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. We show here that comm is required in neurons, not in midline cells as previously thought, and that 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.     

The N-terminal and transmembrane domains of Commissureless are necessary for its function and trafficking within neurons

Commissureless (Comm) 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-localised with Comm intracellularly. Here we show that the ability of Comm to localise intracellularly and hence regulate Robo surface levels requires sequences in both the N-terminal and transmembrane domains. We also show that Comm can dimerise 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.     

Crossing the midline: roles and regulation of Robo receptors

In the Drosophila CNS, the midline repellent Slit acts at short range through its receptor Robo to control midline crossing. Longitudinal axons express high levels of Robo and avoid the midline; commissural axons that cross the midline express only low levels of Robo. Robo levels are in turn regulated by Comm. Here, we show that the Slit receptors Robo2 and Robo3 ensure the fidelity of this crossing decision: rare crossing errors occur in both robo2 and robo3 single mutants. In addition, low levels of either Robo or Robo2 are required to drive commissural axons through the midline: only in robo,robo2 double mutants do axons linger at the midline as they do in slit mutants. Robo2 and Robo3 levels are also tightly regulated, most likely by a mechanism similar to but distinct from the regulation of Robo by Comm.     

Conserved and divergent aspects of Robo receptor signaling and regulation between Drosophila Robo1 and C. elegans SAX-3

The evolutionarily conserved Roundabout (Robo) family of axon guidance receptors control midline crossing of axons in response to the midline repellant ligand Slit in bilaterian animals including insects, nematodes, and vertebrates. Despite this strong evolutionary conservation, it is unclear whether the signaling mechanism(s) downstream of Robo receptors are similarly conserved. To directly compare midline repulsive signaling in Robo family members from different species, here we use a transgenic approach to express the Robo family receptor SAX-3 from the nematode Caenorhabditis elegans in neurons of the fruit fly, Drosophila melanogaster. We examine SAX-3’s ability to repel Drosophila axons from the Slit-expressing midline in gain of function assays, and test SAX-3’s ability to substitute for Drosophila Robo1 during fly embryonic development in genetic rescue experiments. We show that C. elegans SAX-3 is properly translated and localized to neuronal axons when expressed in the Drosophila embryonic CNS, and that SAX-3 can signal midline repulsion in Drosophila embryonic neurons, although not as efficiently as Drosophila Robo1. Using a series of Robo1/SAX-3 chimeras, we show that the SAX-3 cytoplasmic domain can signal midline repulsion to the same extent as Robo1 when combined with the Robo1 ectodomain. We show that SAX-3 is not subject to endosomal sorting by the negative regulator Commissureless (Comm) in Drosophila neurons in vivo, and that peri-membrane and ectodomain sequences are both required for Comm sorting of Drosophila Robo1.     

The divergent Robo family protein rig-1/Robo3 is a negative regulator of slit responsiveness required for midline crossing by commissural axons

Commissural axons in vertebrates and insects are initially attracted to the nervous system midline, but once they reach this intermediate target they undergo a dramatic switch, becoming responsive to repellent Slit proteins at the midline, which expel them onto the next leg of their trajectory. We have unexpectedly implicated a divergent member of the Robo family, Rig-1 (or Robo3), in preventing premature Slit sensitivity in mammals. Expression of Rig-1 protein by commissural axons is inversely correlated with Slit sensitivity. Removal of Rig-1 results in a total failure of commissural axons to cross. Genetic and in vitro analyses indicate that Rig-1 functions to repress Slit responsiveness similarly to Commissureless (Comm) in Drosophila. Unlike Comm, however, Rig-1 does not produce its effect by downregulating Robo receptors on precrossing commissural axon membranes. These results identify a mechanism for regulating Slit repulsion that helps choreograph the precise switch from attraction to repulsion at a key intermediate axonal target.     

Redundant Mechanisms for Regulation of Midline Crossing in Drosophila

During development, all neurons have to decide on whether to cross the longitudinal midline to project on the contralateral side of the body. In vertebrates and invertebrates regulation of crossing is achieved by interfering with Robo signalling either through sorting and degradation of the receptor, in flies, or through silencing of its repulsive activity, in vertebrates. Here I show that in Drosophila a second mechanism of regulation exists that is independent from sorting. Using in vitro and in vivo assays I mapped the region of Robo that is sufficient and required for its interaction with Comm, its sorting receptor. By modifying that region, I generated new forms of Robo that are insensitive to Comm sorting in vitro and in vivo, yet still able to normally translate repulsive activity in vivo. Using gene targeting by homologous recombination I created new conditional alleles of robo that are sorting defective (robo^SD). Surprisingly, expression of these modified proteins results in phenotypically normal flies, unveiling a sorting independent mechanism of regulation.     

