMP2 and aCC extend the adjacent anterior segment were ablated. The posteriorly, fasciculating together over a short distance. (B) The growth cone reaches pCC growth cone extends and stalls at the same a choice point, as it contacts one glia (arrow) and spreads out filopodia (arrowheads) position as in normal segments. Similarly, pCC (choice point 1). (E) The growth cone branches over the glia (arrow) into the aCC axon extends normally and stalls in gcm mutants axon heading to the muscle, and the dMP2 axon heading posteriorly. (Hosoya et al., 1995; Jones et al., 1995). To test (F) Subsequently, the growth cones of dMP2 and vMP2 fasciculate into one whether pCC was attracted jointly by glia and the longitudinal fascicle at the location of two glia (choice point 2). (C) In the top dMP2 growth cone, we drove GAL4 expression hemisegment (asterisk), glia have been ablated, although three remain; the lower both in the MP2 neurons and the glioblast (see hemisegment is normal. The dMP2 growth cone has not extended in the ablated hemisegment as far as the normal one. (D) The top hemisegment is normal, in the Materials and Methods). Ablation of glia, dMP2 lower one glia have been ablated (asterisk). Whereas the axons of dMP2 and vMP2 and vMP2 causes a range of defects in 20% of have established the longitudinal fascicle in the normal hemisegment by stage 13, in cases (n=30) including dramatically enlarged the ablated they do not connect: vMP2 stalls; dMP2 does not defasciculate from aCC. growth cone (Fig. 5A), shorter or longer pCC (G-I) Schematic drawings illustrating defasciculation and fasciculation events. Images axons (Fig. 5B,C), abnormally tortuous trajectory A,B,E,F represent one hemisegment; C,D two; anterior is up. CP, choice point. Glia dictate pioneer axon trajectories 397

(Fig. 5C) and fasciculation with the RP2 axon. These data (Hidalgo and Brand, 1997). Consequently, at stage 17, the suggest that communication between the pCC/vMP2 and axons of dMP2 and pCC extend along the first fasII fascicle dMP2/MP1 growth cones does not drive pathfinding, but it aids and the MP1 axons extend along the second one. We have the encounter between them to form the first longitudinal found that during this transition period at stage 14 the axons fascicle. of MP1, dMP2 and pCC defasciculate from each other transiently to form three thin fascicles (Fig. 4O). These thin fascicles do not correspond to the final stage 17 fas II fascicles, Glia dictate patterns of pioneer axon trajectories since dMP2 will fasciculate with pCC at stage 15 and since the By stage 14, pCC/vMP2 axons defasciculate from MP1/dMP2 pioneer axons only contribute to two of the final three fasII axons to form two fascicles within each segment, the fascicles. pCC/vMP2 fascicle lying closer to the midline, which As these defasciculation events take place, glia are aligned fasciculate again for a short distance posterior to the pCC cell in two rows at either side of the dMP2/MP1 fascicle but not body (Hidalgo and Brand, 1997; Lin et al., 1994). Just before along the pCC/vMP2 pathway (Fig. 4H,N). Slightly later, glia this defasciculation event, glia cluster within the concave side detach from this pathway and appear aligned at either side of of the sinuous pathway of the single fascicle (Fig. 4D). a third thin fascicle (Fig. 4I,O). This third fascicle probably Subsequently, glia are found at choice point 3 where the two corresponds to the MP1 axons, the second fascicle to dMP2 fascicles separate, and also at choice point 4 where the two and the first to pCC (Fig. 4M-O). These fascicles appear only fascicles refasciculate again (Fig. 4G,M). transiently. To achieve the stage 17 pattern of two pioneer fascicles, When glia are ablated, whereas the pCC/vMP2 fascicle is MP1 must defasciculate from dMP2, and pCC must generally present (n=19/21), no dMP2/MP1 fascicle can be defasciculate from vMP2 and refasciculate with dMP2 seen at stage 14 (n=10/21; Fig. 4K,P). Loss of the dMP2/MP1

