Robo Function Research Articles

Reduced synchroneity of intra-islet Ca2+ oscillations in vivo in Robo-deficient β cells.

The spatial architecture of the islets of Langerhans is hypothesized to facilitate synchronized insulin secretion among β cells, yet testing this in vivo in the intact pancreas is challenging. Robo βKO mice, in which the genes Robo1 and Robo2 are deleted selectively in β cells, provide a unique model of altered islet spatial architecture without loss of β cell differentiation or islet damage from diabetes. Combining Robo βKO mice with intravital microscopy, we show here that Robo βKO islets have reduced synchronized intra-islet Ca2+ oscillations among β cells in vivo. We provide evidence that this loss is not due to a β cell-intrinsic function of Robo, mis-expression or mis-localization of Cx36 gap junctions, or changes in islet vascularization or innervation, suggesting that the islet architecture itself is required for synchronized Ca2+ oscillations. These results have implications for understanding structure-function relationships in the islets during progression to diabetes as well as engineering islets from stem cells.

Islet Architecture Controls Synchronous β Cell Response to Glucose in the Intact Mouse Pancreas <i>in vivo</i>

The spatial architecture of the islets of Langerhans is hypothesized to facilitate synchronized insulin secretion between β cells, yet testing this in vivo in the intact pancreas is challenging. Robo βKO mice, in which the genes Robo1 and Robo2 are deleted selectively in β cells, provide a unique model of altered islet architecture without loss of β cell differentiation or islet damage from diabetes. Combining Robo βKO mice with intravital microscopy, we show here that Robo βKO islets lose synchronized intra-islet Ca2+ oscillations between β cells in vivo. We provide evidence that this loss is not due to a β cell-intrinsic function of Robo, loss of Connexin36 gap junctions, or changes in islet vascularization, suggesting that the islet architecture itself is required for synchronized Ca2+ oscillations. These results will have implications for understanding structure-function relationships in the islets during progression to diabetes as well as engineering islets from stem cells.

BmRobo1a and BmRobo1b control axon repulsion in the silkworm Bombyx mori

The development of the nervous system is based on the growth and connection of axons, and axon guidance molecules are the dominant regulators during this course. Robo, as the receptor of axon guidance molecule Slit, plays a key role as a conserved repellent cue for axon guidance during the development of the central nervous system. However, the function of Robo in the silkworm Bombyx mori is unknown. In this study, we cloned two novel robo genes in B. mori (Bmrobo1a and Bmrobo1b). BmRobo1a and BmRobo1b lack an Ig and a FNIII domain in the extracellular region and the CC0 and CC2 motifs in the intracellular region. BmRobo1a and BmRobo1b were colocalized with BmSlit in the neuropil. Knock-down of Bmrobo1a and Bmrobo1b by RNA interference (RNAi) resulted in abnormal development of axons. Our results suggest that BmRobo1a and BmRobo1b have repulsive function in axon guidance, even though their structures are different from Robo1 of other species.

BmRobo2/3 is required for axon guidance in the silkworm Bombyx mori

Axon guidance is critical for proper wiring of the nervous system. During the neural development, the axon guidance molecules play a key role and direct axons to choose the correct way to reach the target. Robo, as the receptor of axon guidance molecule Slit, is evolutionarily conserved from planarians to humans. However, the function of Robo in the silkworm, Bombyx mori, remained unknown. In this study, we cloned robo2/3 from B. mori (Bmrobo2/3), a homologue of robo2/3 in Tribolium castaneum. Moreover, BmRobo2/3 was localized in the neuropil, and RNAi-mediated knockdown of Bmrobo2/3 resulted in the longitudinal connectives forming closer to the midline. These data demonstrate that BmRobo2/3 is required for axon guidance in the silkworm.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Axon Sorting within the Spinal Cord Marginal Zone via Robo-Mediated Inhibition of N-Cadherin Controls Spinocerebellar Tract Formation

