Transcriptional and Post-transcriptional Regulation

Panneural expression of ROBO mRNA does not produce a strong axon scaffold phenotype. Staining with anti-Fasciclin II reveals subtle fasciculation defects, but overall, the axon scaffold looked quite normal. Interestingly, the Robo protein, although expressed at higher levels than in wild type, remains restricted as in wild type, i.e., high levels of expression on the longitudinal portions of axons and very low levels on the commissures. This result indicates that there must be strong regulation of Robo expression, probably posttranslational, that assures its localization to longitudinal axon segments. Such a mechanism could operate by the regulation of protein translation, transport, insertion, internalization, and/or stability (Kidd, 1998a).

The pattern and level of expression of axon guidance proteins must be choreographed with exquisite precision for the nervous system to develop its proper connectivity. Previous work has shown that the transcription factor Lola is required for central nervous system (CNS) axons of Drosophila to extend longitudinally. Lola is simultaneously required to repel these same longitudinal axons away from the midline, and it acts, in part, by augmenting the expression both of the midline repellant, Slit, and of its axonal receptor, Robo. Lola is thus the examplar of a class of axon guidance molecules that control axon patterning by coordinating the regulation of multiple, independent guidance genes, ensuring that they are co-expressed at the correct time, place and relative level (Crowner, 2002).

The reduction of Robo expression seen in lola mutants is relatively modest (~40%). It is known, however, that a 50% diminution in Robo is sufficient by itself to cause some inappropriate midline crossing, and this effect is strongly enhanced by a simultaneous 50% reduction in Slit. Loss of lola causes a greater reduction than this in Slit levels. Thus, it is plausible that the change in Slit and Robo levels could account for much of the midline phenotype observed in embryos that bear strong lola mutations. But why are weaker lola alleles like lola1A4 able to cause extra midline crossing when their effect on target gene expression is presumably proportionately less? It is likely that regulation of Slit and Robo expression is only one part of the control of midline crossing by lola, and that a significant contribution to the phenotype is made by changes in the expression of other, interacting guidance genes that are also controlled by lola. For example, aspects of the lola midline phenotype resemble details of the axon pattern observed upon mutation of genes encoding receptor tyrosine phosphatases, suggesting that these are good candidates for potential lola effectors. Moreover, it is known that the Notch-dependent mechanism that promotes the alternative (longitudinal) trajectory of CNS axons also requires lola. The multiplicity of genes contributing to the midline/longitudinal axon growth decision underscores the need for a gene, like lola, to coordinate the expression of all these cooperating guidance factors. It is suggested that it is the combination of many quantitative effects, each individually modest, which together produce the profound effects of lola on axon patterning (Crowner, 2002).

Many questions remain from these studies. (1) Though Lola itself is a transcriptional regulator, it is not known whether robo and slit are direct Lola targets or whether Lola initiates a longer chain of events leading only indirectly to robo and slit. For example, Lola could regulate other genes that themselves control the stability of robo or slit RNA or protein, or the splicing or translation efficiency of these genes. Analysis of this issue will require unambiguous identification of the exact lola isoforms required for expression of robo and slit, and characterization of their DNA-binding specificities in combination with their appropriate dimerization partner(s). (2) Only the accumulation of Robo and Slit protein has been characterized in lola mutants, and not transcript levels. The inherent variability of whole-mount RNA in situ hybridization has prevented sufficiently precise quantification of robo and slit RNA levels for this purpose. Nonetheless, the observation that ectopic expression of lola 4.7 leads to ectopic expression of slit RNA strongly argues that lola is upstream of slit transcription, though it remains possible that Robo and Slit expression are also subject to lola-dependent regulation at some post-transcriptional level (Crowner, 2002).

The F-actin-microtubule crosslinker Shot is a platform for Krasavietz-mediated translational regulation of midline axon repulsion: Robo is aberrantly detected on commissural axons in kra mutant embryos

Axon extension and guidance require a coordinated assembly of F-actin and microtubules as well as regulated translation. The molecular basis of how the translation of mRNAs encoding guidance proteins could be closely tied to the pace of cytoskeletal assembly is poorly understood. Previous studies have shown that the F-actin-microtubule crosslinker Short stop (Shot) is required for motor and sensory axon extension in the Drosophila embryo. This study provides biochemical and genetic evidence that Shot functions with a novel translation inhibitor, Krasavietz (Kra, Extra bases, Exba), to steer longitudinally directed CNS axons away from the midline. Kra binds directly to the C-terminus of Shot, and this interaction is required for the activity of Shot to support midline axon repulsion. shot and kra mutations lead to weak robo-like phenotypes, and synergistically affect midline avoidance of CNS axons. shot and kra dominantly enhance the frequency of midline crossovers in embryos heterozygous for slit or robo, and in kra mutant embryos, some Robo-positive axons ectopically cross the midline that normally expresses the repellent Slit. Finally, Kra also interacts with the translation initiation factor eIF2β and inhibits translation in vitro. Together, these data suggest that Kra-mediated translational regulation plays important roles in midline axon repulsion and that Shot functions as a direct physical link between translational regulation and cytoskeleton reorganization (Lee, 2007).

Kra and its human homolog BZAP45 contain an N-terminal leucine-zipper domain of unknown function and a C-terminal W2 domain. This study shows that Kra can bind to eIF2ß through its W2 domain and inhibit translation in vitro. It is very likely that Kra competes with eIF5 and eIF2Bepsilon for the common binding partner eIF2ß, thus inhibiting the assembly of functional preinitiation complexes. A similar mode of translation inhibition has been proposed for DAP-5/p97, which may compete with its homolog eIF4G for eIF3 and eIF4A, thus reducing both cap-dependent and -independent translation. However, the step in translation initiation that is regulated by Kra remains to be addressed experimentally (Lee, 2007).

Kra-mediated translational repression appears to be an important mechanism underlying midline axon guidance. In the kra mutant embryos, Fas II-positive CNS axons that normally remain ipsilateral cross the midline ectopically. This phenotype is observed with the pCC axons from early stages (stages 12 and 13) of axogenesis when they pioneer one of the Fas II pathways. The introduction of multiple alanine substitutions (12A and 7A) into Kra significantly reduces its ability to bind eIF2ß and abolishes its activity to rescue the kra mutant phenotype, suggesting that the function of Kra in axon guidance depends on its interaction with eIF2ß. Consistent with this conclusion, mutations in the eIF2ß gene also lead to the ectopic midline crossing of Fas II-positive axons (Lee, 2007).

There is a growing body of evidence that F-actin and microtubules are coordinately assembled to each other during axon extension and guidance. Interactions of filopodial actin bundles and microtubules are key features of filopodial maturation into an axon and of growth cone turning. Shot, a conserved molecule that scaffolds F-actin, microtubules and the microtubule plus end-binding protein EB1, is a strong candidate to bring microtubule plus ends into contact with F-actin bundles. Indeed, Shot is required for the extension of sensory and motor axons, and a mammalian homolog of Shot, ACF7, is required for microtubules to track along F-actin cables towards the leading edge of spreading endodermal cells. Thus, previous studies have suggested that Shot/ACF7 coordinately organizes F-actin and microtubules to support the motility of neuronal growth cones and nonneuronal cells (Lee, 2007).

These findings suggest that Shot also functions together with the translation inhibitor Kra to control midline axon repulsion. Shot physically associates with Kra in vivo. The shot loss-of-function phenotype at the CNS midline is reminiscent of the kra loss-of-function phenotype. The major Kra-binding domain in Shot is required for its role in midline axon repulsion. Moreover, shot and kra genetically interact in a dosage-sensitive manner for the midline phenotype. These data also support the idea that cytoskeletal assembly and translational regulation can occur in a coordinated way. Midline axon repulsion requires both the activity of Kra to recruit eIF2ß and the activity of Shot to bind to F-actin. Thus, it is likely that local levels of eIF2ß available for protein synthesis can be spatially regulated with regard to actin cytoskeleton remodeling during axon guidance (Lee, 2007).

In Drosophila, Slit is the key ligand driving midline axon repulsion, and therefore midline crossing of CNS growth cones is primarily controlled by the Robo receptor of Slit. How then do neurons regulate levels of Robo on the surface of their axons and growth cones? The transmembrane protein Commissureless (Comm) has been shown to dynamically regulate Robo expression. Comm functions as an intracellular sorting receptor to target newly made Robo for lysosomal degradation, thereby blocking its transport to the growth cone that is crossing the midline (Lee, 2007).

Translational regulation has also been shown to alter the responsiveness of growth cones to the midline repellents. In vitro studies of cultured embryonic retinal ganglion cells (RGCs) provided an insight into how translation is regulated in axons and growth cones in response to midline guidance cues. Treatment of these neurons with netrin-1 leads to the rapid activation of signaling pathways that phosphorylate the translation initiation factor eIF4E and its binding protein eIF4E-BP1 and thus induces axonal protein synthesis. The data presented in this study indicate that the role of Kra in midline axon repulsion depends on its ability to recruit the translation initiation factor eIF2ß to Shot. Thus, protein complexes containing Shot, Kra and eIF2ß may function as additional targets for signaling systems that critically control axon guidance at the CNS midline. Regulation of Shot-Kra-eIF2ß complexes may occur in neuronal cell bodies, where Kra is concentrated, or in axons and growth cones, which may require local protein synthesis to meet developmental requirements. In the latter case, since Kra is not detectable in the CNS axons of the Drosophila embryo, even a low amount of Kra may be sufficient for guiding axons (Lee, 2007).

Intriguingly, Robo was aberrantly detected on commissural axons in kra1/kra2 mutant embryos. Given the increased frequency of ectopic crossovers in these embryos, as well as the documented role of Robo in preventing axons from crossing the midline, this finding is somewhat paradoxical. One possibility is that Kra, induced by interaction with Shot and eIF2ß, could repress the synthesis of as yet unidentified proteins that transduce or modulate Robo signaling. In shot or kra mutant embryos, perhaps this translational regulatory circuit is not activated, and thus Robo-expressing growth cones abnormally cross the midline because of a decrease in the strength of Robo signaling output. Alternatively, Kra may function to finely tune the expression levels of their multiple targets that mediate attractive or repulsive responses. In this scenario, impairment of the Shot-Kra-eIF2ß circuit could disturb the precise balance between repulsion and attraction signaling at the midline, thereby decreasing the overall sensitivity of Robo-expressing growth cones to Slit. Therefore, efforts to reveal the direct targets of Kra-mediated repression in the future may provide better insights into the immediate mechanisms by which translational regulation plays an essential role for midline axon repulsion (Lee, 2007).

Midline governs axon pathfinding by coordinating expression of two major guidance systems

Formation of the neural network requires concerted action of multiple axon guidance systems. How neurons orchestrate expression of multiple guidance genes is poorly understood. This study shows that Drosophila T-box protein Midline controls expression of genes encoding components of two major guidance systems: Frazzled, ROBO, and Slit. In midline mutant, expression of all these molecules are reduced, resulting in severe axon guidance defects, whereas misexpression of Midline induces their expression. Midline is present on the promoter regions of these genes, indicating that Midline controls transcription directly. It is proposed that Midline controls axon pathfinding through coordinating the two guidance systems (Liu, 2009).

To address how Mid activates expression of the three axon guidance genes, the binding sequence of Mid was determined using an in vitro binding site selection method. Mid-binding sequence was selected from a pool of random oligonucleotides using Mid protein affinity-purified from an embryonic extract. The consensus sequence deduced from the selected oligonucleotides was (G/A/T)NA(A/T)N(T/G)(A/G)GGTCAAG. This sequence was found in the upstream regions or an intron of slit, frazzled, and robo, and all of these sites were conserved among several Drosophila species. To determine whether Mid binds to these regions in vivo, chromatin immunoprecipitation (ChIP) was performed using anti-Mid antibody. In all three genes, Mid was present around the Mid-binding sites, but not on regions without the binding site. In contrast, a potential Mid-binding site 32-kb upstream of the commisureless gene, whose expression is not affected in mid mutants, was not occupied by Mid (Liu, 2009).

The importance of the Mid-binding sites in frazzled and slit was assessed by transgenic reporter assays. To test the role of the Mid site in frazzled, reporter genes were constructed that contain the transcription start site of frazzled and an upstream region including a wild-type Mid-binding site (fraPlacZ) or a mutated site (fraMPlacZ). Compared with the wild-type reporter gene, the reporter with a mutated binding site showed reduced expression levels (33% reduction). Thus Mid-binding site is indeed required for the proper expression of frazzled. Mutating the Mid-binding site in slit also caused a severe effect on slit expression. The lacZ expression in sliPlacZ is driven by the slit regulatory element and the endogenous promoter. While sliPlacZ with the wild-type binding site recapitulated the slit expression in the midline glia and lateral cells, base substitutions in the Mid-binding site in sliMPlacZ abolished the lacZ expression. It is possible that the Mid-binding site resides in an essential promoter element of slit, and hence, the base substitutions abolished slit transcription in all cells. However, the same results were obtained using sli4.5HHlacZ and sliM4.5HhlacZ in which the slit regulatory element is fused to a heterologous hsp70 promoter. Since mid was expressed in the lateral cells but not in midline glia, these results suggest that Mid-binding sites in slit control slit transcription via binding to multiple factors: Mid in lateral cells and unknown factor(s) in midline glia. Taken together, these results demonstrate a direct role for Mid in the regulation of frazzled and slit, and suggest that Mid governs the expression of multiple axon guidance genes through directly binding of the Mid sites in their regulatory regions (Liu, 2009).

This study has shown that Mid directly controls transcription of key components of the two major axon guidance systems: the Netrin/Frazzled system and the Slit/ROBO system. Because these two systems are considered to have opposing outputs, it is interesting that the expression of both systems are induced by the same transcription factor, Mid. Dynamic expression of Frazzled and ROBO is required for growth cones to simultaneously respond to both attractants and repellents, integrate these signals, and then respond to the relative balance of forces. These molecules also provide nonautonomous functions required for cell motility, such as mediating cell adhesion and promoting axon elongation. The coordination of axon guidance systems by Mid may thus ensure cooperative actions of multiple guidance molecules in growth cone dynamics, axonal adhesion, and elongation. The role of Mid in the transcriptional regulation of axon guidance might be a conserved function, because its orthologs of human, mouse, and zebrafish Tbx20 are also expressed in motor neurons (Liu, 2009).

Intra-axonal patterning: intrinsic compartmentalization of the axonal membrane in Drosophila neurons

In the developing nervous system, distribution of membrane molecules, particularly axon guidance receptors, is often restricted to specific segments of axons. Such localization of membrane molecules can be important for the formation and function of neural networks; however, how this patterning within axons is achieved remains elusive. This study shows that Drosophila neurons in culture establish intra-axonal patterns in a cell-autonomous manner; several membrane molecules localize to either proximal or distal axon segments without cell-cell contacts. This distinct patterning of membrane proteins is not explained by a simple temporal control of expression, and likely involves spatially controlled vesicular targeting or retrieval. Mobility of transmembrane molecules is restricted at the boundary of intra-axonal segments, indicating that the axonal membrane is compartmentalized by a barrier mechanism. It is proposed that this intra-axonal compartmentalization is an intrinsic property of Drosophila neurons that provides a basis for the structural and functional development of the nervous system (Katsuki, 2009).

This study describes a patterning phenomenon that takes place within single axonal processes as a cell-intrinsic event. This patterning involves compartmentalization of the axonal membrane with a diffusion barrier located at a medial point of the axon. The data suggest that this patterning ability is a fundamental property of Drosophila neurons, because the compartment-specific localization of GFP-tagged receptors can be observed in the majority (>90%) of neurons. In the CNS of Drosophila, more than 90% of neurons project their axons to the contralateral side of the nervous system, and the width of the commissural segment or precrossing segment of those neurons is 20-40 μm, which parallels the length of the proximal compartment observed in vitro. This raises the possibility that the intrinsic patterning ability of neurons may serve as the basis of generating the intra-axonal localization of guidance molecules in vivo (Katsuki, 2009).

In addition to these intrinsic abilities of neurons, the results suggest that extrinsic factors may also contribute to the intra-axonal patterning, because not all ROBO receptors examined in this study recapitulated the localization patterns observed in vivo. All three members of ROBO family receptors are localized to distal axon in vivo. Whereas ROBO2 and ROBO3 retained the ability to localize distally when isolated in culture, ROBO was uniformly distributed along axons under such conditions. Localization of ROBO may require extrinsic signals that are absent in the culture system. One of the candidate extrinsic factors are the midline cells, which lie on an axonal region where ROBO expression is downregulated in vivo. It is also possible that the location of the compartment boundary determined by the intrinsic mechanisms is refined by extrinsic signals. It would be interesting to test whether contact with midline cells in culture can induce distal localization of ROBO, or alter the position of the boundary (Katsuki, 2009).

