Slit-Robo signaling mediates lymphangiogenesis and promotes tumor lymphatic metastasis

The Slit family of guidance cues binds to Roundabout (Robo) receptors to modulate neuronal, leukocytic, and endothelial migration. Slit-Robo signaling had been reported to function as chemoattractive signal for vascular endothelial cells during angiogenesis. In this study, we found that Robo1 was expressed in lymphatic endothelial cells to mediate the migration and tube formation of these cells upon Slit2 stimulation, which were specifically inhibited by the function-blocking antibody R5 to Slit2/Robo1 interaction. To further explore the lymphangiogenic effect and significance mediated by Slit-Robo signaling, we intercrossed Slit2 transgenic mice with a non-metastatic RIP1-Tag2 mouse tumor model, and found that transgenic overexpression of Slit2 significantly enhanced tumor lymphangiogenesis and subsequently promoted mesenteric lymph node metastasis of pancreatic islet tumors. Taken together, our findings reveal that through interacting with Robo1, Slit2 is a novel and potent lymphangiogenic factor and contributes to tumor lymphatic metastasis.     

A molecular mechanism for the heparan sulfate dependence of slit-robo signaling

Slit is a large secreted protein that provides important guidance cues in the developing nervous system and in other organs. Signaling by Slit requires two receptors, Robo transmembrane proteins and heparan sulfate (HS) proteoglycans. How HS controls Slit-Robo signaling is unclear. Here we show that the second leucine-rich repeat domain (D2) of Slit, which mediates binding to Robo receptors, also contains a functionally important binding site for heparin, a highly sulfated variant of HS. Heparin markedly enhances the affinity of the Slit-Robo interaction in a solid-phase binding assay. Analytical gel filtration chromatography demonstrates that Slit D2 associates with a soluble Robo fragment and a heparin-derived oligosaccharide to form a ternary complex. Retinal growth cone collapse triggered by Slit D2 requires cell surface HS or exogenously added heparin. Mutation of conserved basic residues in the C-terminal cap region of Slit D2 reduces heparin binding and abolishes biological activity. We conclude that heparin/HS is an integral component of the minimal Slit-Robo signaling complex and serves to stabilize the relatively weak Slit-Robo interaction.     

The role of Slit-Robo signaling in the generation, migration and morphological differentiation of cortical interneurons

Cortical interneurons in rodents are generated in the ventral telencephalon and migrate tangentially into the cortex. This process requires the coordinated action of many intrinsic and extrinsic factors. Here we show that Robo1 and Robo2 receptor proteins are dynamically expressed throughout the period of corticogenesis and colocalize with interneuronal markers, suggesting that they play a role in the migration of these cells. Analysis of Robo mutants showed a marked increase in the number of interneurons in the cortices of Robo1^−/−, but not Robo2^−/−, animals throughout the period of corticogenesis and in adulthood; this excess number of interneurons was observed in all layers of the developing cortex. Using BrdU incorporation in dissociated cell cultures and phosphohistone-3 labeling in vivo, we demonstrated that the increased number of interneurons in Robo1^−/− mice is, at least in part, due to increased proliferation. Interestingly, a similar increase in proliferation was observed in Slit1^−/−/Slit2^−/− mutant mice, suggesting that cell division is influenced by Slit-Robo signaling mechanisms. Morphometric analysis of migrating interneurons in Robo1^−/−, Robo2^−/− and Slit1^−/−/Slit2^−/−, but not in Slit1^−/− mice, showed a differential increase in neuronal process length and branching suggesting that Slit-Robo signaling also plays an important role in the morphological differentiation of these neurons.     

Regulation of cortical dendrite development by Slit-Robo interactions.

Slit proteins have previously been shown to regulate axon guidance, branching, and neural migration. Here we report that, in addition to acting as a chemorepellant for cortical axons, Slit1 regulates dendritic development. Slit1 is expressed in the developing cortex, and exposure to Slit1 leads to increased dendritic growth and branching. Conversely, inhibition of Slit-Robo interactions by Robo-Fc fusion proteins or by a dominant-negative Robo attenuates dendritic branching. Stimulation of neurons transfected with a Met-Robo chimeric receptor with Hepatocyte growth factor leads to a robust induction of dendritic growth and branching, suggesting that Robo-mediated signaling is sufficient to induce dendritic remodeling. These experiments indicate that Slit-Robo interactions may exert a significant influence over the specification of cortical neuron morphology by regulating both axon guidance and dendritic patterning.     

