Cloning and expression of three zebrafish roundabout homologs suggest roles in axon guidance and cell migration.

We report the cloning and expression patterns of three novel zebrafish Roundabout homologs. The Roundabout (robo) gene encodes a transmembrane receptor that is essential for axon guidance in Drosophila and Robo family members have been implicated in cell migration. Analysis of extracellular domains and conserved cytoplasmic motifs shows that zebrafish Robo1 and Robo2 are orthologs of mammalian Robo1 and Robo2, respectively, while zebrafish Robo3 is likely to be an ortholog of mouse Rig-1. The three zebrafish robos are expressed in distinct but overlapping patterns during embryogenesis. They are highly expressed in the developing nervous system, including the olfactory system, visual system, hindbrain, cranial ganglia, spinal cord, and posterior lateral line primordium. They are also expressed in several nonneuronal tissues, including somites and fin buds. The timing and patterns of expression suggest roles for zebrafish robos in axon guidance and cell migration. Wiley-Liss, Inc.     

Cadherin-1, -2, and -11 expression and cadherin-2 function in the pectoral limb bud and fin of the developing zebrafish.

Cadherins are cell adhesion molecules that play important roles in development of a variety of organs, including the vertebrate limb. In this study, we analyze cadherin expression patterns in the embryonic zebrafish pectoral limb buds and larval pectoral fins by using both in situ hybridization and immunocytochemical methods. cadherin-1 is detected in the epidermis of the embryonic limb buds and the larval pectoral fins. Cadherin-2 is expressed in the pectoral limb bud mesenchyme and chondrogenic condensation. As development proceeds, cadherin-2 expression is detected in newly differentiated pectoral fin endoskeleton, but its expression is greatly down-regulated in the fin endoskeleton of larval zebrafish. cadherin-11 is found in the basal region of the embryonic limb buds and in the proximal endoskeleton of the larval pectoral fins. Interfering with cadherin-2 function using two specific antisense morpholino oligonucleotides disrupts formation of the chondrogenic condensation/endoskeleton, suggesting that cadherin-2 is crucial for the normal development of the zebrafish pectoral fins.     

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.     

Slitrk gene duplication and expression in the developing zebrafish nervous system

Results : As a prelude to examining the functional roles of Slitrks, we identified eight slitrk orthologs in zebrafish and observed that seven of the eight orthologs were actively transcribed in the nervous system at embryonic, larval, and adult stages. Similar to previous findings in mice and humans, zebrafish slitrks exhibited unique but overlapping spatial and temporal expression patterns in the developing brain, retina, and spinal cord. 相似文献   

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.     

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 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.     

Cell-surface heparan sulfate is involved in the repulsive guidance activities of Slit2 protein.

Slit proteins are a family of secreted guidance proteins that can repel neuronal migration and axon growth via interaction with their cellular roundabout receptors (Robos). Here it was shown that Slit2-Robo-1 interactions were enhanced by cell-surface heparan sulfate. Removal of heparan sulfate decreased the affinity of Slit for Robo by about threefold. In addition, removal of cell-surface heparan sulfate by heparinase III abolished the chemorepulsive response to Slit2 normally shown by both the migrating neurons and growing axons. These results indicate essential roles for cell-surface heparan sulfate in the repulsive activities of Slit2.     

Expression of sox11 gene duplicates in zebrafish suggests the reciprocal loss of ancestral gene expression patterns in development.

To investigate the role of sox genes in vertebrate development, we have isolated sox11 from zebrafish (Danio rerio). Two distinct classes of sox11-related cDNAs were identified, sox11a and sox11b. The predicted protein sequences shared 75% identity. In a gene phylogeny, both sox11a and sox11b cluster with human, mouse, chick, and Xenopus Sox11, indicating that zebrafish, like Xenopus, has two orthologues of tetrapod Sox11. The work reported here investigates the evolutionary origin of these two gene duplicates and the consequences of their duplication for development. The sox11a and sox11b genes map to linkage groups 17 and 20, respectively, together with other loci whose orthologues are syntenic with human SOX11, suggesting that during the fish lineage, a large chromosome region sharing conserved syntenies with mammals has become duplicated. Studies in mouse and chick have shown that Sox11 is expressed in the central nervous system during development. Expression patterns of zebrafish sox11a and sox11b confirm that they are expressed in the developing nervous system, including the forebrain, midbrain, hindbrain, eyes, and ears from an early stage. Other sites of expression include the fin buds and somites. The two sox genes, sox11a and sox11b, are expressed in both overlapping and distinct sites. Their expression patterns suggest that sox11a and sox11b may share the developmental domains of the single Sox11 gene present in mouse and chick. For example, zebrafish sox11a is expressed in the anterior somites, and zebrafish sox11b is expressed in the posterior somites, but the single Sox11 gene of mouse is expressed in all the somites. Thus, the zebrafish duplicate genes appear to have reciprocally lost expression domains present in the sox11 gene of the last common ancestor of tetrapods and zebrafish. This splitting of the roles of Sox11 between two paralogues suggests that regulatory elements governing the expression of the sox11 gene in the common ancestor of zebrafish and tetrapods may have been reciprocally mutated in the zebrafish gene duplicates. This is consistent with duplicate gene evolution via a duplication-degeneration-complementation process.     