It has been commonly suggested that axon guidance receptors are targeted to the growth cone, and intra-axonal localization patterns of guidance receptors reflect temporal profiles of receptor expression at the growth cone during axonal extension. This study demonstrates that intra-axonal localization patterns that are evident in the culture condition can form regardless of the timing of receptor expression. Although this result does not rule out the involvement of temporal control of expression during axon navigation in vivo, it suggests that critical mechanisms for the intra-axonal localization of receptors described in this study are compartment-specific trafficking pathways. One such trafficking mechanism could involve local translation or targeted membrane transport, which can specifically deposit membrane proteins to either the proximal or distal membrane compartment. It is also possible that membrane proteins are selectively retrieved from one compartment through endocytic pathways (Katsuki, 2009).

A time course experiment in shits1 mutant backgrounds suggests that Derailed (DRL) is preferentially targeted to the proximal compartment. It was also shown that the correct intra-axonal localization of DRL requires Dynamin-dependent endocytosis; however, at present it cannot be distinguished whether or not the endocytosis of DRL is compartment specific. Because fluorescence recovery after photobleaching (FRAP) experiments on CD8-GFP suggest that the barrier between the proximal and distal axon compartments does not completely block the movement of membrane proteins between the compartments, it is possible that the Dynamin-dependent endocytosis is required to remove DRL that leaks into the distal compartment, serving to maintain the pattern generated by targeting. Alternatively, endocytosis itself may be compartment specific, contributing to the establishment of the pattern (Katsuki, 2009).

In contrast to DRL, ROBO3 does not appear to require shibire function for its localization, demonstrating the presence of differential trafficking mechanisms for DRL and ROBO3. Due to this shi-independence of ROBO3, it is not possible to conclusively demonstrate the presence of preferential targeting of ROBO3 by performing a time course experiment. Even if there is preferential targeting, it is likely that ROBO3 also needs to be removed from the incorrect compartment, because ROBO3 shows a level of lateral mobility on the axon similar to that of DRL. Since ROBO3 localization is largely independent of Dynamin function, such a retrieval pathway must be based on Dynamin-independent mechanisms. While the complementary localization patterns of DRL and ROBO3 suggests that intra-axonal compartments are fundamental units for localization of multiple molecules, molecular mechanisms for generating or maintaining their compartmental localization could be diverse (Katsuki, 2009).

Another critical issue raised in the previous studies in vivo is how the intra-axonal localization of guidance receptors is maintained over time. If the guidance receptors are freely diffusible on the axonal membrane, they may spread along the axon, leading to a uniform distribution. FRAP experiments in cultured neurons revealed that localized receptors (ROBO3-EGFP and DRL-EGFP) are indeed mobile within the intra-axonal compartment. Although the mobility of these localized receptors across the compartment boundary was not directly measurable, the mobility of several transmembrane proteins (ROBO-EGFP and CD8-GFP) and lipid-anchored protein (GFPgpi) that distribute along the entire axon length was significantly restricted at the compartment boundary. This restriction is likely due to the diffusion barrier that spans over a 10 μm axon length around the boundary. It is proposed that this barrier is a part of the mechanisms that maintain the pattern of compartment-specific membrane proteins, as shown in different subcellular regions such as the tight junction of epithelial cells, the posterior ring of sperm, the cleavage furrow of dividing yeast and mammalian cells, and the initial segment of mammalian neurons. No significant barrier effect on GAP-GFP, which resides in the inner leaflet of the plasma membrane, was detected. It was also observed that vesicles containing membrane proteins pass through the barrier region. Thus, a model is favored in which the barrier becomes effective only after the molecules are inserted into the axonal membrane. It would be important to test whether or not a diffusion barrier exists in vivo, and whether or not it plays a role in the development of the nervous system (Katsuki, 2009).

An important but yet poorly explored question is the role of the guidance receptors localized on axon shafts. A straightforward explanation can be offered based on non-cell-autonomous functions of guidance receptors or membrane proteins in general; they may 'label' axon pathways through specific adhesion (fasciculation), or through presenting their ligands, thereby providing instructive spatial cues for the navigation of other axons. For example, Fasciclin cell-adhesion molecules have been suggested to provide pathway labels for guiding other growth cones. Drosophila Netrin receptor Frazzled/DCC relocates its ligand Netrin to strategic positions in the nervous system, thereby generating guidance information for a longitudinal pioneer neuron. Other studies reported that guidance receptors can also play non-cell-autonomous roles in cell migration and synaptogenesis. Thus, spatial patterns of molecules on axon shafts likely have direct roles in neuronal circuit formation (Katsuki, 2009).

Lastly, it is proposed that the compartmentalization of the axonal membrane could be a common basis for the structure and function of the nervous system. In the Drosophila ventral nerve cord, formation of the longitudinal axon tracts depends on the expression of ROBO receptors. On the other hand, longitudinal axon tracts are considered as the site for synapse formation, because synaptic proteins such as synaptotagmin and synapsin accumulate on the longitudinal tracts. This study found that in cultured neurons both ROBO receptors and synaptic proteins localize to the distal axon compartment. This may suggest that the spatial distribution of guidance molecules and synaptic proteins can be collectively governed by the compartmentalization of the axonal membrane. Future work to identify the molecular basis of the compartmentalization, and to establish the link between cellular identity and this intracellular pattern, will aid in determining how intra-axonal patterning contributes to tissue organization (Katsuki, 2009).

Protein Interactions

The cytoplasmic domain of Robo homologs varies considerably in length across species and has very little sequence similarity with the exception of three proline-rich motifs of about ten or more amino acids in length. These three short regions are highly conserved and might potentially function as binding sites for SH3 domains or other domains in linker proteins functioning in Robo-mediated signal transduction. The first conserved cytoplasmic motif contains a tyrosine and is a potential site for phosphorylation. The second conserved motif contains the sequence LPPPP and is a potential site for Drosophila Enabled or its mammalian homolog Mena. Given the role of Drosophila Abl tyrosine kinase in midline guidance and the function of enabled as a suppressor of mutations in Abl, it will be of interest to determine whether Enabled binds the Robo cytoplasmic domain (Kidd, 1998a).

Roundabout (Robo) has been shown to be a repulsive guidance receptor on growth cones that binds to an unknown midline ligand. In the original large-scale mutant screen for genes controlling midline axon guidance, 8 alleles were recovered of robo, 2 alleles of comm, and 13 alleles of slit. At the time, because slit had such a similar axon phenotype to sim, which controls midline cell fate and survival, and because of the lack of good midline markers, there was some uncertainty as to whether slit like sim might also control midline cell fate and survival. As a result, initial attention was placed on robo and comm, two genes that clearly control midline axon guidance. Nevertheless, there was always the lingering possibility that Slit might directly control axon guidance. Slit is a large extracellular matrix protein expressed almost exclusively by midline cells; some Slit protein is found on axons, and the slit mutant displays a striking axon pathway phenotype. In slit mutants, growth cones enter the midline but never leave it. With the advent of better markers for midline cells it was shown that midline cell fate and differentiation are relatively normal in slit mutant embryos, thus suggesting that Slit might indeed control axon guidance. The key result that led to the insight that Slit is likely to be the Robo ligand came from a further analysis of Comm. Overexpression of Comm produces a robo-like phenotype in which axons freely cross and recross the midline. If the copy number of the comm transgene is increased, a more severe phenotype results in which axons enter the midline but fail to leave it, leading to a midline collapse of the CNS axon scaffold. The strongest comm gain-of-function phenotype is highly reminiscent of the slit loss-of-function phenotype and led to an evaluation of Slit as a candidate Robo ligand (Kidd, 1999 and references).

Dosage-sensitive genetic interactions between slit and robo are a good indicator that the two gene products are functionally related. The CNS was examined in embryos transheterozygous for slit and robo, that is, embryos carrying one mutant and one wild-type copy of each gene. Would Fas II positive fascicles (those stained with the 1D4 mAb) abnormally cross the midline, particularly the most medial pCC pathway? In either slit or robo heterozygotes, few guidance defects were observed in these pathways. However, depending upon the combination of alleles used, 26%-39% of the segments examined in embryos transheterozygous for slit and robo had Fas II-positive axons inappropriately crossing the midline. Such a dosage-dependent, transheterozygous phenotype is a strong indication that Slit and Robo function in the same pathway. Double mutants for slit and robo were prepared. The genetic distance between the two loci predicted recovery of the double mutant chromosomes at a frequency of 1 in 8: when null alleles of both slit and robo are used instead, the recovery rate is 1 in 35, indicating that removal of one copy of each locus decreases viability (Kidd, 1999).

In a late stage wild-type embryo, the cell bodies of the RP neurons are readily visible between the two commissures. In robo mutants, typically one or both RP cell bodies are obscured by the increased number of axons abnormally crossing in the commissures. However, the longitudinal part of the scaffold always remains outside (lateral to) the RP cell bodies. In slit mutants, this is not the case. The effect of removing one copy of slit on the robo phenotype was tested. When the spacing of the longitudinal axons was examined, slit was found to dominantly enhance the robo phenotype, as judged by the presence of segments displaying greater medial constrictions than are ever seen in robo mutants alone. In some instances, an RP cell body could be seen lateral to the axon scaffold. If Slit is the Robo ligand, then the double robo;slit mutant phenotype would be predicted to resemble that of a slit mutant alone (due to slit having the more severe phenotype). Embryos homozygous for a recombinant chromosome carrying null alleles of both slit and robo resemble the slit null phenotype (Kidd, 1999).

The commissureless phenotype produced by high-level overexpression of Robo suggests that Robo responds to a repulsive cue at the CNS midline. Slit is a large extracellular matrix protein secreted by the midline glia. Slit was reported to be transferred to axons (albeit at a low level). The mAb used for Slit detection displays only a very low level of axon staining, making an analysis of putative transfer in robo mutant embryos inconclusive. Robo is primarily localized to growth cones of the longitudinal portion of the axon scaffold. These expression patterns are consistent with Slit being the repulsive ligand for Robo because Robo-positive axons avoid areas of high Slit expression. slit embryos were stained with anti-Robo mAb 13C9 and it was found that Robo-positive growth cones were then present at the midline. Staining of the mature CNS in slit mutants reveals that Robo protein levels are unaffected (unlike in comm gain-of-function embryos), and thus Robo is expressed at high levels along the midline. In wild-type embryos, Slit and Robo both localize to the muscle attachment sites in complementary dorsoventral gradients, further suggesting the possibility of a functional relationship (Kidd, 1999).

The effect of high-level overexpression of slit in all postmitotic neurons was examined. The resulting phenotype resembles the robo loss-of-function phenotype. However, when individual axon fascicles are examined, the slit overexpression phenotype appears stronger than the robo loss-of-function phenotype. In addition to aberrant midline crossing by axons in the innermost pCC pathway as seen in robo mutants, the medial and lateral pathways are also disrupted, sometimes crossing the midline. These results suggest that when Slit is panneurally expressed throughout the CNS, growth cones are impaired in their ability to respond to Slit at the midline. A similar effect is seen when Netrins are expressed panneurally: the panneural overexpression phenotype resembles the loss-of-function phenotype. In both cases (Slit and Netrins), these results support the notion that the localized distribution of the guidance signal is of crucial importance and that approximating an even distribution throughout the CNS is equivalent to no expression at all (Kidd, 1999 and references).

Slit was ectopically expressed on muscles; the guidance and connectivity of motor axons was then examined. The ISNb motor axons normally innervate muscles 6, 7, 12, and 13. When their muscle targets abnormally express Slit, their innervation is greatly perturbed. Most of these motor growth cones stall in the vicinity of these muscles and fail to innervate them. This lack of innervation is reminiscent of what is observed when the chemorepellent Semaphorin II is ectopically expressed by the same muscles. The morphology of muscles 6, 7, 12, and 13 ectopically expressing Slit was examined and they are normal in attachment sites, size, and position relative to one another and to the epidermis. The motor axon phenotype is not suppressed by removal of robo activity, providing further evidence that there is more than one Slit receptor. Robo2 is a potential candidate for mediating the motor axon response to ectopic expression of Slit (Kidd, 1999).

After gastrulation in Drosophila, many myoblasts migrate laterally at least five to six cell body diameters away from the ventral midline. This migration occurs over the dorsal surface of the neuroepithelium. Later, some ventral body wall muscles extend back toward the midline ventrally under the developing CNS, normally attaching to the epidermis underneath the CNS at some distance from the midline. In contrast, in slit mutant embryos many developing muscles are found near and at the midline, stretching across the midline dorsally over the CNS. This defect is not seen in robo embryos, although very rarely a single muscle can be seen extending inappropriately dorsally across the CNS, suggesting that Robo participates in this process in conjunction with at least one other receptor (possibly Robo2). However, in robo mutant embryos the ventral muscles are frequently found attached closer to the midline than in wild type, suggesting that Robo may in part prevent muscles from extending too close to the midline. When slit mutant embryos are rescued by slit-GAL4 driving UAS-slit, the ventral muscle pattern is restored to near wild type, confirming that Slit expression at the midline is required for migration of muscle precursors away from the midline (Kidd, 1999).

The axon guidance defects seen in robo mutant embryos in Drosophila suggest that the primary function of Slit in controlling Robo-mediated midline guidance is as a short-range repellent. Growth cones that express high levels of Robo do not extend away from the midline, but rather they avoid entering and crossing the midline. For example, the pCC growth cone expresses high levels of Robo, and it extends anteriorly near the edge of the midline. In a robo mutant, the pCC growth cone freely crosses and recrosses the midline; in a slit mutant, the pCC growth cone enters the midline and does not leave it. Although it is possible that Slit might also function as a long-range chemorepellent during axon guidance in Drosophila, causing some growth cones to extend some distance away from the midline, at present the strongest genetic evidence in Drosophila is for a short-range function. This is in contrast to its function during mesoderm migration and muscle formation. After gastrulation in Drosophila, many myoblasts migrate laterally away from the ventral midline. The ventral body wall muscles normally attach to the epidermis underneath the CNS but stay some distance from and do not cross the midline. In contrast, in slit mutant embryos, many developing muscles are found near the midline, stretching across the midline dorsally over the CNS. The slit mutant muscle defects are nearly identical to those seen in single minded mutant embryos in which the midline cells are missing (Lewis, 1994). In contrast, in slit mutants, the midline cells are present but do not secrete Slit into the extracellular environment (Kidd, 1999).

Genetic analysis of sim (Lewis, 1994) shows that after gastrulation the midline cells are required for the migration of muscle precursor cells away from the midline. Many of these mesodermal cells normally migrate at least five to six cell body diameters away from the midline. In the sim mutant, the precursors do not migrate away from the midline, presumably due to the absence of a midline-derived long-range chemorepellent. Moreover, in the sim mutant the muscle precursors that extend ventrally toward the midline are not prevented from crossing the midline, presumably due to the absence of a midline-derived short-range repellent. Rather, when these misplaced muscle precursor cells undergo myogenesis, they form abnormal contacts with each other that freely extend across the dorsal midline of the CNS. slit mutant embryos display the exact same midline mesoderm phenotypes as do sim mutant embryos. This suggests that Slit is both the long-range chemorepellent controlling mesoderm migration away from the midline and the short-range repellent preventing muscles from crossing the midline. The Robo receptor appears to play only a minor role in the ability of Slit to direct the long-range migration of muscle precursors away from the midline. Either Robo2 or some other Slit receptor must function as the major muscle receptor for Slit-mediated long-range chemorepulsion (Kidd, 1999 and references).

