Regulation of axon guidance by slit and netrin signaling in the Drosophila ventral nerve cord

Netrin and Slit signaling systems play opposing roles during the positioning of longitudinal tracts along the midline in the ventral nerve cord of Drosophila embryo. It has been hypothesized that a gradient of Slit from the midline interacts with three different Robo receptors to specify the axon tract positioning. However, no such gradient has been detected. Moreover, overexpression of Slit at the midline has no effect on the positioning of these lateral tracts. In this article, we show that Slit is present outside of the midline along the longitudinal and commissural tracts. Sli from the midline, in a Robo-independent manner, is initially taken up by the commissural axon tracts when they cross the midline and is transported along the commissural tracts into the longitudinal connectives. These results are not consistent with a Sli gradient model. We also find that sli mRNA is maternally deposited and embryos that are genetically null for sli can have weaker guidance defects. Moreover, in robo or robo3 mutants, embryos with normal axon tracts are found and such robo embryos reach pupal stages and die, while robo3 mutant embryos develop into normal individuals and produce eggs. Interestingly, embryos from robo3 homozygous individuals fail to develop but have axon tracts ranging from normal to various defects: robo3 phenotype, robo phenotype, and slit-like phenotype, suggesting a more complex functional role for these genes than what has been proposed. Finally, our previous results indicated that netrin phenotype is epistatic to sli or robo phenotypes. However, it seems likely that this previously reported epistatic relationship might be due to the partial penetrance of the sli, robo, robo3 (or robo2) phenotypes. Our results argue that double mutant epistasis is most definitive only if the penetrance of the phenotypes of the mutants involved is complete.     

Axonal heparan sulfate proteoglycans regulate the distribution and efficiency of the repellent slit during midline axon guidance

The presentation of secreted axon guidance factors plays a major role in shaping central nervous system (CNS) connectivity. Recent work suggests that heparan sulfate (HS) regulates guidance factor activity; however, the in vivo axon guidance roles of its carrier proteins (heparan sulfate proteoglycans, or HSPGs) are largely unknown. Here we demonstrate through genetic analysis in vivo that the HSPG Syndecan (Sdc) is critical for the fidelity of Slit repellent signaling at the midline of the Drosophila CNS, consistent with the localization of Sdc to CNS axons. sdc mutants exhibit consistent defects in midline axon guidance, plus potent and specific genetic interactions supporting a model in which HSPGs improve the efficiency of Slit localization and/or signaling. To test this hypothesis, we show that Slit distribution is altered in sdc mutants and that Slit and its receptor bind to Sdc. However, when we compare the function of the transmembrane Sdc to a different class of HSPG that localizes to CNS axons (Dallylike), we find functional redundancy, suggesting that these proteoglycans act as spatially specific carriers of common HS structures that enable growth cones to interact with and perceive Slit as it diffuses away from its source at the CNS midline.     

Axon repulsion from the midline of the Drosophila CNS requires slit function

Guidance of axons towards or away from the midline of the central nervous system during Drosophila embryogenesis reflects a balance of attractive and repulsive cues originating from the midline. Here we demonstrate that Slit, a protein secreted by the midline glial cells provides a 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 ventral muscles. Transgene expression of slit in the midline restores axon patterning. Ectopic expression of slit inhibits formation of axon tracts at locations of high Slit production and misdirects axon tracts towards the midline. slit interacts genetically with roundabout, which encodes a putative receptor for growth cone repulsion.     

