Effects Of Semaphorin 3A Research Articles

The effects of semaphorin 3A in bone and cartilage metabolism: fundamental mechanism and clinical potential

Semaphorin 3A (Sema3A) is a neuroinformatic protein molecule with widespread expression across various tissues and organs. Recent investigations have unveiled its pivotal role in the skeletal system, primarily through its binding interactions with two co-receptors, neuropilin-1 (Nrp-1) and members of the plexin family. Prior research has confirmed the expression of Sema3A and its receptors in both osteocytes and chondrocytes. Beyond its expression patterns, Sema3A plays a multifaceted role in regulating bone and cartilage metabolism via employing diverse signaling pathways. Additionally, it engages in collaborative interactions with the immune and nervous systems, contributing to the pathophysiological processes underlying a spectrum of bone and joint diseases. In this paper, we undertake a comprehensive review of recent research developments in this field. Our objective is to deepen the understanding of Sema3A within the context of skeletal physiology and pathology. Furthermore, we aim to furnish a valuable reference for potential therapeutic interventions in the realm of bone and joint diseases.

Sodium Valproate Enhances Semaphorin 3A-mediated Anti-angiogenesis and Tumor Growth Inhibition in Human Osteosarcoma Cells.

Class 3 semaphorins, including semaphorin 3A (SEMA3A), are known endogenous angiogenesis inhibitors associated with endothelial cell migration and proliferation, and have been identified in many cancer cells. SEMA3A suppresses tumor angiogenesis by competing with VEGF, but tumors are known to have active angiogenesis, suggesting that expression of SEMA3A and its receptors is epigenetically restrained. To overcome this condition, we aimed to use histone deacetylase (HDAC) inhibitors to enhance the SEMA3A expression in osteosarcoma (OS) cells, thereby suppressing angiogenesis and inhibiting their proliferation and metastasis. OS cell lines and human microvascular endothelial (HMVE) cells were treated with HDAC inhibitors such as sodium valproate (VPA) and Trichostatin A (TSA). Changes in the SEMA3A expression and its related receptors at the mRNA and protein levels, as well as the inhibitory effects on tumor angiogenesis, were investigated. VPA and TSA increased the expression of SEMA3A and its receptor NRP1, without inducing PLXNA1 in OS cells. Similarly, SEMA3A and NRP1 expression was increased in HMVE cells, but no growth inhibition was observed. Furthermore, SEMA3A induced by VPA in OS cell culture medium inhibited vascular tube formation of HMVE cells, and overexpression of SEMA3A enhanced OS cell growth inhibition. This growth-inhibitory effect of SEMA3A induced G1/S cell cycle arrest in OS cells. HDAC inhibitors have anti-angiogenic and anti-tumor activities that may be, in part, mediated via the SEMA3A/NRP1/PLXNA1 autocrine and paracrine pathways.

Effects of Semaphorin3A on the growth of sensory and motor neurons

After peripheral nerve injury, motor and sensory axons can regenerate, but the inaccurate reinnervation of the target leads to poor functional recovery. Schwann cells (SCs) express sensory and motor phenotypes associated with selective regeneration. Semaphorin 3A (Sema3A) is an axonal chemorepellent that plays an essential role in axon growth. SCs can secret Sema3A, and Sema3A presents a different expression pattern at the proximal and distal ends of injured sensory and motor nerves. Hence, in our study, the protein expression and secretion of Sema3A in sensory and motor SCs and the expression of its receptor Neuropilin-1 (Nrp1) in dorsal root ganglia (DRG) sensory neurons (SNs) and spinal cord motor neurons (MNs) were detected by Western blot and ELISA. The effect of Sema3A at different concentrations on neurite growth of sensory and motor neurons was observed by immunostaining. Also, by blocking the Nrp1 receptor on neurons, the effect of Sema3A on neurite growth was observed. Finally, we observed the neurite growth of sensory and motor neurons cocultured with Sema3A siRNA transfected SCs by immunostaining. The results suggested that the expression and secretion of Sema3A in sensory SCs are more significant than that in motor SCs, and the expression of its receptor Nrp1 in SNs is higher than in MNs. Sema3A could inhibit the neurite growth of sensory and motor neurons via Nrp1, and Sema3A has a more substantial effect on the neurite growth of SNs. These data provide evidence that SC-secreted Sema3A might play a role in selective regeneration by a preferential effect on SNs.

Semaphorin 3A in the Immune System: Twenty Years of Study.