Robo4 maintains vessel integrity and inhibits angiogenesis by interacting with UNC5B

Robo4 is an endothelial cell-specific member of the Roundabout axon guidance receptor family. To identify Robo4 binding partners, we performed a protein-protein interaction screen with the Robo4 extracellular domain. We find that Robo4 specifically binds to UNC5B, a vascular Netrin receptor, revealing unexpected interactions between two endothelial guidance receptors. We show that Robo4 maintains vessel integrity by activating UNC5B, which inhibits signaling downstream of vascular endothelial growth factor (VEGF). Function-blocking monoclonal antibodies against Robo4 and UNC5B increase angiogenesis and disrupt vessel integrity. Soluble Robo4 protein inhibits VEGF-induced vessel permeability and rescues barrier defects in Robo4(-/-) mice, but not in mice treated with anti-UNC5B. Thus, Robo4-UNC5B signaling maintains vascular integrity by counteracting VEGF signaling in endothelial cells, identifying a novel function of guidance receptor interactions in the vasculature.     

Receptor tyrosine phosphatases regulate axon guidance across the midline of the Drosophila embryo

Neural receptor-linked protein tyrosine phosphatases (RPTPs) are required for guidance of motoneuron and photoreceptor growth cones in Drosophila. These phosphatases have not been implicated in growth cone responses to specific guidance cues, however, so it is unknown which aspects of axonal pathfinding are controlled by their activities. Three RPTPs, known as DLAR, DPTP69D, and DPTP99A, have been genetically characterized thus far. Here we report the isolation of mutations in the fourth neural RPTP, DPTP10D. The analysis of double mutant phenotypes shows that DPTP10D and DPTP69D are necessary for repulsion of growth cones from the midline of the embryonic central nervous system. Repulsion is thought to be triggered by binding of the secreted protein Slit, which is expressed by midline glia, to Roundabout (Robo) receptors on growth cones. Robo repulsion is downregulated by the Commissureless (Comm) protein, allowing axons to cross the midline. Here we show that the Rptp mutations genetically interact with robo, slit and comm. The nature of these interactions suggests that DPTP10D and DPTP69D are positive regulators of Slit/Roundabout repulsive signaling. We also show that elimination of all four neural RPTPs converts most noncrossing longitudinal pathways into commissures that cross the midline, indicating that tyrosine phosphorylation controls the manner in which growth cones respond to midline signals.     

Short-range and long-range guidance by Slit and its Robo receptors: a combinatorial code of Robo receptors controls lateral position

Slit is secreted by midline glia in Drosophila and functions as a short-range repellent to control midline crossing. Although most Slit stays near the midline, some diffuses laterally, functioning as a long-range chemorepellent. Here we show that a combinatorial code of Robo receptors controls lateral position in the CNS by responding to this presumptive Slit gradient. Medial axons express only Robo, intermediate axons express Robo3 and Robo, while lateral axons express Robo2, Robo3, and Robo. Removal of robo2 or robo3 causes lateral axons to extend medially; ectopic expression of Robo2 or Robo3 on medial axons drives them laterally. Precise topography of longitudinal pathways appears to be controlled by a combination of long-range guidance (the Robo code determining region) and short-range guidance (discrete local cues determining specific location within a region).     

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.     

Short-range and long-range guidance by slit and its Robo receptors. Robo and Robo2 play distinct roles in midline guidance

Previous studies showed that Roundabout (Robo) in Drosophila is a repulsive axon guidance receptor that binds to Slit, a repellent secreted by midline glia. In robo mutants, growth cones cross and recross the midline, while, in slit mutants, growth cones enter the midline but fail to leave it. This difference suggests that Slit must have more than one receptor controlling midline guidance. In the absence of Robo, some other Slit receptor ensures that growth cones do not stay at the midline, even though they cross and recross it. Here we show that the Drosophila genome encodes three Robo receptors and that Robo and Robo2 have distinct functions, which together control repulsive axon guidance at the midline. The robo,robo2 double mutant is largely identical to slit.     

Distinct roles for Robo2 in the regulation of axon and dendrite growth by retinal ganglion cells

Guidance factors act on the tip of a growing axon to direct it to its target. What role these molecules play, however, in the control of the dendrites that extend from that axon’s cell body is poorly understood. Slits, through their Robo receptors, guide many types of axons, including those of retinal ganglion cells (RGCs). Here we assess and contrast the role of Slit/Robo signalling in the growth and guidance of the axon and dendrites extended by RGCs in Xenopus laevis. As Xenopus RGCs extend dendrites, they express robo2 and robo3, while slit1 and slit2 are expressed in RGCs and in the adjacent inner nuclear layer. Interestingly, our functional data with antisense knockdown and dominant negative forms of Robo2 (dnRobo2) and Robo3 (dnRobo3) indicate that Slit/Robo signalling has no role in RGC dendrite guidance, and instead is necessary to stimulate dendrite branching, primarily via Robo2. Our in vitro culture data argue that Slits are the ligands involved. In contrast, both dnRobo2 and dnRobo3 inhibited the extension of axons and caused the misrouting of some axons. Based on these data, we propose that Robo signalling can have distinct functions in the axon and dendrites of the same cell, and that the specific combinations of Robo receptors could underlie these differences. Slit acts via Robo2 in dendrites as a branching/growth factor but not in guidance, while Robo2 and Robo3 function in concert in axons to mediate axonal interactions and respond to Slits as guidance factors. These data underscore the likelihood that a limited number of extrinsic factors regulate the distinct morphologies of axons and dendrites.     