Fig. 4. No pioneer axon defasciculation when glia are ablated. Stage 13 to 15 transition. Axons are visualised with fasII (brown) and glia with anti-Repo (black) in two hemisegments, the midline is to the right. (A,D,G,H,I,J) Controls in which glia have not been ablated; (B,C,E,F,K,L) glia have been ablated. (A-F) White arrowhead, pCC cell body. Feathered arrowheads, aCC cell body and axon. (A) pCC axon extends anteriorly (stage 12.3; black arrowhead). (B) Ablated. The lower hemisegment lacks most glia: both pCC and aCC axons extend normally. Ricin (C) Ablated, same stage embryo as in B. Glia are visualised with anti-Heartless. Glia are present in the upper hemisegment, in the lower one they have been ablated. pCC axon extends normally in the absence of glial cytoplasm projections. (D) Control. At stage 13, the growth cones of dMP2/MP1 and pCC/vMP2 meet and fasciculate together into one single fascicle. The sinuous trajectory of the fascicle (arrowheads) is delimited by glia. (E) Ablation of glia leads to absence of the longitudinal fascicle at stage 13, although the axon of pCC extends unaffected. (F) Slightly later, when glia are ablated the axon of pCC makes contact further anteriorly with other glia and fasII-positive neurons, although the normal fascicle is not established yet (arrow). (G) Control. Progressing into stage 14, the normal longitudinal fascicle defasciculates into the two pCC/vMP2 (black arrowhead) and MP1/dMP2 (white arrowhead) pathways at the location of a glial cell (choice point 3, arrows) and refasciculates again at the position of another glia (choice point 4, arrows), near the pCC cell bodies. Feathered arrowhead, aCC axon. (H) At stage 14, glia move away from the pCC/vMP2 pathway and align along and on both sides of the MP1/dMP2 pathway (arrow). (I) Slightly later, glia (arrows) move away from the MP1/dMP2 pathway and line up along both sides of a third pathway (feathered arrowhead). dMP2 has defasciculated from MP1 and now there are three separate axons: the third pathway corresponds to MP1 (feathered arrowhead), the second to dMP2 (white arrowhead) and the first to pCC (black arrowhead). (J) Subsequently, the fascicles stretch out, overlain by glia (arrow). The three distinct pioneer axons are visible. (K) When glia are ablated, the stage 14 pCC pathway forms, but not the MP2/MP1 pathway (arrow). (L) Also at stage 14, there are only thick pCC (arrowhead) and aCC (feathered arrowhead) fascicles present. There is no MP2/MP1 fascicle (arrow), as it does not defasciculate from pCC/vMP2 in the absence of glia. (M-P) Schematic drawings showing defasciculation events. M-O are normal cases, in P glia have been ablated. CP, choice point. Anterior is up. 398 A. Hidalgo and G. E. Booth

Fig. 5. Ablation of glia, dMP2 and vMP2. Axons visualised with fasII (brown) and glia with anti-Repo (black). (A) Glia are virtually missing from the lower hemisegment, depleted in the upper one Fig. 6. The three major fascicles do not separate when glia are (asterisk), MP1 neurons are present but not MP2s. pCC extends a ablated. (A-C) are wild-type embryos; (D-F) are ablated embryos. normal axon, but has a remarkably large growth cone (arrow). Black arrowheads indicate area of ablation. (A,D) Stage 15. (B) Glia have been partially ablated from the two hemisegments on (D) When glia are ablated, there is a single fascicle, slightly the right, but are present on the left, slightly out of focus. The pCC defasciculated, in the absence of glia (white arrow), instead of a