The axons of spinal projection neurons transmit sensory information to the brain by ascending within highly organized longitudinal tracts. However, the molecular mechanisms that control the sorting of these axons within the spinal cord and their directed growth to poorly defined targets are not understood. Here, we show that an interplay between Robo and the cell adhesion molecule, N-cadherin, sorts spinal commissural axons into appropriate longitudinal tracts within the spinal cord, and thereby facilitates their brain targeting. Specifically, we show that d1 and d2 spinal commissural axons join the lateral funiculus within the spinal cord and target the cerebellum in chick embryos, and that these axons contribute to the spinocerebellar projection in transgenic reporter mice. Disabling Robo signaling or overexpressing N-cadherin on these axons prevents the formation of the lateral funiculus and the spinocerebellar tract, and simultaneously perturbing Robo and N-cadherin function rescues both phenotypes in chick embryos. Consistent with these observations, disabling Robo function in conditional N-cadherin knock-out mice results in a wild-type-like lateral funiculus. Together, these findings suggest that spinal projection axons must be sorted into distinct longitudinal tracts within the spinal cord proper to project to their brain targets.

The Actin-Binding Protein Canoe/AF-6 Forms a Complex with Robo and Is Required for Slit-Robo Signaling during Axon Pathfinding at the CNS Midline

Axon guidance is a key process during nervous system development and regeneration. One of the best established paradigms to study the mechanisms underlying this process is the axon decision of whether or not to cross the midline in the Drosophila CNS. An essential regulator of that decision is the well conserved Slit-Robo signaling pathway. Slit guidance cues act through Robo receptors to repel axons from the midline. Despite good progress in our knowledge about these proteins, the intracellular mechanisms associated with Robo function remain poorly defined. In this work, we found that the scaffolding protein Canoe (Cno), the Drosophila orthologue of AF-6/Afadin, is essential for Slit-Robo signaling. Cno is expressed along longitudinal axonal pioneer tracts, and longitudinal Robo/Fasciclin2-positive axons aberrantly cross the midline in cno mutant embryos. cno mutant primary neurons show a significant reduction of Robo localized in growth cone filopodia and Cno forms a complex with Robo in vivo. Moreover, the commissureless (comm) phenotype (i.e., lack of commissures due to constitutive surface presentation of Robo in all neurons) is suppressed in comm, cno double-mutant embryos. Specific genetic interactions between cno, slit, robo, and genes encoding other components of the Robo pathway, such as Neurexin-IV, Syndecan, and Rac GTPases, further confirm that Cno functionally interacts with the Slit-Robo pathway. Our data argue that Cno is a novel regulator of the Slit-Robo signaling pathway, crucial for regulating the subcellular localization of Robo and for transducing its signaling to the actin cytoskeleton during axon guidance at the midline.

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

In this study we examined Roundabout signaling in the Drosophila embryonic hindgut. 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. 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.

Slit‐Robo signals regulate pioneer axon pathfinding of the tract of the postoptic commissure in the mammalian forebrain

During early vertebrate forebrain development, pioneer axons establish a symmetrical scaffold descending longitudinally through the rostral forebrain, thus forming the tract of the postoptic commissure (TPOC). In mouse embryos, this tract begins to appear at embryonic day 9.5 (E9.5) as a bundle of axons tightly constrained at a specific dorsoventral level. We have characterized the participation of the Slit chemorepellants and their Robo receptors in the control of TPOC axon projection. In E9.5-E11.5 mouse embryos, Robo1 and Robo2 are expressed in the nucleus origin of the TPOC (nTPOC), and Slit expression domains flank the TPOC trajectory. These findings suggested that these proteins are important factors in the dorsoventral positioning of the TPOC axons. Consistently with this role, Slit2 inhibited TPOC axon growth in collagen gel cultures, and interfering with Robo function in cultured embryos induced projection errors in TPOC axons. Moreover, absence of both Slit1 and Slit2 or Robo1 and Robo2 in mutant mouse embryos revealed aberrant TPOC trajectories, resulting in abnormal spreading of the tract and misprojections into both ventral and dorsal tissues. These results reveal that Slit-Robo signaling regulates the dorsoventral position of this pioneer tract in the developing forebrain.

