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1.
Slit is the midline repellent for the robo receptor in Drosophila   总被引:22,自引:0,他引:22  
Kidd T  Bland KS  Goodman CS 《Cell》1999,96(6):785-794
Previous studies suggested that Roundabout (Robo) is a repulsive guidance receptor on growth cones that binds to an unknown midline ligand. Here we present genetic evidence that Slit is the midline Robo ligand; a companion paper presents biochemical evidence that Slit binds Robo. Slit is a large extracellular matrix protein expressed by midline glia. In slit mutants, growth cones enter the midline but never leave it; they abnormally continue to express high levels of Robo while at the midline. slit and robo display dosage-sensitive genetic interactions, indicating that they function in the same pathway. slit is also required for migration of muscle precursors away from the midline. Slit appears to function as a short-range repellent controlling axon crossing of the midline and as a long-range chemorepellent controlling mesoderm migration away from the midline.  相似文献   

2.
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.  相似文献   

3.
Crossing the midline: roles and regulation of Robo receptors   总被引:12,自引:0,他引:12  
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.  相似文献   

4.
roundabout (robo) family genes play key roles in axon guidance in a wide variety of animals. We have investigated the roles of the robo family members, robo, robo2, and robo3, in the guidance of sensory axons in the Drosophila embryo. In robo(-/-), slit(-/-), and robo(-/+) slit(-/+) mutants, lateral cluster sensory neurons misproject to cells and axons in the nearby ventral' (v') cluster. These phenotypes, together with the normal expression pattern of Slit and Robo, suggest that Slit ligand secreted from the epidermis interacts with Robo receptors on lateral cluster sensory growth cones to limit their exploration of nearby attractive substrates. The most common sensory axon phenotype seen in robo2(-/-) mutants was misprojection of dorsal cluster sensory axons away from their normal growth substrate, the transverse connective of the trachea. slit appears to play no role in this aspect of sensory axon growth. Robo2 is expressed, not on the dorsal sensory axons, but on the transverse connective. These results suggest a novel, non-cell-autonomous mechanism for axon guidance by robo family genes: Robo2 expressed on the trachea acts as an attractant for the dorsal sensory growth cones.  相似文献   

5.
The slit (sli) gene, encoding a secreted glycoprotein, has been demonstrated to play a vital role in axonal guidance in Drosophila melanogaster by acting as a signalling ligand for the robo receptor (Rothberg, J.M., Jacobs, J.R., Goodman, C.S., Artavanis-Tsakonas, S., 1990. slit: an extracellular protein necessary for development of midline glia and commissural axon pathways contains both EGF and LRR domains. Genes Dev. 4, 2169-2187; Kidd, T., Bland, K.S., Goodman, C. S., 1999. Slit is the midline repellent for the robo receptor in Drosophila. Cell 96, 785-794). Multiple homologs of both sli and robo have been identified in vertebrates and are thought to play similar roles to their fly counterparts in neural development (Brose, K., Bland, K.S., Wang, K.H., Arnott, D., Henzel, W., Goodman, C.S., Tessier-Lavigne, M., Kidd, T., 1999. Slit proteins bind Robo receptors and have an evolutionarily conserved role in repulsive axon guidance. Cell 96, 795-806). Slit2 has been shown to bind Robo1, mediating both neuronal and axonal guidance in the developing central nervous system (CNS), (Brose et al., 1999; Hu, H., 1999. Chemorepulsion of neuronal migration by Slit2 in the developing mammalian forebrain. Neuron 23, 703-711). Importantly, both gene families display distinct expression patterns outside the CNS (Holmes, G.P., Negus, K., Burridge, L., Raman, S., Algar, E., Yamada, T., Little, M.H., 1998. Distinct but overlapping expression patterns of two vertebrate slit homologs implies functional roles in CNS development and organogenesis. Mech. Dev. 79, 57-72; Yuan, W., Zhou, L., Chen, J.H., Wu, J.Y., Rao, Y., Ornitz, D.M., 1999. The mouse SLIT family: secreted ligands for ROBO expressed in patterns that suggest a role in morphogenesis and axon guidance. Dev. Biol. 212, 290-306). Using in situ hybridization on metanephric explant cultures and urogenital tract sections, the expression patterns of Slit1, 2, 3 and Robo1 and 2 were investigated during murine metanephric development. Slit1 was expressed in the metanephric mesenchyme (MM) surrounding the invading ureteric tree (UT). Slit2 was expressed at the tips of the UT and both Slit2 and Slit3 were expressed at the far proximal end of the comma shaped and S-shaped bodies. Expression of Robo1 was initially diffuse throughout the MM, then upregulated in the pretubular aggregates, and maintained at the distal end of the comma and S-shaped bodies. Robo2 was detected in the induced MM surrounding the arborizing UT tips and later in the proximal end of the S-shaped bodies. Coincident expression of Robo1 with Slit1 in the metanephric mesenchyme and Robo2, Slit2 and Slit3 in the far proximal end of the S-shaped bodies was observed during metanephric development.  相似文献   