If commissural growth cones are so attracted to Netrin, if the highest concentration of Netrin is at the midline, and if when growth cones arrive at the midline they meet their homologs from the other side for which they have a high affinity, why do these growth cones ever leave the midline? Although the mechanism is not fully understood, the answer to this question has something to do with the qualitatively different ways in which growth cones respond to Slit. For growth cones near the midline that do not cross it, Slit forms a strong repulsive barrier. But for growth cones that do cross the midline, Slit cannot be such a strong repellent, rather functioning in a more subtle fashion, somehow preventing them from lingering at the midline and driving them across. In the absence of Slit, growth cones enter the midline but do not leave it, extending in a single fused longitudinal tract at the midline. Thus, Slit must be part of the anti-linger mechanism. One thing is certain: the ability of Slit to form a repulsive barrier requires the Robo receptor. Any growth cone that expresses high levels of Robo cannot cross the midline. So in a robo mutant, growth cones freely cross and recross the midline, but they do not stay at the midline. Two inferences follow from these observations: (1) there must be at least one additional Slit receptor that controls midline guidance, and at present Robo2 is the best candidate; (2) because Slit appears to have two different functions (one as a midline repulsive barrier and the second as a midline anti-linger signal), it follows that either Robo2 signals differently from Robo, or alternatively, that the low levels of Robo2 alone (or Robo2 and Robo together) on growth cones crossing the midline give rise to a qualitatively different response as compared to high levels of Robo. Whether these are two qualitatively different negative responses, or alternatively, quantitative differences in a common repulsive mechanism, is not yet clear. Teasing this mystery apart in the future should shed some light on how growth cones make stereotyped and divergent decisions at complex choice points (Kidd, 1999).

The availability of expressed Slit proteins enabled an examination of their interactions with Robo proteins. The interaction between Slit and Robo proteins was further demonstrated using soluble forms of the ectodomains of rRobo1, rRobo2, and dRobo1 fused to either the constant region (Fc) of the human immunoglobulin molecule or to alkaline phosphatase. rRobo1-Fc and rRobo2-Fc bind transfected cells expressing hSlit2, and Drosophila Robo bound cells expressing Drosophila Slit. These results indicate a high degree of specificity in the interaction between Slit and Robo proteins. In addition, in cross-species experiments it was found that the binding interactions are evolutionarily conserved. Thus, Drosophila Slit binds cells expressing either rRobo1 or rRobo2, and hSlit2 binds cells expressing dRobo1, although these interactions appeared weaker than those observed within species. Similarly, Robo1-Fc and Robo2-Fc bind cells expressing Drosophila Slit, and Drosophila Robo binds hSlit2-expressing cells. The amino-terminal LRRs of Drosophila and mammalian Slit proteins have homology to a number of ECM molecules, including the laminin-binding molecule biglycan. This prompted an examination of whether Slit proteins can also bind laminins. hSlit2, applied in conditioned medium from transfected cells, binds to a substrate with laminin 1, but not a substrate coated with fibronectin. Since Netrin proteins show homology to a portion of the laminin molecule, whether Netrin 1 and Slit2 can bind one another was examined. Netrin 1 binds to COS cells expressing hSlit2 in a pattern that was indistinguishable from binding observed with Robo1-Fc and Robo2-Fc (Brose, 1999).

Frazzled (Fra) is the DCC-like Netrin receptor in Drosophila that mediates attraction; Roundabout (Robo) is a Slit receptor that mediates repulsion. Both ligands, Netrin and Slit, are expressed at the midline; both receptors have related structures and are often expressed by the same neurons. To determine if attraction versus repulsion is a modular function encoded in the cytoplasmic domain of these receptors, chimeras were created carrying the ectodomain of one receptor and the cytoplasmic domain of the other and their function in transgenic Drosophila was tested. Fra-Robo (Fra's ectodomain and Robo's cytoplasmic domain) functions as a repulsive Netrin receptor; neurons expressing Fra-Robo avoid the Netrin-expressing midline and muscles. Robo-Fra (Robo's ectodomain and Fra's cytoplasmic domain) is an attractive Slit receptor; neurons and muscle precursors expressing Robo-Fra are attracted to the Slit-expressing midline (Bashaw, 1999).

In Drosophila, the same midline cells normally secrete both Netrins and Slit. Growth cones can simultaneously respond to both ligands in a cell-specific fashion. Some growth cones express high levels of Fra and low levels of Robo, and they extend toward and across the midline. Other growth cones appear to express high levels of both receptors, and they can extend toward the midline, but they do not cross it. Growth cones can dramatically change their levels of Robo expression; once they cross the midline, growth cones increase their level of Robo, a change that prevents them from crossing the midline again. Such complex and dynamic behavior requires growth cones to be able to simultaneously respond to both attractants and repellents and to integrate these signals and respond to the relative balance of forces. Introducing a chimeric receptor into this finely tuned system leads to dramatic phenotypes. Adding a receptor that responds to Netrin as a repellent leads to a comm-like phenotype in which too few axons cross the midline. Adding a receptor that responds to Slit as an attractant leads to the opposite robo- or slit-like phenotypes, in which too many axons cross the midline or remain at the midline, respectively. These phenotypes are dose dependent, suggesting that by adding more chimeric receptor, the relative balance can be tipped and in tis way the growth cone's response is selectively controlled. This striking dosage sensitivity raises the possibility of using these phenotypes as the basis for genetic suppressor screens to identify signaling components that function downstream of attractive and repulsive guidance receptors (Bashaw, 1999).

Another finding of this study is that the signal transduction machinery for attraction and repulsion downstream of these receptors appears to be present in all neurons, and probably in all migrating muscle precursors as well. All neurons expressing either Fra-Robo or Robo-Fra appear to behave the same, regardless of their environment: if they express Fra-Robo, they stay away from the midline; if they express Robo-Fra, they extend toward the midline. No other factor appears to intrinsically commit one growth cone or another to only one kind of response. The same is true for migrating muscle precursors. Normally, many of them express Robo and migrate away from the Slit-expressing midline. However, given the opportunity (by transgenic expression of Robo-Fra), they clearly contain the full machinery for the opposite response. In all these transgenic experiments, the growth cone or muscle response always correlated with the level of receptor (Brashaw, 1999 and references).

The finding that the cytoplasmic sequence determines the response of a guidance receptor raises a number of interesting questions. Attraction might lead to a local change favoring actin polymerization over depolymerization, while repulsion might lead to the opposite change. But is guidance that simple? The cytoplasmic sequences of five different families of repulsive guidance receptors are now known: UNC-5s, Eph receptors, Neuropilins, Plexins, and Robos. Interestingly, they appear to share little if any sequence similarity to one another in their cytoplasmic domains. It is possible, of course, that they bind different adapter proteins that converge on the same repulsive motility machinery. But it is equally likely that not all repulsion is the same and that different classes of repulsive receptors mediate different types of responses in the growth cone. It could be that what is lumped together under the term 'repulsion' actually represents several molecularly distinct mechanisms that negatively influence local growth cone behavior. Just what these different cytoplasmic domains do, and how many different types of repulsion exist, awaits future investigation (Bashaw, 1999 and references).

The extracellular sequence similarity between Robo, Robo2, and Robo3 and the robo,robo2 double mutant phenotype, strongly suggests that Robo2 and Robo3 also bind Slit. This was tested in cell culture. Full-length and the N-terminal cleavage fragment of Slit remain predominantly cell associated when transgenically expressed in culture. AP-tagged Robo2 and Robo3 ectodomains bind to Slit-expressing COS cells but not mock-transfected or untransfected cells. The relative affinities of the receptors for Slit were examined using equilibrium binding experiments. The three Robo receptors have dissocation constants (Kds) in the range of 10-40 nM (Simpson, 2000a).

Repulsive axon guidance: Abelson and Enabled play opposing roles downstream of the Roundabout receptor

Drosophila Roundabout (Robo) is the founding member of a conserved family of repulsive axon guidance receptors that respond to secreted Slit proteins. Little is known about the signaling mechanisms that function downstream of Robo to mediate repulsion. Genetic and biochemical evidence is presented that the Abelson (Abl) tyrosine kinase and its substrate Enabled (Ena) play direct and opposing roles in Robo signal transduction. Genetic interactions support a model in which Abl functions to antagonize Robo signaling, while Ena is required in part for Robo's repulsive output. Both Abl and Ena can directly bind to Robo's cytoplasmic domain. A mutant form of Robo that interferes with Ena binding is partially impaired in Robo function, while a mutation in a conserved cytoplasmic tyrosine that can be phosphorylated by Abl generates a hyperactive Robo receptor (Bashaw, 2000).

Abl and Ena are complementary components of the signaling machinery downstream of the Robo repulsive axon guidance receptor. Genetic interactions indicate that loss of ena function partially disrupts Slit- and Robo-mediated repulsion from the midline. Limiting or removing ena function enhances partial loss-of-function robo phenotypes and suppresses robo gain-of-function phenotypes. In contrast, reduction of abl has the opposite consequence, suppressing the effects of a partial loss of robo function, while panneural overexpression of Abl antagonizes Robo function, leading to a phenotype resembling that of robo mutants (Bashaw, 2000).

Both Abl and Ena bind directly to Robo's cytoplasmic domain in vitro and Robo can act as a substrate for Abl kinase activity in vitro and in cell culture. Robo and Ena also show in vivo physical interactions. Furthermore, cytoplasmic domain mutants that reduce Ena binding to Robo result in impaired ability to rescue robo loss of function, while a Y-F mutation in a conserved tyrosine that can be phosphorylated by Abl in vitro has the opposite consequence, generating a hyperactive Robo receptor. These genetic and biochemical data support a model in which Abl and Ena play direct and opposing roles in the transmission of Robo's repulsive signal (Bashaw, 2000).

The implication of Ena in repulsive axon guidance is somewhat surprising in light of the previous results from the pathogen Listeria monocytogenes indicating that Mena is required for Listeria's actin-polymerization dependent motility. The Listeria data, together with the in vitro effects on actin of the Ena/VASP proteins has frequently been interpreted to suggest that Ena/VASP proteins function to promote actin polymerization, thereby promoting motility. On the contrary, the results presented here indicate that Ena is partially required for axon repulsion from the midline. These data suggest that Ena may have the opposite function, namely, to inhibit forward growth cone motility at sites where Robo encounters Slit (Bashaw, 2000).

In a companion paper (Bear, 2000), an independent study in mammalian cell culture has reached a similar conclusion. By expressing a multimerized EVH1 domain binding site attached to specific subcellular localization sequences, Ena/VASP family members can be efficiently targeted to different areas of cultured fibroblasts. This system has allowed a direct examination of the role of Ena/VASP proteins in cell motility. Surprisingly, when Ena/VASP proteins are directed away from the cell membrane, using a mitochondrial targeting sequence, the cells actually migrate more quickly. Conversely, targeting Ena/VASP proteins to the membrane, or overexpressing Mena, leads to a dose-dependent decrease in the rate of cell migration. A major conclusion of this study is that Ena/VASP proteins function in part to decrease the rate of whole cell motility. Whether Ena/VASP proteins achieve the observed in vivo effects on whole cell and growth cone motility by stimulating or inhibiting actin polymerization awaits future investigation (Bashaw, 2000).

While the dosage-sensitive genetic interactions between ena and robo support a role for Ena in midline repulsion, Ena clearly can not explain all of Robo's repulsive output. Indeed, although mild midline crossing defects are observed in ena mutants, on the whole, Robo-mediated repulsion works fairly well in the absence of Ena. In this light, it is perhaps not surprising that the Robo DeltaCC2 mutant receptor (in which the Ena binding site is deleted) still provides some repulsive activity and can partially rescue robo loss-of-function mutants. These results indicate that there must be other proteins that function downstream of Robo to mediate repulsion. One would predict that simultaneously removing ena and the as yet unknown additional factors would reveal stronger disruptions of midline repulsion (Bashaw, 2000).

Thus, Ena is only part of what must be a more complex repulsive output from Robo. Ena helps strengthen the output (perhaps by locally putting the break on the actin-based motility machinery), but is only part of the output. In this light, it is interesting to note that Robo2 also binds Slit and mediates repulsion (albeit apparently more weakly than Robo), but Robo2 does not have the Ena binding site and does not bind Ena (J. Simpson, personal communication to Bashaw, 2000).

An important question for future studies concerns whether Ena is always docked on Robo, or alternatively, whether Slit binding to Robo leads to the recruitment of Ena to Robo's cytoplasmic domain. From what is known about other receptor systems, this second alternative seems more likely, but it remains an open question and needs to be directly tested (Bashaw, 2000).

Genetic analysis shows that Abl antagonizes Robo-mediated repulsion. The two most likely possibilities are that Abl functions to antagonize this pathway by phosphorylating Robo or by phosphorylating Ena. Three results argue in favor of a direct interaction with Robo. (1) Certain kinds of dose-dependent genetic interactions between abl and robo are observed that are not observed between abl and ena, suggesting that the Abl and Robo proteins might directly interact. (2) Biochemical experiments have shown that Abl can directly phosphorylate Robo's cytoplasmic domain at one or more tyrosine residues. (3) A Y-F mutation in a conserved tyrosine that can be phosphorylated by Abl in vitro generates a hyperactive Robo receptor. Taken together, these genetic and biochemical data suggest that it is the dephosphorylated form of Robo that is most active (Bashaw, 2000).

How might Abl normally regulate the output of Robo signaling? Abl-mediated phosphorylation might normally modulate the output of Robo signaling. Alternatively, this phosphorylation might participate more directly in the ligand-gated signal. It is interesting to speculate that it is the binding of Robo to its ligand Slit that triggers dephosphorylation, and that this in turn activates the repulsive response (Bashaw, 2000).

The CNS-specific receptor protein tyrosine phosphatases (RPTPs) RPTP10D and 69D are candidates to be additional factors that contribute to Robo repulsion. Simultaneous removal of these two RPTPs results in substantial ectopic midline crossing, and the double mutant shows dose-sensitive genetic interactions with slit. Whether these two phosphatases interact directly with Robo and whether their phosphatase activity is required for their observed roles in repulsion await future investigation (Bashaw, 2000 and references therein).

In the model presented above, it is attractive to speculate that these two RPTPs function in opposition to the Abl kinase activity by directly dephosphorylating Robo upon Robo's interaction with Slit. Interestingly, the other two Robo family members in Drosophila (Robo2 and Robo3; J. Simpson, personal communication to Bashaw, 2000) share the phosphorylation sites in Robo that are phosphorylated by Abl in vitro. In addition, genetic interactions are observed between the RPTPs and Robo2. Together these observations suggest that perhaps a common mechanism is employed to regulate the signaling output of the three Robo receptors. It will be of interest to determine the in vivo significance of the conserved tyrosine phosphorylation sites in the three Robo receptors. The future elucidation of the events set in motion by ligand binding will require the development of cell culture systems that will allow analysis of the phosphorylation state and cytoplasmic domain associations of the Robo receptors before and after Slit stimulation (Bashaw, 2000).

In addition to their function during Robo signaling shown here, it is clear that both Abl and Ena function in multiple guidance signaling pathways, and thus that they are not committed to repulsion downstream of Robo. In the nematode C. elegans, ena acts as a suppressor of the axon migration defects associated with ectopic expression of the UNC5 repulsive Netrin receptor. This raises the possibility that ena functions downstream of diverse repulsive guidance receptors. In Drosophila, during motor axon pathfinding, ena and abl play roles in ISNb choice point control. Overexpression of abl or loss of ena generates an ISNb 'bypass' phenotype, where the ISNb fails to defasciculate and branch off at the appropriate location to enter its muscle target region. This phenotype is also observed in mutations in Dlar, the gene encoding a receptor protein tyrosine phosphatase (RPTP). Mutations in all three of these genes (ena, abl,) and Dlar) give rise to only partially penetrant ISNb guidance phenotypes and appear to modulate guidance decisions at this choice point. At the midline, mutations in the genes encoding the ligand (Slit) and a key receptor (Robo) have strong and highly penetrant midline guidance phenotypes. In contrast, mutations in the genes encoding Ena, Abl, and RPTP10D and RPTP69D on their own have weaker and less penetrant phenotypes. This is consistent with Abl and the RPTPs modulating Robo receptor output, and Ena mediating only part of Robo output (Bashaw, 2000 and references therein).

If this same logic is applied to the motor axon ISNb choice point, then it is likely that some of the key components are still missing. At present, the only gene with a nearly 100% penetrant bypass phenotype at this choice point is sidestep. Side is an Ig superfamily transmembrane protein that is expressed on muscle surfaces and appears to function as an attractive ligand for motor axons. The Side receptor is not known. Whether the key receptor is the Side receptor or not, it is likely that the major growth cone receptor for the ISNb choice point has not yet been identified (Bashaw, 2000 and references therein).