Author index

Down syndrome cell adhesion molecules (DSCAMs) are broadly expressed in nervous systems and play conserved roles in programmed cell death, neuronal migration, axon guidance, neurite branching and spacing, and synaptic targeting. However, DSCAMs appear to have distinct functions in different vertebrate animals, and little is known about their functions outside the retina. We leveraged the genetic tractability and optical accessibility of larval zebrafish to investigate the expression and function of a DSCAM family member, dscamb. Using targeted genome editing to create transgenic reporters and loss-of-function mutant alleles, we discovered that dscamb is expressed broadly throughout the brain, spinal cord, and peripheral nervous system, but is not required for overall structural organization of the brain. Despite the absence of obvious anatomical defects, homozygous dscamb mutants were deficient in their ability to ingest food and rarely survived to adulthood. Thus, we have discovered a novel function for dscamb in feeding behavior. The mutant and transgenic lines generated in these studies will provide valuable tools for identifying the molecular and cellular bases of these behaviors.     

Two Frodo/Dapper homologs are expressed in the developing brain and mesoderm of zebrafish.

Members of the Wnt family of extracellular proteins play essential roles during many phases of vertebrate embryonic development. The molecular mechanism of their action involves a complex cascade of intracellular signaling events, which remains to be understood completely. Recently, two novel cytoplasmic modulators of Wnt signaling, Frodo and Dapper, were identified in Xenopus. We report isolation of their homologs in zebrafish, and show that these genes, frd1 and frd2, are expressed in restricted domains during embryogenesis. Both genes are expressed during early gastrulation in the future mesendoderm, and continue to be expressed in distinct patterns in the forming neurectoderm and mesoderm. Comparative sequence analysis and similar expression patterns argue that frd1 is the zebrafish ortholog of Frodo and Dapper, whereas frd2 is a more divergent member of the same family. Our data suggest important roles for zebrafish frd1 and frd2 in patterning the neural plate and several mesodermal derivatives.     

Six cadm/SynCAM genes are expressed in the nervous system of developing zebrafish.

The Cadm (cell adhesion molecule) family of cell adhesion molecules (also known as IGSF4, SynCAM, Necl and TSLC) has been implicated in a multitude of physiological and pathological processes, such as spermatogenesis, synapse formation and lung cancer. The precise mechanisms by which these adhesion molecules mediate these diverse functions remain unknown. To investigate mechanisms of action of these molecules during development, we have identified zebrafish orthologs of Cadm family members and have examined their expression patterns during development and in the adult. Zebrafish possess six cadm genes. Sequence comparisons and phylogenetic analysis suggest that four of the zebrafish cadm genes represent duplicates of two tetrapod Cadm genes, whereas the other two cadm genes are single orthologs of tetrapod Cadm genes. All six zebrafish cadms are expressed throughout the nervous system both during development and in the adult. The spatial and temporal patterns of expression suggest multiple roles for Cadms during nervous system development.     

Conservation,expression, and knockdown of zebrafish plxnb2a and plxnb2b

In mice lacking Plexin B2, a receptor of the axon guidance molecules Semaphorin 4C and Semaphorin 4D, the closure of the neural tube and structural organization of the cerebellum are severely impaired. We cloned two Plexin B2 orthologs, plxnb2a and plxnb2b, in zebrafish, which is a widely used model for the development of the vertebrate central nervous system (CNS). The predicted proteins, Plexin B2a and Plexin B2b, contain all the conserved and functional domains of the plexin B‐subfamily. During embryonic development, plxnb2a is expressed, e.g., in pharyngeal arches while plxnb2b expression is more confined to neuronal structures like the cerebellum. However, both plxnb2a and plxnb2b are expressed at the midbrain–hindbrain boundary, in the otic vesicles, facial ganglia, and pectoral fins. Knockdown of both plxnb2a and plxnb2b simultaneously (>95% and 45%, respectively) resulted in normal CNS structure, axon guidance and swimming performance of the morphants. Developmental Dynamics 239:2722–2734, 2010. © 2010 Wiley‐Liss, Inc.     

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.     

Expression of slit-2 and slit-3 during chick development.

The Drosophila and vertebrate slit proteins have been characterized as secreted chemorepellents recognized by the robo receptor proteins that function principally for the guidance of neuronal axons and neurons. slit genes are also expressed in the limb. To provide a basis for the determination of slit functions in the limb we have isolated and characterized the expression of chick slit-2 and slit-3 in the developing limb and other tissues of the chick embryo. Both genes share similar expression profiles in the chick embryo when compared to that of their mammalian homologues, particularly in the neural tube. In the limb, their expression patterns suggest their involvement in many aspects of limb development. In the early limb bud, slit-2 is expressed in the peripheral mesenchyme and invading muscle precursors, while slit-3 expression is restricted to the future chondrogenic core of the limb bud. At later stages, both slit genes are expressed in interdigital mesenchyme, in inner periosteal cells, and in mesenchyme immediately radial to the periosteum and under the epidermis. slit-3 is also expressed in proliferating chondrocytes during cartilage development, while slit-2 is expressed in later muscle masses and peripherally to joints in the autopod.     

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.