Longitudinal axons are guided by Slit/Robo signals from the floor plate

Longitudinal axons grow long distances along precise pathways to connect major CNS regions. However, during embryonic development, it remains largely undefined how the first longitudinal axons choose specific positions and grow along them. Here, we review recent evidence identifying a critical role for Slit/Robo signals to guide pioneer longitudinal axons in the embryonic brain stem. These studies indicate that Slit/Robo signals from the floor plate have dual functions: to repel longitudinal axons away from the ventral midline, and also to maintain straight longitudinal growth. These dual functions likely cooperate with other guidance cues to establish the major longitudinal tracts in the brain.Key words: Slit, Robo, longitudinal axon, hindbrain, axon guidance     

Conserved roles for Slit and Robo proteins in midline commissural axon guidance

In Drosophila, Slit at the midline activates Robo receptors on commissural axons, thereby repelling them out of the midline into distinct longitudinal tracts on the contralateral side of the central nervous system. In the vertebrate spinal cord, Robo1 and Robo2 are expressed by commissural neurons, whereas all three Slit homologs are expressed at the ventral midline. Previous analysis of Slit1;Slit2 double mutant spinal cords failed to reveal a defect in commissural axon guidance. We report here that when all six Slit alleles are removed, many commissural axons fail to leave the midline, while others recross it. In addition, Robo1 and Robo2 single mutants show guidance defects that reveal a role for these two receptors in guiding commissural axons to different positions within the ventral and lateral funiculi. These results demonstrate a key role for Slit/Robo signaling in midline commissural axon guidance in vertebrates.     

Slit proteins prevent midline crossing and determine the dorsoventral position of major axonal pathways in the mammalian forebrain.

We report that Slit proteins, a family of secreted chemorepellents, are crucial for the proper development of several major forebrain tracts. Mice deficient in Slit2 and, even more so, mice deficient in both Slit1 and Slit2 show significant axon guidance errors in a variety of pathways, including corticofugal, callosal, and thalamocortical tracts. Analysis of multiple pathways suggests several generalizations regarding the functions of Slit proteins in the brain, which appear to contribute to (1) the maintenance of dorsal position by prevention of axonal growth into ventral regions, (2) the prevention of axonal extension toward and across the midline, and (3) the channeling of axons toward particular regions.     

The function of leak and kuzbanian during growth cone and cell migration

Axonal growth cones require an evolutionary conserved repulsive guidance system to ensure proper crossing of the CNS midline. In Drosophila, the Slit protein is a repulsive signal secreted by the midline glial cells. It binds to the Roundabout receptors, which are expressed on CNS axons in the longitudinal tracts but not in the commissural tracts. Here we present an analysis of the genes leak and kuzbanian and show that both genes are involved in the repulsive guidance system operating at the CNS midline. Mutations in leak, which encodes the Roundabout-2 Slit receptor, were first recovered by Nüsslein-Volhard and co-workers based on defects in the larval cuticle. Analysis of the head phenotype suggests that slit may be able to act as an attractive guidance cue while directing the movements of the dorsal ectodermal cell sheath. kuzbanian also regulates midline crossing of CNS axons. It encodes a metalloprotease of the ADAM family and genetically interacts with slit. Expression of a dominant negative Kuzbanian protein in the CNS midline cells results in an abnormal midline crossing of axons and prevents the clearance of the Roundabout receptor from commissural axons. Our analyses support a model in which Kuzbanian mediates the proteolytic activation of the Slit/Roundabout receptor complex.     

Patched regulation of axon guidance is by specifying neural identity in the<Emphasis Type="Italic"> Drosophila</Emphasis> nerve cord

Within an axon bundle, one or two are pioneering axons and the rest are follower axons. Pioneering axons are projected first and the follower axons are projected later but follow a pioneering axon(s) pathway. It is not clear whether the pioneering axons have a guidance role for follower axons. In this paper, we have investigated the role of Patched (Ptc) in regulating the guidance of medial tract, one of the longitudinal tracts in the nerve cord. In patched mutants the medial longitudinal tract fails to fasciculate on its own side along the nerve cord, instead it abnormally crosses the midline and fasciculates with the contralateral tract. Interestingly, the medial tracts cross the midline ignoring the axon-repellant Slit on the midline and Roundabout on growth cones. The medial tract is pioneered by neurons pCC and vMP2. Our results show that guidance defects of this tract are due to loss and mis-specification of vMP2, which results in the projection from pCC to either stall or project outward near the location of vMP2. Thus, both pioneering neurons are necessary for the proper guidance of pioneering and follower axons. We also show that the loss of Ptc activity in the neuroectoderm prior to the formation of S1 and S2 neuroblasts causes the majority of axon guidance defects. These results provide insight into how mis-specification and loss of neurons can non-autonomously contribute to defects in axon pathfinding.     