In this context, it is tempting to speculate, by analogy with the proposed model for Robo signaling, that at the ISNb motor axon choice point, DLAR and Abl play complementary roles in modulating the output activity of the hypothetical guidance receptor, while Ena functions as part of the receptor output. In this way, the two guidance decisions -- to cross or not to cross the midline, and to fasciculate or defasciculate from other motor axons -- use different signals on the outside of the growth cone, but similar signaling and regulatory mechanisms on the inside. It is suggested that once the signal crosses the membrane, in both cases the output is regulated in opposing directions by Abl vs. one or more RPTPs, and that the output is partially mediated by Ena. This model provides a unifying way of viewing signal transduction during these two different guidance decisions. It will be interesting in the future to see to what degree this model holds up in terms of both the role of phosphorylation in modulating receptor output, and the role of Ena in mediating part of repulsive signaling (Bashaw, 2000).

Repellent signaling by Slit requires the leucine-rich repeats

Slit is a repellent axon guidance cue produced by the midline glia in Drosophila that is required to regulate the formation of contralateral projections and the lateral position of longitudinal tracts. Four sequence motifs comprise the structure of Slit: a leucine-rich repeat (LRR), epidermal growth factor-like (EGF) repeats, a laminin-like globular (G)-domain, and a cysteine domain. The LRR is required for repellent signaling and in vitro binding to Robo. Repellent signaling by slit is reduced by point mutations that encode single amino acid changes in the LRR domain. By contrast to the EGF or G-domains, the LRR domain is required in transgenes to affect axon guidance. The midline repellent receptor, Robo, binds Slit proteins with internal deletions that also retain repellent activity. However, Robo does not bind Slit protein missing the LRR. Taken together, these data demonstrate that Robo binding and repellent signaling by Slit require the LRR region (Battye, 2001).

To learn more about the structural requirements for repellent signaling by Slit, attempts were made to rescue slit mutants with midline expression of slit transgenes lacking internal sequences. A slit transgene lacking the LRR fails to restore midline guidance and fails to generate effects after ectopic expression. Furthermore, in vitro translated Slit, which lacks a full LRR, does not bind to Robo, the repellent receptor. Point mutations encoding single amino acid changes in the LRR also greatly reduced repellent signaling. These data indicate that the LRR of Slit is required for receptor binding and repellent signaling (Battye, 2001).

Comm sorts Robo to control axon guidance at the Drosophila midline

Axon growth across the Drosophila midline requires Comm to downregulate Robo, the receptor for the midline repellent Slit. comm is required in neurons, not in midline cells as previously thought, and it is expressed specifically and transiently in commissural neurons. Comm acts as a sorting receptor for Robo, diverting it from the synthetic to the late endocytic pathway. A conserved cytoplasmic LPSY motif is required for endosomal sorting of Comm in vitro and for Comm to downregulate Robo and promote midline crossing in vivo. Axon traffic at the CNS midline is thus controlled by the intracellular trafficking of the Robo guidance receptor, which in turn depends on the precisely regulated expression of the Comm sorting receptor (Keleman, 2002).

comm is required in commissural neurons for crossing. Conversely, forced expression of comm in ipsilateral neurons is sufficient to reroute them across the midline. It is therefore anticipated that comm is expressed in commissural neurons but not in ipsilateral neurons. Furthermore, since comm is expressed in such a dynamic pattern, it is of interest to enquire whether commissural neurons might only express comm since their axons grow across the midline. To explore these ideas, comm expression was surveyed in a set of identifiable neurons for which specific axonal markers are available, thus allowing comm expression to be correlated with growth cone behavior (Keleman, 2002).

The set of neurons examined included both commissural and ipsilateral neurons, and in each class both motor neurons and interneurons. The commissural neurons examined were (1) the RP1, 3, 4, and 5 motor neurons, which express the lim3A-taumyc reporter; (2) the cluster of 10–15 lateral EG interneurons labeled by eg-GAL4; (3) the three EW interneurons, also labeled with eg-GAL4; (4) the drlU intersegmental interneuron, identified with drlU-taumyc; and (5) the Sema2b intersegmental interneuron in each of the A4-A8 hemisegments, identified with Sema2b-taumyc. The ipsilateral neurons examined were (1) the aCC motor neuron and (2) the pCC intersegmental interneuron (both of which are labeled by anti-FasciclinII MAb 1D4) and (3) the dorsal Ap intersegmental interneuron, labeled with ap-GAL4 (Keleman, 2002).

Analysis of comm expression in these neurons reveals a striking correlation between comm expression and a contralateral projection: all of the commissural neurons and none of the ipsilateral neurons express comm. At least for this set of neurons, the correlation is perfect, with just two minor caveats: (1) for the EG neurons, and to a lesser extent the RP and EW neurons, it cannot be entirely certain that every single neuron in these clusters expresses comm, though the impression was gained that this is likely to be the case; (2) the Sema2b neuron can only be identified after its axon has crossed the midline, at which time comm expression appears to be stochastic. Stochastic expression of comm is also seen in the Ap ipsilateral neuron at later stages, although it is consistently negative for comm, as its axon first contacts the midline and turns to avoid it (Keleman, 2002).

There is a striking temporal correlaton between comm expression and midline crossing. The RP, drlU, EG, and EW neurons all extinguish their comm expression shortly after their axons have crossed the midline. Interestingly, the EG and EW neurons, which are the only commmissural neurons that could be identified before their axons reach the midline, are also clearly negative for comm prior to crossing. In particular, the EW axons grow anteriorly for a short distance before turning medially to cross the midline. These neurons do not appear to express comm until they make this medial turn (Keleman, 2002).

Ipsilateral neurons and postcrossing commissural neurons express robo but not comm, and Robo levels are high in the growth cone, whereas crossing commissural neurons express both robo and comm, and Robo levels are low. How does coexpression of Comm prevent Robo from accumulating in the growth cone? To address this question, studies sought to mimic these two situations by expressing comm and robo alone or together in cultured cells (Keleman, 2002).

In COS cells that express robo alone, Robo protein is present mainly at the plasma membrane, as well as in the Golgi and endoplasmic reticulum. Only a small amount of Robo can be detected in endosomes. In contrast, in cells that express both robo and comm, most Robo protein is in late endosomes and lysosomes, where it colocalizes with Comm. This endosomal staining of Comm is also seen in the absence of Robo and is reminiscent of the punctate distribution of endogenous Comm in neurons. Comm can also usually be detected in the Golgi but not at the plasma membrane. Plasma membrane staining is normally only seen in cells expressing particularly high levels of Comm, suggesting that the machinery that sorts Comm to the endosomal compartment can be saturated (Keleman, 2002).

It is concluded that Comm is normally sorted to the late endosomal-lysosomal system and can also recruit Robo to this compartment. To test whether this effect might be specific for Robo, it was asked whether Comm could also recruit the Netrin receptor Frazzled (Fra) to endosomes. It can not. Using Fra-Robo and Robo-Fra chimeric receptors, in which the cytoplasmic domains of the two receptors had been swapped, it was further shown that the ability of Comm to recruit Robo to endosomes requires only the extracellular and/or transmembrane domains of Robo (Keleman, 2002).

To test for a physical association between Robo and Comm, lysates from cells expressing both proteins were immunoprecipitated with antibodies against either the HA tag on Robo or the myc tag on Comm, and then probed on Western blots with anti-myc. Comm protein precipitated with anti-myc appears to exist in three major forms, one that migrates at around 40 kDa, the predicted size of the unmodified protein, and two slower-migrating forms of about 52 kDa and 55 kDa that presumably carry some posttranslational modification. Comm can also be detected in the anti-HA precipitates, indicating that Robo indeed associates with Comm. Interestingly, Robo associates preferentially with the modified forms of Comm. Using Fra and the chimeric receptors, it was shown that this association is specific and that it also requires the extracellular and/or transmembrane domains of Robo. The association between Robo and Comm does not require their colocalization in endosomes, since Robo and the Robo-Fra chimera also associate with a mutant form of Comm (L229A,P230A) that is not sorted to endosomes but is instead delivered to the plasma membrane. Additional data provide evidence that, in the presence of Comm, very little if any Robo is trafficked to endosomes via the cell surface. It is concluded that Comm does not collect Robo at the plasma membrane, but rather sorts it directly from the trans-Golgi network to late endosomes (Keleman, 2002).

Attempts were made to identify the endosomal sorting signal in the Comm cytoplasmic domain. Comm contains a predicted binding site for heterotetrameric adaptor (AP) proteins, which could potentially mediate endosomal sorting. Otherwise, there is no obvious candidate sorting signal, nor any region of significant homology to other known proteins. Additional comm-like genes were sought in Drosophila and in the mosquito Anopheles gambiae. Drosophila has two other genes with some similarity to comm, which are referred to as comm2 and comm3, and for both of these full-length cDNAs were recovered. In the Anopheles genome a single predicted gene related to comm was identified. Functional characterization of these genes is still in progress, but preliminary data indicate that Drosophila comm2 is also able to downregulate Robo proteins in vivo. These four predicted insect Comm proteins are of a similar size and structure but are poorly conserved, with only 15%–20% identity between any pair. Their cytoplasmic domains do, however, contain a highly conserved region of 22 amino acids (residues 215–236 in Comm). The putative AP binding site in Comm (YPSL, residues 251–254) is conserved in Comm2 (YPSV) but not in Comm3 or Anopheles Comm (Keleman, 2002).

To map Comm's endosomal sorting signal, a series of deletion and alanine-scanning mutations were generated within the cytoplasmic domain. The localization of these mutant Comm proteins was examined in COS cells, both in the absence and presence of Robo. In all cases where Comm was correctly targeted to endosomes, Robo goes with it, consistent with the view that the interaction between Comm and Robo does not require their respective cytoplasmic domains. These studies defined a region of 25 amino acids that, together with the 15 amino acids that were left untouched in the juxtamembrane domain, is sufficient for targeting Comm (and Robo, if coexpressed) to endosomes. This region excludes the putative AP binding site, but contains most of the highly conserved residues. Within this region, an LPSY motif is critical for endosomal sorting. If it is mutated, then Comm is found mainly at the plasma membrane. Only a minor fraction of the mutant Comm protein is found in endosomes, possibly reaching this compartment by endocytosis from the plasma membrane rather than direct trafficking from the Golgi. This LPSY motif is also present in each of the other Comms (as LPTY in Comm3). Comm3 and Anopheles Comm even have a second LPSY motif within the conserved region, where Comm has PPCY (Keleman, 2002).

Each of the mutant Comm proteins was tested for its ability to downregulate Robo and promote midline crossing in vivo. It was reasoned that if Comm also sorts Robo to late endosomes in vivo and this is its only function in midline crossing, then the LPSY sorting motif should be the only part of Comm's cytoplasmic domain needed for its function in vivo. The mutant Comm proteins that were mislocalized to the plasma membrane should all be nonfunctional in vivo, while those that are correctly sorted should still be functional, even though they lack most of the cytoplasmic domain (Keleman, 2002).

To test these predictions, flies were generated carrying UAS transgenes encoding each of the mutant Comm proteins that had been tested in COS cells. These transgenes were then expressed in all CNS neurons using the elav-GAL4 driver. These embryos were examined with anti-myc antibodies to determine the expression and localization of the transgenic Comm protein, with anti-Robo MAb 13C9 to test for the ability of the mutant Comm protein to downregulate Robo, and with MAb 1D4 to detect any misrouting of longitudinal axons across or along the midline, the hallmarks of the robo and slit loss-of-function phenotypes (Keleman, 2002).

Consistent with the hypothesis that Comm sorts Robo to endosomes in vivo, a striking correlation was found between the sorting of a mutant Comm protein to endosomes in COS cells and its function in vivo. In particular, the 25 amino acid region of Comm's cytoplasmic domain that is sufficient for endosomal sorting in vitro is also sufficient for Comm to downregulate Robo and promote midline crossing in vivo. Conversely, point mutations in the LPSY motif completely abolish Comm function in vivo, just as they prevent endosomal sorting in vitro. Comm mutants that were sorted to endosomes in COS cells also showed a punctate intracellular localization in vivo, but in general were difficult to detect, even though they were fully functional. Indeed, it was only the nonfunctional Comm proteins that could readily be detected in vivo. This seemingly paradoxical result is, however, in complete agreement with the view that the essential function of Comm in vivo is to be degraded in lysosomes, taking Robo with it. This function is critically dependent on the same LPSY motif that targets Comm (and Robo) directly from the Golgi to late endosomes and lysosomes in vitro (Keleman, 2002).

What is the basis for the specificity of Comm's action? Why are only commissural axons allowed across the midline, and why only once? Previous models have proposed that Robo levels may initially be lower in commissural neurons than in ipsilateral neurons, or that only commissural neurons might express a cell surface receptor needed for the uptake of Comm from midline glia. Analysis of comm expression in the CNS offers a much simpler explanation for its specificity: in general, only commissural neurons express comm, and only as they cross (Keleman, 2002).

These data thus suggest a simple model in which comm expression is the intrinsic switch that specifies an ipsilateral versus a contralateral projection—OFF for ipsilateral, ON for contralateral. This switch appears to be regulated not only spatially but also temporally, since comm generally goes OFF in a commissural neuron after crossing. Early markers needed to determine whether comm is usually ON or OFF before crossing are lacking, but it is noted that for the few commissural neurons that can be identified early (the EW and EG neurons), comm is initially OFF. What turns comm ON and then OFF again to allow just a single passage across the midline? One possibility is that each commissural neuron is intrinsically programmed for a brief pulse of comm expression. Alternatively, comm expression might be controlled by retrograde signals sent from the growth cone to inform the nucleus of its arrival at the midline and its successful passage across. This is an appealing idea, since such a mechanism would uncouple the ability of the growth cone to cross the midline from the precise time of arrival and duration of transit (Keleman, 2002).

Understanding Comm's function in midline crossing has also been hindered by the fact that its molecular function was unknown and its amino acid sequence provided no obvious clues. The data suggest that Comm is a sorting receptor, recognizing Robo via its lumenal and/or transmembrane domain and consigning it for delivery from the trans-Golgi network to late endosomes. Robo may not be the only cargo for Comm. From gain-of-function genetic experiments, it is inferred that Comm also selects Robo2 and Robo3 for delivery to endosomes. Analogous gain-of-function studies suggest that Comm2 also sorts Robo receptors, with a preference for Robo2, while Comm3 may not sort any of the three Robos. The Comm proteins thus define a new family of sorting receptors, the cargo of which include, but may not be limited to, the Robo family of guidance receptors (Keleman, 2002).

The view that Comm is an endosomal sorting receptor and that comm expression is a cell-autonomous switch for midline crossing leads to a model in which axon traffic at the midline is controlled by regulating the intracellular trafficking of Robo, the receptor for the midline repellent Slit. If comm is OFF, Robo is packaged into vesicles for delivery to the growth cone. The insertion of these vesicles at the growth cone confers sensitivity to Slit, thereby preventing growth across the midline. Conversely, if comm is ON, Comm sorts Robo into vesicles destined for late endosomes and lysosomes. Membrane vesicles delivered to the growth cone contain only very low levels of Robo, and so the axon can grow unimpeded across the midline by inserting these vesicles at its tip (Keleman, 2002).

One requirement for this model is that, in order to prevent a commissural axon from recrossing, Comm protein, like comm mRNA, should rapidly disappear after crossing, or at least lose its ability to sort Robo. It is believed that Comm is indeed rapidly degraded in vivo. In contrast to COS cells, very little Comm protein can be detected in vivo, even when the GAL4-UAS system is used to express high levels of comm mRNA throughout the CNS. Only if the LPSY endosomal sorting motif is mutated can Comm accumulate to appreciable levels in vivo, in this case at the plasma membrane. This suggests that, unlike other sorting receptors, Comm may not be recycled back to the Golgi for repeated rounds of sorting but instead be degraded along with its cargo in lysosomes. Other mechanisms may also exist to inactivate Comm after crossing, for example by altering the posttranslational modifications that appear to be necessary for it to recognize Robo (Keleman, 2002).

Drosophila Nedd4, a ubiquitin ligase, is recruited by Commissureless to control cell surface levels of the roundabout receptor

Crossing the midline produces changes in axons such that they are no longer attracted to the midline. In Drosophila, Roundabout reaches high levels on axons once they have crossed the midline, and this prohibits recrossing. Roundabout protein levels are regulated by Commissureless. Commissureless binds to and is regulated by the ubiquitin ligase DNedd4. The ability of Commissureless to regulate Roundabout protein levels requires an intact DNedd4 binding site and ubiquitin acceptor sites within the Commissureless protein. The ability of Commissureless to regulate Robo in the embryo also requires a Commissureless/DNedd4 interaction. These results show that changes in axonal sensitivity to external cues during pathfinding across the midline makes use of ubiquitin-dependent mechanisms to regulate transmembrane protein levels (Myat, 2002).

