Characterization of a novel member of murine semaphorin family

Semaphorin gene family contains a large number of secreted and transmembrane proteins, and some of them are functioning as the repulsive and attractive cues of the axon guidance during development. Here we report murine orthologues of a novel member of class 6 semaphorin gene, semaphorin 6D (Sema6D), mapped on the chromosome 2. Sema6D is mainly expressed in the brain and lung, and the ubiquitous expression in the brain continues from embryonic late stage to adulthood, as determined by Northern blot and in situ hybridization. We also found that Sema6D has five different splicing variants, and the expression patterns of individual isoforms differ depending on the tissues. Thus, Sema6D may play important roles in various functions including the axon guidance during development and neuronal plasticity.     

Identification of a member of mouse semaphorin family

Grasshopper semaphorin I (Sema I) and its related proteins, chick collapsin and mouse Sema III contribute to the axon guidance by their repellent actions [5,9,12]. We have identified a member of semaphorin gene family from the mouse brain and named it M-Sema F. The N-terminal encodes a semaphorin domain that is similar between Sema I–III [6] followed by a single putative immunoglobulin-like domain, a transmembrane domain, and a proline-rich intracellular domain. M-Sema F mRNA is expressed widely in the nervous tissues during development. These suggest that M-Sema F is a transmembrane member of the semaphorin family of the vertebrate which may function in the developing neuronal network.     

Cis interaction between Semaphorin6A and Plexin‐A4 modulates the repulsive response to Sema6A

The correct navigation of axons to their targets depends on guidance molecules in the extra‐cellular environment. Differential responsiveness to a particular guidance cue is largely an outcome of disparity in the expression of its receptors on the reacting axons. Here, we show that the differential responsiveness of sympathetic and sensory neurons to the transmembrane Semaphorin Sema6A is mainly determined by its co‐expression in the responding neurons. Both sympathetic and sensory neurons express the Sema6A receptor Plexin‐A4, but only sympathetic neurons respond to it. The expression of Sema6A counteracts this responsiveness and is detected only in sensory neurons. Remarkably, sensory neurons that lack Sema6A gain sensitivity to it in a Plexin‐A4‐dependent manner. Using heterologus systems, we show that the co‐expression of Sema6A and Plexin‐A4 hinders the binding of exogenous ligand, suggesting that a Sema6A–Plexin‐A4 cis interaction serves as an inhibitory mechanism. Finally, we provide evidence for differential modes of interaction in cis versus in trans. Thus, co‐expression of a transmembrane cue together with its receptor can serve as a guidance response modulator.     

Plexin-A3 and plexin-A4 restrict the migration of sympathetic neurons but not their neural crest precursors

During development, the semaphorin family of guidance molecules is required for proper formation of the sympathetic nervous system. Plexins are receptors that mediate semaphorin signaling, but how plexins function during sympathetic development is not fully understood. Using phenotypic analyses of mutant mice in vivo, expression pattern studies, and in vitro assays, we show that plexin-A3 and plexin-A4 are essential for normal sympathetic development. This study confirms our previous in vitro findings that the two plexins differentially regulate the guidance of sympathetic axons. In addition, we find that semaphorin signaling through plexin-A3 and plexin-A4 restricts the migration of sympathetic neurons, but these two plexins function redundantly since migration defects are only observed in plexin-A3/-A4 double mutants. Surprisingly, our analysis also indicates that plexin-A3 and plexin-A4 are not required for guiding neural crest precursors prior to reaching the sympathetic anlagen. Immunoprecipitation studies suggest that these two plexins independently mediate secreted semaphorin signaling. Thus, plexin-A3 and plexin-A4 are expressed in newly-differentiated sympathetic neurons, but not their neural crest precursors. They function cooperatively to regulate the migration of sympathetic neurons and then differentially to guide the sympathetic axons.     

Diverse functions for the semaphorin receptor PlexinD1 in development and disease

Plexins are a family of single-pass transmembrane proteins that serve as cell surface receptors for Semaphorins during the embryonic development of animals. Semaphorin–Plexin signaling is critical for many cellular aspects of organogenesis, including cell migration, proliferation and survival. Until recently, little was known about the function of PlexinD1, the sole member of the vertebrate-specific PlexinD (PlxnD1) subfamily. Here we review novel findings about PlxnD1's roles in the development of the cardiovascular, nervous and immune systems and salivary gland branching morphogenesis and discuss new insights concerning the molecular mechanisms of PlxnD1 activity.     

