Development 126, 2475-2481 (1999) 2475 Printed in Great Britain © The Company of Biologists Limited 1999 DEV8595

Axon repulsion from the midline of the CNS requires slit function

Robin Battye, Adrienne Stevens and J. Roger Jacobs* Department of Biology, McMaster University, 1280 Main St W., Hamilton, Ontario L8S 4K1, Canada *Author for correspondence (e-mail: [email protected])

Accepted 3 March; published on WWW 4 May 1999

SUMMARY Guidance of axons towards or away from the midline of the restores axon patterning. Ectopic expression of slit inhibits during Drosophila embryogenesis formation of axon tracts at locations of high Slit production reflects a balance of attractive and repulsive cues and misdirects axon tracts towards the midline. slit originating from the midline. Here we demonstrate that interacts genetically with roundabout, which encodes a Slit, a protein secreted by the midline glial cells provides a putative receptor for growth cone repulsion. repulsive cue for the growth cones of axons and muscle cells. Embryos lacking slit function show a medial collapse of lateral axon tracts and ectopic midline crossing of Key words: slit, roundabout (robo), Midline growth cone, Glia, Axon ventral muscles. Transgene expression of slit in the midline guidance,

INTRODUCTION produce the complementary phenotype, further supporting this interpretation (Kidd et al., 1998a,b, 1999). The navigation of axonal growth cones through the developing The comm, and robo genes have been cloned and nervous system is guided by short and long range signals in the characterised. Homologues for some of these genes have been extracellular environment. These signals act as attractive or identified in other model organisms, suggesting a conserved repellent cues depending upon whether they facilitate growth mechanism regulating midline guidance in the CNS. cone extension or inhibit it (Goodman, 1996). A major feature Noteworthy are homologues to C. elegans UNC-6, vertebrate of in the developing CNS is the establishment Netrin-1 and its fly homologue, Netrin-A, expressed by the of a contralateral projection, or conversely, avoidance of the floorplate and midline glia (MG) respectively (Kennedy et al., midline of the CNS. In vertebrates, guidance of axons towards 1994; Mitchell et al., 1996; Serafini et al., 1994). are or away from the midline is communicated by attractive and secreted proteins with -like motifs that can attract repulsive signals originating from the floorplate (Bernhardt et DCC- or Frazzled-expressing growth cones (Chan et al., 1996; al., 1992; Colamarino and Tessier-Lavigne, 1995). The midline Keino-Masu et al., 1996; Kolodziej et al., 1996). Homologues glia (MG) cells of the Drosophila nervous system appear to of C. elegans sax-3, the vertebrate and Drosophila robo communicate similar information (Flanagan and van Vactor, genes, encode a transmembrane receptor with multiple 1998; Kidd et al., 1998a; Mitchell et al., 1996). immunoglobulin and fibronectin repeats, and is also expressed Molecular and genetic studies in Drosophila are revealing on growth cones (Kidd et al., 1998a; Zallen et al., 1998). Like how midline cells direct the formation of contralateral Frazzled, it appears that Robo responds to a signal produced projections. A number of mutations that disrupt midline axon by the midline, except that Robo signaling apparently guidance have been isolated in large screens for axon tract communicates a repulsive signal. Another group has recently malformations in Drosophila (Hummel et al., 1999; Seeger et identified Drosophila Slit as the repellent ligand for Robo al., 1993). The diversity of mutant phenotypes observed (Kidd et al., 1999), and in collaboration with a second group, suggest that multiple mechanisms regulate midline guidance. has shown that in Drosophila and mammals, Slit orthologs bind Among these mutants, the phenotypes of embryos mutant for Robo (Brose et al., 1999). the commissureless (comm) or netrin genes are distinctive, as A logical candidate for a repulsive signal for midline they lack most or all commissural axons (Harris et al., 1996; guidance would be a secreted molecule, produced by the MG, Mitchell et al., 1996). This mutant phenotype suggests a which, when