pair growth cone from the ablated side extends more slowly (black arrow) of closely but not tightly associated fascicles. (B) Stage 16, two than the one on the left (white arrow). (C) Glia have been ablated fascicles (white arrows) are clearly separated by two columns of glia. from two adjacent hemisegments. pCC extends an abnormal axon Third fascicle appears. (E) When glia are ablated (hemisegment on (arrow) in the absence of MP2 neurons and glia from its own, as well the right), the two-to-three fascicles fuse into a single thick fascicle as from the next anterior segment. (D) Ablation of both glia and MP2 (white arrow). The hemisegment on the left has a subtle fusion of neurons leads to fasciculation defects in the stage 17 fascicles, fascicles two and three, it also has fewer glia, but many remain affecting also the first fascicle (arrow). Arrowheads point to area of (white arrowheads). (C) Stage 17, with three clear fascicles (white ablation. Anterior is up. Two hemisegments are shown in A,C, two arrows) separated by two columns of glia. (F) Stage 17 ablated segments in B and three in D. embryos, where second fascicle fuses with first (feathered arrowhead) or misroutes to the nerve (white arrows). Anterior is up. fascicle is not due to neuronal death, since we did not observe loss of pioneer neuron cell bodies (see Booth et al., 2000). fused into one single fascicle along the pCC pathway (41%, Instead, the pCC fascicle and the aCC axons are thicker, Fig. 6E). Fasciculation defects may affect only the second or suggesting that the dMP2/MP1 fascicle fails to defasciculate third fascicles, fascicles can be missing, may misroute towards from the pCC/vMP2 or aCC fascicles in the absence of glia the muscle (Fig. 6F) or midline. In the most severe cases, the (Fig. 4L,P). The three thin fascicles described above are not first fascicle is also damaged (9.8%). When glia, dMP2 and present either. Hence, glia control defasciculation of MP1, vMP2 are all ablated, the first fascicle had defasciculated, dMP2 and pCC at the stage 14 to 15 transition. broken, misrouted or was missing in 42% of cases (n=13; Fig. At stage 15 the three thin fascicles stretch out under two 5D). This implies that dMP2 and vMP2 contribute to the single tightly packed columns of glia (Figs 4J, 6A), and dMP2 fused fascicle resulting from glial ablation. It also implies that fasciculates with pCC. Following this last fasciculation, elimination of dMP2 and vMP2, whose axons run along the pioneer axons run along two fasII fascicles: pCC/dMP2 along pCC pathway, further depletes axons of fasciculation cues. the first one, and MP1 along the second one. Ablation of glia These data demonstrate that glia lead the defasciculation and leads to a single fascicle (Fig. 6D). refasciculation events that drive the establishment of pioneer From stage 16 to 17, glia are located in two discrete rows in axon trajectories. between the three forming fascicles, as visualised with fasII (Fig. 6B,C). When glia are ablated, the separation of the three Severe fasciculation problems in glial cells missing longitudinal fascicles is damaged in 90% of examined (gcm) mutants segments (n=83). The three longitudinal fascicles can appear glial cells missing (gcm) mutant embryos virtually lack glia Glia dictate pioneer axon trajectories 399

Fig. 8. Effect of glial ablation on follower neurons. Follower neurons are visualised at stage 16 with BP102 (brown) and glia with anti- Repo (black). (A) Wild type, arrow indicates longitudinal connectives. (B) Ablated embryo. Arrowheads indicate area of ablation. In the absence of longitudinal glia follower axons misroute to the intersegmental nerve (arrow), at the location of a glia. No longitudinal tract forms. Anterior is up.

