Floor plate-derived neuropilin-2 functions as a secreted semaphorin sink to facilitate commissural axon midline crossing

Commissural axon guidance depends on a myriad of cues expressed by intermediate targets. Secreted semaphorins signal through neuropilin-2/plexin-A1 receptor complexes on post-crossing commissural axons to mediate floor plate repulsion in the mouse spinal cord. Here, we show that neuropilin-2/plexin-A1 are also coexpressed on commissural axons prior to midline crossing and can mediate precrossing semaphorin-induced repulsion in vitro. How premature semaphorin-induced repulsion of precrossing axons is suppressed in vivo is not known. We discovered that a novel source of floor plate-derived, but not axon-derived, neuropilin-2 is required for precrossing axon pathfinding. Floor plate-specific deletion of neuropilin-2 significantly reduces the presence of precrossing axons in the ventral spinal cord, which can be rescued by inhibiting plexin-A1 signaling in vivo. Our results show that floor plate-derived neuropilin-2 is developmentally regulated, functioning as a molecular sink to sequester semaphorins, preventing premature repulsion of precrossing axons prior to subsequent down-regulation, and allowing for semaphorin-mediated repulsion of post-crossing axons.     

Plexin-A4 negatively regulates T lymphocyte responses

Semaphorins and their receptors play crucial roles not only in axon guidance during neuronal development but also in the regulation of immune responses. Plexin-A4, a member of the plexin-A subfamily, forms a receptor complex with neuropilins and transduces signals for class III semaphorins in the nervous system. Although plexin-A4 is also expressed in the lymphoid tissues, the involvement of plexin-A4 in immune responses remains unknown. To explore the role of plexin-A4 in the immune system, we analyzed immune responses in plexin-A4-deficient (plexin-A4-/-) mice. Among immune cells, plexin-A4 mRNA was detected in T cells, dendritic cells and macrophages but not in B cells and NK cells. Plexin-A4-/- mice had normal numbers and cell surface markers for each lymphocyte subset, suggesting that plexin-A4 is not essential for lymphocyte development. However, plexin-A4-/- mice exhibited enhanced antigen-specific T cell responses and heightened sensitivity to experimental autoimmune encephalomyelitis. Plexin-A4-/- T cells exhibited hyperproliferative responses to anti-CD3 stimulation and to allogeneic dendritic cells in vitro. Furthermore, this hyperproliferation was also observed in both T cells from neuropilin-1 mutant (npn-1(Sema-)) mice, in which the binding site of class III semaphorins is disrupted, and T cells from Sema3A-deficient (Sema-3A-/-) mice. Collectively, these results suggest that plexin-A4, as a component of the receptor complex for class III semaphorins, negatively regulates T cell-mediated immune responses.     

Plexin-A2 and its ligand, Sema6A, control nucleus-centrosome coupling in migrating granule cells

During their migration, cerebellar granule cells switch from a tangential to a radial mode of migration. We have previously demonstrated that this involves the transmembrane semaphorin Sema6A. We show here that plexin-A2 is the receptor that controls Sema6A function in migrating granule cells. In plexin-A2-deficient (Plxna2(-/-)) mice, which were generated by homologous recombination, many granule cells remained in the molecular layer, as we saw in Sema6a mutants. A similar phenotype was observed in mutant mice that were generated by mutagenesis with N-ethyl-N-nitrosourea and had a single amino-acid substitution in the semaphorin domain of plexin-A2. We found that this mutation abolished the ability of Sema6A to bind to plexin-A2. Mouse chimera studies further suggested that plexin-A2 acts in a cell-autonomous manner. We also provide genetic evidence for a ligand-receptor relationship between Sema6A and plexin-A2 in this system. Using time-lapse video microscopy, we found that centrosome-nucleus coupling and coordinated motility were strongly perturbed in Sema6a(-/-) and Plxna2(-/-) granule cells. This suggests that semaphorin-plexin signaling modulates cell migration by controlling centrosome positioning.     

Segregation of arterial and venous markers in subpopulations of blood islands before vessel formation.