absent, resulted in a loss of midline axon function for these genes in directing commissural axons to the guidance. These criteria are met by slit, a gene encoding a large midline. In contrast, mutation of the roundabout (robo) or glycoprotein secreted by the MG, which can be detected on the karussell genes results in an increase in commissural axon surface of the MG as well as upon the axon tracts of the CNS. number, generated by re-crossing of decussated axons The 200 kDa Slit protein is composed of leucine rich repeats, (Hummel et al., 1999; Tear et al., 1996). This suggests that EGF repeats and a domain similar to the G domain of laminin robo and karussell act to prevent axons from crossing the and agrin (Rothberg et al., 1988, 1990). A loss of slit function midline. The phenotype of over-expression of comm and robo results in medial fusion of longitudinal and commissural axon 2476 R. Battye, A. Stevens and J. R. Jacobs tracts, and errors in ipsilateral or contralateral axon projection. CAC TTG CGC 3′ and 5′ AGC ACG GCC AGC GCG GCC GCT Cells of the midline, in particular the MG, are present in slit ACT 3′) to an MluI- and SfiI-digested cDNA clone (Rothberg et al., mutant embryos, however they are ventrally displaced within 1990) which was then inserted into the KpnI and XbaI sites of pUAST the CNS (Sonnenfeld and Jacobs, 1994). Here we demonstrate (Brand and Perrimon, 1993) with 3 bp of both 3′ and 5′ slit UTR. Transformant lines were generated and mapped by standard that reductions in slit function result in ectopic projections of 2 2 axons and ventral muscles over the ventral midline. Normal procedures. P[sim-GAL4], slit /P[sim-GAL4], slit ; P[UAS- slit]/P[UAS-slit] was generated to rescue the phenotype. Over- and ectopic expression of a slit transgene correlates with expression was produced by crosses generating P[sim-GAL4[/P[sim- repulsion of axon extension. We describe a strong genetic GAL4]; P[UAS-slit]/P[UAS-slit] and similarly using P[.0-GAL4], interaction of slit with mutations in robo, suggesting further P[scab-GAL4] and P[eng-GAL4] lines. Embryos were assayed by that Slit may act as a ligand for Robo, mediating axon repulsion staining with mAbs BP102 and 1D4. Transcription of transgenic slit from commissural projection. was monitored with an antisense mRNA in situ probe generated from a 600 bp region at the N-terminal of sli cDNA, and synthesis of protein was confirmed with Slit antibody (provided by D. Hartley). MATERIALS AND METHODS

Genetic stocks RESULTS 2 IG107 slit (formerly slit ), was isolated by Nüsslein-Volhard (Nüsslein- Slit is required for midline guidance Volhard 1984) robo1 was isolated on a background deficient for fasciclin III and fasciclin I described by Seeger (Seeger et al., 1993). Previous studies of slit function characterise the mutant CNS A wild-type genetic background was restored prior to study. A phenotype as a midline fusion of all axon tracts, and a ventral homozygous viable insert of P[sim-GAL4] obtained from S. Crews displacement of midline cells (Rothberg et al., 1988, 1990; (generated by J. Nambu), P[eng-GAL4] (T. Bössing), P[scab-GAL4] Sonnenfeld and Jacobs, 1994). These studies did not establish (C. Goodman) and P[sli1.0-GAL4] (C. Klämbt) were used in the over- whether the axon tract phenotype reflects a deficiency in axon expression and rescue experiments. slit2, P[sim-GAL4] and other 2nd guidance, or a secondary effect of disrupting midline recombinants were generated and then screened using cytoarchitecture. We have re-examined axon tract architecture P[UAS-tau-lacZ], BP102 and mAb 1D4 (anti-Fasciclin II). All mutant of all CNS axons (labeled with BP102 antibody) and subsets stocks were isogenised and maintained on a yw mutant background of longitudinal tract fascicles (with antibodies to Fasciclin II) balanced with CyO-P[eng-lacZ]. in embryos homozygous for a hypomorphic and a null allele Immunocytochemistry of slit (Fig. 1). In contrast to the ladder-like axon tract Immunocytochemistry was adapted from Patel (Patel, 1994). Embryos organisation of a wild-type CNS (Fig. 1A,G), all axons are were collected at 22¡C (or 29¡C for some GAL4 experiments) and displaced towards a single midline tract in a slit amorph, slit2 fixed