Semaphorin 2a secreted by oenocytes signals through plexin B and plexin A to guide sensory axons in the Drosophila embryo

The semaphorin gene family has been shown to play important roles in axonal guidance in both vertebrates and invertebrates. Both transmembrane (Sema1a, Sema1b, Sema5c) and secreted (Sema2a, Sema2b) forms of semaphorins exist in Drosophila. Two Sema receptors, plexins (Plex) A and B, have also been identified. Many questions remain concerning the axon guidance functions of the secreted semaphorins, including the identity of their receptors. We have used the well-characterized sensory system of the Drosophila embryo to address these problems. We find novel sensory axon defects in sema2a loss-of-function mutants in which particular axons misproject and follow inappropriate pathways to the CNS. plexB loss-of-function mutants show similar phenotypes to sema2a mutants and sema2a interacts genetically with plexB, supporting the hypothesis that Sema2a signals through PlexB receptors. Sema2a protein is expressed by larval oenocytes, a cluster of secretory cells in the lateral region of the embryo and the sema2a mutant phenotype can be rescued by driving Sema2a in these cells. Ablation of oenocytes results in sensory axon defects similar to the sema2a mutant phenotype. These data support a model in which Sema2a, while being secreted from oenocytes, acts in a highly localized fashion: It represses axon extension from the sensory neuron cell body, but only in regions in direct contact with oenocytes.     

Plexin-A1 and plexin-B1 specifically interact at their cytoplasmic domains

Semaphorin 3A (Sema3A) is a member of semaphorins and functions as an axonal repulsive guidance molecule. Neuropilin-1 and plexin-As form receptor complexes for Sema3A and plexin-As are thought to initiate the intracellular signaling cascade. However, the molecule by which plexin-As transduce their signal is not well understood. We searched molecules that interact with intracellular domains of plexin-A1 by yeast two-hybrid screening and identified a 349 amino acid fragment of plexin-B1 as a plexin-A1 interacting protein. We, then, cloned mouse plexin-B1 and confirmed their interaction in a mammalian expression system. Plexin-B1 physically associated with plexin-A1, but not with plexin-A2 or A3. Northern blot analysis showed the expression of both plexin-A1 and B1 in adult brain. We propose that plexin-A1 and B1 interact in the adult brain and transduce Sema3A signaling in cooperation.     

Repulsive and attractive semaphorins cooperate to direct the navigation of cardiac neural crest cells

The cardiac neural crest, a subpopulation of the neural crest, contributes to the cardiac outflow tract formation during development. However, how it follows the defined long-range migratory pathway remains unclear. We show here that the migrating cardiac neural crest cells (NCCs) express Plexin-A2, Plexin-D1 and Neuropilin. The membrane-bound ligands for Plexin-A2, Semaphorin (Sema)6A and Sema6B, are expressed in the dorsal neural tube and the lateral pharyngeal arch mesenchyme (the NCC “routes”). Sema3C, a ligand for Plexin-D1/neuropilin-1, is expressed in the cardiac outflow tract (the NCC “target”). Sema6A and Sema6B repel neural crest cells, while Sema3C attracts neural crest cells. Sema6A and Sema6B repulsion and Sema3C attraction are diminished either when Plexin-A2 and Neuropilin-1, or when Plexin-D1, respectively, are knocked down in NCCs. When RNAi knockdown diminishes each receptor in NCCs, the NCCs fail to migrate into the cardiac outflow tract in the developing chick embryo. Furthermore, Plexin-A2-deficient mice exhibit defects of cardiac outflow tract formation. We therefore conclude that the coordination of repulsive cues provided by Sema6A/Sema6B through Plexin-A2 paired with the attractive cue by Sema3C through Plexin-D1 is required for the precise navigation of migrating cardiac NCCs.     