The neuropilin-1 (np1) and the neuropilin-2 (np2) receptors bind vascular endothelial growth factor (VEGF) and class-3 semaphorins. They form complexes with VEGF tyrosine-kinase receptors or alternatively with type-A plexins to transduce respective VEGF or semaphorin signals. We have compared the expression patterns of np1, np2, plexin-A1, and plexin-A2 in the emerging vasculature of chick embryos. Double in situ hybridization reveals that six-somite embryos contain intermingled extraembryonic blood island (BI) subpopulations that express np1 or np2 as well as a BI subpopulation that coexpresses both neuropilins. In 13-somite embryos, which already contain an extraembryonic vascular plexus, the expression of np1 and np2 is segregated between the arterial and venous parts of the plexus, despite the absence of blood flow. However, the arterial marker ephrin-B2 was not yet expressed in the plexus at this stage. In 26-somite embryos, which possess a functional vascular system, np1 and np2 are differentially expressed in arteries and veins as previously reported. At this stage, posterior BIs expressing np2 appear to undergo fusion to form the posterior sinus vein and its tributaries, suggesting that the venous identity of these veins may be established before their formation. The neuropilin coreceptor plexin-A2 was expressed in extraembryonic veins but not in extraembryonic arteries. In contrast, within the embryo, plexin-A2 expression was observed in the dorsal aorta as well as in the cardinal vein. Semaphorin-3F (s3f), an np2 ligand, bound to np2-expressing cells in 26-somite embryos regardless of the presence or absence of plexin-A1 or plexin-A2. Of interest, even though s3f binds to np1 in vitro, np1-expressing arteries fail to bind s3f in whole-mount binding experiments.     

Sema3A and neuropilin-1 expression and distribution in rat white adipose tissue

Semaphorins are cell surface and/or soluble signals that exert an inhibitory control on axon guidance. Sema3A, the vertebrate-secreted semaphorin, binds to neuropilin-1, which together with plexins constitutes the functional receptor. To verify whether Sema3A is produced by white adipocytes and, in that case, to detect its targets in white adipose tissue, we studied the cell production and tissue distribution of Sema3A and neuropilin-1 in rat retroperitoneal and epididymal adipose depots. Sema3A and neuropilin-1 were detected in these depots by Western blotting. The immunohistochemical results showed that Sema3A is produced in, and possibly secreted by, smooth muscle cells of arteries and white adipocytes. Accordingly, neuropilin-1 was found on perivascular and parenchymal nerves. Such a pattern of distribution is in line with a role for secreted Sema3A in the growth and plasticity of white adipose tissue nerves. Indeed, after fasting, when white adipocytes are believed to be overstimulated by noradrenaline and rearrangement of the parenchymal nerve supply may occur, adipocytic expression of Sema3A is reduced. Finally, the presence of neuropilin-1 in some white adipocytes raises the interesting possibility that Sema3A also exerts an autocrine-paracrine role on these cells.     

miR-124 acts through CoREST to control onset of Sema3A sensitivity in navigating retinal growth cones

During axon pathfinding, growth cones commonly show changes in sensitivity to guidance cues that follow a cell-intrinsic timetable. The cellular timer mechanisms that regulate such changes are, however, poorly understood. Here we have investigated microRNAs (miRNAs) in the timing control of sensitivity to the semaphorin Sema3A in Xenopus laevis retinal ganglion cell (RGC) growth cones. A developmental profiling screen identified miR-124 as a candidate timer. Loss of miR-124 delayed the onset of Sema3A sensitivity and concomitant neuropilin-1 (NRP1) receptor expression and caused cell-autonomous pathfinding errors. CoREST, a cofactor of a NRP1 repressor, was newly identified as a target and mediator of miR-124 for this highly specific temporal aspect of RGC growth cone responsiveness. Our findings indicate that miR-124 is important in regulating the intrinsic temporal changes in RGC growth cone sensitivity and suggest that miRNAs may act broadly as linear timers in vertebrate neuronal development.     

Neuropilin-1a is involved in trunk motor axon outgrowth in embryonic zebrafish.

Neuropilin-1, a receptor for axon-repellent semaphorins and vascular endothelial growth factor (VEGF), functions both in angiogenesis and axon growth. Here, we show strong expression of neuropilin-1a in primary motor neurons in the trunk of embryonic zebrafish. Reducing the expression of neuropilin-1a using antisense morpholino oligonucleotides induced aberrant branching of motor nerves, additional exit points of motor nerves from the spinal cord, and migration of neurons out of the spinal cord along the motor axon pathway in a dose-dependent manner. These phenotypes could be partially rescued by co-injecting neuropilin-1a mRNA. Other axons in the spinal cord and head appeared unaffected by the morpholino treatment. In addition, neuropilin-1a morpholino treatment disturbed normal formation of blood vessels in the trunk of 24 hours postfertilization embryos, as shown by microangiography. Morpholinos to VEGF also disturbed formation of blood vessels but did not affect motor axons, indicating that correct formation of blood vessels is not needed for the growth of primary motor axons. Morpholinos to the semaphorin 3A homologs semaphorin 3A1 and semaphorin 3A2 also had no effect on motor axon growth. However, combined injections of neuropilin-1a morpholino, at a concentration that did not elicit axonal aberrations when injected alone, with VEGF, semaphorin 3A1, or semaphorin 3A2 morpholinos synergistically increased the proportion of embryos showing aberrant motor axon growth. Thus, neuropilin-1a in primary motor neurons may integrate signals from several ligands and is needed for proper segmental growth of primary motor nerves in zebrafish.     