at 16 hour intervals. BP102, mAb 1D4, α-sim or mAb C1.427 (Fig. 1C,I). During stage 12/0, Fasciclin II immunolabeling (provided by N. Patel, C. Goodman, S. Crews and R. White, identifies pioneers of the longitudinal tracts, the pCC, vMP2 respectively) were diluted 1:4 in phosphate-buffered saline (PBS) with and MP1 axons (Grenningloh et al., 1991; Jacobs and 0.1% Triton X-100 and incubated at room temperature for 6 hours Goodman, 1989; Fig. 1D). MP pioneers are medially displaced followed by 2 hours incubation in goat anti-mouse conjugated with in stage 12/0 slit2 mutant embryos, and the growth cone of the HRP (Jackson Immunological) at a 1:1000 dilution. Reactions with more lateral pCC neuron extends towards the midline (Fig. 1F). most embryos were carried out in the presence of 0.03% cobalt chloride. Nerve cords were dissected in methyl salicylate prior to Are contralaterally projecting axons also trapped in the mounting in DPX (Sigma 31761-6) and visualised on a Zeiss midline axon fascicle labeled with BP102 antibody, or are Axiophot microscope. contralateral axon projections still possible in slit amorphs? Ultrastructural examination of the midline of the CNS reveals Fluorescence microscopy that contralaterally projecting axons persist in slit mutant Manually devitellinised embryos were dissected on glass in PBS and embryos despite midline fusion of longitudinal tracts. A cross fixed for 10 minutes in 4% paraformaldehyde. The dissections were section of a wild-type nerve cord at the posterior limit of the washed in buffer and incubated for 30 minutes in rhodamine-labeled anterior commissure reveals bundles of commissural axons phalloidin (Verheyen and Cooley, 1994) (Molecular Probes R415) and ensheathed by processes of the MG. Longitudinal axon tracts mounted in glycerol with p-phenylene d-amine (Sigma P-6001) as an are located within the developing neuropil of each neuromere anti-bleaching agent. Projections were made from confocal images were collected with a Zeiss 310. (Fig. 2A). In contrast, at an equivalent position in the nerve cord of a slit mutant, the longitudinal tracts are fused at the Electron microscopy dorsal midline of the CNS in a single bundle. Underneath the Dechorionated embryos were fixed in heptane equilibrated with 25% longitudinal tract, fascicles of contralaterally projecting axons glutaraldehyde (Fluka) in 0.1 M sodium cacodylate. Embryos were are prominent (Fig. 2B). MG are displaced to the ventral limit manually devitellinised in 4% paraformaldehyde and 2.5% of the neuropil and still maintain contact with commissural glutaraldehyde in cacodylate buffer, post-fixed in 1% osmium axons. In locations anterior and posterior to the commissural tetroxide, and stained before embedding in uranyl acetate (Jacobs and axons, the neuropil of slit mutant nerve cords contains many µ Goodman, 1989). Lead stained 0.1 m sections were examined on a more growth cone filopodia, and fewer axons than wild type JEOL 1200EXII microscope. (data not shown). Germline transformation Longitudinal tract axons in slit2 mutants appear to re-cross A 4.2 kb fragment of sli cDNA was engineered by ligation of a C- the midline as they project anteriorly or posteriorly (Fig. 1I) in terminal PCR fragment (primers 5′ CTT CAG AGC TCT GCC ACA a manner comparable to robo mutants (Fig. 5A). The ventral ATG GCC 3′ and 5′ CCC TTG AAG ACG CGT CTA CCC ACG 3′) displacement of midline cells clearly contributes to the and N-terminal PCR fragment (primers 5′ TTC TTA CTA GTT CCG midline fusion phenotype. The position of midline cells of slit is required for midline axon guidance 2477