Jones et al., 1995): longitudinal tracts are fuzzy (Fig. 7C), dramatically defasciculated (Fig. 7D), fused into a single Fig. 7. Fasciculation problems in gcm mutants. Axons are visualised fascicle (Fig. 7D), misrouted towards the muscle (Fig. 7D) or with (A) 22c10 or (B-D) fasII in brown; these embryos have also across the midline (83% hemisegments affected, n=196). At been stained with anti-Repo (black). (A) At stage 13, the axons of stages 15/17 as well as earlier on, fasII reveals multiple round dMP2 (black arrowhead) and vMP2 (white arrowhead) are delayed in cells remarkably reminiscent in their location of the their extension, although in some hemisegments they make contact longitudinal glia, distributed along the longitudinal fascicles (right). (B) At stage 14 there is only one fasII fascicle (arrowheads) (Fig. 7C). This suggests that the transformed cells are still instead of two, sometimes it is missing (white arrowhead). There are present in the places normally occupied by glia and therefore many extra cell bodies expressing fasII along the longitudinal they could provide information to extending growth cones. pathways (arrows). (C) Fasciculation defects at stage 16. Ectopic This may explain why in some cases the fascicles appear to round cell bodies expressing fasII are located along the fascicles form better in gcm mutants than when glia are ablated. (arrowheads), reminiscent of the positions of longitudinal glia. (D) Stage 17 example: only one, first, fascicle may be present Nevertheless, the fasciculation defects also recapitulate earlier (arrowheads). Axons from fascicles two and three may defasciculate fasciculation patterns and reveal missed defasciculation and (arrow) or misroute towards the muscle (white arrowhead). Anterior refasciculation of axons. is up. Ablation of glia affects follower neuron trajectories What are the consequences of the absence of glia and abnormal fasciculation of pioneer axons to the pathfinding by the and sometimes lack, but can also form, longitudinal tracts majority of CNS neurons? (Hosoya et al., 1995; Jones et al., 1995; Vincent et al., 1996). Follower axons were monitored using BP102 antibodies, Whereas the pCC growth cone extends and then stalls which label many CNS axons running along the longitudinal normally in gcm mutants, the axon of dMP2 extends but it may pathways from stage 14 but do not label the pioneer axons. In or may not contact vMP2 (Fig. 7A; see also Hosoya et al., 56% of the cases in which glia had been ablated, the 1995; Jones et al., 1995). Eventually, the first fascicle does longitudinal axon tracts were missing, thin or broken, or had form at stage 13. Although this may suggest that glia play no misrouted towards the muscle (Fig. 8B) or towards the midline role in guidance, in gcm mutants, there are many extra fasII (n=85). At stages 16-17, axonal damage was seen in 53% of neurons along the route of the longitudinal axons (Fig. 7B). the hemisegments examined, versus 62% at earlier stages 14- Fas II is thought to play a role in fasciculation of pioneer axons 15. The expressivity of this phenotype is higher than that of and their contact with glia (Grenningloh et al., 1991; Lin et al., gcm mutants (40% abnormal hemisegments in Hosoya et al., 1994). This means that, although the transformed cells do not 1995), suggesting that gcm mutants retain more information express glial markers, they are located along the longitudinal than ablated embryos. Whereas neuronal apoptosis induced pathways and may be instructive to extending growth cones, upon glial ablation is likely to contribute to phenotypes of by which they are contacted. By stage 14, both pCC/vMP2 and missing, thin or broken BP102 axons (see Booth et al., 2000), dMP2/MP1 fascicles may be formed normally (6.8% of diversion of BP102 axons towards the muscle along the hemisegments, n=44), but in most cases (93%) they are either intersegmental nerve or towards the midline reflect a change in missing or fused into one single thick fascicle (Fig. 7B). By axonal pathway. These data show that ablation of glia alters the stages 16/17, longitudinal fascicles can also be formed, even if pioneer axon and glia scaffold diverting the trajectories of with fasciculation problems (Fig. 7C,D; Hosoya et al., 1995; follower axons. 400 A. Hidalgo and G. E. Booth

Fig. 9. Roles of glia in patterning pioneer axon trajectories. Two segments are represented. Colour code: yellow, vMP2; green, dMP2; red, MP1; blue, pCC; purple,: aCC; brown, third fascicle of unknown origin. Glia are located at fasciculation, defasciculation and turning points of axons; at the end of embryogenesis glia separate the three longitudinal fascicles. CP, choice point, anterior is up.