6.
Extending axons in the developing nervous system are guided in part by repulsive cues. Genetic analysis in Drosophila, reported in a companion to this paper, identifies the Slit protein as a candidate ligand for the repulsive guidance receptor Roundabout (Robo). Here we describe the characterization of three mammalian Slit homologs and show that the Drosophila Slit protein and at least one of the mammalian Slit proteins, Slit2, are proteolytically processed and show specific, high-affinity binding to Robo proteins. Furthermore, recombinant Slit2 can repel embryonic spinal motor axons in cell culture. These results support the hypothesis that Slit proteins have an evolutionarily conserved role in axon guidance as repulsive ligands for Robo receptors.  相似文献   

7.
The developing optic pathway has proven one of the most informative model systems for studying mechanisms of axon guidance. The first step in this process is the directed extension of retinal ganglion cell (RGC) axons within the optic fibre layer (OFL) of the retina towards their exit point from the eye, the optic disc. Previously, we have shown that the inhibitory guidance molecules, Slit1 and Slit2, regulate two distinct aspects of intraretinal axon guidance in a region-specific manner. Using knockout mice, we have found that both of these guidance activities are mediated via Robo2. Of the four vertebrate Robos, only Robo1 and Robo2 are expressed by RGCs. In mice lacking robo1 intraretinal axon guidance occurs normally. However, in mice lacking robo2 RGC axons make qualitatively and quantitatively identical intraretinal pathfinding errors to those reported previously in Slit mutants. This demonstrates clearly that, as in other regions of the optic pathway, Robo2 is the major receptor required for intraretinal axon guidance. Furthermore, the results suggest strongly that redundancy with other guidance signals rather than different receptor utilisation is the most likely explanation for the regional specificity of Slit function during intraretinal axon pathfinding.  相似文献   

8.
Glia are required for axon pathfinding along longitudinal trajectories, but it is unknown how this relates to the molecular paradigm of axon guidance across the midline. Most interneuron axons in bilateral organisms cross the midline only once. Preventing them from recrossing the midline requires the expression of Robo receptors on the axons. These sense the repulsive signal Slit, which is produced by the midline. The lateral positioning of longitudinal axons depends on the response to Slit by the combination of Robo receptors expressed by the axons, on selective fasciculation, and on longitudinal (lateral) glia. Here, we analyse how longitudinal glia influence reading of the 'Robo code' by axons. We show that whereas loss of robo1 alone only affects the most medial axons, loss of both glial cells missing (gcm) and robo1 causes a severe midline collapse of longitudinal axons, similar to that caused by the loss of multiple Robo receptors. Furthermore, whereas ectopic expression of robo2 is sufficient to displace the medial MP2 axons along a more lateral trajectory, this does not occur in gcm-robo1 double-mutant embryos, where axons either do not extend at all or they misroute exiting the CNS. Hence, lateral neuron-glia interactions steer the response of axons to the Robo code.  相似文献   

9.
Rajagopalan S  Vivancos V  Nicolas E  Dickson BJ 《Cell》2000,103(7):1033-1045
On each side of the midline of the Drosophila CNS, axons are organized into a series of parallel pathways. Here we show that the midline repellent Slit, previously identified as a short-range signal that regulates midline crossing, also functions at long range to pattern these longitudinal pathways. In this long-range function, Slit signals through the receptors Robo2 and Robo3. Axons expressing neither, one, or both of these receptors project in one of three discrete lateral zones, each successively further from the midline. Loss of robo2 or robo3 function repositions axons closer to the midline, while gain of robo2 or robo3 function shifts axons further from the midline. Local cues further refine the lateral position. Together, these long- and short-range guidance cues allow growth cones to select with precision a specific longitudinal pathway.  相似文献   