Structural and functional analysis of slit and heparin binding to immunoglobulin-like domains 1 and 2 of Drosophila Robo

Recognition of the secreted protein Slit by transmembrane receptors of the Robo family provides important signals in the development of the nervous system and other organs, as well as in tumor metastasis and angiogenesis. Heparan sulfate (HS) proteoglycans serve as essential co-receptors in Slit-Robo signaling. Previous studies have shown that the second leucinerich repeat domain of Slit, D2, binds to the N-terminal immunoglobulin-like domains of Robo, IG1-2. Here we present two crystal structures of Drosophila Robo IG1-2, one of which contains a bound heparin-derived oligosaccharide. Using structure-based mutagenesis of a Robo IG1-5 construct we identified key Slit binding residues (Thr-74, Phe-114, Arg-117) forming a conserved patch on the surface of IG1; mutation of similarly conserved residues in IG2 had no effect on Slit binding. Mutation of conserved basic residues in IG1 (Lys-69, Arg-117, Lys-122, Lys-123), but not in IG2, reduced binding of Robo IG1-5 to heparin, in full agreement with the Robo-heparin co-crystal structure. Our collective results, together with a recent crystal structure of a minimal human Slit-Robo complex ( Morlot, C., Thielens, N. M., Ravelli, R. B., Hemrika, W., Romijn, R. A., Gros, P., Cusack, S., and McCarthy, A. A. (2007) Proc. Natl. Acad. Sci. U.S.A. 104, 14923-14928 ), reveal a contiguous HS/heparin binding surface extending across the Slit-Robo interface. Based on the size of this composite binding site, we predict that at least five HS disaccharide units are required to support Slit-Robo signaling.     

Jelly belly: a Drosophila LDL receptor repeat-containing signal required for mesoderm migration and differentiation.

Inductive interactions subdivide the Drosophila mesoderm into visceral, somatic, and heart muscle precursors. The muscle precursors form organs by executing tissue-specific migrations and cell fusions. We identified a novel gene, jelly belly (jeb), which is required for visceral mesoderm development. jeb encodes a secreted protein that contains an LDL receptor repeat. In jeb mutants, visceral mesoderm precursors form, but they fail to migrate or differentiate normally; no visceral muscles develop. Jeb protein is produced in somatic muscle precursors and taken up by visceral muscle precursors. jeb reveals a signaling process in which somatic muscle precursors support the proper migration and differentiation of visceral muscle cells. Later in embryogenesis, jeb is transcribed in neurons and Jeb protein is found in axons.     

Cooperative signaling between Slit2 and Ephrin-A1 regulates a balance between angiogenesis and angiostasis

Slit proteins induce cytoskeletal remodeling through interaction with roundabout (Robo) receptors, regulating migration of neurons and nonneuronal cells, including leukocytes, tumor cells, and endothelium. The role of Slit2 in vascular remodeling, however, remains controversial, with reports of both pro- and antiangiogenic activity. We report here that cooperation between Slit2 and ephrin-A1 regulates a balance between the pro- and antiangiogenic functions of Slit2. While Slit2 promotes angiogenesis in culture and in vivo as a single agent, Slit2 potently inhibits angiogenic remodeling in the presence of ephrin-A1. Slit2 stimulates angiogenesis through mTORC2-dependent activation of Akt and Rac GTPase, the activities of which are inhibited in the presence of ephrin-A1. Activated Rac or Akt partially rescues vascular assembly and motility in costimulated endothelium. Taken together, these data suggest that Slit2 differentially regulates angiogenesis in the context of ephrin-A1, providing a plausible mechanism for the pro- versus antiangiogenic functions of Slit2. Our results suggest that the complex roles of Slit-Robo signaling in angiogenesis involve context-dependent mechanisms.     

Evidence for a role of srGAP3 in the positioning of commissural axons within the ventrolateral funiculus of the mouse spinal cord

Slit-Robo signaling guides commissural axons away from the floor-plate of the spinal cord and into the longitudinal axis after crossing the midline. In this study we have evaluated the role of the Slit-Robo GTPase activating protein 3 (srGAP3) in commissural axon guidance using a knockout (KO) mouse model. Co-immunoprecipitation experiments confirmed that srGAP3 interacts with the Slit receptors Robo1 and Robo2 and immunohistochemistry studies showed that srGAP3 co-localises with Robo1 in the ventral and lateral funiculus and with Robo2 in the lateral funiculus. Stalling axons have been reported in the floor-plate of Slit and Robo mutant spinal cords but our axon tracing experiments revealed no dorsal commissural axon stalling in the floor plate of the srGAP3 KO mouse. Interestingly we observed a significant thickening of the ventral funiculus and a thinning of the lateral funiculus in the srGAP3 KO spinal cord, which has also recently been reported in the Robo2 KO. However, axons in the enlarged ventral funiculus of the srGAP3 KO are Robo1 positive but do not express Robo2, indicating that the thickening of the ventral funiculus in the srGAP3 KO is not a Robo2 mediated effect. We suggest a role for srGAP3 in the lateral positioning of post crossing axons within the ventrolateral funiculus.     