Semaphorin-3A guides radial migration of cortical neurons during development

Postmitotic neurons in the developing cortex migrate along radial glial fibers to their proper location in the cortical plate and form the layered structure. Here we report that the radial migration of rat layer II/III cortical neurons requires guidance by the extracellular diffusible factor Semaphorin-3A (Sema3A). This factor is expressed in a descending gradient across the cortical layers, whereas its receptor neuropilin-1 (NP1) is highly expressed in migrating neurons. Downregulation or conditional knockout of NP1 in newborn cortical neurons impedes their radial migration by disrupting their radial orientation during migration without altering their cell fate. Studies in cultured cortical slices further show that the endogenous gradient of Sema3A is required for the proper migration of newborn neurons. In addition, transwell chemotaxis assays show that isolated newborn neurons are attracted by Sema3A. Thus, Sema3A may function as a chemoattractive guidance signal for the radial migration of newborn cortical neurons toward upper layers.     

Identification and characterization of a novel member of murine semaphorin family

The semaphorin gene family contains a large number of secreted type or transmembrane type proteins, and some of them function as the repulsive and attractive cues of axon guidance during development. Here we report a novel member of murine class 3 semaphorin genes, semaphorin 3G (Sema3G), mapped on chromosome 14. In adulthood, Sema3G is mainly expressed in the lung and kidney, and a little in the brain. Interestingly, in the adult rodent brain Sema3G is expressed only in the granular layer of the cerebellum, as determined by Northern blot and in situ hybridization analyses. We also found that Sema3G binds Neuropilin-2, but not Neuropilin-1, and induces the repulsion of sympathetic axons, but not dorsal root ganglion axons, indicating that Sema3G utilizes Neuropilin-2 as a receptor to repel specific types of axons.     

Vascular endothelial growth factor controls neuronal migration and cooperates with Sema3A to pattern distinct compartments of the facial nerve

Developing neurons accurately position their somata within the neural tube to make contact with appropriate neighbors and project axons to their preferred targets. Taking advantage of a collection of genetically engineered mouse mutants, we now demonstrate that the behavior of somata and axons of the facial nerve is regulated independently by two secreted ligands for the transmembrane receptor neuropilin 1 (Nrp1), the semaphorin Sema3A and the VEGF164 isoform of Vascular Endothelial Growth Factor. Although Sema3A is known to control the guidance of facial nerve axons, we now show that it is not required for the pathfinding of their somata. Vice versa, we find that VEGF164 is not required for axon guidance of facial motor neurons, but is essential for the correct migration of their somata. These observations demonstrate, for the first time, that VEGF contributes to neuronal patterning in vivo, and that different compartments of one cell can be co-ordinately patterned by structurally distinct ligands for a shared receptor.     

重组人信号素3A抑制胃癌血管生成及其机制

目的 探讨信号素3A（Sema3A）及其受体神经激-1（NRP-1）在胃癌中的表达及其与微血管密度（MVD）的相关性;并探讨重组人Sema3A对胃癌血管生成的影响及其相关的机制。方法 选取40例手术切除胃癌组织及其癌旁正常组织,免疫组织化学法检测组织中Sema3A、NRP-1的表达及MVD。ELISA检测胃癌患者组及正常对照组血清Sema3A的表达水平。Western blotting检测5株胃癌细胞系（MGC-803、HGC-27、MKN-28、SGC-7901、MKN-45）、人正常胃黏膜细胞（GES-1）中Sema3A和NRP-1的表达。应用Transwell小室构建非接触式体外共培养体系,小管形成实验初步研究不同浓度重组人Sema3A对胃癌血管生成的影响;Western blotting检测共培养体系中血管内皮生长因子受体2（VEGFR2）、NRP-1的表达水平。结果 胃癌组织、细胞和患者血清中Sema3A的表达显著低于对照组（P<0.05）,胃癌组织和MKN 28细胞中的NRP-1表达明显升高（P<0.05）,两者均与胃癌TNM分期相关（P<0.05）,且Sema3A与微血管密度存在负相关（P<0.05）。在体外共培养体系中,重组人Sema3A处理组人脐静脉内皮细胞（HUVEC）小管形成能力下降,且具有浓度依赖性;重组人Sema3A可下调VEGFR2蛋白的表达。结论 Sema3A在胃癌组织、细胞和患者血清中均表达降低,与微血管密度成负相关,重组人Sema3A具有体外抑制胃癌血管生成的作用,可能与下调VEGFR2蛋白表达有关。     