Within the embryo, Comm protein is located both at the cell surface and within intracellular vesicles in midline cells and commissural axons. This distribution is suggestive of a protein that can move between different locations in the cell. Robo, however, is expressed on the surface of longitudinal axons. Comm can regulate Robo protein levels, and the proteins are occasionally coexpressed in the same cell in the embryo when the Robo protein is found within intracellular vesicles with Comm. Thus, Comm may internalize Robo as part of its regulation of Robo. When Comm is expressed in Drosophila S2 cells, the protein displays a similar distribution to that seen in the embryos with the majority of the protein within intracellular vesicles. Robo, as expected, is expressed on the cell surface when expressed alone in S2 cells. However, when Comm and Robo are expressed together in S2 cells, the Robo protein is no longer found at the cell surface but is now colocalized with Comm within intracellular vesicles within the cell. Thus, Comm is able to change the site of Robo localization within the cell. This ability correlates with the observation in the embryo that overexpression of Comm results in the reduction of Robo protein at the cell surface. This study shows that the normal intracellular distribution of Comm requires an interaction with DNedd4. Removal or disruption of either the DNedd4 binding sites or the intracellular lysines in Comm or the reduction of DNedd4 levels in S2 cells results in Comm accumulating at the cell surface. Comm is no longer brought into the cell and is unable to remove coexpressed Robo from the cell surface. Thus, DNedd4 is a key cofactor that allows Comm to harness the ubiquitination pathway to target its removal from the cell surface together with other membrane receptors it may bind (Myat, 2002).

To test whether DNedd4 has an important role in Comm function, neural overexpression of Comm was used as a sensitive assay. Overexpression of a single copy of comm within all CNS neurons results in the downregulation of Robo in these cells and the production of a robo phenocopy where axons recross the midline. When the level of overexpressed Comm is increased, the phenotype becomes more severe and many axons remain at the CNS midline. Overexpression of DNedd4 alongside one copy of comm produces a phenotype similar to that seen when greater levels of comm are overexpressed. The presence of additional DNedd4 makes the overexpressed Comm more effective at downregulating Robo activity, suggesting it does indeed act with Comm to regulate Robo levels in the embryo. This is supported by the observation that the overexpression of a catalytically inactive form of DNedd4 partially suppresses the ability of overexpressed comm to cause a robo phenocopy. Thus, one activity of DNedd4 in Drosophila is to function with Comm to regulate Robo protein levels. Extrapolating from S2 cell observations, it is assumed that a similar process is taking place in the embryo whereby Comm acts with DNedd4 to internalize Robo into the cell. This suggests that normally Comm and DNedd4 function together in commissural neurons to reduce Robo activity and allow axons to cross the midline. Comm accumulates within commissural axons, and recent experiments have revealed that comm is expressed within these axons with Comm protein only reaching high levels at the midline (Myat, 2002).

Nedd4 family proteins regulate the internalization of a number of cell surface proteins. Nedd4 regulates levels of the epithelial Na+ channel, while the yeast homolog Rsp5 catalyzes the internalization of a number of membrane transporters. Although Nedd4 was identified in a screen for transcripts expressed in the mouse nervous system during embryonic development, no targets for this molecule during neural development have yet been identified. DNedd4 can regulate Comm and consequently, Robo. Yet, removal of DNedd4 function in the embryo does not give rise to the same phenotype as a loss of comm. If DNedd4 was acting purely within the Comm pathway to regulate Robo protein levels, then one might expect that a loss of DNedd4 function would give rise to a comm-like phenotype, since Robo protein levels may stay high. However, inhibition of DNedd4 could result in the stabilization of Comm on neuronal membranes where it can bind Robo and possibly interfere with Robo function to produce a partial robo-like phenotype. RNA interference with DNedd4 gives rise to a phenotype where axons stall at the junction of the longitudinal and commissural axon tracts, resulting in thinner longitudinal and commissural axon tracts (i.e., neither a comm nor robo phenocopy). This phenotype suggests that DNedd4 may also regulate the cell surface levels of other axon guidance molecules. Additionally, DNedd4 may also affect neuronal fate decisions since a close homolog, Su(dx), acts as a regulator of Notch signaling. The isolation of DNedd4 loss-of-function mutations will aid full evaluation of the exact roles of DNedd4 in the embryo (Myat, 2002).

Local caspase activation interacts with Slit-Robo signaling to restrict axonal arborization

In addition to being critical for apoptosis, components of the apoptotic pathway, such as caspases, are involved in other physiological processes in many types of cells, including neurons. However, very little is known about their role in dynamic, nonphysically destructive processes, such as axonal arborization and synaptogenesis. This study shows that caspases are locally active in vivo at the branch points of young, dynamic retinal ganglion cell axonal arbors but not in the cell body or in stable mature arbors. Caspase activation, dependent on Caspase-3, Caspase-9, and p38 mitogen-activated protein kinase (MAPK), rapidly increased at branch points corresponding with branch tip addition. Time-lapse imaging revealed that knockdown of Caspase-3 and Caspase-9 led to more stable arbors and presynaptic sites. Genetic analysis showed that Caspase-3, Caspase-9, and p38 MAPK interacted with Slit1a-Robo2 signaling, suggesting that localized activation of caspases lie downstream of a ligand receptor system, acting as key promoters of axonal branch tip and synaptic dynamics to restrict arbor growth in vivo in the central nervous system (Campbell, 2013).

Slit stimulation recruits Dock and Pak to the Roundabout receptor and increases Rac activity to regulate axon repulsion at the CNS midline

Drosophila Roundabout is the founding member of a conserved family of repulsive axon guidance receptors that respond to secreted Slit proteins. Evidence is presented that the SH3-SH2 adaptor protein Dreadlocks (Dock), the p21-activated serine-threonine kinase (Pak), and the Rac1/Rac2/Mtl small GTPases can function during Robo repulsion. Loss-of-function and genetic interaction experiments suggest that limiting the function of Dock, Pak, or Rac partially disrupts Robo repulsion. In addition, Dock can directly bind to Robo's cytoplasmic domain, and the association of Dock and Robo is enhanced by stimulation with Slit. Furthermore, Slit stimulation can recruit a complex of Dock and Pak to the Robo receptor and trigger an increase in Rac1 activity. These results provide a direct physical link between the Robo receptor and an important cytoskeletal regulatory protein complex and suggest that Rac can function in both attractive and repulsive axon guidance (Fan, 2003).

Strong defects in embryonic axon guidance are observed only when both the maternal and zygotic components of dock function are removed. In these maternal minus dock mutants (dockmat), phenotypes reminiscent of loss of robo function can often be seen. dockmat embryos examined with an antibody that labels all axons frequently show thickening of commissural axon bundles and a commensurate reduction in the thickness of longitudinal axon bundles. Staining these embryos with an antibody that selectively labels noncrossing axons (anti-fasII) reveals a significant degree of ectopic midline crossing. These phenotypes are similar to, but considerably less severe than, those observed in robo mutants. The similarity in mutant phenotypes that is observed provides genetic support for the idea that dock could contribute to Robo repulsion (Fan, 2003).

If dock and robo function together during midline guidance, they should be coexpressed in embryonic axons. This is indeed the case. Double labeling of embryos with antibodies raised against Dock and Robo reveals substantial coexpression of the two proteins. Both Dock and Robo show enriched expression on CNS axons beginning as early as stage 12, corresponding to the time of initial axon outgrowth. At these early stages of axon growth, Dock is detected in the pCC axon, a cell known to express Robo, as revealed by double labeling with FasII. Interestingly, while Robo shows a regionally restricted expression pattern with high levels of expression on longitudinal portions of axons and low levels in commissural axons, Dock is expressed equivalently in both commissural and longitudinal axon segments. This observation raises the possibility that Dock could have additional roles in the guidance of commissural axons not shared by Robo. These observations show that Dock and Robo are both present at the right time and place to function together during midline repulsion (Fan, 2003).

Biochemical data suggests that the interaction between Dock and Robo is an SH3-dependent interaction and that the first two SH3 domains of Dock are most important for mediating Robo binding. Based on the observations that a three-protein interaction can be detected between Robo, Dock, and Pak and that Pak has been shown to interact with the SH3-2 domain of Dock, it is believed that the SH3-1 domain is the most important for Robo and Dock binding. Furthermore, Slit stimulation enhances Dock's ability to bind to Robo, suggesting a ligand-regulated SH3 domain interaction. This represents a different kind of adaptor interaction to many that have been observed previously, where Nck appears to interact with a number of tyrosine-kinase receptors through an SH2 domain/phosphotyrosine interaction. In the latter case, how ligand binding to the receptor regulates the Nck SH2 domain interaction is quite well understood. The observation that the Robo receptor shows a ligand-regulated SH3 domain interaction with Dock/Nck suggests that somehow ligand binding results in an increased availability of the SH3 binding sites in the receptor (Fan, 2003).

The regions of Robo that appear to be most important for the interaction are the proline-rich regions CC2 and CC3. Individual mutations in these motifs strongly reduce the amount of Dock that coimmunoprecipitates with Robo in cell culture, while removing both of these motifs completely abolishes binding. Furthermore, expression of Robo receptors that lack the CC2 and CC3 motifs in transgenic Drosophila disrupt the in vivo function of the receptor. It is important to stress that the CC2 and CC3 sequences are not only involved in Dock binding, but also bind Ena, Abl, and potentially other proteins as well. In addition, CC2 and CC3 are also required for the observed upregulation of Rac activity. The fact that many proteins bind Robo at these sites prevents clear conclusions about why the ΔCC2ΔCC3 mutant receptor is nonfunctional. In the future, more precisely defining the binding requirements of the many proteins that interact with Robo may allow forms of Robo to be created that specifically disrupt the binding of some partners and not others, which in turn should provide insight into the relative roles of different Robo signaling outputs (Fan, 2003).

The implication of Rac in Robo repulsion (dominant negative Rac1 shows a strong enhancement of slit;robo/+ defects) was unexpected in view of the well-established role of Rac as a positive regulator of axon outgrowth. On the surface, this finding appears quite contradictory to the function of Rac to promote actin polymerization at the leading edge of motile cells and axons. One possible explanation of this finding is that perhaps Rac can have different or even opposite effects on the actin cytoskeleton, depending on the molecular context in which it is activated and its overall level of activity. For example, depending on the coordinate local function of other small GTPases and actin regulatory proteins, the consequences of Rac function could be different. It is interesting to note that in addition to a role for Rac, genetic analysis and previously published data also support an important role for Rho in midline repulsion. Furthermore, in addition to strongly stimulating Rac activity, Slit has been shown have a modest stimulatory effect on Rho activity. The implication of both Rac and Rho in mediating repulsive responses has also been suggested to explain the output of the Plexin receptor. It will be interesting in the future to determine the interrelationship between Rac and Rho outputs in the context of Robo repulsion as well as in signaling downstream of other attractive and repulsive axon guidance receptors (Fan, 2003).

As an alternative to the context- and level-dependent explanation of the role of Rac in Robo repulsion, the observed axon steering defects in embryos where both Rac and Slit function are reduced, or in embryos deficient for multiple rac genes, could be explained as a secondary consequence of defects in the rate of axon extension. In this scenario, Rac's role in repulsive axon guidance would be intimately coupled with its role in axon outgrowth. That is to say, that appropriate steering decisions go hand and hand with the appropriate regulation of the rate of axon outgrowth (e.g., you are more likely to miss your exit if you are driving too fast). In this regard, it is important to emphasize that even repulsive cues can have stimulatory effects on axon extension. For example, in addition to repelling Xenopus spinal neurons, Slit also has a stimulatory effect on the rate of axon extension (Fan, 2003 and references therein).

Perhaps the most difficult observation to explain is how reciprocal shifts in Pak levels can lead to similar consequences for Robo repulsion. Since the enhancing effects of Pak overexpression in partial loss-of-function robo backgrounds are more dramatic with the membrane-tethered form of Pak, it is tempting to speculate that in order to signal properly, turning Pak activity on and off needs to be tightly controlled. Little is known about how Pak signaling is terminated and it seems quite possible that the membrane-tethered version of pak is not as effectively regulated as the wild-type form of pak. Interestingly, in genetic backgrounds where robo signaling is specifically compromised in its output, through reduction of rac, introducing the UASPakMyr transgene can partially suppress the midline crossing defects. Given the clear ability of alterations in pak expression to modulate midline repulsion and the observation that Slit can promote the formation of a Robo, Dock, and Pak protein complex, it is somewhat surprising that complete removal of zygotic pak does not have major consequences for embryonic axon guidance. Indeed, in the absence of clear loss-of-function phenotypes in pak mutants, it is difficult to argue unequivocally for a critical role of endogenous pak in robo function. There are a number of potential explanations for these observations including, but not limited to, maternal pak contribution and the potential redundant function of a second pak-like gene. Future experiments should address these possibilities in order to link pak more firmly to robo (Fan, 2003).

Dock has been suggested to act downstream of the Dscam axon guidance receptor during pathfinding of Bolwig's nerve, and the vertebrate homolog of Dock, Nck, has also been linked to several guidance receptors in vitro, including Eph receptors and c-Met receptors. More recently, Nck has been shown to directly interact with the cytoplasmic domain of the vertebrate attractive Netrin receptor DCC. The Nck and DCC interaction is important for DCC's function to stimulate axon extension in vitro. Together these observations raise the question of whether a similar DCC/Nck interaction occurs in Drosophila, and if so whether the interaction is important for the in vivo function of Drosophila DCC (encoded in the fly by the frazzled gene) to attract commissural axons across the midline. Interestingly, in addition to its substantial overlap in expression with the Robo receptor, Dock protein is also expressed in commissural portions of axons, as is the Frazzled receptor. While the dock mutant phenotype is most consistent with a role in midline repulsion, an additional function in attraction cannot be ruled out. In the future it will be interesting to test for genetic interactions between frazzled, dock, Rac, and pak to determine if this signaling module is also employed during midline axon attraction in Drosophila (Fan, 2003).

The implication of Dock/Nck and Rac in both DCC-mediated attraction and Robo-mediated repulsion raises the obvious question of how the specificity of attraction and repulsion is controlled and argues against a committed role of either of these signaling molecules to either one or the other type of responses. This is perhaps not too surprising, given the fact that Robo and DCC receptors themselves are intimately connected through their ability to form a heteromeric receptor complex with potentially unique signaling properties. Although it remains possible that signaling molecules or adaptors will be identified that can account for the specificity, an alternative possibility is that it is the coordinate regulation, relative activity levels, and combinatorial action of a core group of common signaling molecules that makes the difference in attraction versus repulsion (Fan, 2003).

Biochemical data support the idea that Slit stimulation of Robo can regulate the recruitment of Dock and Pak to the Robo receptor and also trigger an increase in Rac activity. Both of these events are dependent on the CC2 and CC3 sequences in Robo's cytoplasmic domain. Thus, the observations are consistent with either a Dock-dependent or a Dock-independent recruitment of Rac to Robo. Based on the known physical interactions between Dock and Pak and between Pak and Rac, it is likely that the recruitment of Rac is dependent on Dock. Alternatively, another protein interacting through CC2 and/or CC3 could function to recruit Rac in a Dock-independent fashion (Fan, 2003).

Regardless of whether the recruitment of Rac to Robo is dependent on Dock and Pak or is an independent event, the data cannot explain how Slit stimulation of Robo results in increased Rac activity. Two obvious types of molecules that are missing from the model and the protein complex are the upstream regulators of Rac, the GEF and GAP proteins. Intriguingly, in the course of a genome-wide analysis of all RhoGEFs and RhoGAPs in Drosophila, one Rac-specific GAP has been identified that when overexpressed results in phenotypes reminiscent of robo loss of function (H. Hu et al., submitted, reported in Fan, 2003). There are a number of candidate GEFs that could explain how Rac activity is upregulated by Slit activation of Robo, most notably Sos, rtGEF (pix), and Trio. It will be interesting to determine which if any of these molecules could play such a role in Robo signaling (Fan, 2003).