Hedgehog regulated Slit expression determines commissure and glial cell position in the zebrafish forebrain

Three major axon pathways cross the midline of the vertebrate forebrain early in embryonic development: the postoptic commissure (POC), the anterior commissure (AC) and the optic nerve. We show that a small population of Gfap+ astroglia spans the midline of the zebrafish forebrain in the position of, and prior to, commissural and retinal axon crossing. These glial ;bridges' form in regions devoid of the guidance molecules slit2 and slit3, although a subset of these glial cells express slit1a. We show that Hh signaling is required for commissure formation, glial bridge formation, and the restricted expression of the guidance molecules slit1a, slit2, slit3 and sema3d, but that Hh does not appear to play a direct role in commissural and retinal axon guidance. Reducing Slit2 and/or Slit3 function expanded the glial bridges and caused defasciculation of the POC, consistent with a ;channeling' role for these repellent molecules. By contrast, reducing Slit1a function led to reduced midline axon crossing, suggesting a distinct role for Slit1a in midline axon guidance. Blocking Slit2 and Slit3, but not Slit1a, function in the Hh pathway mutant yot (gli2DR) dramatically rescued POC axon crossing and glial bridge formation at the midline, indicating that expanded Slit2 and Slit3 repellent function is largely responsible for the lack of midline crossing in these mutants. This analysis shows that Hh signaling helps to pattern the expression of Slit guidance molecules that then help to regulate glial cell position and axon guidance across the midline of the forebrain.     

The midline glial cells are required for regionalization of commissural axons in the embryonic CNS of Drosophila

 The ventral nerve cord of arthropods is characterised by the organisation of major axon tracts in a ladder-like pattern. The individual neuromeres are connected by longitudinal connectives whereas the contra-lateral connections are brought about through segmental commissures. In each neuromere of the embryonic central nervous system (CNS) of Drosophila an anterior and a posterior commissure is found. The development of these commissures requires a set of neurone-glia interactions at the midline. Here we show that both the anterior as well as the posterior commissures are subdivided into three axon-containing regions. Electron microscopy of the ventral nerve cord of mutations affecting CNS midline cells indicates that the midline glial cells are required for this subdivision. In addition the midline glial cells appear required for a crossing of commissural growth cones perpendicular to the longitudinal tracts, since in mutants with defective midline glial cells commissural axons frequently cross the midline at aberrant angles. Received: 6 July 1997 / Accepted: 27 August 1997     

Constitutively active myosin light chain kinase alters axon guidance decisions in Drosophila embryos

Conventional myosin II activity provides the motile force for axon outgrowth, but to achieve directional movement during axon pathway formation, myosin activity should be regulated by the attractive and repulsive guidance cues that guide an axon to its target. Here, evidence for this regulation is obtained by using a constitutively active Myosin Light Chain Kinase (ctMLCK) to selectively elevate myosin II activity in Drosophila CNS neurons. Expression of ctMLCK pan-neurally or in primarily pCC/MP2 neurons causes these axons to cross the midline incorrectly. This occurs without altering cell fates and is sensitive to mutations in the regulatory light chains. These results confirm the importance of regulating myosin II activity during axon pathway formation. Mutations in the midline repulsive ligand Slit, or its receptor Roundabout, enhance the number of ctMLCK-induced crossovers, but ctMLCK expression also partially rescues commissure formation in commissureless mutants, where repulsive signals remain high. Overexpression of Frazzled, the receptor for midline attractive Netrins, enhances ctMLCK-dependent crossovers, but crossovers are suppressed when Frazzled activity is reduced by using loss-of-function mutations. These results confirm that proper pathway formation requires careful regulation of MLCK and/or myosin II activity and suggest that regulation occurs in direct response to attractive and repulsive cues.     