Independent Functions of Slit–Robo Repulsion and Netrin–Frazzled Attraction Regulate Axon Crossing at the Midline inDrosophila

Slit and Netrin and their respective neuronal receptors play critical roles in patterning axonal connections in the developing nervous system by regulating the decision of whether or not to cross the midline. Studies of both invertebrate and vertebrate systems support the idea that Netrin, secreted by midline cells, signals through DCC (Deleted in Colorectal Carcinoma)/UNC40/Frazzled receptors to attract commissural axons toward and across the midline, whereas Slit signals through Robo family receptors to prevent commissural axons from recrossing the midline, as well as to prevent ipsilateral axons from ever crossing. Recent evidence from both Xenopus neuronal cell culture and Drosophila genetics have suggested that these signals may interact more directly in a hierarchical relationship, such that one response extinguishes the other. Here we present loss- and gain-of-function genetic evidence showing that the influence of Slit and Netrin on midline axon crossing is dictated by both independent and interdependent signaling functions of the Robo and Frazzled (Fra) receptors. Our results are not consistent with the proposal based on genetic analysis in Drosophila that the sole function of Slit and Robo during midline guidance is to repress Netrin attraction.

Slit-mediated repulsion is a key regulator of motor axon pathfinding in the hindbrain

The floor plate is known to be a source of repellent signals for cranial motor axons, preventing them from crossing the midline of the hindbrain. However, it is unknown which molecules mediate this effect in vivo. We show that Slit and Robo proteins are candidate motor axon guidance molecules, as Robo proteins are expressed by cranial motoneurons, and Slit proteins are expressed by the tissues that delimit motor axon trajectories, i.e. the floor plate and the rhombic lip. We present in vitro evidence showing that Slit1 and Slit2 proteins are selective inhibitors and repellents for dorsally projecting, but not for ventrally projecting, cranial motor axons. Analysis of mice deficient in Slit and Robo function shows that cranial motor axons aberrantly enter the midline, while ectopic expression of Slit1 in chick embryos leads to specific motor axon projection errors. Expression of dominant-negative Robo receptors within cranial motoneurons in chick embryos strikingly perturbs their projections, causing some motor axons to enter the midline, and preventing dorsally projecting motor axons from exiting the hindbrain. These data suggest that Slit proteins play a key role in guiding dorsally projecting cranial motoneurons and in facilitating their neural tube exit.

Robo3 isoforms have distinct roles during zebrafish development

Roundabout (Robo) receptors and their secreted ligand Slits have been shown to function in a number of developmental events both inside and outside of the nervous system. We previously cloned zebrafish robo orthologs to gain a better understanding of Robo function in vertebrates. Further characterization of one of these orthologs, robo3, has unveiled the presence of two distinct isoforms, robo3 variant 1 ( robo3var1) and robo3 variant 2 ( robo3var2). These two isoforms differ only in their 5′-ends with robo3var1, but not robo3var2, containing a canonical signal sequence. Despite this difference, both forms accumulate on the cell surface. Both isoforms are contributed maternally and exhibit unique and dynamic gene expression patterns during development. Functional analysis of robo3 isoforms using an antisense gene knockdown strategy suggests that Robo3var1 functions in motor axon pathfinding, whereas Robo3var2 appears to function in dorsoventral cell fate specification. This study reveals a novel function for Robo receptors in specifying ventral cell fates during vertebrate development.

Axon Sorting in the Optic Tract Requires HSPG Synthesis by ext2 (dackel) and extl3 (boxer)

Retinal ganglion cell (RGC) axons are topographically ordered in the optic tract according to their retinal origin. In zebrafish dackel (dak) and boxer (box) mutants, some dorsal RGC axons missort in the optic tract but innervate the tectum topographically. Molecular cloning reveals that dak and box encode ext2 and extl3, glycosyltransferases implicated in heparan sulfate (HS) biosynthesis. Both genes are required for HS synthesis, as shown by biochemical and immunohistochemical analysis, and are expressed maternally and then ubiquitously, likely playing permissive roles. Missorting in box can be rescued by overexpression of extl3. dak;box double mutants show synthetic pathfinding phenotypes that phenocopy robo2 mutants, suggesting that Robo2 function requires HS in vivo; however, tract sorting does not require Robo function, since it is normal in robo2 null mutants. This genetic evidence that heparan sulfate proteoglycan function is required for optic tract sorting provides clues to begin understanding the underlying molecular mechanisms.