Robo interaction with Comm: The N-terminal and transmembrane domains of Comm are necessary for its function and trafficking within neurons

Commissureless is a novel transmembrane molecule necessary both for commissural axons to cross the midline of the Drosophila central nervous system and normal synaptogenesis. Comm is able to reduce cell surface levels of Roundabout (Robo), a receptor for the midline repellent Slit, on commissural axons and unknown inhibitors of synaptogenesis expressed on muscle cells. Comm is expressed dynamically and is found at the cell surface and within intracellular vesicles. Comm can bind Robo and when the proteins are co-expressed Robo is found co-localized with Comm intracellularly. The ability of Comm to localize intracellularly and hence regulate Robo surface levels requires sequences in both the N-terminal and transmembrane domains. Comm can dimerize via its N-terminal domain. Furthermore, absence of the Comm N-terminal and transmembrane regions results in the protein being restricted to the neuron soma (Georgiou, 2003).

Comm is necessary for axons to cross the CNS midline in Drosophila, where it plays an important role in regulation of cell surface levels of the transmembrane receptor protein Robo. By controlling the surface levels of Robo, Comm regulates the sensitivity of axons to the midline repellent Slit. Comm is present at highest levels at the midline where it is expressed on midline cells and commissural axons, precisely the area where Robo protein is excluded. The distribution of Comm allows the downregulation of Robo on commissural axons and permits them to cross the midline. Comm also functions at the neuromuscular junction where it is expressed in the post-synaptic cell and acts to remove unknown inhibitors of synapse formation from the cell surface. Comm activity at the neuromuscular junction requires that Comm can endocytose into the cell, when it presumably co-endocytoses the synaptogenesis inhibitors. The intracellular region of Comm has been shown to be essential for function both at the midline and at the neuromuscular junction. The intracellular region of Comm includes binding sites for the ubiquitin ligase, DNedd4, and a possible adaptin binding site. The interaction between DNedd4 and Comm is necessary for Comm to localize within intracellular vesicles. In the absence of this interaction Comm is expressed at the cell surface and is unable to prevent Robo accumulation at the plasma membrane. In this paper, it has been shown that the N-terminal and transmembrane domains of Comm are also essential for its function. Both these regions of the protein appear to be necessary for Comm to localize efficiently to endosomes within the cell. In the absence of the N-terminal Comm is less efficiently targeted to the endosomes: this reduces the efficiency of the molecule to sort Robo away from the cell surface. When the N-terminal and transmembrane domains of Comm are replaced, the chimeric protein localizes to the cell surface and is unable to reduce surface levels of Robo. It is further found that the Comm transmembrane domain is required both for targeting Comm to intracellular vesicles and also its transport along CNS axons. When deletion constructs lacking the Comm transmembrane domain are expressed within neurons at the midline or in the CNS they remain in the soma and are not trafficked to the axon. When the deletion constructs CommDelta2 and CommDeltaEC are expressed at the midline, little Comm protein is seen in the axons of the midline neurons in contrast to their expression throughout the CNS when they can be seen in axons. It is suspected that these forms can reach the axons inefficiently and the protein cannot be detected in the axons when it is expressed in the small number of midline neurons as compared to when it is expressed in all CNS neurons (Georgiou, 2003).

The N-terminal domain of Comm is required for Comm's correct function in both in vivo and in vitro experiments. CommDeltaEC , lacking the N-terminal domain, shows some residual function but its ability to prevent Robo reaching the cell surface is severely disrupted. If increased proportions of the N-terminal are provided, an improvement in Comm function results. CommDelta1 is more often targeted to the same intracellular localization as Comm whereas CommDelta2 and CommDeltaEC are less often found within vesicles, suggesting an essential trafficking signal exists within the sequence between amino acids 62 and 99 (Georgiou, 2003).

When Robo is co-transfected with the Comm variants, its distribution follows that of Comm. Removal of the N-terminal of Comm results in a greater proportion of Robo localized to the plasma membrane. When driven in neurons, some CommDeltaEC can localize correctly to the commissure, however, the molecule's reduced function is evident in its failure to generate a strong robo phenocopy. This suggests that the appropriate trafficking of Comm requires both the DNedd4 binding site in the cytosolic domain of Comm and sequences in the N-terminal. Two models have been proposed for Comm mediated regulation of Robo whereby Comm retrieves Robo from the cell surface or prevents Robo transport to the cell surface. One model suggest that Comm acts as a protein chaperone to sort Robo away from a plasma membrane pathway to an intracellular endocytic destination. Perhaps the appropriate sorting of Comm itself to an endocytic location involves elements resident in the trans-Golgi network or endoplasmic reticulum that recognise the N-terminal region of Comm to traffic it appropriately in addition to the ubiquitin tag signal added by D-Nedd4. Alternatively, the second model suggests that the N-terminal sequence may be necessary for the receipt of an extracellular signal required for the efficient internalization of Comm via the intracellular addition of ubiquitin (Georgiou, 2003).

The normal intracellular targeting of Comm is completely disrupted if the Comm transmembrane domain is not present or is replaced. Comm variants that replace the Comm transmembrane region localize to the plasma membrane when expressed in S2 cells, at the midline or in neurons. This disruption is most striking when the proteins are expressed throughout the nervous system. Here, the mutant protein localizes to the cell surface of neuronal cell bodies and does not exit the cell bodies. This manipulation of the Comm protein disrupts two aspects of its normal intracellular localization, a vesicular distribution within the cell body and distribution to the axon (Georgiou, 2003).

In the absence of both the N-terminal and transmembrane regions of Comm, the protein cannot localize intracellularly and therefore cannot target Robo away from the cell surface. The replacement of the Comm transmembrane region results in a complete abrogation of Comm's ability to localize within intracellular vesicles, suggesting that the Comm transmembrane region provides some information necessary for its targeting in addition to that provided by the N-terminal regions (Georgiou, 2003).

Intriguingly the replacement of the normal Comm transmembrane region results in targeting specifically to the cell body plasma membrane and the manipulated Comm molecule is not transported along the axon. This suggests that Comm is translated in the cell body and requires a transport mechanism to reach distal regions of the axon. Targeting of Comm to the intracellular vesicles it normally occupies may be a requisite for Comm to extend along the axon (Georgiou, 2003).

The Comm protein has a specific spatial location within the commissural axons where it is expressed at high levels on the commissural stretches of these neurons. This is precisely the region where Robo protein levels are at their lowest throughout axon outgrowth. Even after the neurons have crossed the midline they maintain low levels of Robo protein on the commissures until stage 16 when the Comm protein levels eventually decline. This localization/stabilization of Comm at the commissures may act to ensure continued downregulation of Robo levels there and ensure Robo is only present on the distal regions of the axons. The failure to observe Robo within the commissural regions suggests that Robo may be added to the axon surface distally, perhaps at the growth cone and that a mechanism exists at the midline to prevent this protein diffusing back to the soma (Georgiou, 2003).

Neurons are highly specialized cells that can both send and receive intercellular signals at different regions within the cell. This property requires that membrane proteins be targeted to specific locations within the cell. Different proteins may be found either throughout the cell or targeted to the axon, growth cone or the somatodendritic region. Comm displays a specific distribution within neurons where it accumulates along the commissural regions at the midline. This distribution is not unique to Comm as a similar distribution is also taken up by the Drosophila Derailed protein. To date no general targeting signals or mechanisms have been identified that specify a neuronal protein's intracellular address. Studies on neuronal protein targeting have suggested that the trans-Golgi network sorts membrane proteins into different vesicles that traffic the protein to their correct location or that proteins are selectively retained in particular neuronal domains. Attempts have been made to identify the neuronal sorting signals that target proteins to the somatodendritic region or the axon. Coarse locations for these sorting signals have been defined for several neuronal proteins. The amyloid precursor protein (APP) contains a signal in its N-terminal or transmembrane domain necessary for targeting to the axon whereas Synaptobevin contains a signal in its cytoplasmic region. The transferrin receptor, polymeric immunoglobulin receptor and the low density lipoprotein receptor all have their somatodendritic sorting signals in their cytoplasmic domains. In addition to targeting signals that direct neuronal proteins to particular locations there are also selective retention signals that allow proteins to be maintained or lost from particular locations. Transcytosis can occur whereby a protein is initially located throughout the neuron but subsequently relocates via the endocytic machinery to its appropriate location, e.g., APP moves from axon to the soma. Also, diffusion barriers exist that serve to maintain localized neuronal proteins to their correct locale. In the case of Comm it appears to be specifically localized to the commissural region of the axon. Is the protein selectively trafficked to this region of the axon or do components of the endocytic machinery prevent Comm from remaining distal to the midline? Interestingly it has emerged that differential lipid domains within the membrane may also play a role in targeting proteins within a cell. Detergent-insoluble glycolipid domains or rafts may serve as sorting platforms to direct proteins such as Thy-1 to the axon. Perhaps the commissural region of axons contains a differential lipid composition that mediates Comm localization. Interestingly Nedd4 associates with lipid rafts and thus may play a role in targeting Comm (Georgiou, 2003).

Since Comm is expressed at high levels on the surface of the midline cells that contact the commissural axons at precisely the point where Comm accumulates within the axons it was wondered whether there may be an interaction between Comm on the midline cells and Comm on the commissural axons. A Comm:Comm interaction has been shown to take place within S2 cells using a co-immunoprecipitation assay. A strong interaction is dependent on the region N-terminal to the transmembrane region being intact while no interaction is observed without this region and the Comm transmembrane domain. This assay suggests that a cis-interaction between Comm molecules can take place and since these regions are necessary for Comm trafficking this interaction may also be necessary for Comm to be located intracellularly (Georgiou, 2003).

It is suggested that the Comm N-terminal region and transmembrane domain, possibly through a cis-homophilic interaction or by some other interaction, is promoting the internalization of Comm. This internal vesicular location of Comm enables it to be transported away from the cell body to the commissure. Once Comm reaches the commissural region it is sequestered there and does not travel further distally. Perhaps a signal from the midline cells triggers the sequestration of the Comm protein at the commissure. Whether this signal affects a membrane specialization of the commissural axon or endocytic trafficking processes or allows a trans-homophilic interaction between Comm on the surface of the midline cells and any Comm that reaches the surface of the axon is not yet known (Georgiou, 2003).

Heparan sulfate proteoglycan syndecan promotes axonal and myotube guidance by slit/robo signaling

Biochemical studies suggest that axon guidance activity requires cell-surface heparan sulfate to promote binding of mammalian Slit/Robo homologs. Drosophila Syndecan, a heparan sulfate proteoglycan (HSPG), is required for proper Slit signaling. Slit, the ligand for the Roundabout (Robo) receptors, is secreted from midline cells of the Drosophila central nervous system (CNS). It acts as a short-range repellent that controls midline crossing of axons and allows growth cones to select specific pathways along each side of the midline. In addition, Slit directs the migration of muscle precursors and ventral branches of the tracheal system, showing that it provides long-range activity beyond the limit of the developing CNS. Syndecan (Sdc) mutations have been generated; they affect all aspects of Slit activity and cause robo-like phenotypes. sdc interacts genetically with robo and slit, and double mutations cause a synergistic strengthening of the single-mutant phenotypes. The results suggest that Syndecan is a necessary component of Slit/Robo signaling and is required in the Slit target cells (Steigemann, 2003).

Genetic assays provide a sensitive means of detecting an in vivo interaction between different components in a pathway, but they do not show that the association is direct. Thus, a biochemical assay was developed to determine whether Sdc binds to Slit and/or Robo in cellular extracts in which all three proteins are endogenously expressed. Immunoprecipitation of either Slit or Robo and subsequent detection with anti-Sdc antibodies reveals that Sdc associates with both Slit and its receptor, suggesting the possibility of a ternary complex. This association is specific because no Sdc is trapped by nonspecific IgG or N-Cadherin antibodies that successfully IP other signaling molecules. Thus, Sdc participates directly in a complex with Slit and Robo (Johnson, 2004).

Homozygous sdc mutants are semilethal and show identical phenotypes in the CNS and in the muscle pattern. In order to unambiguously demonstrate that the lack of sdc activity is responsible for the mutant phenotype observed, Sdc-RA was panneurally expressed using the GAL4/UAS system in sdc mutant individuals. The neural phenotype of the mutants was rescued, indicating that the mutant phenotype was caused by the lack of Sdc and that the transgene provides functional Sdc activity (Steigemann, 2003).

In order to examine the possible defects in axonal guidance and muscle patterning, sdc mutant embryos were stained with both Fasciclin II (FasII; mAB 1D4) antibodies, which label three longitudinal axon tracts at each side of the midline, and anti-Mhc antibodies, which visualize the muscle pattern. The results show that the lack of sdc activity causes phenocopies of robo and robo2 mutants; i.e., it affects both midline guidance of axons and the establishment of the muscle pattern. The defects in CNS axon guidance were strikingly similar to robo2 mutants but less pronounced than in robo mutants. The muscle and CNS phenotypes were also weaker than in slit mutants, in which signaling through all Robo receptors is impaired (Steigemann, 2003).

In order to link embryonic Sdc requirement genetically to Slit/Robo signaling, it was next asked whether sdc mutations can enhance loss-of-function slit and robo phenotypes. It was found that the number of ventral muscles, which cross the midline dorsal of the CNS in homozygous sdc and robo2 single mutants, is significantly increased in double mutant combinations of sdc and robo2, resulting in a muscle phenotype indistinguishable from slit and homozygous robo, robo2 double mutants. In the CNS, the FasII-expressing longitudinal fascicles of robo2, sdc double mutants converged into a single thick axon bundle at the ventral midline, resembling the effects seen with slit mutants. Similar observations were obtained with the mAb BP102 against all CNS axons, showing strongly condensed fascicles in robo2, sdc double mutant embryos. The synergistic strengthening of both the muscle and the CNS phenotypes in robo2, sdc double mutants, which are similar to a weak slit mutant phenotype, indicates that only some Slit-derived repellent activity is received along the midline. In contrast to robo2, the robo mutant phenotype was not significantly enhanced by the simultaneous lack of sdc. The data suggest that Robo can, in part, compensate for the lack of Robo2 and vice versa and that Robo is more sensitive to reduced Sdc-dependent Slit activity than Robo2 (Steigemann, 2003).

The results imply that sdc, slit, and robo are components of the same genetic circuitry. This proposal was tested by genetic means, asking whether the gene activities interact in vivo. Loss of only one copy of sdc led to the development of a normal muscle pattern, whereas the simultaneous absence of one copy of both slit and sdc in slit/+, sdc/+ double heterozygous embryos caused an increase in the number of longitudinal transverse muscles. Furthermore, the number of FasII-expressing inner fascicles that cross the midline is increased (3.3%) as compared to slit heterozygous embryos (0.6%;). More clearly, homozygous sdc mutant embryos, which also lack one copy of either robo, robo2, or slit, show an enhanced axonal guidance defect with multiple midline crossings of the fascicles (72%, 47%, and 95%, respectively), a phenotype very similar to the robo mutant. These results establish that Sdc acts in the same genetic circuitry as Slit and the Robo receptor family and represents a critical component of the Slit/Robo signaling pathway (Steigemann, 2003).

Vilse, a conserved Rac/Cdc42 GAP mediating Robo repulsion in tracheal cells and axons

Slit proteins steer the migration of many cell types through their binding to Robo receptors, but how Robo controls cell motility is not clear. vilse is a Drosophila gene required for Robo repulsion in epithelial cells and axons. Vilse defines a conserved family of RhoGAPs (Rho GTPase-activating proteins), with representatives in flies and vertebrates. The phenotypes of vilse mutants resemble the tracheal and axonal phenotypes of Slit and Robo mutants at the CNS midline. Dosage-sensitive genetic interactions between vilse, slit, and robo mutants suggest that vilse is a component of robo signaling. Moreover, overexpression of Vilse in the trachea of robo mutants ameliorates the phenotypes of robo, indicating that Vilse acts downstream of Robo to mediate midline repulsion. Vilse and its human homolog bind directly to the conserved intracellular domains CC0, CC1, and CC2 of the corresponding Robo receptors and promote the hydrolysis of RacGTP and, less efficiently, of Cdc42GTP. These results together with genetic interaction experiments with robo, vilse, and rac mutants suggest a mechanism whereby Robo repulsion is mediated by the localized inactivation of Rac through Vilse (Lundström, 2004).