The Neuropilin-1 Ligand,Sema3A,Acts as a Tumor Suppressor in the Pathogenesis of Acute Leukemia

Semaphorin-3A (Sema3A) and vascular endothelial growth factor (VEGF165) are ligands of neuropilin-1 (NRP-1 or CD304) and are related to immunoregulation and tumor angiogenesis, respectively. However, possible interactions between NRP-1 and Sema3A and VEGF165 in acute leukemia remain unclear, especially whether Sema3A plays a role in acute leukemia. In this study, both of the proportion of regulatory T cells (Tregs) and their expression of NRP-1 were found to increase in acute leukemia patients compared with healthy controls. In contrast, lower mRNA and plasma levels of Sema3A were detected in the acute leukemia patients. In vitro, the addition of exogenous Sema3A inhibited the expression of NRP-1 on Tregs and it promoted apoptosis of leukemia cells. However, in the presence of anti-Sema3A antibody, the effect of rhSema3A on NRP-1 expression was reversed. These results suggest that Sema3A promotes apoptosis in leukemia cells by inhibiting expression of NRP-1, and thus, represents a tumor suppressor protein with a role in the pathogenesis of acute leukemia. Consequently, NRP-1/Sema3A signaling may represent a novel target for the treatment of acute leukemia and should be further studied. Anat Rec, 302:1127–1135, 2019. © 2018 Wiley Periodicals, Inc.     

Autism spectrum disorder susceptibility gene TAOK2 affects basal dendrite formation in the neocortex

How neurons develop their morphology is an important question in neurobiology. Here we describe a new pathway that specifically affects the formation of basal dendrites and axonal projections in cortical pyramidal neurons. We report that thousand-and-one-amino acid 2 kinase (TAOK2), also known as TAO2, is essential for dendrite morphogenesis. TAOK2 downregulation impairs basal dendrite formation in vivo without affecting apical dendrites. Moreover, TAOK2 interacts with Neuropilin 1 (Nrp1), a receptor protein that binds the secreted guidance cue Semaphorin 3A (Sema3A). TAOK2 overexpression restores dendrite formation in cultured cortical neurons from Nrp1(Sema-) mice, which express Nrp1 receptors incapable of binding Sema3A. TAOK2 overexpression also ameliorates the basal dendrite impairment resulting from Nrp1 downregulation in vivo. Finally, Sema3A and TAOK2 modulate the formation of basal dendrites through the activation of the c-Jun N-terminal kinase (JNK). These results delineate a pathway whereby Sema3A and Nrp1 transduce signals through TAOK2 and JNK to regulate basal dendrite development in cortical neurons.     

Membrane potential shifts caused by diffusible guidance signals direct growth-cone turning

Plasma membrane potentials gate the ion channel conductance that controls external signal-induced neuronal functions. We found that diffusible guidance molecules caused membrane potential shifts that resulted in repulsion or attraction of Xenopus laevis spinal neuron growth cones. The repellents Sema3A and Slit2 caused hyperpolarization, and the attractants netrin-1 and BDNF caused depolarization. Clamping the growth-cone potential at the resting state prevented Sema3A-induced repulsion; depolarizing potentials converted the repulsion to attraction, whereas hyperpolarizing potentials had no effect. Sema3A increased the intracellular concentration of guanosine 3',5'-cyclic monophosphate ([cGMP]i) by soluble guanylyl cyclase, resulting in fast onset and long-lasting hyperpolarization. Pharmacological increase of [cGMP](i) caused protein kinase G (PKG)-mediated depolarization, switching Sema3A-induced repulsion to attraction. This bimodal switch required activation of either Cl(-) or Na+ channels, which, in turn, regulated the differential intracellular Ca2+ concentration increase across the growth cone. Thus, the polarity of growth-cone potential shifts imposes either attraction or repulsion, and Sema3A achieves this through cGMP signaling.     