Slit2 and Robo3 modulate the migration of GnRH-secreting neurons

Gonadotropin-releasing hormone (GnRH) neurons are born in the nasal placode and migrate along olfactory and vomeronasal axons to reach the forebrain and settle in the hypothalamus, where they control reproduction. The molecular cues that guide their migration have not been fully identified, but are thought to control either cell movement directly or the patterning of their axonal substrates. Using genetically altered mouse models we show that the migration of GnRH neurons is directly modulated by Slit2 and Robo3, members of the axon guidance Slit ligand and Robo receptor families. Mice lacking Slit2 or Robo3 have a reduced number of GnRH neurons in the forebrain, but a normal complement of their supporting axons, pointing to a direct role for these molecules in GnRH neuron migration.     

Slit2 regulates attractive eosinophil and repulsive neutrophil chemotaxis through differential srGAP1 expression during lung inflammation

Directional migration of leukocytes is an essential step in leukocyte trafficking during inflammatory responses. However, the molecular mechanisms governing directional chemotaxis of leukocytes remain poorly understood. The Slit family of guidance cues has been implicated for inhibition of leuocyte migration. We report that Clara cells in the bronchial epithelium secreted Slit2, whereas eosinophils and neutrophils expressed its cell-surface receptor, Robo1. Compared to neutrophils, eosinophils exhibited a significantly lower level of Slit-Robo GTPase-activating protein 1 (srGAP1), leading to activation of Cdc42, recruitment of PI3K to Robo1, enhancment of eotaxin-induced eosinophil chemotaxis, and exaggeration of allergic airway inflammation. Notably, OVA sensitization elicited a Slit2 gradient at so-called bronchus-alveoli axis, with a higher level of Slit2 in the bronchial epithelium and a lower level in the alveolar tissue. Aerosol administration of rSlit2 accelerated eosinophil infiltration, whereas i.v. administered Slit2 reduced eosinophil deposition. In contrast, Slit2 inactivated Cdc42 and suppressed stromal cell-derived factor-1α-induced chemotaxis of neutrophils for inhibiting endotoxin-induced lung inflammation, which were reversed by blockade of srGAP1 binding to Robo1. These results indicate that the newly identified Slit2 gradient at the bronchus-alveoli axis induces attractive PI3K signaling in eosinophils and repulsive srGAP1 signaling in neutrophils through differential srGAP1 expression during lung inflammation.     

Slit-Robo signalling prevents sensory cells from crossing the midline in Drosophila

Maintenance of bilateral symmetry throughout animal development requires that both left and right halves of the body follow nearly identical patterns of cell proliferation, differentiation, death and migration. During formation of the perfectly bilateral Drosophila larval peripheral nervous system (PNS), the sensory precursor cells of the ventral multidendritic neuron vmd1a originating from each hemisegment migrate away from the ventral midline. Our observations indicate that in slit mutant embryos, as well as in robo, robo2 double mutants, sensory precursor cells of the left and right vmd1a neurons aberrantly cluster at the midline and then the pair of vmd1a neurons migrate to their final position on the same side of the embryo. This results in disruption of PNS bilateral symmetry. Expression of slit at the midline rescues the slit mutant vmd1a phenotype, suggesting that midline-secreted Slit activates Robo/Robo2 signalling to control the migration of the vmd1a sensory precursor cells. Our study indicates that midline-secreted Slit prevents vmd1a sensory cells from crossing the midline and thereby maintains PNS bilateral symmetry during development.     

The first "Slit" is the deepest: the secret to a hollow heart

Tubular organs are essential for life, but lumen formation in nonepithelial tissues such as the vascular system or heart is poorly understood. Two studies in this issue (Medioni, C., M. Astier, M. Zmojdzian, K. Jagla, and M. Sémériva. 2008. J. Cell Biol. 182:249-261; Santiago-Martínez, E., N.H. Soplop, R. Patel, and S.G. Kramer. 2008. J. Cell Biol. 182:241-248) reveal unexpected roles for the Slit-Robo signaling system during Drosophila melanogaster heart morphogenesis. In cardioblasts, Slit and Robo modulate the cell shape changes and domains of E-cadherin-based adhesion that drive lumen formation. Furthermore, in contrast to the well-known paracrine role of Slit and Robo in guiding cell migrations, here Slit and Robo may act by autocrine signaling. In addition, the two groups demonstrate that heart lumen formation is even more distinct from typical epithelial tubulogenesis mechanisms because the heart lumen is bounded by membrane surfaces that have basal rather than apical attributes. As the D. melanogaster cardioblasts are thought to have significant evolutionary similarity to vertebrate endothelial and cardiac lineages, these findings are likely to provide insights into mechanisms of vertebrate heart and vascular morphogenesis.     