Fgf19 is required for zebrafish lens and retina development

Fgf signaling plays crucial roles in morphogenesis. Fgf19 is required for zebrafish forebrain development. Here, we examined the roles of Fgf19 in the formation of the lens and retina in zebrafish. Knockdown of Fgf19 caused a size reduction of the lens and the retina, failure of closure of the choroids fissure, and a progressive expansion of the retinal tissue to the midline of the forebrain. Fgf19 expressed in the nasal retina and lens was involved in cell survival but not cell proliferation during embryonic lens and retina development. Fgf19 was essential for the differentiation of lens fiber cells in the lens but not for the neuronal differentiation and lamination in the retina. Loss of nasal fate in the retina caused by the knockdown of Fgf19, expansion of nasal fate in the retina caused by the overexpression of Fgf19 and eye transplantation indicated that Fgf19 in the retina was crucial for the nasal-temporal patterning of the retina that is critical for the guidance of retinal ganglion cell axons. Knockdown of Fgf19 also caused incorrect axon pathfinding. The present findings indicate that Fgf19 positively regulates the patterning and growth of the retina, and the differentiation and growth of the lens in zebrafish.     

B-plexins control microtubule dynamics and dendrite morphology of hippocampal neurons

Semaphorins and their receptors plexins are implicated in various processes in the nervous system, but how B-plexins regulate the growth of dendrites remains poorly characterized. We had previously observed that Plexin-B1 and B3 interact with microtubule end-binding proteins (EBs) that are central adapters at growing microtubule tips, and this interaction is involved in neurite growth. Therefore, we hypothesized that plexins regulate microtubule dynamics and through that also dendritogenesis. The role of all three B-plexins was systematically examined in these processes. B-plexins and their ligand Semaphorin-4D influence the dynamics of microtubule tips both EB-dependently and independendently. EB3 as well as Plexin-B1, B2 and B3 turned out to have a significant role in the development of dendritic arbor of rat hippocampal neurons. Our results clearly indicate that semaphorin-plexin-EB pathway is one molecular mechanism how extracellular guidance cues are translated into intracellular mechanics. Taken together, Semaphorin-4D and B-plexins modulate the dynamic behavior of microtubule tips, and are therefore important in neurite growth.     

人Semaphorin 4D慢病毒载体的构建及鉴定

目的:构建并制备能够有效表达Semaphorin 4D的重组慢病毒。方法:从人急性T细胞白血病Jurkat细胞DNA 扩增人Semaphorin 4D基因,克隆至pWPI GW慢病毒载体上,与pVSVG及pSPAX质粒共转染人胚肾293T细胞,包装出重组慢病毒,将纯化后的重组病毒直接感染293T和HUVEC细胞,通过免疫印迹、免疫荧光染色和血管内皮细胞迁移分析等方法检测Semaphorin 4D的表达和诱导血管内皮细胞迁移的作用。结果: 重组慢病毒介导Semaphorin 4D在293T和HUVEC内获得表达,能介导血管内皮细胞迁移。结论:成功构建了表达Semaphorin 4D的重组慢病毒载体。     

Plexin-A4 is expressed in oligodendrocyte precursor cells and acts as a mediator of semaphorin signals

Class 3 semaphorin acts as a guidance clue for both cell migration and nerve fiber projection. The signal of class 3 semaphorin travels via a receptor complex consisting of neuropilins and Plexin-A subfamily. Although it has been reported that class 3 semaphorin acts as a repellent for oligodendrocyte precursor cells (OPCs), which migrate actively during brain development, the expression of Plexin-A subfamily has not been reported in OPCs yet. Therefore, it is currently unclear how semaphorin signals can travel in OPCs. In the present study, the expression of Plexin-A4 (PlexA4) was first demonstrated in a newly established OPC line and OPCs in developing brain. In the OPC line, repulsion for process extension was caused by both Sema3A and Sema6A, and the effect of the semaphorins was diminished in cells expressing PlexA4 lacking the cytoplasmic domain. These results strongly suggest that PlexA4 expressed in OPCs acts as a mediator of semaphorin signals.     