Fig. 1. Axon tracts and ventral muscle are medially misplaced in embryos lacking slit function. Wild type (A,D,G), sli2 (C,F,I) and sli532 (B,E,H) nerve cords are labeled with BP102 (A- C) and anti- Fasciclin II (D-I) antibodies. Wild-type embryos have a ladder-like scaffold of axon tracts in the ventral nerve cord (A). Arrows in A, B and C indicate the separation between anterior and posterior commissures; arrowheads identify the intersegmental portion of the longitudinal tract. Embryos homozygous for a null allele of slit, slit2, show a complete fusion of axon tracts at the midline (C). Embryos homozygous for a hypomorphic allele of slit, slit532 (B), have a medial narrowing of the axon tract scaffold, and poor definition of the anterior and posterior commissures of each segment (arrow). In late embryogenesis, three well defined longitudinal fascicles expressing FasII are seen in wild type (G). Fusion of FasII- expressing axons into a single medial fascicle is evident in a slit amorph (I). In a slit hypomorph, Fas II-expressing fascicles are medially displaced, and may cross over the midline (H). The major longitudinal tracts are established during stage 12/0, when Fasciclin II labeling identifies the anterior projection of pCC (arrowhead in D,E,F). Both axons and neuronal somas (MPs identified by arrow) are medially displaced by stage 12 in slit amorphs (F). Early medial displacement is significantly less in a slit hypomorph (E), however, ectopic midline crossing of Fasciclin II-labeled axons are evident (arrow). Muscles and axon tracts may also be visualised with fluorescently tagged phalloidin, which binds f-actin (J,K). In a wild- type dissected embryo (J), the ventral oblique muscles insert in the ectoderm underneath the lateral edge of the nerve cord (arrowhead). In a dissected embryo homozygous for slit2 (K), the ventral oblique muscles cross over the dorsal surface of the cord (arrowhead), to insert together with the ventral longitudinal muscles. Anterior is at top in all panels.

pattern in slit mutants after labeling dissected embryos with fluorescently tagged phalloidin, which binds F-actin. In wild type, the most medial muscles, the ventral oblique, avoid the ventral midline and insert underneath the edge of the nerve cord. In embryos mutant for slit, the ventral oblique muscles do not insert below the cord. Instead, they cross the dorsal surface of the cord to insert contralaterally with the ventral longitudinal muscles (Fig. 1J,K). Fewer muscles cross the mesectodermal origin can be followed by assessing the dorsal surface of the cord in embryos mutant for hypomorphic expression pattern of Single-minded, a transcription factor alleles of slit (data not shown). These data suggest that the required for determination of the Drosophila mesectoderm. ventral oblique muscles may also respond to a midline Mesectodermal cells (MECs) demonstrate stereotyped repulsive signal missing in slit mutants. positioning in the midline of the wild-type nervous system (Fig. 3A). In contrast, most MECs line the ventral midline in Does slit act as a repulsive signal? slit amorphs (Fig. 3B). Drosophila mutant for hypomorphic If Slit provides a signal that repels the growth cones of neurons alleles of slit, such as slit532, have a nearly normal pattern of and muscles, then ectopic expression of slit during MEC distribution (Fig. 3C). Nevertheless, axon trajectories in development should misdirect their growth. The molecular and the CNS continue to show misdirection towards the midline. genetic structure of slit has been previously described The commissural tracts of slit hypomorphs are thickened, and (Rothberg et al., 1988, 1990). We have generated a complete show poor definition of anterior and posterior commissures cDNA from clones isolated in this earlier study, and generated (Fig. 1B). The thinning of the longitudinal tracts is comparable transgenic Drosophila carrying P[UAS-slit]. To verify that this to that seen in a slit amorph. During stage 12/0, pioneers of the construct generates functional Slit protein, we then employed longitudinal tracts show misdirection and crossing of the the UAS-GAL4 expression system to direct expression of slit midline, resulting at later stages in a phenotype reminiscent of in midline cells with P[simGAL4] and P[slit1.0-GAL4]. robo amorphs (Fig. 1E,H). This suggests that disorders in Midline expression of P[UAS-slit] in embryos mutant for slit2 midline axon guidance in slit mutants cannot be solely generates a partial rescue of the amorphic phenotype, to one attributed to changes in MEC position. resembling a slit or robo hypomorph. Midline structures, We have also noted that the nerve cord of slit mutants is including commissural tracts, have been partially restored, often kinked. Upon closer examination, kinks appear to be however, errant Fasciclin II-expressing axons continue to cross formed by muscle cells extending across the dorsal surface of the midline (Fig. 4A, arrow in D). The same level of midline the cord. We have further characterised the ventral muscle expression of the slit transgene with P[UAS-sim] in slit2 mutant 2478 R. Battye, A. Stevens and J. R. Jacobs