The vilse locus was identified in a P-element screen for genes with pathfinding defects in the tracheal ganglionic branch (GB). The vilse lacZ strain contained a single P[w + mC = lacW] transposon in chromosomal position 93B10-11 that caused misroutings in GB outgrowth and, at lower frequency, crossing of the VNC midline; the gene was name "vilse" (which means "lost" in Swedish). Additional mutants and revertants of this phenotype were generated by excision of the P-element, and the allele vilse1 was chosen for study because the analysis of the genomic region in this mutant and in situ hybridization indicated that it represents the zygotic null condition for the gene (Lundström, 2004).

In wild-type midstage-16 embryos, the GB1 cell, the tip cell of the ganglionic branch, has reached the ventral side of the neuropil, and it turns posteriorly as it migrates in the proximity of the ventral longitudinal glia. Then, just before it reaches the midline, it abruptly turns to migrate dorsally to reach its final target on the dorsal side of the neuropil. In vilse1 mutants, 20% of the GBs migrated normally to the midline, but stalled once they reached it. An additional 14% of GBs failed to turn posteriorly; instead, they extended straight toward the ventral midline, where most of them stalled or, occasionally, continued to migrate across the midline. In wild-type embryos, <1% of the GBs migrated straight toward the midline, and none of them crossed it. Despite the low penetrance, this misguidance phenotype was interesting because it was similar to the tracheal phenotype seen in robo mutants, where GBs also migrate straight toward the midline but instead of halting there, they often cross it (Lundström, 2004).

Given these similarities between vilse and robo mutants in GB1 guidance, it was of interest to discover whether vilse mutants might also show defects in CNS axon guidance similar to those in robo mutants. In wild-type embryos, Fasciclin II-positive axons project along specific pathways in the longitudinal connectives; they never cross the CNS midline. In robo mutants, many of these axons project along or across the midline. A similar phenotype is also seen in vilse mutants, albeit at much lower frequency (1 in 30 embryos). This phenotype too suggested a link between vilse and the slit signaling pathway (Lundström, 2004).

Database searches with the sequence from genomic DNA flanking the vilse lacZ P-element showed that it was inserted in the 5'-untranslated region of the predicted gene CG3421 (RhoGap93B in GadFly). The search also identified several cDNAs deriving from this gene, and the longest available clone, LD10379 (BDGP), was sequenced. The predicted Vilse protein contains a number of conserved domains: two N-terminal WW domains (residues 6-36 and 45-75), a more C-terminal myosin tail homology 4 (MyTH4) domain (amino acids 997-1124), a RhoGAP domain (amino acids 1154-1303), and a Pfam-B 53745 domain (amino acids 1304-1330). A predicted human protein, KIAA1688 has an identical domain structure and overall 29% identity and 51% similarity to Drosophila Vilse, which is in turn the closest match to KIAA1688 in Drosophila. KIAA1688 is therefore referred to as the human Vilse protein. In addition, the human genome encodes a second Vilse homolog with an additional extensin-2 domain (48% similarity, 26% identity, GenBank accession no. gi|37574693|r). Vilse homologs can also be found in the mosquito (66% similarity, 29% identity for gi|30176853|) and mouse (50% similarity, 28% identity, gi|28380066|) genomes. No protein with the same modular structure as Vilse was identified in the Caenorhabditis elegans genome, the closest relative in worms (33% similarity, 13% identity) is encoded by C38D4.5 CE and contains a WW, a PH, and a RhoGAP domain (Lundström, 2004).

In situ hybridization revealed that vilse transcript is ubiquitous during the first stages of development, suggesting a robust maternal contribution. Zygotic transcripts were prominent at stage 15 in the tip cells of all tracheal branches, the muscles, and midline cells of the VNC. This pattern was the same as the ß-gal marker expression in the enhancer trap strain, and was absent in vilse1 mutants, indicating that vilse1 is a strong loss-of-function mutant in RhoGap93B. To analyze the expression of Vilse protein, anti-Vilse antisera were raised. Immunostainings of whole-mount wild-type embryos detected Vilse protein expression in a pattern that mirrored the pattern of the vilse transcript and that of ß-gal expression in the vilse LacZ enhancer trap. In addition, Vilse antisera stained the epidermis, the peripheral nervous system (PNS) segmental and intersegmental nerves, and the CNS longitudinal connectives and commissures. This expression was not detected in the enhancer trap or by in situ hybridization, and may in part reflect the maternal protein. The antiserum is specific for Vilse; the staining was much reduced in vilse1 mutants. Vilse staining showed a subcellular distribution consistent with a cytoplasmic localization of the protein (Lundström, 2004).

Vilse promotes the hydrolysis of RacGTP and to a lesser extent that of Cdc42GTP. It is thus expected to antagonize the activity of these GTPases on their known effectors. Increasing experimental evidence indicates that Rac and Cdc42 regulate a multitude of cellular responses ranging from establishment of epithelial polarity and integrity to membrane trafficking and the control of planar polarity, in addition to their well known function in modulating the actin cytoskeleton. The tracheal phenotypes of vilse embryos are remarkably specific; all branches form, fuse, and grow toward their targets without any apparent defects on epithelial polarity, integrity, or shape. In addition, the rest of the terminal cells that target other internal organs concurrently with GB1 migrate correctly and associate with their targets. It is therefore concluded that the primary function of Vilse in the trachea is in the guidance of GB1 migration (Lundström, 2004).

How then does Vilse fulfill its role in cell navigation? Both of its target GTPases are key regulators of the actin cytoskeleton in several cell types. In fibroblasts, GTP-bound Cdc42 generates actin bundles and characteristic filopodial extensions, possibly through its association with the WASP protein and subsequent stimulation of the actin polymerizing activity of the Arp2/3 complex. RacGTP, in contrast, generates distinct cytoskeletal attributes, membrane ruffling, and lamellipodial protrusions. Rac controls actin polymerization through the intermediary protein IRSp53, which associates to the SCAR/WAVE regulator of Arp2/3 activity. Cdc42 also binds to the IRSp53 adaptor, suggesting that both GTPases regulate actin polymerization through SCAR/WAVE (Lundström, 2004).

An additional regulatory role for Rac and Cdc42 in cytoskeletal dynamics is exerted through their activating role on PAK (p21 activated kinase). PAK in turn activates the LIM kinase, which can phosphorylate the actin depolymerization factor (ADF/cofilin). Cofilin mediates depolymerization of actin filaments and can also function as a filament-severing factor. Its phosphorylation by LIM-kinase down-regulates its activity and inhibits F-actin depolymerization. Thus, the two GTPases in their active form promote the growth of actin filaments by both enhancing polymerization through the Arp2/3 complex and inhibiting severing and depolymerization at the minus end of the filaments. The phenotypic analysis of vilse and GTPase mutants leads to a proposal that Vilse antagonizes the function of Rac in promoting actin polymerization locally at the migrating tip of GB1 (Lundström, 2004).

Cell guidance through Slit signaling, apart from its role in axonal pathfinding, has been implicated in a large number of morphogenetic events involving cell migration. It is involved in the migration of leukocytes in vertebrates, epithelial sheets and muscle cells in Drosophila, and in the branching of the lung epithelium in mice, has been found to control movements of endothelial cells during angiogenesis. Slit signal interpretation and the cellular responses it elicits depend on the intracellular domains of Robo receptors. The best characterized examples of Robo signal transduction derive from studies of migrating neurons. These studies highlight two basic mechanisms for Slit signaling through Robo. In the first paradigm, derived from studies of early-stage-22 Xenopus spinal neurons, Robo silences the attractive Netrin signal in response to Slit through the binding of its CC1 domain to the intracellular part of the DCC receptor. This Netrin-silencing function of Robo is different from the repulsive response to Slit, which is acquired by the same neurons only later, at stage 28 (Lundström, 2004).

Slit-mediated repulsive responses involve the regulation of cytoskeletal organization in the growth cone. In Drosophila, the Abelson kinase (Abl) binds to CC3 and phosphorylates a tyrosine in CC1, thereby modulating Robo activity. In contrast, the Abl substrate Enabled (Ena), a member of the profilin-binding family of proteins, associates with CC2 and mediates the repulsive role of Robo through an unknown mechanism that may involve control of cytoskeletal organization. More recently, Abl was found to collaborate with the cyclase-associated protein CAP, this time to mediate Robo repulsion. srGAPs bind to the CC3 domain of Robo in response to Slit and aid Cdc42GTP hydrolysis to directly mediate the repulsive response to Slit in cultured anterior subventricular rat neurons. This Cdc42GTP hydrolysis at the site of Robo activation would then result in actin filament depolymerization and severing, thus promoting the turn of the growth cone to the opposite direction. The functional analysis of Vilse identifies a direct transducer of the Slit signal to the inactivation of Rac. vilse, robo, and slit mutants show qualitatively the same phenotypes of midline crossings of tracheal cells and axons. The effect of vilse overexpression on the robo tracheal phenotypes and the dose-dependent interaction between slit, robo, and vilse, combined with the biochemical analysis indicate that Vilse acts downstream of Robo. Hence, Vilse may play an analogous role to srGAP in locally down-regulating actin polymerization through the hydrolysis of RacGTP and facilitating turning away from the midline (Lundström, 2004).

Paradoxically, both activation and inactivation of Rac appear to interfere with midline crossing and Slit signaling. Expression of constitutively activated Rac causes longitudinal axons to cross the midline, and reduction of Robo signaling enhances this phenotype. In contrast, rac mutants show strong phenotypes in axonal growth and guidance, including midline crosses, and the latter phenotype becomes more prominent by reduction of Slit. One possible explanation is that Rac might be involved in multiple cellular processes affecting different aspects of the Slit/Robo pathway. For example Rac might mediate Slit secretion by midline cells or intracellular trafficking of Robo in the axons, in addition to its effect on cytoskeletal dynamics downstream of Robo. The protein adaptor Dock has also been implicated in midline repulsion downstream of Robo. In response to Slit, Dock's binding to the intracellular domain of Robo is enhanced, leading to the recruitment of the Rac effector kinase Pak. This chain of events has been proposed to bring activated Rac to Robo in response to Slit. Yet, it is not clear how Rac becomes activated in response to Slit, or how the recruitment of active Rac and Pak might translate in the cellular events that lead to repulsion from Slit. The contradicting models of the function of rac downstream of robo may be reconciled by considering a sequential interaction of the effectors with the receptor. For example, Vilse may be required initially for severing of actin filaments at the cell extensions that first encounter Slit. The inducible recruitment of Pak to Robo might occur subsequently, perhaps in response to higher concentrations of Slit, promoting cytoskeletal reorganizations that lead to a sustained turning response. This involves a new function of Rac in the context of repulsion from the signal source. The genetic analysis of midline repulsion reveals that Slit signaling relies on the dynamic and spatially coordinated control of Rac activity. Vilse provides both the first direct link from Robo to the inactivation of Rac, and a molecular handle to address the complex interactions that control repulsion during cell migration (Lundström, 2004).

Son of sevenless directly links the Robo receptor to rac activation to control axon repulsion at the midline

Son of sevenless (Sos) is a dual specificity guanine nucleotide exchange factor (GEF) that regulates both Ras and Rho family GTPases and thus is uniquely poised to integrate signals that affect both gene expression and cytoskeletal reorganization. Sos is recruited to the plasma membrane, where it forms a ternary complex with the Roundabout receptor and the SH3-SH2 adaptor protein Dreadlocks (Dock) to regulate Rac-dependent cytoskeletal rearrangement in response to the Slit ligand. Intriguingly, the Ras and Rac-GEF activities of Sos can be uncoupled during Robo-mediated axon repulsion; Sos axon guidance function depends on its Rac-GEF activity, but not its Ras-GEF activity. These results provide in vivo evidence that the Ras and RhoGEF domains of Sos are separable signaling modules and support a model in which Robo recruits Sos to the membrane via Dock to activate Rac during midline repulsion (Yang, 2006).

Sos was identified in Drosophila as a GEF for Ras in the sevenless signaling pathway during the development of the Drosophila compound eye, where it activates the Ras signaling cascade to determine R7 photoreceptor specification. Studies in mammalian cell culture demonstrated that Sos functions as a GEF for both Ras and Rac in the growth factor-induced receptor tyrosine kinase (RTK) signaling cascade. Upon RTK activation, the SH3/SH2 adaptor protein Grb2/Drk recruits Sos to autophosphorylated receptors at the plasma membrane, where Sos activates membrane-bound Ras. In a later event downstream of RTK activation, Sos is thought to be targeted to submembrane actin filaments by interaction with another SH3 adaptor, E3b1(Abi-1), where Sos activates Rac . Whether the activation of Rac by Sos is strictly dependent on prior activation of Ras remains controversial, nor is it clear how Sos coordinates the activity of its two GEF domains in vivo (Yang, 2006 and references therein).

Evidence is provided that Sos functions as a Rac-specific GEF during Drosophila midline guidance. Sos is enriched in developing axons, and sos exhibits dosage-sensitive genetic interactions with slit and robo. Strikingly, genetic rescue experiments show that the Dbl homology (DH) RhoGEF domain of Sos, but not its RasGEF domain, is required for its midline guidance function. Biochemical experiments show that Sos physically associates with the Robo receptor through Dock in both mammalian cells and Drosophila embryos. Furthermore, Slit stimulation of cultured cells results in the rapid recruitment of Sos to membrane Robo receptors. These results provide a molecular link between the Robo receptor and Rac activation, reveal an independent in vivo axon guidance function of the DH RhoGEF domain of Sos, and support the model that Slit stimulation recruits Sos to the membrane Robo receptor via Dock to activate Rac-dependent cytoskeletal changes within the growth cone during axon repulsion (Yang, 2006).

These data support the idea that Sos provides a direct molecular link between the Robo receptor and the activation of Rac during Drosophila midline guidance. Genetic interactions between sos, robo, dock, crGAP/vilse, and the Rho family of small GTPases strongly suggest that Sos functions in vivo to regulate Rac activity during Robo signaling. Genetic rescue experiments indicate that sos is required specifically in neurons to mediate its axon guidance function. Furthermore, genetic data establish that, in the context of midline axon guidance, the Ras-GEF and Rac-GEF activities of Sos can be functionally uncoupled. Biochemical experiments in cultured cells and Drosophila embryos show that Sos is recruited into a multiprotein complex consisting of the Robo receptor, the SH3-SH2 adaptor protein Dock, and Sos, in which Dock bridges the physical association between Robo and Sos. Finally, experiments in cultured cells support the idea that Slit activation of Robo can recruit Sos to the submembrane actin cytoskeleton to regulate cell morphology. Together, these results suggest a model in which Slit stimulation recruits Sos to the Robo receptor via Dock to regulate Rac-dependent cytoskeletal changes within the growth cone during axon repulsion (Yang, 2006).

Based on previous work implicating rac in Robo repulsion, as well as in vitro studies demonstrating that Sos exhibits GEF activity for Rac, but not Rho or Cdc42, Rac seemed the most likely Sos substrate. However, rho has also been implicated in mediating Robo repulsion, and genetic interactions between sos and dominant-negative Rho have been interpreted to suggest that Sos could act as a GEF for Rho. This question was investigated further, and two types of genetic evidence have been presented that suggest that indeed Rac is the favored substrate of Sos. First, ectopic expression experiments in the eye reveal interactions exclusively between sos and rac. Second, genetic interaction experiments using loss of function mutations in rac and rho (rather than the more problematic dominant-negative forms of the GTPases) reveal strong dose-dependent interactions between sos and rac, but not sos and rho during midline axon guidance. Together, these observations argue in favor of Rac as the primary in vivo Sos substrate. Nevertheless, the possibilities that Sos also contributes to Rho activation and that the combined activation of Rac and Rho is instrumental in mediating the Robo response cannot be excluded (Yang, 2006).

Previous studies have demonstrated that Slit stimulation of the Robo receptor leads to a rapid increase in Rac activity in cultured cells. However, the mechanism by which Rac is activated downstream of Robo was not clear. This study provides direct genetic and biochemical evidence that Sos is coupled to the Robo receptor through the Dock/Nck SH3-SH2 adaptor, where it can regulate local Rac activation. Studies in cultured mammalian cells have highlighted the importance of distinct Sos/adaptor protein complexes in controlling the subcellular localization and substrate specificity of Sos. In the context of Rac activation, the E3b1 (Abi-1) adaptor has been shown to play a critical and rate-limiting role in Sos-dependent Rac activation and subsequent formation of membrane ruffles (Innocenti, 2002). Could Sos regulation of Rac activity during Robo repulsion be similarly limited by the availability of specific adaptor proteins? It is interesting to note in this context that overexpression of dock does not lead to ectopic axon repulsion, suggesting that Dock may not be limiting for Robo signaling. However, although dock mutants do have phenotypes indicative of reduced Robo repulsion, their phenotype is considerably milder than that seen in robo mutants, raising the possibility that there may be additional links between Robo and Sos (Yang, 2006).