Unidirectional startle responses and disrupted left–right co-ordination of motor behaviors in robo3 mutant zebrafish

The Roundabout (Robo) family of receptors and their Slit ligands play well-established roles in axonal guidance, including in humans where horizontal gaze palsy with progressive scoliosis (HGPPS) is caused by mutations in the robo3 gene. Although significant progress has been made toward understanding the mechanism by which Robo receptors establish commissural projections in the central nervous system, less is known about how these projections contribute to neural circuits mediating behavior. In this study, we report cloning of the zebrafish behavioral mutant twitch twice and show that twitch twice encodes robo3 . We show that in mutant hindbrains the axons of an identified pair of neurons, the Mauthner cells, fail to cross the midline. The Mauthner neurons are essential for the startle response, and in twitch twice / robo3 mutants misguidance of the Mauthner axons results in a unidirectional startle response. Moreover, we show that twitch twice mutants exhibit normal visual acuity but display defects in horizontal eye movements, suggesting a specific and critical role for twitch twice / robo3 in sensory-guided behavior.     

Expression and characterization of a brain-specific protein kinase BSK146 from zebrafish

We have previously identified a novel protein kinase, pk146, in the brain of Tetraodon. In the present study, we cloned the homologous protein kinase gene encoding a protein of 385 amino acid residues from zebrafish. The overall amino acid sequence and the kinase domain of zebrafish BSK146 shows 48% and 69% identity to that of rat sbk, a SH3-containing serine/threonine protein kinase. By whole-mount in situ hybridization and RT-PCR, the expression of bsk146 mRNA was mainly in the brain. To explore the in vivo function of BSK146 during zebrafish development, we used morpholino knockdown approach and found that BSK146 morphants displayed enlarged hindbrain ventricle and smaller eyes. Whole-mount in situ hybridization was further performed to analyze the brain defects in BSK146-MO-injected embryos. The expression of brain-specific markers, such as otx2, pax2.1, and krox20, was found normal in morphant embryos at 24hpf, while expression of pax2.1 exerted changes in midbrain-hindbrain boundary and hindbrain in morphant embryos at 48hpf. These data suggest that BSK146 may play an important role in later ventricle expansion in zebrafish brain development. Although the recombinant BSK146 protein produced in insect cells was active and could phosphorylate both histone H1 and histone 2B, the endogenous substrate of BSK146 in the embryonic brain of zebrafish is not clear at the present time and needs further investigation.     

Identification and characterization of roundabout orthologs in zebrafish

The Roundabout (Robo) family of receptors and their extracellular ligands, the Slit protein family, play important roles in repulsive axon guidance. First identified in Drosophila, Robo receptors form an evolutionarily conserved sub-family of the immunoglobulin (Ig) superfamily that are characterized by the presence of five Ig repeats and three fibronectin-type III repeats in the extracellular domain, a transmembrane domain, and a cytoplasmic domain with several conserved motifs that play important roles in Robo-mediated signaling (Cell 92 (1998) 205; Cell 101 (2000) 703). Robo family members have now been identified in C. elegans, Xenopus, rat, mouse, and human (Cell 92 (1998) 205; Cell 92 (1998) 217; Cell 96 (1999) 807; Dev. Biol. 207 (1999) 62). Furthermore, multiple robo genes have been described in Drosophila, rat, mouse and humans, raising the possibility of potential redundancy and diversity in robo gene function. As a first step in elucidating the role of Robo receptors during vertebrate development, we identified and characterized two Robo family members from zebrafish. We named these zebrafish genes robo1 and robo3, reflecting their amino acid sequence similarity to other vertebrate robo genes. Both genes are dynamically expressed in the developing nervous system in distinct patterns. robo3 is expressed during the first day of development in the hindbrain and spinal cord and is later expressed in the tectum and retina. robo1 nervous system expression appears later in development and is more restricted. Moreover, both genes are expressed in non-neuronal tissues consistent with additional roles for these genes during development.     