Fig. 3. Slit function is required for mesectodermal cytoarchitecture. Antibodies to Single-minded label the nuclei of MECs in stage 15 embryos. Neurons dominate the ventral half of the midline in wild- type nerve cords (arrow, A) while the MG are dorsally located (arrowhead). All cells are ventrally displaced in the slit amorph, sli2 (B), however, MEC cell position is similar to wild type in sli532 hypomorphs (C). Transgene expression of slit in the midline (in P[simGAL4], sli2/P[simGAL4], sli2; P[UAS-slit]/P[UAS-slit] embryos) partially restores midline cell position (D).

Slit protein (Fig. 4H). Midline over-expression with a more effective midline enhancer (P[slit1.0-GAL4], Fig. 4B,E), leads to defasciculation of commissural tracts, some missing commissures and ectopic lateral axon extension. To further assess the repulsive property of Slit during axon guidance, we directed ectopic P[UAS-slit] expression in embryos. Transgene expression with P[engrailed-GAL4] is Fig. 2. Midline fusion of commissural and longitudinal axons in restricted to the posterior compartment of the segment (just embryos mutant for slit. A cross section of a wild-type nerve cord at anterior to the anterior commissure). slit expression here the posterior limit of the anterior commissure of an abdominal results in breaks in longitudinal tracts, including the MP segment at stage 16 (A) outlines the normal architecture of the fascicle (arrow, Fig. 4F) and the later forming SP1 fascicle commissural axons (c) which traverse the midline, here in (arrow, Fig. 4I). Furthermore, redirected Fasciclin II-labeled association with the midline glia (MG). Longitudinal tract axons (l) axons now inappropriately cross the midline (arrowhead, Fig. dominate the dorsal aspect of the lateral neuropil. A cross-section at 4F). Broad expression in the neuroectoderm with P[scabrous- an equivalent location of a homozygous slit2 mutant nerve cord (B) demonstrates the dorsal midline fusion of the longitudinal tract, GAL4] decreased the number of axons traversing the segment overlying a bundle of medially compacted commissurally projecting boundary in the longitudinal tracts, thinning and delaying axons. Scale bar, 5 µm. development of Fasciclin II-expressing fascicles (data not shown). embryos was not sufficient to restore MEC cytoarchitecture, in Slit function interacts with robo spite of significant restoration of longitudinal tract structure The axon tract phenotype of hypomorphs of slit, and partially (Fig. 3D). This further indicates a role for slit in axon guidance rescued function of slit amorphs resembles that of amorphic independent of its requirement to establish MEC alleles of robo, suggesting at least one component of slit cytoarchitecture. function also requires robo function. If the functions of slit and If P[UAS-slit] expression in the midline is superimposed robo are inter-dependent, for instance, both functioning in rate upon wild-type expression, fewer than normal numbers of limiting steps in signaling midline repulsion, then mutant axons cross the commissural tracts (Fig. 4C). Midline over- alleles of these two genes should demonstrate a phenotypic expression also results in thicker longitudinal tracts within interaction (Kidd et al., 1999). Fasciclin II-labeled axon segments, and a thinning of intersegmental connections. A fascicles show deviation towards the midline in embryos more restricted subset of midline and longitudinal fascicles can heterozygous for either robo1 or slit2, in less than 5% of be visualised with an antibody to Connectin, which labels the segments examined, indicating that these alleles lack a SP1 projection. In wild type, the SP1 neuron migrates medially penetrant semi-dominant phenotype (Fig. 5B,C). An to within one cell diameter of the midline as its axon extends interaction between these genes is clearly evident when axon across the midline to contact its contralateral partner, and