Robo family of proteins exhibit differential expression in mouse spinal cord and Robo-Slit interaction is required for midline crossing in vertebrate spinal cord.

The ventral midline of the central nervous system is an important intermediate target where growing commissural axons either cross and project contralaterally or remain on the same side of the body. New studies on mice and humans show that this decision by commissural axons is largely dependent on Slits, extracellular matrix proteins that are widely expressed in the midline of the nervous system, and their receptors, Robos (Long et al. [2004] Neuron 42:213-223; Sabatier et al. [2004] Cell 117:157-169; Jen et al. [2004] Science 304:1509-1513). Here, we show that the Robo family proteins Robo1 and Rig-1 exhibit differential expression patterns on commissural axons as they approach, cross, and leave the midline of the developing mouse spinal cord and demonstrate that Robo1 and Robo2 bind Slit1 and Slit2, but Rig-1 does not. In addition, we show that cultured chick commissural axons are repelled by a source of Slit protein, and the soluble Robo-Fc proteins are capable of neutralizing this repulsion. Finally, we exploit the large size and accessibility of the early chick embryo to analyze the function of Slit/Robo signaling in midline commissural axon guidance, and we demonstrate that the in vivo perturbation of Robo-Slit interaction at the floor plate causes consistent guidance defects of commissural axons during midline crossing. These findings demonstrate the evolutionarily conserved role for Robo-Slit interaction in the control of midline crossing axons in vertebrates.     

Shared receptors in axon guidance: SAX-3/Robo signals via UNC-34/Enabled and a Netrin-independent UNC-40/DCC function

The C. elegans SAX-3/Robo receptor acts in anterior-posterior, dorsal-ventral and midline guidance decisions. Here we show that SAX-3 signaling involves the C. elegans Enabled protein UNC-34 and an unexpected Netrin-independent function of the Netrin receptor UNC-40/DCC. Genetic interactions with gain- and loss-of-function mutations suggest that unc-34 and unc-40 act together with sax-3 in several guidance decisions, but the C. elegans Netrin gene unc-6 does not act in the same genetic pathways. Within the migrating axon, sax-3, unc-34 and unc-40 all act cell-autonomously. Our results support a role for UNC-34/Enabled proteins in SAX-3-mediated repulsion, and show that UNC-40/DCC can potentiate SAX-3/Robo signaling via a mechanism that may involve direct binding of the two guidance receptors. A combinatorial logic dictates alternative functions for UNC-40/DCC, which can act in attraction to UNC-6/Netrin, repulsion from Netrin (with UNC-5), or repulsion from Slit (with SAX-3).     

Slit1 promotes regenerative neurite outgrowth of adult dorsal root ganglion neurons in vitro via binding to the Robo receptor

Secreted Slit proteins have previously been shown to signal through Roundabout (Robo) receptors to negatively regulate axon guidance and cell migration. During vertebrate development, Slit proteins have also been shown to stimulate branching and elongation of sensory axons and cortical dendrites. In this study, Slit1/Robo2 mRNA and protein expressions were detected in adult rat dorsal root ganglion (DRG) and in cultured DRG neurons. Treatment of both models with recombinant, soluble Slit1 protein was found to promote neurite outgrowth and elongation. In contrast, treatment with a recombinant human Robo2/Fc chimera inhibited neurite outgrowth and elongation. When adult DRG and cultured DRG neurons were pretreated with soluble recombinant human Robo2/Fc chimera, neurite outgrowth and elongation was not induced. These findings indicate that Slit1/Robo2 signaling may have a role in regulating peripheral nerve regeneration.     

Roundabout 4 is expressed on hematopoietic stem cells and potentially involved in the niche-mediated regulation of the side population phenotype

Roundabout (Robo) family proteins are immunoglobulin-type cell surface receptors that are expressed predominantly in the nervous system. The fourth member of this family, Robo4, is distinct from the other family members in that it is expressed specifically in endothelial cells. In this study, we examined the expression of Robo4 in hematopoietic stem cells (HSCs) and its possible role in HSC regulation. Robo4 mRNA was specifically expressed in murine HSCs and the immature progenitor cell fraction but not in lineage-positive cells or differentiated progenitors. Moreover, flow cytometry showed a correlation between higher expression of Robo4 and immature phenotypes of hematopoietic cells. Robo4(high) hematopoietic stem/progenitor cells presented higher clonogenic activity or long-term repopulating activity by colony assays or transplantation assays, respectively. A ligand for Robo4, Slit2, is specifically expressed in bone marrow stromal cells, and its expression was induced in osteoblasts in response to myelosuppressive stress. Interestingly, overexpression of Robo4 or Slit2 in HSCs resulted in their decreased residence in the c-Kit(+)Sca-1(+)Lineage(-)-side population fraction. These results indicate that Robo4 is expressed in HSCs, and Robo4/Slit2 signaling may play a role in HSC homeostasis in the bone marrow niche.     