Slit coordinates cardiac morphogenesis in Drosophila

Slit is a secreted guidance cue that conveys repellent or attractive signals from target and guidepost cells. In Drosophila, responsive cells express one or more of three Robo receptors. The cardial cells of the developing heart express both Slit and Robo2. This is the first report of coincident expression of a Robo and its ligand. In slit mutants, cardial cell alignment, polarization and uniform migration are disrupted. The heart phenotype of robo2 mutants is similar, with fewer migration defects. In the guidance of neuronal growth cones in Drosophila, there is a phenotypic interaction between slit and robo heterozygotes, and also with genes required for Robo signaling. In contrast, in the heart, slit has little or no phenotypic interaction with Robo-related genes, including Robo2, Nck2, and Disabled. However, there is a strong phenotypic interaction with Integrin genes and their ligands, including Laminin and Collagen, and intracellular messengers, including Talin and ILK. This indicates that Slit participates in adhesion or adhesion signaling during heart development.     

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

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

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

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

Compartmentalization of visual centers in the Drosophila brain requires Slit and Robo proteins

Brain morphogenesis depends on the maintenance of boundaries between populations of non-intermingling cells. We used molecular markers to characterize a boundary within the optic lobe of the Drosophila brain and found that Slit and the Robo family of receptors, well-known regulators of axon guidance and neuronal migration, inhibit the mixing of adjacent cell populations in the developing optic lobe. Our data suggest that Slit is needed in the lamina to prevent inappropriate invasion of Robo-expressing neurons from the lobula cortex. We show that Slit protein surrounds lamina glia, while the distal cell neurons in the lobula cortex express all three Drosophila Robos. We examine the function of these proteins in the visual system by isolating a novel allele of slit that preferentially disrupts visual system expression of Slit and by creating transgenic RNA interference flies to inhibit the function of each Drosophila Robo in a tissue-specific fashion. We find that loss of Slit or simultaneous knockdown of Robo, Robo2 and Robo3 causes distal cell neurons to invade the lamina, resulting in cell mixing across the lamina/lobula cortex boundary. This boundary disruption appears to lead to alterations in patterns of axon navigation in the visual system. We propose that Slit and Robo-family proteins act to maintain the distinct cellular composition of the lamina and the lobula cortex.     

Slit1a inhibits retinal ganglion cell arborization and synaptogenesis via Robo2-dependent and -independent pathways

Upon arriving at their targets, developing axons cease pathfinding and begin instead to arborize and form synapses. To test whether CNS arborization and synaptogenesis are controlled by Slit-Robo signaling, we followed single retinal ganglion cell (RGC) arbors over time. ast (robo2) mutant and slit1a morphant arbors had more branch tips and greater arbor area and complexity compared to wild-type and concomitantly more presumptive presynaptic sites labeled with YFP-Rab3. Increased arborization in ast was phenocopied by dominant-negative Robo2 expressed in single RGCs and rescued by full-length Robo2, indicating that Robo2 acts cell-autonomously. Time-lapse imaging revealed that ast and slit1a morphant arbors stabilized earlier than wild-type, suggesting a role for Slit-Robo signaling in preventing arbor maturation. Genetic analysis showed that Slit1a acts both through Robo2 and Robo2-independent mechanisms. Unlike previous PNS studies showing that Slits promote branching, our results show that Slits inhibit arborization and synaptogenesis in the CNS.     

Signal transduction in neuronal migration: roles of GTPase activating proteins and the small GTPase Cdc42 in the Slit-Robo pathway.

The Slit protein guides neuronal and leukocyte migration through the transmembrane receptor Roundabout (Robo). We report here that the intracellular domain of Robo interacts with a novel family of Rho GTPase activating proteins (GAPs). Two of the Slit-Robo GAPs (srGAPs) are expressed in regions responsive to Slit. Slit increased srGAP1-Robo1 interaction and inactivated Cdc42. A dominant negative srGAP1 blocked Slit inactivation of Cdc42 and Slit repulsion of migratory cells from the anterior subventricular zone (SVZa) of the forebrain. A constitutively active Cdc42 blocked the repulsive effect of Slit. These results have demonstrated important roles for GAPs and Cdc42 in neuronal migration. We propose a signal transduction pathway from the extracellular guidance cue to intracellular actin polymerization.