10.
Bhat KM  Gaziova I  Krishnan S 《Genetics》2007,176(4):2235-2246
Netrin and Slit signaling systems play opposing roles during the positioning of longitudinal tracts along the midline in the ventral nerve cord of Drosophila embryo. It has been hypothesized that a gradient of Slit from the midline interacts with three different Robo receptors to specify the axon tract positioning. However, no such gradient has been detected. Moreover, overexpression of Slit at the midline has no effect on the positioning of these lateral tracts. In this article, we show that Slit is present outside of the midline along the longitudinal and commissural tracts. Sli from the midline, in a Robo-independent manner, is initially taken up by the commissural axon tracts when they cross the midline and is transported along the commissural tracts into the longitudinal connectives. These results are not consistent with a Sli gradient model. We also find that sli mRNA is maternally deposited and embryos that are genetically null for sli can have weaker guidance defects. Moreover, in robo or robo3 mutants, embryos with normal axon tracts are found and such robo embryos reach pupal stages and die, while robo3 mutant embryos develop into normal individuals and produce eggs. Interestingly, embryos from robo3 homozygous individuals fail to develop but have axon tracts ranging from normal to various defects: robo3 phenotype, robo phenotype, and slit-like phenotype, suggesting a more complex functional role for these genes than what has been proposed. Finally, our previous results indicated that netrin phenotype is epistatic to sli or robo phenotypes. However, it seems likely that this previously reported epistatic relationship might be due to the partial penetrance of the sli, robo, robo3 (or robo2) phenotypes. Our results argue that double mutant epistasis is most definitive only if the penetrance of the phenotypes of the mutants involved is complete.  相似文献   

11.
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.  相似文献   

12.
Simpson JH  Bland KS  Fetter RD  Goodman CS 《Cell》2000,103(7):1019-1032
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).  相似文献   

13.
Slit, the ligand for the Roundabout (Robo) receptors, is secreted from midline cells of the Drosophila central nervous system (CNS). It acts as a short-range repellent that controls midline crossing of axons and allows growth cones to select specific pathways along each side of the midline. In addition, Slit directs the migration of muscle precursors and ventral branches of the tracheal system, showing that it provides long-range activity beyond the limit of the developing CNS. Biochemical studies suggest that guidance activity requires cell-surface heparan sulfate to promote binding of mammalian Slit/Robo homologs. Here, we report that the Drosophila homolog of Syndecan (reviewed in ), a heparan sulfate proteoglycan (HSPG), is required for proper Slit signaling. We generated syndecan (sdc) mutations and show that they affect all aspects of Slit activity and cause robo-like phenotypes. sdc interacts genetically with robo and slit, and double mutations cause a synergistic strengthening of the single-mutant phenotypes. The results suggest that Syndecan is a necessary component of Slit/Robo signaling and is required in the Slit target cells.  相似文献   

14.
Qian L  Liu J  Bodmer R 《Current biology : CB》2005,15(24):2271-2278
Basic aspects of heart morphogenesis involving migration, cell polarization, tissue alignment, and lumen formation may be conserved between Drosophila and humans, but little is known about the mechanisms that orchestrate the assembly of the heart tube in either organism. The extracellular-matrix molecule Slit and its Robo-family receptors are conserved regulators of axonal guidance. Here, we report a novel role of the Drosophila slit, robo, and robo2 genes in heart morphogenesis. Slit and Robo proteins specifically accumulate at the dorsal midline between the bilateral myocardial progenitors forming a linear tube. Manipulation of Slit localization or its overexpression causes disruption in heart tube alignment and assembly, and slit-deficient hearts show disruptions in cell-polarity marker localization within the myocardium. Similar phenotypes are observed when Robo and Robo2 are manipulated. Rescue experiments suggest that Slit is secreted from the myocardial progenitors and that Robo and Robo2 act in myocardial and pericardial cells, respectively. Genetic interactions suggest a cardiac morphogenesis network involving Slit/Robo, cell-polarity proteins, and other membrane-associated proteins. We conclude that Slit and Robo proteins contribute significantly to Drosophila heart morphogenesis by guiding heart cell alignment and adhesion and/or by inhibiting cell mixing between the bilateral compartments of heart cell progenitors and ensuring proper polarity of the myocardial epithelium.  相似文献   