Binding site for Robo receptors revealed by dissection of the leucine-rich repeat region of Slit

Recognition of the large secreted protein Slit by receptors of the Robo family provides fundamental signals in axon guidance and other developmental processes. In Drosophila, Slit-Robo signalling regulates midline crossing and the lateral position of longitudinal axon tracts. We report the functional dissection of Drosophila Slit, using structure analysis, site-directed mutagenesis and in vitro assays. The N-terminal region of Slit consists of a tandem array of four independently folded leucine-rich repeat (LRR) domains, connected by disulphide-tethered linkers. All three Drosophila Robos were found to compete for a single highly conserved site on the concave face of the second LRR domain of Slit. We also found that this domain is sufficient for biological activity in a chemotaxis assay. Other Slit activities may require Slit dimerisation mediated by the fourth LRR domain. Our results show that a small portion of Slit is able to induce Robo signalling and indicate that the distinct functions of Drosophila Robos are encoded in their divergent cytosolic domains.     

Robo1 and Robo2 have distinct roles in pioneer longitudinal axon guidance

Pioneer longitudinal axons grow long distances parallel to the floor plate and precisely maintain their positions using guidance molecules released from the floor plate. Two receptors, Robo1 and Robo2, are critical for longitudinal axon guidance by the Slit family of chemorepellents. Previous studies showed that Robo1^−/−;2^−/− double mutant mouse embryos have disruptions in both ventral and dorsal longitudinal tracts. However, the role of each Robo isoform remained unclear, because Robo1 or 2 single mutants have mild or no errors. Here we utilized a more sensitive genetic strategy to reduce Robo levels for determining any separate functions of the Robo1 and 2 isoforms. We found that Robo1 is the predominant receptor for guiding axons in ventral tracts and prevents midline crossing. In contrast, Robo2 is the main receptor for directing axons within dorsal tracts. Robo2 also has a distinct function in repelling neuron cell bodies from the floor plate. Therefore, while Robo1 and 2 have some genetic overlap to cooperate in guiding longitudinal axons, each isoform has distinct functions in specific longitudinal axon populations.     

Netrin, Slit and Wnt receptors allow axons to choose the axis of migration

One of the challenges to understanding nervous system development has been to establish how a fairly limited number of axon guidance cues can set up the patterning of very complex nervous systems. Studies on organisms with relatively simple nervous systems such as Drosophila melanogaster and C. elegans have provided many insights into axon guidance mechanisms. The axons of many neurons migrate along both the dorsal-ventral (DV) and the anterior-posterior (AP) axes at different phases of development, and in addition they may also cross the midline. Axon migration in the dorsal-ventral (DV) direction is mainly controlled by Netrins with their receptors; UNC-40/DCC and UNC-5, and the Slits with their receptors; Robo/SAX-3. Axon guidance in the anterior-posterior (AP) axis is mainly controlled by Wnts with their receptors; the Frizzleds/Fz. An individual axon may be subjected to opposing attractive and repulsive forces coming from opposite sides in the same axis but there may also be opposing cues in the other axis of migration. All the information from the cues has to be integrated within the growth cone at the leading edge of the migrating axon to elicit a response. Recent studies have provided insight into how this is achieved.Evidence suggests that the axis of axon migration is determined by the manner in which Netrin, Slit and Wnt receptors are polarized (localized) within the neuron prior to axon outgrowth. The same molecules are involved in both axon outgrowth and axon guidance, for at least some neurons in C. elegans, whether the cue is the attractive cue UNC-6/Netrin working though UNC-40/DCC or the repulsive cue SLT-1/Slit working though the receptor SAX-3/Robo (Adler et al., 2006, Chang et al., 2006, Quinn et al., 2006, 2008). The molecules involved in cell signaling in this case are polarized within the cell body of the neuron before process outgrowth and direct the axon outgrowth. Expression of the Netrin receptor UNC-40/DCC or the Slit receptor SAX-3/Robo in axons that normally migrate in the AP direction causes neuronal polarity reversal in a Netrin and Slit independent manner (Levy-Strumpf and Culotti 2007, Watari-Goshima et al., 2007). Localization of the receptors in this case is caused by the kinesin-related VAB-8L which appears to govern the site of axon outgrowth in these neurons by causing receptor localization. Therefore, asymmetric localization of axon guidance receptors is followed by axon outgrowth in vivo using the receptor's normal cue, either attractive, repulsive or unknown cues.     

