Two divergent slit1 genes in zebrafish.

Members of the Slit family regulate axon guidance and cell migration. To date, three vertebrate slit1 genes have been identified in mammals and orthologs of two, slit2 and slit3, have been identified in zebrafish. Here, we describe the cloning of full-length cDNAs for two zebrafish slit orthologs, slit1a and slit1b. Both predicted proteins contain the conserved motifs that characterize other vertebrate Slits. slit1a and slit1b are both expressed in the midline, hypochord, telencephalon, and hindbrain. Apart from these shared expression domains, however, their expression patterns largely differ. Whereas slit1a is expressed broadly in the central nervous system (CNS) and in the somites, pectoral fin buds, tail bud, and caudal fin folds, slit1b is expressed in the olfactory system throughout embryonic and larval development, and in the retina during larval stages. Their expression patterns, particularly that of slit1a, suggest that Slit proteins may have roles in tissue morphogenesis in addition to their established roles in axon guidance and cell migration.     

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

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

Expression and role of Roundabout-1 in embryonic Xenopus forebrain.

The receptor Roundabout-1 (Robo1) and its ligand Slit are known to influence axon guidance and central nervous system (CNS) patterning in both vertebrate and nonvertebrate systems. Although Robo-Slit interactions mediate axon guidance in the Drosophila CNS, their role in establishing the early axon scaffold in the embryonic vertebrate brain remains unclear. We report here the identification and expression of a Xenopus Robo1 orthologue that is highly homologous to mammalian Robo1. By using overexpression studies and immunohistochemical and in situ hybridization techniques, we have investigated the role of Robo1 in the development of a subset of neurons and axon tracts in the Xenopus forebrain. Robo1 is expressed in forebrain nuclei and in neuroepithelial cells underlying the main axon tracts. Misexpression of Robo1 led to aberrant development of axon tracts as well as the ectopic differentiation of forebrain neurons. These results implicate Robo1 in both neuronal differentiation and axon guidance in embryonic vertebrate forebrain.     

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

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

Slit and robo expression in the developing mouse lung.

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

Expression pattern for unc5b, an axon guidance gene in embryonic zebrafish development

Branching processes such as nerves and vessels share molecular mechanisms of path determination. Our study focuses on unc5b, a member of the unc5 axon guidance gene family. Here, we have cloned the full-length zebrafish ortholog of unc5b, mapped its chromosome location in the zebrafish genome, and compared its expression patterns to robo4, another axon guidance family member. In situ show that unc5b is expressed predominantly in sensory structures such as the eye, ear, and brain. Both unc5b and robo4 show robust expression in all three compartments of the embryonic brain, namely forebrain, midbrain, and hindbrain. In particular, the hindbrain rhombomere expression displays interesting patterns in that robo4 is expressed in medial rhombomere cell clusters when compared to unc5b expressed in lateral rhombomere clusters. A similar medial-lateral theme is observed in other neural structures such as the neural tube. Our expression analysis provides a starting point for studying the role of axon guidance genes in embryonic hindbrain patterning.     

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

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

Robo家族蛋白在神经元轴突发育和肿瘤血管生成中的作用

Robo家族（Roundabout family）是与发育有关的保守跨膜蛋白家族,主要表达于神经系统并在神经元轴突发育中发挥重要作用。近期,人们研究发现Robo家族的一个新成员Robo4(又称magic roundabout,MRB)特异表达于血管生成活跃的内皮细胞表面,并参与调控内皮细胞迁移。成年人正常组织中不表达MRB,而肿瘤组织相对特异性高表达,MRB有望成为抑制血管生成治疗肿瘤的新靶点。     

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

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

Slit-Robo interactions during cortical development

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

大鼠坐骨神经切断后Robo2在脊髓及背根节的表达变化

目的:研究坐骨神经损伤后Roundabout 2(Robo2)在成年大鼠背根节和脊髓的表达变化。方法:健康成年雌性SD大鼠坐骨神经切断后分别存活3～28d,取其L_(4～6)背根节(DRG)和脊髓;利用RT-PCR和免疫组织化学技术检测Robo2在上述组织中的表达变化。图像分析技术对阳性细胞的灰度值进行测定。结果:正常DRG感觉神经元表达Robo2 mRNA和蛋白质,脊髓前角运动神经元不表达。坐骨神经切断后3 d DRG内Robo2表达增加,7～14 d达高峰,21～28 d恢复到正常水平。结论:坐骨神经切断可导致DRG内Robo2的表达上调,可能与早期的感觉轴突再生有关。     

The E3 ubiquitin ligase Mycbp2 genetically interacts with Robo2 to modulate axon guidance in the mouse olfactory system

The E3 ubiquitin ligase Mycbp2 and it homologues play an important role in axon guidance and synaptogenesis in Drosophila, Caenorhabditis elegans, zebrafish and mouse. Despite this conserved function, the molecular and cellular basis of Mycbp2-dependent axon guidance remains largely unclear. We have examined here the effect of the loss-of-MYCBP2 function on the topography of the olfactory sensory neuron projection from the nasal cavity to the olfactory bulb in mice. A subpopulation of olfactory sensory axons failed to project to the dorsal surface of the olfactory bulb causing abnormal topography in this neural pathway. These defects were similar to the olfactory bulb phenotype in loss-of-ROBO2 function mice. While mice heterozygous for either Mycbp2 or Robo2 were normal, mice double heterozygous for these two genes produced severe defects in the olfactory system. Therefore, Mycbp2 and Robo2 were found to cooperate within a genetic network that has profound effects on axon guidance. The Mycbp2 phenotype could be partly explained by aberrant patterning of olfactory sensory neurons residing in the dorsal compartment of the nasal cavity. Some of these neurons fail to appropriately express Robo2 which is consistent with their aberrant projection to the ventral olfactory bulb. These results provide the first evidence linking an ubiquitin ligase to an axon guidance receptor during pathfinding in the developing mammalian nervous system.     