A number of studies in cultured mammalian cells have suggested that Rac activation induced by activated growth factor receptors requires the prior activation of Ras. For example, PDGF-induced membrane ruffling can be promoted or inhibited by expression of constitutively active or dominant-negative Ras, respectively. However, other studies have suggested that in Swiss 3T3 cell lines RTK activation of Rac is Ras independent. In addition, the observation that Ras activation and Rac activation display very different kinetics, with Rac activation persisting long after Ras activity has returned to basal levels, has been used to argue against an obligate role for Ras in Rac activation. In this study, using a genetic rescue approach, whether the ability of Sos to activate Rac during axon guidance in an intact organism requires its Ras-GEF function was directly tested. Genetic data indicate that the RasGEF domain of Sos is dispensable for axon guidance, while the DH RhoGEF domain is strictly required. This observation argues strongly in favor of the model that in vivo Sos activation of Rac does not strictly require Sos activation of Ras (Yang, 2006).

It is clear that subcellular localization plays a major role in regulating Sos activity and that different protein complexes containing Sos exist in different locations in the cell. This study has shown that activation of the Robo receptor by Slit triggers the recruitment of Sos to Robo receptors at the plasma membrane. Biochemical data argue that the adaptor Dock/Nck is instrumental in bridging this interaction, and given the diverse interactions between Dock/Nck and guidance receptors, it seems likely that Dock/Nck could fulfill this role in many guidance receptor contexts. This bridging function of Dock/Nck and guidance receptors is analogous to the role of Grb2 for growth factor receptors only insomuch as it brings signaling molecules to the receptor—the mechanism of interaction is distinct, since it is mediated through SH3 domain contacts rather than SH2/phosphotyrosine interactions. These observations suggest that there may be an additional pool of Sos that can function in a distinct adaptor protein/guidance receptor complex to regulate cell morphology in response to extracellular guidance cues (Yang, 2006).

Is regulating subcellular localization the only mechanism by which Sos activity is controlled? This seems unlikely. Indeed, a recent study has implicated tyrosine phosphorylation of Sos by Abl as an additional mechanism to activate the Rac-specific GEF activity of Sos in vertebrate cell culture models. This raises the intriguing possibility that Abl may fulfill a similar role for Robo signaling. This is a particularly appealing idea given the well-documented genetic and physical interactions between Robo and Abl. Indeed, sos and abl exhibit dose-dependent genetic interactions during midline axon guidance. A clear genetic test of whether Abl activates the Rac-GEF activity of Sos downstream of Robo may be complicated by the fact that Abl appears to play a dual role in Robo repulsion: both increasing and decreasing abl function lead to disruptions in Robo function. Nevertheless, it should be possible in the future to generate mutant versions of Sos that are refractory to Abl activation and to test whether these alterations disrupt the Sos guidance function. It will also be of great interest to determine whether the redistribution of Sos can also be observed in response to guidance receptor signaling in navigating growth cones, and if so, then what changes in actin dynamics and growth cone behavior are elicited (Yang, 2006).

LRT, a tendon-specific leucine-rich repeat protein, promotes muscle-tendon targeting through its interaction with Robo

Correct muscle migration towards tendon cells, and the adhesion of these two cell types, form the basis for contractile tissue assembly in the Drosophila embryo. While molecules promoting the attraction of muscles towards tendon cells have been described, signals involved in the arrest of muscle migration following the arrival of myotubes at their corresponding tendon cells have yet to be elucidated. This study describes a novel tendon-specific transmembrane protein, which has been named LRT due to the presence of a leucine-rich repeat domain (LRR) in its extracellular region. The gene was identified in a microarray screen designed to identify genes that are expressed downstream of Stripe. This analysis suggests that LRT acts non-autonomously to better target the muscle and/or arrest its migration upon arrival at its corresponding tendon cell. Muscles in embryos lacking LRT exhibited continuous formation of membrane extensions despite arrival at their corresponding tendon cells, and a partial failure of muscles to target their correct tendon cells. In addition, overexpression of LRT in tendon cells often stalled muscles located close to the tendon cells. LRT formed a protein complex with Robo, a functional genetic interaction was detected between Robo and LRT at the level of muscle migration behavior. Taken together, these data suggest a novel mechanism by which muscles are targeted towards tendon cells as a result of LRT-Robo interactions. This mechanism may apply to the Robo-dependent migration of a wide variety of cell types (Wayburn, 2009).

When migrating cells encounter their target tissue they must arrest their migration behavior. Signals promoting arrest of cell migration are yet to be elucidated. This study describes the identification of a novel leucine-rich repeat protein, LRT, expressed specifically by tendon cells downstream of Stripe activity, and capable of associating with muscle-expressed Robo receptors. LRT acts non-autonomously on muscles to promote proper muscle-tendon assembly by arresting muscle filopodia formation following the arrival of the muscle at the tendon cell. This activity is presumably achieved by an interaction between LRT on the tendon cell and Robo receptors on the migrating muscle cell (Wayburn, 2009).

LRT protein was detected at relatively late stages of embryonic development following muscle attachment to tendon cells. This suggests that it does not function primarily to guide muscles to their attachment sites, but rather is required for the arrest of muscle extension and/or migration behavior. Two possible mechanisms may account for the arrest of muscle migration behavior: a specific signal provided by the tendon cell promoting the end of filopodia formation, or alternatively the establishment of the myotendinuous junction, which alters the leading edge morphology of the muscle cell so that it is no longer forming filopodia. Lack of LRT led to ectopic membrane extensions in various ventral oblique and acute muscles as well as in muscle 12, whereas in some muscles (e.g., VO4 or VA3), the continuous formation of muscle extensions in mutant embryos led to a failure to form proper muscle-tendon contacts. In other muscles (e.g., muscle 12) these extra-muscle extensions did not affect contact formation with the tendon cells. This latter phenotype supports a model by which a contact-independent signal promotes the arrest of muscle migration behavior. The expression of LRT on the surface of the tendon cell might represent such a signal (Wayburn, 2009).

LRT may mediate the arrest of these extraneous membrane extensions by interacting with a receptor on the muscle, which, upon binding, represses the formation of further membrane extensions. Alternatively, LRT may compete for binding to a common receptor with an attractive signal provided by the tendon cell. Association of LRT with such a receptor may dampen further signaling required for the continuous extension of the muscle leading edge. In both scenarios, overexpression of LRT in tendon cells is likely to result in a stronger association of the muscle with the proximal tendon cell, inhibiting muscle extension to the more distal tendon by arresting its filopodia formation. This phenotype was indeed observed when LRT was overexpressed in tendon cells. The muscles with extension that was arrested failed to form a proper myotendinous junction (Wayburn, 2009).

A possible mechanism by which LRT might act is through its interaction with the Robo receptors. Robo mediates attractive activity between the muscles and tendon cells. This study demonstrated that LRT associates with the extracellular domain of Robo. In addition, a reciprocal interaction between LRT and Robo was detected, in which reducing Robo levels could rescue the muscle-stalling phenotype of LRT overexpression. This experiment suggests that LRT promotes muscle stalling through an interaction with Robo receptors. In addition, overexpression of LRT in muscles partially rescued the Robo knockdown phenotype, which is consistent with LRT potentiating Robo activity when co-expressed in the muscle. Whether LRT affects the distribution of Robo receptors along the muscle membrane was tested; however, no major difference was detected between Robo distribution or expression levels in Df(2R)BSC403, LRT-overexpressing, or wild-type embryos. Alternatively, LRT may inhibit ectopic membrane extensions by reducing the levels of free Robo receptors by either direct interaction with these receptors, or by association with additional muscle surface proteins required for Robo-Slit signaling (Wayburn, 2009).

If LRT inhibits Robo-Slit interactions, its expression at the ventral midline should inhibit muscle repulsion from this domain. Ectopic expression of LRT-GFP using the midline driver slit-gal4 slightly affected the pattern of the ventral muscles, and these muscles appeared to extend closer to the midline, suggesting that LRT counteracts Robo-mediated repulsion of the muscles in the midline. No notable effect was observed on the pattern of the central nervous system axons following LRT ectopic expression in the midline. This could be due to additional mechanisms regulating Robo distribution on the axonal membrane (Wayburn, 2009).

LRR proteins expressed on distinct tissues share a common function in cell-cell recognition events as well as in the regulation of membrane extensions. In recent years, several Drosophila LRR proteins were demonstrated to affect targeting of motor axons and of specific muscles. Intriguingly, simultaneous labeling of the presynaptic and postsynaptic sites showed that, before synapse formation, muscle membrane extensions called myopodia express the LRR protein Capricious at their tips. These myopodia tips interact with axonal tips in order to form a properly located synapse. The axonal receptor for Capricious has not yet been identified. Based on the current studies, Robo receptors at the axon tips may interact directly with Capricious at the muscle myopodia. Similarly to LRT-mediated arrest of filopodia formation at the muscle tips, LRR proteins on the muscle myopodia may promote the arrest of the axonal tip formation to enable synapse formation (Wayburn, 2009).

In summary, this study has identified a novel tendon-specific surface protein, which is essential for arresting ectopic muscle filopodia formation following the arrival of the muscle at its targeted tendon cell, thereby promoting targeting of muscles to tendon cells. LRT may act by modulating Robo-Slit interactions, which are essential for the correct guidance of muscles to their specific tendon-mediated insertion sites (Wayburn, 2009).

The Adam family metalloprotease Kuzbanian regulates the cleavage of the roundabout receptor to control axon repulsion at the midline

Slits and their Roundabout (Robo) receptors mediate repulsive axon guidance at the Drosophila ventral midline and in the vertebrate spinal cord. Slit is cleaved to produce fragments with distinct signaling properties. In a screen for genes involved in Slit-Robo repulsion, the Adam family metalloprotease Kuzbanian (Kuz) was identified. Kuz does not regulate midline repulsion through cleavage of Slit, nor is Slit cleavage essential for repulsion. Instead, Kuz acts in neurons to regulate repulsion and Kuz can cleave the Robo extracellular domain in Drosophila cells. Genetic rescue experiments using an uncleavable form of Robo show that this receptor does not maintain normal repellent activity. Finally, Kuz activity is required for Robo to recruit its downstream signaling partner, Son of sevenless (Sos). These observations support the model that Kuz-directed cleavage is important for Robo receptor activation (Coleman, 2010).

Genetic and biochemical findings support the hypothesis that cleavage of the Robo receptor (rather than its Slit ligand) by the metalloprotease Kuz is important in the context of midline guidance. Loss of Kuz protease activity or of the cleavage site of Robo in vivo results in ectopic crossing of ipsilateral axons because of the loss of Robo-mediated repulsion, whereas an uncleavable form of Slit is able to rescue guidance defects in slit mutants as well as does Slit-FL. Furthermore, biochemical analyses have demonstrated that Robo is a substrate of KuzADAM10 in vitro. Finally, an Sos recruitment assay demonstrates that reduction of endogenous Kuz protease activity attenuates Slit-dependent relocalization of Sos to the plasma membrane, where it acts as a regulator of actin cytoskeletal rearrangement and, presumably, growth cone retraction (Coleman, 2010).

The data suggest a model in which Kuz promotes Robo ectodomain shedding as a mechanism of Robo activation. It is proposed that Kuz cleavage of Robo is initiated by binding of Slit, and that the release of the ectodomain of Robo causes a conformational change in Robo that allows its cytoplasmic domain to associate with Sos via the SH3-SH2 adaptor protein Dreadlocks. Sos is then properly localized in order to exert its effect on cytoskeletal rearrangement (Coleman, 2010).

In light of the strong evidence from vertebrate studies indicating that the different Slit cleavage products have distinct properties, it was surprising to find that an uncleavable form of Slit can rescue slit mutants as effectively as can wild-type Slit. What then is the significance of Slit cleavage? Although the cleavage fragments are clearly present in western blots of total embryonic protein, it is not known where in the embryo this cleavage is occurring. Proteolysis might be important for developmental events other than axon guidance that involve Slit; for instance, muscle migration or attachment, or formation of the heart. Future experiments might shed light on the significance, if any, for Slit proteolysis in these contexts. Even though Slit-FL and Slit-U (incleavable Slite) appear to be largely interchangeable in the experiments presented in this study, it cannot be ruled out that Slit proteolysis plays a role in fine-tuning axon guidance. Slit-FL does not fully rescue slit mutant axon guidance defects, which leaves open the question of whether cleavage is important for those guidance events that are not rescued; for example, the fine-tuning of the lateral positioning of axons (Coleman, 2010).

Although it seems evident that Kuz activity is important for Robo-mediated growth cone retraction, it is unclear how Robo ectodomain shedding is involved in the repulsive process. Both Notch and ephrins are known to be substrates of Kuz, but the role that Kuz plays in their signaling is very different. GPI-linked Ephrin A2 forms a stable complex with ADAM10, although ADAM10 proteolytic activity is only initiated when EphA3, the transmembrane Eph receptor, is present. ADAM10 cleavage of Ephrin A2 can be considered a permissive event, in that it releases the strong Eph-ephrin tether that attaches the two cell surfaces, thereby allowing the EphA3-expressing growth cone to retract. The role of Kuz in Notch signaling is more directly linked to Notch activation. Although the genetic data cannot distinguish whether Kuz acts in a permissive or an activating capacity with respect to Robo signaling, the observation that the expression of dominant-negative ADAM10 blocks Slit-induced recruitment of Sos to the plasma membrane suggests that Kuz/ADAM10 is likely to be important for the association of Robo with its signaling effectors. In other words, it appears that Kuz/ADAM10 contributes to the initiation of Robo signaling events (Coleman, 2010).

If Kuz is indeed playing an activating role in Robo signaling, it should be regulated in a way to prevent continuous repulsive signaling. The most parsimonious explanation for regulation of Kuz activity is that it is Slit dependent. Indeed Notch and Ephrin proteolysis by Kuz is known to be dependent upon ligand binding. Additionally, other studies have demonstrated that ADAM10 substrates, including APP and Notch, are cleaved upon receptor-ligand binding. Tests were performed to see whether Kuz proteolysis of Robo was also dependent upon ligand binding, but unfortunately it was not possible to detect a Slit-induced effect on Kuz-dependent Robo ectodomain shedding in vitro. However, these experiments were performed in Drosophila S2 cells in which both Robo and Kuz were overexpressed, and the normal regulation of cleavage might not be maintained in this context. The possibility also exists that Kuz processing of Robo might be regulated by calcium influx, differential substrate glycosylation events, or substrate oligomerization, as is observed with some ADAM10 substrates. In the future, it will be important to determine if Robo proteolysis is dependent on Slit binding, perhaps by examining, both in mammalian cells and in vivo, the processing of a Robo receptor that cannot bind Slit (Coleman, 2010).

High structural resolution hydroxyl radical protein footprinting reveals an extended Robo1-heparin binding interface

Interaction of transmembrane receptors of the Robo family and the secreted protein Slit provides important signals in the development of the central nervous system and regulation of axonal midline crossing. Heparan sulfate (HS), a sulfated linear polysaccharide modified in a complex variety of ways, serves as an essential co-receptor in Slit-Robo signaling. Previous studies have shown that closely-related heparin octasaccharides bind to Drosophila Robo directly, and Surface plasmon resonance (SPR) analysis revealed that Robo1 binds more tightly to full-length unfractionated heparin. This study utilized electron-transfer dissociation- (ETD-) based high spatial resolution hydroxyl radical protein footprinting to identify two separate binding sites for heparin interaction with Robo1: one binding site at the previously-identified site for heparin dp8, and a second binding site at the N-terminus of Robo1 that is disordered in the X-ray crystal structure. Mutagenesis of the identified N-terminal binding site exhibited a decrease in binding affinity as measured by SPR and heparin affinity chromatography. Footprinting also indicated heparin binding induces a minor change in the conformation and/or dynamics of the Ig2 domain, but no major conformational changes were detected. These results indicate a second low-affinity binding site in the Robo-Slit complex, as well as suggesting the role of the Ig2 domain of Robo1 in heparin-mediated signal transduction. This study also marks the first use of ETD-based high spatial resolution hydroxyl radical protein footprinting, which shows great utility for the characterization of protein-carbohydrate complexes (Li, 2015).

roundabout: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

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