Lateral neuron--glia interactions steer the response of axons to the Robo code

Glia are required for axon pathfinding along longitudinal trajectories, but it is unknown how this relates to the molecular paradigm of axon guidance across the midline. Most interneuron axons in bilateral organisms cross the midline only once. Preventing them from recrossing the midline requires the expression of Robo receptors on the axons. These sense the repulsive signal Slit, which is produced by the midline. The lateral positioning of longitudinal axons depends on the response to Slit by the combination of Robo receptors expressed by the axons, on selective fasciculation, and on longitudinal (lateral) glia. Here, we analyse how longitudinal glia influence reading of the 'Robo code' by axons. We show that whereas loss of robo1 alone only affects the most medial axons, loss of both glial cells missing (gcm) and robo1 causes a severe midline collapse of longitudinal axons, similar to that caused by the loss of multiple Robo receptors. Furthermore, whereas ectopic expression of robo2 is sufficient to displace the medial MP2 axons along a more lateral trajectory, this does not occur in gcm-robo1 double-mutant embryos, where axons either do not extend at all or they misroute exiting the CNS. Hence, lateral neuron-glia interactions steer the response of axons to the Robo code.     

Slit-roundabout signaling neutralizes netrin-Frazzled-mediated attractant cue to specify the lateral positioning of longitudinal axon pathways

An extending axon growth cone is subjected to attractant and repellent cues. It is not clear how these growth cones discriminate the two opposing forces and select their projection paths. Here, we report that in the Drosophila nerve cord the growth cones of longitudinal tracts are subjected to attraction by the Netrin-Frazzled pathway. However, the midline Slit neutralizes this pathway in a Robo-dependent manner and prevents Netrin-Frazzled-mediated attraction of longitudinal tracts. Our results suggest that the loss of a neutralizing effect on the Netrin-mediated attraction is responsible for the longitudinal tracts entering the midline in slit mutants as opposed to a loss of repulsion as is currently believed. This effect is not via a direct inhibition of Frazzled by Robo; instead, it is at a level downstream of Frazzled. Thus, the growth cones of longitudinal tracts subjected to two opposing forces are able to block one with the other and specify their correct lateral positioning along the midline.     

Heparan sulfate proteoglycan specificity during axon pathway formation in the Drosophila embryo

Axon guidance is influenced by the presence of heparan sulfate (HS) proteoglycans (HSPGs) on the surface of axons and growth cones (Hu, [2001]: Nat Neurosci 4:695-701; Irie et al. [2002]: Development 129:61-70; Inatani et al. [2003]: Science 302:1044-1046; Johnson et al. [2004]: Curr Biol 14:499-504; Steigemann et al. [2004]: Curr Biol 14:225-230). Multiple HSPGs, including Syndecans, Glypicans and Perlecans, carry the same carbohydrate polymer backbones, raising the question of how these molecules display functional specificity during nervous system development. Here we use the Drosophila central nervous system (CNS) as a model to compare the impact of eliminating Syndecan (Sdc) and/or the Glypican Dally-like (Dlp). We show that Dlp and Sdc share a role in promoting accurate patterns of axon fasciculation in the lateral longitudinal neuropil; however, unlike mutations in sdc, which disrupt the ability of the secreted repellent Slit to prevent inappropriate passage of axons across the midline, mutations in dlp show neither midline defects nor genetic interactions with Slit and its Roundabout (Robo) receptors at the midline. Dlp mutants do show genetic interactions with Slit and Robo in lateral fascicle formation. In addition, simultaneous loss of Dlp and Sdc demonstrates an important role for Dlp in midline repulsion, reminiscent of the functional overlap between Robo receptors. A comparison of HSPG distribution reveals a pattern that leaves midline proximal axons with relatively little Dlp. Finally, the loss of Dlp alters Slit distribution distal but not proximal to the midline, suggesting that distinct yet overlapping pattern of HSPG expression provides a spatial system that regulates axon guidance decisions.     