DISCUSSION to follow. Given this complex cellular mesh it is not surprising that elimination of longitudinal glia either through ablation or We have shown that glia are required for axon guidance during gcm mutations does not completely prevent formation of the formation of the embryonic Drosophila CNS. Pathfinding longitudinal fascicles (see also Hidalgo et al., 1995; Hosoya et along the longitudinal pathways encounters two problems: (1) al., 1995; Jones et al., 1995; Vincent et al., 1996). Similarly, ascending and descending growth cones have to navigate and we had previously shown that ablation of individual pioneer finally meet to form the first longitudinal fascicle, and (2) a neurons did not prevent their formation either (Hidalgo and final pattern of three longitudinal fascicles, of which two are Brand, 1997). Hence, neither glia nor individual pioneer pioneered by pCC, vMP2, dMP2 and MP1, has to emerge from neurons are absolutely required for growth cone guidance, but an initial single fascicle (Fig. 9). We have shown that four types instead their interaction is crucial to form axonal pathways. In of events require glia: (1) glia-to-glia and glia-to-neuron this context, we have found an interesting difference in the contact (through projections across the segment), (2) growth behaviour of the pCC/vMP2 versus the dMP2/MP1 growth cone attraction (e.g., of dMP2), (3) defasciculation (e.g. of cone. Whereas the latter seems more sensitive to glial dMP2 from aCC, of dMP2 from MP1, of pCC from vMP2), depletion, neither glial ablation nor gcm mutations had major and (4) fasciculation (e.g. contact between the pCC/vMP2 and consequences on the pCC axon(this work; (Hosoya et al., 1995; the dMP2/MP1 fascicles, fasciculation of dMP2 with pCC). Jones et al., 1995). Interestingly, when we ablated the dMP2 and vMP2 neurons as well as glia, the pCC growth cone was Growth cone guidance depends on neuronal and slightly affected. This suggests that the pCC/vMP2 growth glial cues cone may sense signals from the dMP2/MP1 growth cone prior It has been previously suggested that glia form a prepattern to contact. prior to growth cone extension (Jacobs and Goodman, 1989; Jacobs et al., 1989; see also Bate and Grunewald, 1989). We Glia direct defasciculation and refasciculation have observed that glia do not form a prepattern, but instead events migrate together with growth cone extension. Nevertheless, Patterns of pioneer axons change with time (Hidalgo and glia do migrate ahead of the dMP2/MP1 growth cone and Brand, 1997). We have shown that these changes in axonal growth cones appear to be attracted to glia, since they project fasciculation are controlled by glia (Fig. 9). In the wild-type filopodia towards them. When glia are ablated, the growth cone CNS, we have found glia at the choice points where of the dMP2/MP1 fascicle grows more slowly. fasciculation and defasciculation of axons take place (Fig. 9). The distance across a segment covered by the descending In the absence of glia from these positions, either through dMP2/MP1 and the ascending pCC/vMP2 growth cones is rich ablation or gcm mutations, pioneer neurons do not in both neuronal filopodia and glial projections. Both pioneer defasciculate from sister axons. This leads to axonal growth cones and migrating glia also contact intermediate phenotypes that recapitulate the fasciculation patterns typical neurons, located half way along the distance between of earlier developmental stages. For instance, at choice point 1 neuromeres (see also Lin et al., 1994). Filopodia and large glial where dMP2 normally separates from aCC to head posteriorly, projections had also been observed ahead of MP1 growth cones ablation of glia leads to the misrouting of dMP2 towards the in grasshopper (Bastiani et al., 1986; Bastiani and Goodman, muscle, as it does not defasciculate from aCC. Similarly, 1986). This implies that long before growth cones contact each whereas normally at choice point 2 the two dMP2/MP1 and other, intermediate neurons and cytoplasmic processes from vMP2/pCC growth cones meet and fasciculate together, both neurons and glia provide a cellular route for growth cones absence of glia can prevent contact between the two growth Glia dictate pioneer axon trajectories 401 cones. Hence, when glial function is compromised, more axons REFERENCES take the route of the intersegmental nerve, towards the muscle. Glia are also located at choice points 3 and 4, where Auld, V. (1999). 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