15.
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.  相似文献   

16.
Diffusible chemorepellents play a major role in guiding developing axons towards their correct targets by preventing them from entering or steering them away from certain regions. Genetic studies in Drosophila revealed a novel repulsive guidance system that prevents inappropriate axons from crossing the CNS midline; this repulsive system is mediated by the Roundabout (Robo) receptors and their secreted ligand Slits. Three distinct slit genes (slit1, slit2 and slit3) and three distinct robo genes (robo1, robo2 and rig-1) have been cloned in mammals. In collagen gel co-cultures, Slit1 and Slit2 can repel and collapse olfactory axons. However, there is also some positive effect associated with Slits, as Slit2 stimulates the formation of axon collateral branches by NGF-responsive neurons of the dorsal root ganglia (DRG). Slit2 is a large ECM glycoproteins of about 200 kD, which is proteolytically processed into 140 kD N-terminal and 55-60 kD C-terminal fragments. Slit2 cleavage fragments appear to have different cell association characteristics, with the smaller C-terminal fragment being more diffusible and the larger N-terminal and uncleaved fragments being more tightly cell associated. This suggested that the different fragments might have different functional activities in vivo. We have begun to explore these questions by engineering mutant and truncated versions of hSlit2 representing the two cleavage fragments, N- and C-, and the uncleavable molecule and examining the activities of these mutants in binding and functional assays. We found that an axon's response to Slit2 is not absolute, but rather is reflective of the context in which the protein is encountered.  相似文献   

17.
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.  相似文献   

18.
Guidance of axons towards or away from the midline of the central nervous system during Drosophila embryogenesis reflects a balance of attractive and repulsive cues originating from the midline. Here we demonstrate that Slit, a protein secreted by the midline glial cells provides a repulsive cue for the growth cones of axons and muscle cells. Embryos lacking slit function show a medial collapse of lateral axon tracts and ectopic midline crossing of ventral muscles. Transgene expression of slit in the midline restores axon patterning. Ectopic expression of slit inhibits formation of axon tracts at locations of high Slit production and misdirects axon tracts towards the midline. slit interacts genetically with roundabout, which encodes a putative receptor for growth cone repulsion.  相似文献   

19.
The Slit genes encode secreted ligands that regulate axon branching, commissural axon pathfinding and neuronal migration. The principal identified receptor for Slit is Robo (Roundabout in Drosophila). To investigate Slit signalling in forebrain development, we generated Robo1 knockout mice by targeted deletion of exon 5 of the Robo1 gene. Homozygote knockout mice died at birth, but prenatally displayed major defects in axon pathfinding and cortical interneuron migration. Axon pathfinding defects included dysgenesis of the corpus callosum and hippocampal commissure, and abnormalities in corticothalamic and thalamocortical targeting. Slit2 and Slit1/2 double mutants display malformations in callosal development, and in corticothalamic and thalamocortical targeting, as well as optic tract defects. In these animals, corticothalamic axons form large fasciculated bundles that aberrantly cross the midline at the level of the hippocampal and anterior commissures, and more caudally at the medial preoptic area. Such phenotypes of corticothalamic targeting were not observed in Robo1 knockout mice but, instead, both corticothalamic and thalamocortical axons aberrantly arrived at their respective targets at least 1 day earlier than controls. By contrast, in Slit mutants, fewer thalamic axons actually arrive in the cortex during development. Finally, significantly more interneurons (up to twice as many at E12.5 and E15.5) migrated into the cortex of Robo1 knockout mice, particularly in both rostral and parietal regions, but not caudal cortex. These results indicate that Robo1 mutants have distinct phenotypes, some of which are different from those described in Slit mutants, suggesting that additional ligands, receptors or receptor partners are likely to be involved in Slit/Robo signalling.  相似文献   

20.
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.  相似文献   

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