Roundabout is required in the visceral mesoderm for proper microvillus length in the hindgut epithelium

Introduction: In this study we examined Roundabout signaling in the Drosophila embryonic hindgut. Results: Slit and its receptors Roundabout (Robo) and Roundabout 2 (Robo2) localize to discrete regions in the hindgut epithelium and surrounding visceral mesoderm. Loss of robo, robo2 or slit did not disrupt overall hindgut patterning. However, slit and robo mutants showed a decrease in microvillus length on the boundary cells of the hindgut epithelium. Rescue and overexpression analysis revealed that robo is specifically required in the visceral mesoderm for correct microvillus length in the underlying hindgut epithelium. Expression of robo in the visceral mesoderm of robo mutant embryos restored normal microvillus length, while overexpression of robo resulted in an increase in microvillus length. Microvillus length was also increased in robo2 mutants suggesting that robo2 may antagonize robo function in the hindgut. Conclusion: Together, these results establish a novel, dose‐dependent role for Robo in regulating microvilli growth and provide in vivo evidence for the role of the visceral mesoderm in controlling morphological changes in the underlying intestinal epithelium. Developmental Dynamics 241:759–769, 2012. © 2012 Wiley Periodicals, Inc     

Axonal growth regulation of fetal and embryonic stem cell-derived dopaminergic neurons by Netrin-1 and Slits

The physical restoration of dopamine circuits damaged or lost in Parkinson disease by implanting embryonic stem (ES)-derived cells may become a treatment. It is critical to understand responses of ES-derived dopamine (DA) neurons to guidance signals that determine axonal path and targeting. Using a collagen gel culture system, we examined effects of secreted molecules Netrin-1 and Slits on neurite outgrowth of fetal DA neurons and murine ES-differentiated DA neurons. We have previously shown that fetal DA neurons express DCC and Robo1/2 receptors and that Netrin-1 and Slit2 function as an attractant and a repellent for DA neurite outgrowth. In the present study, we observe that both Slit1 and Slit3 repel and inhibit neurite growth of fetal DA neurons. Here, we also demonstrate that ES-differentiated neurons including DA neurons express the Netrin receptor DCC and Slit receptor Robo proteins. In the gel culture system of ES cells, Netrin-1 promoted neurite outgrowth mediated by DCC receptor, and Slit1 and Slit3 were inhibitory for neurite outgrowth through Robo receptors. Slit2 appeared to exert inhibitory as well as repulsive effects in the coculture assay. However, unlike fetal DA neurites, no directed neurite outgrowth was observed in the cocultures of ES-derived DA neurons with Netrin-1-, Slit1-, and Slit3-producing cells. The findings suggest that ES-derived DA neurons generated by current protocols can respond to guidance cues in vitro in a similar manner to fetal cells but also exhibit distinct responses. This may result from developmental differences generated by present in vitro methods of cell patterning or conditioning during ES cell differentiation.     

Slit3 inhibits activator protein 1-mediated migration of malignant melanoma cells

The repellent factor family of Slit molecules has been described to have repulsive function in the developing nervous system on growing axons expressing the Robo receptors. Alterations of the Slit/Robo system have been observed in various pathological conditions and in cancer. However, until today no detailed studies on Slit function on melanoma migration are available. Therefore, we analysed the mRNA expression in melanoma cells and found induction of Robo3 expression compared to normal melanocytes. Functional assays performed with melanoma cells revealed that treatment with Slit3 led to strong inhibition of migration. Interestingly, we observed down-regulation of AP-1 activity and target gene expression after Slit3 treatment contributing to the negative regulation of migration. Taken together, our data showed that Slit3 reduces the migratory activity of melanoma cells, potentially by repulsion of the cells in analogy to the neuronal system. Further studies will be necessary to prove Slit activity in?vivo, but due to its function, Slit3 activity may be helpful in the treatment of melanoma.     