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

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

Embryonic expression and extracellular secretion of Xenopus slit

The slit genes have recently been found to encode proteins with a conserved chemorepulsive activity for axons in invertebrates and vertebrates. We have determined the expression pattern of a slit gene in Xenopus embryos. In the neural tube, slit is expressed at the ventral and dorsal midlines, and the motor neurons. slit is also expressed in a changing pattern in the retina. The full-length Xenopus Slit protein is secreted extracellularly, whereas its receptor Roundabout can not be secreted. Using a myc-tagged secreted Slit protein, we confirmed the binding of Slit to Roundabout expressed on the cell surface.These results confirm Slit-Roundabout interactions and the biochemical properties of Slit and Roundabout proteins, and further support the idea that Slit may guide axon projections in multiple regions of the embryo.     

Plexin A3 is involved in semaphorin 3F-mediated oligodendrocyte precursor cell migration

Class 3 semaphorins are expressed in the neurodevelopmental or damage repair phase of the central nervous system (CNS). They play an important role in guiding axon growth and directing cell migration, including the migration of oligodendrocyte precursor cells (OPCs). As co-receptors for semaphorin 3F(sema3F), the expression and role of neuropilin-2 (NRP2) and plexin A3 in OPC migration are unclear. Using RT-PCR, Western blot analysis, and immunofluorescence, we demonstrated that primary OPCs and immature oligodendrocytes from neonatal rats express NRP2 and plexin A3. After transfection with NRP2 siRNA and plexin A3 siRNA, the number of migrating OPCs attracted to sema3F remarkably decreased. These results suggest that plexin A3 is expressed in OPCs and immature oligodendrocytes and is involved in OPC migration.     

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

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

Identification and expression of voltage‐gated calcium channel β subunits in Zebrafish

Voltage‐gated calcium channels (VGCC) play important roles in electrically excitable cells and embryonic development. The VGCC β subunits are essential for membrane localization of the channel and exert modulatory effects on channel functions. In mammals, the VGCC β subunit gene family contains four members. In zebrafish, there appear to be seven VGCC β subunits including the previously identified β1 subunit. cDNAs for six additional VGCC β subunit homologs were identified in zebrafish, their chromosomal locations determined and their expression patterns characterized during embryonic development. These six genes are primarily expressed in the nervous system with cacnb4a also expressed in the developing heart. Sequence homology, genomic synteny and expression patterns suggest that there are three pairs of duplicate genes for β2, β3, and β4 in zebrafish with distinct expression patterns during embryonic development. Developmental Dynamics 237:3842–3852, 2008. © 2008 Wiley‐Liss, Inc.     

Zebrafish bashful/laminin-alpha 1 mutants exhibit multiple axon guidance defects.

Laminin is known to provide a highly permissive substratum and in some cases directional information for axon outgrowth in vitro. However, there is still little known about laminin function in guiding axons in vivo. We investigated the axon guidance role of laminin-alpha1 in the developing zebrafish nervous system. Analysis of zebrafish bashful (bal)/laminin-a1 mutants revealed multiple functions for laminin-alpha1 in the outgrowth and guidance of central nervous system (CNS) axons. Most CNS axon pathways are defective in bal embryos. Some axon types, including retinal ganglion cell axons, early forebrain axons, and hindbrain reticulospinal axons, make specific pathfinding errors, suggesting laminin-alpha1 is required for directional decisions. Other axon tracts are defasciculated or not fully extended in bal embryos, suggesting a function for laminin-alpha1 in regulating adhesion or providing a permissive substratum for growth. In addition, some neurons have excessively branched axons in bal, indicating a potential role for laminin-alpha1 in branching. In contrast to CNS axons, most peripheral axons appear normal in bal mutants. Our results, thus, reveal important and diverse functions for laminin-alpha1 in guiding developing axons in vivo.     

Genome-wide association studies of adolescent idiopathic scoliosis suggest candidate susceptibility genes

Adolescent idiopathic scoliosis (AIS) is an unexplained and common spinal deformity seen in otherwise healthy children. Its pathophysiology is poorly understood despite intensive investigation. Although genetic underpinnings are clear, replicated susceptibility loci that could provide insight into etiology have not been forthcoming. To address these issues, we performed genome-wide association studies (GWAS) of ～327 000 single nucleotide polymorphisms (SNPs) in 419 AIS families. We found strongest evidence of association with chromosome 3p26.3 SNPs in the proximity of the CHL1 gene (P < 8 × 10(-8) for rs1400180). We genotyped additional chromosome 3p26.3 SNPs and tested replication in two follow-up case-control cohorts, obtaining strongest results when all three cohorts were combined (rs10510181 odds ratio = 1.49, 95% confidence interval = 1.29-1.73, P = 2.58 × 10(-8)), but these were not confirmed in a separate GWAS. CHL1 is of interest, as it encodes an axon guidance protein related to Robo3. Mutations in the Robo3 protein cause horizontal gaze palsy with progressive scoliosis (HGPPS), a rare disease marked by severe scoliosis. Other top associations in our GWAS were with SNPs in the DSCAM gene encoding an axon guidance protein in the same structural class with Chl1 and Robo3. We additionally found AIS associations with loci in CNTNAP2, supporting a previous study linking this gene with AIS. Cntnap2 is also of functional interest, as it interacts directly with L1 and Robo class proteins and participates in axon pathfinding. Our results suggest the relevance of axon guidance pathways in AIS susceptibility, although these findings require further study, particularly given the apparent genetic heterogeneity in this disease.