Comm Keeps Robo Down

Some axons will cross the midline to innervate the contralateral side of the animal. The developing Drosophila nervous system has provided a model system for studying the molecular mechanisms underlying the movement of axons at the midline. Neurons that express the Robo receptor are repelled from crossing the midline by the Slit ligand produced by the midline cells; whereas neurons migrate across the midline in response to the chemoattractant Netrins, which interact with the Frazzled receptor. To prevent recrossing of the midline, Robo is up-regulated once the neurons reach the contralateral side. Kelemann et al. provide evidence that the regulation of Robo occurs through the actions of the commissureless ( comm ) gene product and involves the regulation of the intracellular trafficking of Robo by Comm. Using cell transplantation studies, the authors determined that Comm function was important in the neuron and not the midline glia for allowing midline crossing. Neurons that crossed the midline displayed high levels of comm expression at the time the axons were crossing the midline, and expression was much lower before and after midline crossing. Neurons that did not cross the midline did not express comm . When expressed in cultured COS cells, Robo was detected at the cell surface, but when coexpressed with Comm, Robo was detected in late endosomes and lysosomes along with Comm. Comm and Comm-deletion constructs that were competent to sort Robo in transfected cultured cells into the endosomal-lysosomal compartment also produced robo-like phenotypes in Drosophila . Thus, Comm appears to control Robo function by preventing cell-surface expression of Robo during the period of midline crossing. K. Keleman, S. Rajagopalan, D. Cleppien, D. Teis, K. Paiha, L. A. Huber, G. M. Tachnau, B. J. Dickson, Comm sorts Robo to control axon guidance at the Drosophila midline. Cell 110 , 415-427 (2002). [Online Journal]

Comm Keeps Robo Down

Some axons will cross the midline to innervate the contralateral side of the animal. The developing Drosophila nervous system has provided a model system for studying the molecular mechanisms underlying the movement of axons at the midline. Neurons that express the Robo receptor are repelled from crossing the midline by the Slit ligand produced by the midline cells; whereas neurons migrate across the midline in response to the chemoattractant Netrins, which interact with the Frazzled receptor. To prevent recrossing of the midline, Robo is up-regulated once the neurons reach the contralateral side. Kelemann et al. provide evidence that the regulation of Robo occurs through the actions of the commissureless (comm) gene product and involves the regulation of the intracellular trafficking of Robo by Comm. Using cell transplantation studies, the authors determined that Comm function was important in the neuron and not the midline glia for allowing midline crossing. Neurons that crossed the midline displayed high levels of comm expression at the time the axons were crossing the midline, and expression was much lower before and after midline crossing. Neurons that did not cross the midline did not express comm. When expressed in cultured COS cells, Robo was detected at the cell surface, but when coexpressed with Comm, Robo was detected in late endosomes and lysosomes along with Comm. Comm and Comm-deletion constructs that were competent to sort Robo in transfected cultured cells into the endosomal-lysosomal compartment also produced robo-like phenotypes in Drosophila. Thus, Comm appears to control Robo function by preventing cell-surface expression of Robo during the period of midline crossing.K. Keleman, S. Rajagopalan, D. Cleppien, D. Teis, K. Paiha, L. A. Huber, G. M. Tachnau, B. J. Dickson, Comm sorts Robo to control axon guidance at the Drosophila midline. Cell 110, 415-427 (2002). [Online Journal]