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

Drosophila Roundabout (Robo) is the founding member of a conserved family of repulsive axon guidance receptors that respond to secreted Slit proteins. Here we present evidence 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.     

ROBO4 与缺血性脑血管病相关的研究进展

Roundabout(Robo)蛋白是神经轴突导向分子家族Slit蛋白的单次跨膜受体,属于一种神经细胞粘附分子。Robo蛋白在神经系统已被确认具有重要轴突导向功能。近年来研究发现,血管新生的内皮细胞表面只特异性地表达Robo4,且Robo4对内皮细胞迁移、病理性血管生成和血管完整性都具有调节作用。缺血性脑血管病是人类致残甚至死亡的主要疾病之一,由于短暂或持续的脑血流减少而造成脑细胞损伤,因此,恢复脑血流、促进血管再生对脑功能恢复至关重要。Robo4对血管方面的作用为我们进一步研究及了解其在血管生成中的机制提供重要依据,也为缺血性脑血管病的治疗提供新的发展方向。     

Pioneer longitudinal axons navigate using floor plate and Slit/Robo signals

Longitudinal axons transmit all signals between the brain and spinal cord. Their axon tracts through the brain stem are established by a simple set of pioneer axons with precise trajectories parallel to the floor plate. To identify longitudinal guidance mechanisms in vivo, the overall role of floor plate tissue and the specific roles of Slit/Robo signals were tested. Ectopic induction or genetic deletion of the floor plate diverted longitudinal axons into abnormal trajectories. The expression patterns of the diffusible cues of the Slit family were altered in the floor plate experiments, suggesting their involvement in longitudinal guidance. Genetic tests of Slit1 and Slit2, and the Slit receptors Robo1 and Robo2 were carried out in mutant mice. Slit1;Slit2 double mutants had severe longitudinal errors, particularly for ventral axons, including midline crossing and wandering longitudinal trajectories. Robo1 and Robo2 were largely genetically redundant, and neither appeared to specify specific tract positions. However, combined Robo1 and Robo2 mutations strongly disrupted each pioneer tract. Thus, pioneer axons depend on long-range floor plate cues, with Slit/Robo signaling required for precise longitudinal trajectories.     

Robo1 and Robo2 have distinct roles in pioneer longitudinal axon guidance

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

Robo-Slit interactions regulate longitudinal axon pathfinding in the embryonic vertebrate brain

The early network of axons in the embryonic brain provides connectivity between functionally distinct regions of the nervous system. While many of the molecular interactions driving commissural pathway formation have been deciphered, the mechanisms underlying the development of longitudinal tracts remain unclear. We have identified here a role for the Roundabout (Robo) family of axon guidance receptors in the positioning of longitudinally projecting axons along the dorsoventral axis in the embryonic zebrafish forebrain. Using a loss-of-function approach, we established that Robo family members exhibit complementary functions in the tract of the postoptic commissure (TPOC), the major longitudinal tract in the forebrain. Robo2 acted initially to split the TPOC into discrete fascicles upon entering a broad domain of Slit1a expression in the ventrocaudal diencephalon. In contrast, Robo1 and Robo3 restricted the extent of defasciculation of the TPOC. In this way, the complementary roles of Robo family members balance levels of fasciculation and defasciculation along this trajectory. These results demonstrate a key role for Robo-Slit signaling in vertebrate longitudinal axon guidance and highlight the importance of context-specific guidance cues during navigation within complex pathways.     

Rig-1 a new member of Robo family genes exhibits distinct pattern of expression during mouse development

The Robo genes encode a family of proteins that are the receptors for the midline repellent Slits and play a role in axon guidance. In addition to Robo1 and Robo2, Rig-1 has been recently identified in mouse as a novel member of the Robo family of proteins. As a first step in elucidating the role of Rig-1 during vertebrate development, we characterised the expression of Rig-1 by in situ hybridisation together with Robo1 and Robo2 in the spinal cord and other tissues of the mouse embryo. Our results show that Rig-1 has a dynamic pattern of expression in the developing CNS. In the spinal cord Rig-1 shows overlapping but distinct pattern of expression with Robo1 and Robo2.     

Attractive and repulsive functions of Slit are mediated by different receptors in the Drosophila trachea

Oxygen delivery in many animals is enabled by the formation of unicellular capillary tubes that penetrate target tissues to facilitate gas exchange. We show that the tortuous outgrowth of tracheal unicellular branches towards their target tissues is controlled by complex local interactions with target cells. Slit, a phylogenetically conserved axonal guidance signal, is expressed in several tracheal targets and is required both for attraction and repulsion of tracheal branches. Robo and Robo2 are expressed in different branches, and are both necessary for the correct orientation of branch outgrowth. At the CNS midline, Slit functions as a repellent for tracheal branches and this function is mediated primarily by Robo. Robo2 is necessary for the tracheal response to the attractive Slit signal and its function is antagonized by Robo. We propose that the attractive and repulsive tracheal responses to Slit are mediated by different combinations of Robo and Robo2 receptors on the cell surface.