Autocrine/juxtaparacrine regulation of axon fasciculation by Slit-Robo signaling

Axons travel to their targets in bundles or fascicles, but the molecules regulating fasciculation remain incompletely characterized. We found that Slit2 and its Robo receptors are expressed by motor axons, and that inactivation of Slit2 or Robo1 and Robo2 in mice caused axons to defasciculate prematurely at muscle targets. In vitro, Slit2 secreted by motoneurons regulated fasciculation through Robo1 and Robo2. These results support the idea that Slit2 promotes axon fasciculation via an autocrine and/or juxtaparacrine mechanism.     

Control of human hematopoietic stem/progenitor cell migration by the extracellular matrix protein Slit3

Patients whose hematopoietic system is compromised by chemo- and/or radiotherapy require transplantation of hematopoietic stem and progenitor cells (HSPCs) to restore hematopoiesis. Successful homing of transplanted HSPCs to the bone marrow (BM) largely depends on their migratory potential, which is critically regulated by the chemokine CXCL12. In this study, we have investigated the expression and function of Slit proteins and their corresponding Roundabout (Robo) receptors in human HSPC migration. Slit proteins are extracellular matrix proteins that can modulate the (chemoattractant-induced) migration of mature leukocytes. We show that mRNAs for all Slits (Slit1-3) are expressed in primary BM stroma and BM-derived endothelial and stromal cell lines, but not in CD34? HSPCs. Human CD34? HSPCs expressed mRNAs for all Robos (Robo1-4), but only the Robo1 protein was detected on their cell surface. Functionally, Slit3 treatment increased the in vivo homing efficiency of CD34? HSPCs to the BM in NOD/SCID mice, whereas Slit3-exposed HSPC migration in vitro was inhibited. These effects do not appear to result from modulated CXCL12 responsiveness as CXCR4 expression, CXCL12-induced actin polymerization or the basal and CXCL12-induced adhesion to fibronectin or BM-derived endothelial cells of CD34? HSPC were not altered by Slit3 exposure. However, we show that Slit3 rapidly reduced the levels of active RhoA in HL60 cells and primary CD34? HSPC, directly affecting a pathway involved in actin cytoskeleton remodeling and HSPC migration. Together, our results support a role for Slit3 in human HSPC migration in vitro and homing in vivo and might contribute to the design of future approaches aimed at improving transplantation efficiency of human CD34? HSPCs.     

Cell-surface heparan sulfate is involved in the repulsive guidance activities of Slit2 protein.

Slit proteins are a family of secreted guidance proteins that can repel neuronal migration and axon growth via interaction with their cellular roundabout receptors (Robos). Here it was shown that Slit2-Robo-1 interactions were enhanced by cell-surface heparan sulfate. Removal of heparan sulfate decreased the affinity of Slit for Robo by about threefold. In addition, removal of cell-surface heparan sulfate by heparinase III abolished the chemorepulsive response to Slit2 normally shown by both the migrating neurons and growing axons. These results indicate essential roles for cell-surface heparan sulfate in the repulsive activities of Slit2.     

Comm function in commissural axon guidance: cell-autonomous sorting of Robo in vivo

Commissureless (Comm) controls axon guidance across the Drosophila melanogaster midline by regulating surface levels of Robo, the receptor for the midline repellent Slit. Two different models have been proposed for how Comm regulates Robo: a 'sorting' model and a 'clearance' model, both based on studies using heterologous cells in vitro. Here, we test these two models in vivo. We establish a genetic rescue assay for Comm, and use this assay to show that midline crossing does not require the presence of Comm in midline cells, as proposed by the clearance model. Moreover, by monitoring the trafficking of a Robo-green fluorescent protein (GFP) fusion in living embryos, we demonstrate that Comm prevents the delivery of Robo-GFP to the growth cone, as predicted by the sorting model. It has also been suggested that Comm must be ubiquitinated by the Nedd4 ubiquitin ligase. We show here, however, that ubiquitination of Comm is not required for its function in vitro or in vivo, and that Nedd4 is unlikely to function in axon guidance at the midline.     