The Robo Code

Robo (expressed on neurons) and Slit (expressed by midline glia) are a receptor and repulsive ligand, respectively, that help control midline crossing and cellular migration of neurons during development (and mesodermal cells during the development of other organs and tissues). However, Drosophila slit and robo mutants have different phenotypes, suggesting that there may be additional receptors for Slit: slit causes all axons to enter the midline and not leave, robo causes aberrant crossing and recrossing of the midline. Two groups (Rajagopalan et al. and Simpson et al. ) have characterized two additional Robo receptors, Robo2 and Robo3, in Drosophila for their roles in controlling neuronal migration and midline crossing and the formation of the longitudinal fascicles. Both groups showed that Robo and Robo2 double mutants recapitulate the slit phenotype, and they analyze the expression patterns of the robo genes. The three robo genes are expressed at different stages of development with unique and overlapping patterns of expression in different cells. This differential expression of Robo proteins or "Robo code" provides a complex interplay and regulatory mechanism for cellular interpretation of the repulsive Slit signal. Simpson et al. also identified a biphasic effect of Robo2 dosage: low doses cause inappropriate crossing of the midline, high doses cause a commissureless phenotype where no axons cross the midline. Simpson et al. suggest that low doses of Robo2 interfere with Robo signaling resulting in a partial robo loss-of-function phenotype, whereas high enough levels of Robo2 are sufficient to transmit a repulsive signal. Kinrade et al. focused their attention on the role of Robo in glial migration along the midline in Drosophila embryos. They found that Robo is expressed transiently by glia early in development and that loss of Robo function results in the glia of the most medial fascicle inappropriately crossing the midline. Furthermore, whereas the initial trajectory of the most medial, longitudinal fascicle is directed in a Robo-dependent manner by the pioneer neuron, the maintenance of the longitudinal trajectory of the follower neurons is dependent on trophic interactions between the longitudinal glia and the neurons. When the pioneer neuron is ablated, then both the longitudinal glia and the axons of the longitudinal fascicle cross the midline despite the expression of Robo by the longitudinal neurons. As Rusch and Van Vactor put it, neuronal migration along the midline is regulated by the Robo code and, according to Kinrade et al ., enforced by trophic interactions between glia and neuron. S. Rajagopalan, E. Nicolas, V. Vivancos, J. Berger, B. J. Dickson, Crossing the midline: Roles and regulation of Robo receptors. Neuron 28, 767-777 (2000). [Online Journal] J. H. Simpson, T. Kidd, K. S. Bland, C. S. Goodman, Short-range and long-range guidance by slit and its Robo receptors: Robo and Robo2 play distinct roles in midline guidance. Neuron 28 , 753-766 (2000). [Online Journal] E.F.V. Kinrade, T. Brates, G. Tear, A. Hidalgo, Roundabout signalling, cell contact and trophic support confine longitudinal glia and axons in the Drosophila CNS. Development 128, 207-216 (2000). [Online Journal] J. Rusch, D. Van Vactor, New roundabouts send axons into the fas lane. Neuron 28 , 637-640 (2000). [Online Journal] J. H. Simpson, K. S. Bland, R. D. Fetter, C. S. Goodman, Short-range and long-range guidance by Slit and its Robo receptors: A combinatorial code of Robo receptors controls lateral position. Cell 103 , 1019-1032 (2000). [Online Journal] S. Rajagopalan, V. Vivancos, E. Nicolas, B. J. Dickson, Selecting a longitudinal pathway: Robo receptors specify the lateral position of axons in the Drosophila CNS. Cell 103 , 1033-1045 (2000). [Online Journal]

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 which function downstream of Robo to mediate repulsion. Here, we present genetic and biochemical evidence 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.

Dosage-Sensitive and Complementary Functions of Roundabout and Commissureless Control Axon Crossing of the CNS Midline

commissureless and roundabout lead to complementary mutant phenotypes in which either too few or too many axons cross the midline. The robo; comm double-mutant phenotype is identical to robo alone, suggesting that in the absence of robo, comm is no longer required. Comm is expressed on midline cells; Robo is expressed in a dynamic fashion on growth cones and appears to function as an axon guidance receptor. robo function is dosage-sensitive. Overexpression of comm is also dosage-sensitive and leads to a phenotype identical to robo loss-of-function. Comm controls Robo expression; increasing Comm leads to a reduction of Robo protein. The levels of Comm and Robo appear to be tightly regulated to assure that only certain growth cones cross the midline and that those growth cones that do cross never do so again.