Phosphatidylinositol-4-phosphate 5-kinase and GEP100/Brag2 protein mediate antiangiogenic signaling by semaphorin 3E-plexin-D1 through Arf6 protein

The semaphorins are a family of secreted or membrane-bound proteins that are known to guide axons in the developing nervous system. Genetic evidence revealed that a class III semaphorin, semaphorin 3E (Sema3E), and its receptor Plexin-D1 also control the vascular patterning during development. At the molecular level, we have recently shown that Sema3E acts on Plexin-D1 expressed in endothelial cells, thus initiating a novel antiangiogenic signaling pathway that results in the retraction of filopodia in endothelial tip cells. Sema3E induces the rapid disassembly of integrin-mediated adhesive structures, thereby inhibiting endothelial cell adhesion to the extracellular matrix. This process requires the activation of small GTPase Arf6 (ADP-ribosylation factor 6), which regulates intracellular trafficking of β1 integrin. However, the molecular mechanisms by which Sema3E-Plexin-D1 activates Arf6 remained to be identified. Here we show that GEP100 (guanine nucleotide exchange protein 100)/Brag2, a guanine nucleotide exchange factor for Arf6, mediates Sema3E-induced Arf6 activation in endothelial cells. We provide evidence that upon activation by Sema3E, Plexin-D1 recruits phosphatidylinositol-4-phosphate 5-kinase, and its enzymatic lipid product, phosphatidylinositol 4,5-bisphosphate, binds to the pleckstrin homology domain of GEP100. Phosphatidylinositol 4,5-bisphosphate binding to GEP100 enhances its guanine nucleotide exchange factor activity toward Arf6, thus resulting in the disassembly of integrin-mediated focal adhesions and endothelial cell collapse. Our present study reveals a novel phospholipid-regulated antiangiogenic signaling pathway whereby Sema3E activates Arf6 through Plexin-D1 and consequently controls integrin-mediated endothelial cell attachment to the extracellular matrix and migration.     

A systematic expression analysis implicates Plexin-B2 and its ligand Sema4C in the regulation of the vascular and endocrine system

Plexins serve as receptors for semaphorins and play important roles in the developing nervous system. Plexin-B2 controls decisive developmental programs in the neural tube and cerebellum. However, whether Plexin-B2 also regulates biological functions in adult nonneuronal tissues is unknown. Here we show by two methodologically independent approaches that Plexin-B2 is expressed in discrete cell types of several nonneuronal tissues in the adult mouse. In the vasculature, Plexin-B2 is selectively expressed in functionally specialized endothelial cells. In endocrine organs, Plexin-B2 localizes to the pancreatic islets of Langerhans and to both cortex and medulla of the adrenal gland. Plexin-B2 expression is also detected in certain types of immune and epithelial cells. In addition, we report on a systematic comparison of the expression patterns of Plexin-B2 and its ligand Sema4C, which show complementarity or overlap in some but not all tissues. Furthermore, we demonstrate that Plexin-B2 and its family member Plexin-B1 display largely nonredundant expression patterns. This work establishes Plexin-B2 and Sema4C as potential regulators of the vascular and endocrine system and provides an anatomical basis to understand the biological functions of this ligand-receptor pair.     

Identification and characterization of zebrafish ocular formation genes.

To study genes that are specifically expressed in the eyes, we employed microarray and in situ hybridization analyses to identify and characterize differentially expressed ocular genes in eyeless masterblind (mbl-/-) zebrafish (Danio rerio). Among 70 differentially expressed genes in the mbl-/- mutant identified by microarray analysis, 8 down-regulated genes were characterized, including 4 eye-specific genes, opsin 1 short-wave-sensitive 1 (opn1sw1), crystallinbetaa1b (cryba1b), crystallinbetaa2b (cryba2b), and crystallingamma M2d3 (crygm2d3); 2 eye and brain genes, ATPase, H+ transporting, lysosomal, V0 subunit c (atp6v0c) and basic leucine zipper and W2 domains 1a (bzw1a); and 2 constitutive genes, heat shock protein 8 (hspa8) and ribosomal protein L7a (rpl7a). In situ hybridization experiments confirmed down-regulation of these 8 ocular formation genes in mbl-/- zebrafish and showed their ocular and dynamic temporal expression patterns during zebrafish early development. Further, an automated literature analysis of the 70 differentially expressed genes identified a sub-network of genes with known associations, either with each other or with ocular structures or development, and shows how this study contributes to the current body of knowledge.