Dynamic changes in Robo2 and Slit1 expression in adult rat dorsal root ganglion and sciatic nerve after peripheral and central axonal injury

Robos are transmembrane receptors that mediate Slit signaling to repel growth cone outgrowth and neural migration in the developing central nervous system. Their distribution and function in the peripheral nervous system remains unclear. In the present study, we examined expression of Slit1 and Robo2 in adult rat dorsal root ganglion (DRG), spinal cord and sciatic nerve after peripheral nerve injury (axotomy). In control rats, Slit1 and Robo2 mRNA and protein were expressed at basic levels in the L5 and L6 DRGs. Sciatic transection resulted in a significant up-regulation of both Robo2 and Slit1 mRNA and protein (p<0.05 versus control). The peak of Slit1 and Robo2 expression occurred at days 7 and 14, respectively, and returned to control levels at days 28 and 21 post-axotomy, respectively. By contrast, injury to the central axons of the DRG by dorsal rhizotomy did not up-regulate Slit1 and Robo2 expression. Robo2 staining was stronger in small diameter neurons than in large diameter neurons in control DRG. Interestingly, post-axotomy, Robo2 immunostaining increased in the large diameter neurons and the number of Robo2 positive large diameter neurons increased significantly relative to controls. Non-neuronal cells surrounding the primary sensory neurons, including the satellite cells, were Slit1-positive, and Slit1 protein was expressed in the myelin sheath and non-neural cells in both intact and degenerating sciatic nerve axons. Sciatic nerve transection also led to an accumulation of Slit1 protein in peripheral region of the traumatic neuroma. In conclusion, we report an altered expression and redistribution of Robo2 and Slit1 in the DRG and sciatic nerve trunk after peripheral axotomy. Our results indicate that Slit1 and Robo2 likely play an important role in regeneration after peripheral nerve injury.     

Fragments of SLIT3 inhibit cellular migration

The repellent factor family of Slit molecules has been described as having a repulsive function in the developing nervous system on growing axons expressing the Roundabout (Robo) receptors. Recent studies determined the effects of Slit molecules on the migratory and invasive potential of several types of tumor cells but also on synovial fibroblasts (SFs) derived from rheumatoid arthritis (RA) patients. To optimize a potential therapeutic application we aimed at generatingfragments of Slit3 showing the same functional ability as the full-length molecule but having the advantage of a smaller size. Recombinant Slit3 proteins were expressed and analyzed by western blotting. Their activity was defined by functional assays such as migration assays with RASF and melanoma cells. Recombinant Slit3 containing only leucine rich repeat domain?2 (D2), the domain important for Robo binding and the minimal functional unit D2 dNC were both able to inhibit migration of RASFs as effectively as Slit3 with all 4?repeats. Collectively, our data showed that the ability of Slit3 to reduce the migratory activity of synovial cells from patients with RA and melanoma cells can be mimicked by small protein fragments derived from Slit3. Slit3 fragments may be helpful in therapeutic attempts; however, further studies are necessary in order to elucidate their activity in vivo.     

Cloning and expression of three zebrafish roundabout homologs suggest roles in axon guidance and cell migration.

We report the cloning and expression patterns of three novel zebrafish Roundabout homologs. The Roundabout (robo) gene encodes a transmembrane receptor that is essential for axon guidance in Drosophila and Robo family members have been implicated in cell migration. Analysis of extracellular domains and conserved cytoplasmic motifs shows that zebrafish Robo1 and Robo2 are orthologs of mammalian Robo1 and Robo2, respectively, while zebrafish Robo3 is likely to be an ortholog of mouse Rig-1. The three zebrafish robos are expressed in distinct but overlapping patterns during embryogenesis. They are highly expressed in the developing nervous system, including the olfactory system, visual system, hindbrain, cranial ganglia, spinal cord, and posterior lateral line primordium. They are also expressed in several nonneuronal tissues, including somites and fin buds. The timing and patterns of expression suggest roles for zebrafish robos in axon guidance and cell migration. Wiley-Liss, Inc.     

Slit-Robo interactions during cortical development

Interneurons are an integral part of cortical neuronal circuits. During the past decade, numerous studies have shown that these cells, unlike their pyramidal counterparts that are derived from the neuroepithelium along the lumen of the lateral ventricles, are generated in the ganglionic eminences in the subpallium. They use tangential migratory paths to reach the cortex, guided by intrinsic and extrinsic cues. Evidence is now emerging which suggests that the family of Slit proteins, acting through Robo receptors, play a role not only in axon guidance in the developing forebrain, but also as guiding signals in the migration of cortical interneurons. Here we describe the patterns of expression of Slit and Robo at different stages of forebrain development and review the evidence in support of their role in cortical interneuron migration. Slit-Robo signal transduction mechanisms are also important during normal development in a number of systems in the body and in disease states, making them potential therapeutic targets for the treatment of neurological disorders and certain types of cancer.     