then tract organisation in embryos heterozygous for both slit2 and turns to extend anteriorly (Fig. 4G). When midline cells robo1 are examined (Fig. 5D). Although these embryos have produce greater than normal levels of Slit, some commissural one functional copy of each gene, the combined effect of a axons take circuitous routes across the midline, and SP1 cell reduction of expression of both slit and robo is reduced midline position is more irregular, suggesting that medial movement of repulsion of Fasciclin II-expressing axons, resulting in a weak both growth cones and a neuron soma are affected by levels of robo-like phenotype. In slit2/robo1 embryos, 74% of all slit is required for midline axon guidance 2479 segments examined (n=175 segments in 20 embryos) show CNS midline. Differing levels of robo expression generate a mild to severe perturbation of FasII-expressing axon fascicle comparable phenotype (Kidd et al., 1998a), suggesting, in guidance near the midline. To further assess whether the addition to the demonstrated genetic interaction between robo genetic interaction between robo and slit reflects a rate limiting and slit, that Robo and Slit proteins function in the same function, we examined transheterozygotes of robo1 with a pathway (Kidd et al., 1999). hypomorph of slit (slitE158), believed to express lower levels of Slit is a large glycoprotein secreted by the MG, which can Slit because of a transposon insertion 105 bp upstream of the be detected at low levels on longitudinal tract axons (Rothberg transcription initiation site (Rothberg et al., 1990). We detected et al., 1990). Robo is a transmembrane receptor expressed on midline deviations or crossovers in 32% of all segments CNS growth cones, at highest levels in the longitudinal tracts examined in robo1/slitE158 transheterozygotes (n=221 (Kidd et al., 1998a). It is possible that Slit in the extracellular segments in 28 embryos). matrix is required to make a repulsive signal available to Robo. Alternatively, Slit may represent the repulsive signal generated at the midline, which binds to Robo on growth cones and DISCUSSION directs them to avoid the commissural tracts. Establishment of a commissural projection must therefore proceed by Our analysis of the slit mutant phenotype and the consequences suppression of the repulsive signal. This may proceed by of transgene expression indicate that Slit acts to repel the removal of Robo from growth cones, which is found at growth cones of axons and ventral muscles away from the relatively low levels on commissural axons (Kidd et al., ventral midline. Reduced slit function of a slit hypomorph 1998a). results in ectopic projections of longitudinal tract axons across Vertebrate homologues for the receptor of the midline the midline. Removal of all slit function results further in the repulsive signal, Robo, have been identified, and are expressed medial fusion of longitudinal tracts, large numbers of by neurons which must cross or avoid the floorplate (Kidd et decussating axons, ventral displacement of midline cells and al., 1998a). Putative and rat homologues of slit have mis-projection of muscles across the dorsal surface of the nervous system. Ectopic expression of slit not only inhibits axon tract formation and cell migration close to the point of secretion, but also results in the misdirection of axonogenesis to midline domains where Fasciclin II expression is normally excluded. This suggests that sli- mediated repulsion is dose dependent, and may clearly communicate a gradient of positional cues relative to the

Fig. 4. Axons fail to extend into domains of slit misexpression. Partial restoration of midline commissure architecture (BP102, A) and separation of lateral FasII-expressing axon fascicles (α-FasII, D) is obtained in a slit2 mutant expressing a slit transgene in midline cells (P[simGAL4, sli2/P[simGAL4], sli2; P[UAS- slit]/P[UAS-slit]). Arrow in D indicates ectopic midline crossing of a Fasciclin II-labeled axon. (C) Expression of a slit transgene in midline cells, superimposed upon wild-type slit expression (P[simGAL4/P[simGAL4]; P[UAS-slit]/P[UAS-slit]) results in a thinning of commissural tracts labeled with BP102, a thickening of the lateral neuropil (arrow) and