Involvement of semaphorins in immunoregulation]

Semaphorins were identified originally as guidance cues for developing neuronal axons. However, it is becoming clear that several semaphorins play important roles in the immune system. For instance, Sema4D (CD100) enhances activation of B cells and dendritic cells through its receptor, CD72. Sema4A is crucially involved not only in T cell priming but also in Th1/Th2 regulation. Additional semaphorins and related molecules also have distinct biological activities in the immune system. We here review the current topic of immunoregulatory semaphorins which could be targets for potenciation of, and intervention for immune response.     

Distinct roles for Sema3A, Sema3F, and an unidentified trophic factor in controlling the advance of geniculate axons to gustatory lingual epithelium

Geniculate ganglion axons arrive in the lingual mesenchyme on embryonic day 13 (E13), 3–4 days before penetrating fungiform papilla epithelium (E17). This latency may result from chemorepulsion by epithelial Sema3A (Dillon et al. (2004) Journal of Comparative Neurology 470, 13–24), or Sema3F, which we report is also expressed in this epithelium. Sema3A and Sema3F repelled or suppressed geniculate neurite outgrowth, respectively, and these effects were stage and neurotrophic factor dependent. BDNF-stimulated outgrowth is repelled by Sema3A until E17, but insensitive to Sema3F from E16. NT-4-stimulated neurite outgrowth is sensitive to Sema3A and Sema3F through E18, but NT-4 has not been detected in E15–18 tongue. E15–18 tongue explants did not exhibit net chemorepulsion of geniculate neurites, but the ability of tongue explants to support geniculate neurite outgrowth fluctuates: E12–13 (Rochlin et al. (2000), Journal of Comparative Neurology, 422, 579–593) and E17–18 explants promote and may attract geniculate neurites, but stages corresponding to intralingual arborization do not. The E18 trophic and tropic effects were evident even in the presence of BDNF or NT-4, suggesting that some other factor is responsible. Intrinsic neurite outgrowth capability (without exogenous neurotrophic factors) fluctuated similarly: ganglia deteriorated at E15, but exhibited moderate outgrowth at E18. The chemorepulsion studies are consistent with a role for Sema3A, not Sema3F, in restricting geniculate axons from the epithelium until E17, when axons penetrate the epithelium. The transient inability of tongue explants to promote geniculate neurite outgrowth may signify an alternative mechanism for restricting geniculate axons from the epithelium: limiting trophic factor access. This work was supported by a research grant from the NIDCD, 1 R03 DC04965-01 to MWR.     

Biology and function of neuroimmune semaphorins 4A and 4D

Semaphorins belong to a family of membrane-bound and secreted molecules that regulate the functional activity of axons in the nervous system. Sema4A and Sema4D were the first semaphorins also found to be expressed in immune cells and were, therefore, termed “immune semaphorins”. It is known that Sema4A has three functional receptors, namely Plexin D1, Plexin B1, and Tim-2, whereas Sema4D binds to Plexin B1 and CD72. Recent studies suggest that immune semaphorins play critical roles in many physiological and pathological processes and such. In this review, we summarize the current knowledge on the biology of neuroimmune semaphorins and their corresponding receptors, their distribution in organs and tissues, function in the immune response, and critical regulatory roles in various diseases.     

Longest neurite‐specific activation of Rap1B in hippocampal neurons contributes to polarity formation through RalA and Nore1A in addition to PI3‐kinase

In a developing nervous system, axon‐dendrite formation is instructed by extrinsic cues, and the mechanism whereby a developing neuron interprets these cues using intracellular signaling is particularly important. Studies using dissociated hippocampal neurons have identified many signaling pathways underlying neuronal polarization. Among the components of these pathways, Rap1B is essential for axon specification in hippocampal cultures. However, spatiotemporal regulation of Rap1B activity in polarizing neurons and how it affects neuronal polarization remain unclear. Herein, we investigated spatiotemporal activity‐change of Rap1B and its target molecules in hippocampal neurons. FRET imaging showed that specific activation of Rap1B was observed at the tip of a future axon. To dissect downstream signaling, we used three effector mutants of Rap1B. Expression of Rap1B‐G12V/E37G and G12V/Y40C mutants resulted in supernumerary axons. The targets of Rap1B‐G12V/E37G were RalA and Nore1A, whereas Rap1B‐G12V/Y40C activated PI3‐kinase. RalA was activated in the tip of stage 3 axons, and RalA‐S28N expression reduced the fraction of neurons with supernumerary axons induced by Rap1B‐G12V/E37G. Furthermore, Nore1A depletion reduced the number of cells without axons. These results indicate that specific activation of Rap1B contributes to neuronal polarization via interaction with RalA and Nore1A in addition to PI3‐kinase.