Expression of the vertebrate Slit gene family and their putative receptors, the Robo genes, in the developing murine kidney

The slit (sli) gene, encoding a secreted glycoprotein, has been demonstrated to play a vital role in axonal guidance in Drosophila melanogaster by acting as a signalling ligand for the robo receptor (Rothberg, J.M., Jacobs, J.R., Goodman, C.S., Artavanis-Tsakonas, S., 1990. slit: an extracellular protein necessary for development of midline glia and commissural axon pathways contains both EGF and LRR domains. Genes Dev. 4, 2169-2187; Kidd, T., Bland, K.S., Goodman, C. S., 1999. Slit is the midline repellent for the robo receptor in Drosophila. Cell 96, 785-794). Multiple homologs of both sli and robo have been identified in vertebrates and are thought to play similar roles to their fly counterparts in neural development (Brose, K., Bland, K.S., Wang, K.H., Arnott, D., Henzel, W., Goodman, C.S., Tessier-Lavigne, M., Kidd, T., 1999. Slit proteins bind Robo receptors and have an evolutionarily conserved role in repulsive axon guidance. Cell 96, 795-806). Slit2 has been shown to bind Robo1, mediating both neuronal and axonal guidance in the developing central nervous system (CNS), (Brose et al., 1999; Hu, H., 1999. Chemorepulsion of neuronal migration by Slit2 in the developing mammalian forebrain. Neuron 23, 703-711). Importantly, both gene families display distinct expression patterns outside the CNS (Holmes, G.P., Negus, K., Burridge, L., Raman, S., Algar, E., Yamada, T., Little, M.H., 1998. Distinct but overlapping expression patterns of two vertebrate slit homologs implies functional roles in CNS development and organogenesis. Mech. Dev. 79, 57-72; Yuan, W., Zhou, L., Chen, J.H., Wu, J.Y., Rao, Y., Ornitz, D.M., 1999. The mouse SLIT family: secreted ligands for ROBO expressed in patterns that suggest a role in morphogenesis and axon guidance. Dev. Biol. 212, 290-306). Using in situ hybridization on metanephric explant cultures and urogenital tract sections, the expression patterns of Slit1, 2, 3 and Robo1 and 2 were investigated during murine metanephric development. Slit1 was expressed in the metanephric mesenchyme (MM) surrounding the invading ureteric tree (UT). Slit2 was expressed at the tips of the UT and both Slit2 and Slit3 were expressed at the far proximal end of the comma shaped and S-shaped bodies. Expression of Robo1 was initially diffuse throughout the MM, then upregulated in the pretubular aggregates, and maintained at the distal end of the comma and S-shaped bodies. Robo2 was detected in the induced MM surrounding the arborizing UT tips and later in the proximal end of the S-shaped bodies. Coincident expression of Robo1 with Slit1 in the metanephric mesenchyme and Robo2, Slit2 and Slit3 in the far proximal end of the S-shaped bodies was observed during metanephric development.     

Slit1 and Slit2 cooperate to prevent premature midline crossing of retinal axons in the mouse visual system.

During development, retinal ganglion cell (RGC) axons either cross or avoid the midline at the optic chiasm. In Drosophila, the Slit protein regulates midline axon crossing through repulsion. To determine the role of Slit proteins in RGC axon guidance, we disrupted Slit1 and Slit2, two of three known mouse Slit genes. Mice defective in either gene alone exhibited few RGC axon guidance defects, but in double mutant mice a large additional chiasm developed anterior to the true chiasm, many retinal axons projected into the contralateral optic nerve, and some extended ectopically-dorsal and lateral to the chiasm. Our results indicate that Slit proteins repel retinal axons in vivo and cooperate to establish a corridor through which the axons are channeled, thereby helping define the site in the ventral diencephalon where the optic chiasm forms.