Slit-2/Robo-1 modulates the CXCL12/CXCR4-induced chemotaxis of T cells

Slit, which mediates its function by binding to the Roundabout (Robo) receptor, has been shown to regulate neuronal, dendritic, and leukocyte migration. However, the molecular mechanism by which the Slit/Robo complex inhibits the migration of cells is not well defined. Here, we showed that Slit-2 can inhibit the CXCL12-induced chemotaxis and transendothelial migration of T cells and monocytes. We observed that CXCR4 associates with Robo-1 and that Slit-2 treatment enhances this association with the Robo-1 receptor. Robo-1 is a single-pass transmembrane receptor whose intracellular region contains four conserved motifs designated as CC0, CC1, CC2, and CC3. Structural and functional analyses of Robo receptors revealed that interaction of the CC3 motif with the CXCR4 receptor may regulate the CXCL12-induced chemotaxis of T cells. We further characterized Slit-2-mediated inhibition of the CXCL12/CXCR4 chemotactic pathway and found that Slit-2 can block the CXCL12-induced activation of the Src and Lck kinases but not Lyn kinase. Although Slit-2 did not inhibit the CXCL12-induced activation of MAPKs, it did inhibit the Akt phosphorylation and Rac activation induced by this chemokine. Altogether, our studies indicate a novel mechanism by which the Slit/Robo complex may inhibit the CXCR4/CXCL12-mediated chemotaxis of T cells.     

Slit and robo expression in the developing mouse lung.

Mammalian lung development is mediated through complex interactions between foregut endoderm and surrounding mesenchyme. As airway branching progresses, the mesenchyme undergoes dramatic remodeling and differentiation. Little is understood about the mechanisms that direct mesenchymal organization during lung development. A screen for candidate genes mediating this process identified Slit, a ligand for the Roundabout (Robo) receptor previously associated with guidance of axonal projections during central nervous system development. Here, we demonstrate by in situ hybridization that two Slit genes (Slit-2 and Slit-3) and two Robo genes (Robo-1 and Robo-2) are expressed in fetal lung mesenchyme. Slit-2 and Robo-1 expression is present throughout mesenchyme at midgestation and is not detectable by newborn day 1. Slit-3 and Robo-2 expression is restricted to specific, complementary subsets of mesenchyme. Robo-2 is expressed in mesenchymal cells immediately adjacent to large airways, whereas Slit-3 expression predominates in mesenchyme remote from airway epithelium. The temporal and spatial distribution of Slit and Robo mRNAs indicate that these genes may direct the functional organization and differentiation of fetal lung mesenchyme.     

A genetic screen for sleep and circadian mutants reveals mechanisms underlying regulation of sleep in Drosophila

STUDY OBJECTIVES: In order to characterize the genetic mechanisms underlying sleep, we have carried out a large-scale screen in Drosophila to identify short-sleeping mutants. The objectives of this study were as follows: (1) characterize the phenotypes of the shortest-sleeping mutants; (2) examine whether changes in arousal threshold or sleep homeostasis underlie short-sleeping phenotypes; (3) clone a gene affected in one of the shortest sleepers; and (4) investigate whether circadian mutants can be identified using light:dark (L:D) locomotor data obtained for studying sleep behavior. DESIGN: Locomotor activity was measured using the Drosophila Activity Monitoring System in a 12:12 L:D cycle. SETTING: Drosophila research laboratory. Participants: Adult flies from the 2nd chromosome Zuker collection, which contain mutations in most of the nonessential genes on the Drosophila 2nd chromosome. MEASUREMENTS AND RESULTS: Our analysis of sleep characteristics suggests that daily activity (but not waking activity) correlates with daily sleep time and that defects in sleep maintenance are more common than defects in sleep initiation. Our shortest sleepers have intact or increased sleep rebound following sleep deprivation but show reduced thresholds for arousal. Molecular analysis of one of the short-sleeping lines indicates that it is a novel allele of a dopamine transporter (DAT). Finally, we describe a novel approach for identifying circadian mutants using L:D data. CONCLUSIONS: Our data suggest that most short-sleeping mutant phenotypes in Drosophila are characterized by an inability to stay asleep, most likely because of a reduced arousal threshold.