thinning of longitudinal connectives (arrowhead). (B,E) Higher levels of midline expression (generated with P[slit1.0-GAL4]; P[UAS-slit]) disrupts and reduces commissural projections labeled with BP102 (arrow) and generates ectopic projections laterally in the nerve cord (arrowhead). (E) Fas II-labeled axon fascicles in P[slit1.0- GAL4]; P[UAS-slit] embryos reveal interruptions and lateral extensions of longitudinal tracts (arrowhead and arrow). (F) Ectopic expression of the slit transgene was superimposed upon wild-type expression of slit, employing P[engrailed-GAL4]. Labeling of FasII axon fascicles demonstrates ectopic projections towards the midline (arrowhead) and thinning or loss of longitudinal tracts in locations of high transgene expression (arrow). (G) Antibody to Connectin labels the SP1 neuron (arrowhead), whose axon extends contralaterally across the anterior commissure, then turning to extend anteriorly in the contralateral longitudinal tract (arrow). (H) The SP1 fascicle in the longitudinal tract is missing in some segments after over- expression of slit in the midline (arrowhead). Other commissural fascicles take circuitous routes across the midline (arrow). (I) Many segments lack a contralateral SP1 fascicle if slit is mis- expressed in the engrailed pattern (arrow). 2480 R. Battye, A. Stevens and J. R. Jacobs

have shown that elevated levels of slit expression in a normal pattern, or ectopic expression in the posterior domain of each segment inhibits the establishment of axon tracts in those territories. Slit transcript has been identified in the MG and the mature dorsal vessel, but not in muscle (Rothberg et al., 1990; R. A. B., J. R. J., unpublished). However, Slit antigenicity has been detected in muscle insertions. It is possible that some if not all of the Slit detected here is originally secreted by the MG, and is bound by a receptor at muscle insertions. The unexpected role for slit in muscle patterning recollects Michael Bate’s seminal characterisation of embryonic muscle patterning in which he observes ‘the first [muscle] precursors appear over the CNS, and it is only later by a lateral migration that they Fig. 5. slit interacts genetically with robo. (A) Labeling of FasII- come to span the epidermis. The CNS, like the epidermis, expressing axon fascicles in robo1 homozygotes identifies axons might be capable of specifying an overlying pattern of muscle crossing and re-crossing the midline (arrowhead). Embryos precursors’ (Bate, 1990). Our observations suggest that 1 2 heterozygous for robo (B) and sli (C) are similar in appearance to secretion of Slit from the midline contributes to this pattern. wild type, indicating no semi-dominant effect of these alleles. 1 2 (D) Embryos doubly heterozygous for robo and slit (robo /slit ) The authors thank C. Smith and G. Zhao for technical assistance demonstrate midline crossing and disruption of FasII-expressing and G. Boulianne and A. Campos for comments on the manuscript. longitudinal axon fascicles (arrowhead). We are grateful for Drosophila stocks provided by C. Goodman, C. Klämbt, S. Crews and the Indiana fly stock centre. Molecular and antibody reagents were provided by S. Artavanis-Tsakonas, G. also been described (Holmes et al., 1998; Itoh et al., 1998), Boulianne, C. Goodman, R. White, S. Crews and G. Tear. This work suggesting that the mechanism of midline guidance has been was supported by the MRC of Canada. conserved. If Slit and Robo function in the same pathway, why does a null mutation in slit generate a more severe phenotype? Two REFERENCES possible reasons are evident. First, it is possible that repulsion mediated by Slit is transduced through a Robo-independent Bate, M. (1990). The embryonic development of larval muscles in Drosophila. pathway. The karussell mutation, for instance, which generates Development 110,791-804. Bernhardt, R., Nguyen, N. and Kuwada, J. (1992). Growth cone guidance a similar phenotype may function in a Robo-independent by floor plate cells in the spinal cord of zebrafish embryos. Neuron 8, 869- repulsion from the midline (Hummel et al., 1999) 882. Secondly, the similarity of the slit hypomorph (slit532) Brand, A. 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