Semaphorin 3A is a secreted glycoprotein, which was originally identified as axon guidance factor in the neuronal system, but it also possesses immunoregulatory properties. Here, the effect of semaphorin 3A on T-lymphocytes, myeloid dendritic cells and macrophages is systematically analyzed on the bases of all publications available in the literature for 20 years. Expression of semaphorin 3A receptors – neuropilin-1 and plexins A – in these cells is described in details. The data obtained on human and murine cells is described comparatively. A comprehensive overview of the interaction of semaphorin 3A with mononuclear phagocyte system is presented for the first time. Semaphorin 3A signaling mostly results in changes of the cytoskeletal machinery and cellular morphology that regulate pathways involved in migration, adhesion, and cell–cell cooperation of immune cells. Accumulating evidence indicates that this factor is crucially involved in various phases of immune responses, including initiation phase, antigen presentation, effector T cell function, inflammation phase, macrophage activation, and polarization. In recent years, interest in this field has increased significantly because semaphorin 3A is associated with many human diseases and therefore can be used as a target for their treatment. Its involvement in the immune responses is important to study, because semaphorin 3A and its receptors turn to be a promising new therapeutic tools to be applied in many autoimmune, allergic, and oncology diseases.

SEMA3A protects against hyperoxia-induced lung injury in a bronchopulmonary dysplasia model of newborn rat by inhibiting ERK pathway.

Hyperoxia induces lung injury through lung inflammation in premature infants, leading to bronchopulmonary dysplasia (BPD). Semaphorin 3A (SEMA3A) participates in diverse biological processes, including cell migration, angiogenesis, and inflammation. The effect of SEMA3A on hyperoxic lung injury of neonatal rats with BPD was investigated in this study. Neonatal rats with BPD were established through hyperoxia treatment. Hematoxylin-eosin staining was used to evaluate histopathological analysis in lung tissues. SEMA3A expression was assessed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot assay. Adeno-associated virus (AAV)-mediated over-expression of SEMA3A (AAV-SEMA3A) was administrated into hyperoxia-induced rats, and apoptosis was evaluated by TUNEL staining. Levels of inflammatory cytokines were investigated by enzyme-linked-immunosorbent serologic assay (ELISA). Hyperoxia-induced histopathological changes in lung tissue reduced alveolar number and enhanced alveolar interval and alveolar volume. SEMA3A was downregulated in lung tissue of hyperoxia-induced rats. AAV-SEMA3A injection attenuated hyperoxia-induced cell apoptosis in lung tissues by increasing Bcl-2 and decreasing Bax and cleaved caspase-3. Moreover, the enhanced levels of Interleukin (IL)-1β, monocyte chemoattractant protein (MCP)-1, and tumor necrosis factor-α (TNF-α) in hyperoxia-induced rats were restored by AAV-SEMA3A injection by the downregulation of nuclear factor kappa B (NF-κB) phosphorylation. AAV-SEMA3A injection also ameliorated histopathological changes in lung tissues of hyperoxia-induced rats by increasing the number of radial alveolar count and decreasing the volume of mean linear intercept. Besides, the protein expression levels of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) phosphorylation were reduced in hyperoxia-induced rats post-AAV-SEMA3A injection. Ectopical expression of SEMA3A suppressed hyperoxia-induced apoptosis and inflammation in neonatal rats, and ameliorated the histopathological changes through inactivation of ERK/JNK pathway.

Effect of semaphorin-3A on the cellular stability of CD4+CD25+ regulatory T cells induced by lipopolysaccharide

To investigate the effect and mechanism of semaphorin-3A (Sema3A) in maintaining the cellular stability of CD4+CD25+ regulatory T cells (Tregs) induced by lipopolysaccharide (LPS). In vitro, using immunomagnetic beads, splenic CD4+CD25+ Tregs of C57BL/6J mice were isolated and cultured. According to the random number table, the isolated cells were divided into control group (treated with anti-CD3e and anti-CD28 for polyclonal activation), LPS group (on the basis of control group, treated with LPS at the dose of 100 μg/L), LPS + nuclear factor kappa B (NF-κB) inhibitor pyrrolidine dithiocarbamate (PDTC) group (treated with LPS at the dose of 100 μg/L and PDTC at the dose of 25 mg/L), LPS + phosphate buffer solution (PBS) group (treated with LPS at the dose of 100 μg/L and PBS at the volume of 10 μL), LPS + PDTC + recombinant Sema3A (rSema3A) group (treated with LPS at the dose of 100 μg/L, PDTC at the dose of 25 mg/L and rSema3A at the dose of 300 μg/L), and LPS + PBS + rSema3A group (treated with LPS at the dose of 100 μg/L, PBS at the volume of 10 μL and rSema3A at the dose of 300 μg/L). mRNA and protein expressions of the specific markers of CD4+CD25+ Tregs, including forkhead box protein P-3 (Foxp-3), cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) and membrane-associated transforming growth factor-β1 (TGF-β1m+) were detected by reverse transcription-polymerase chain reaction (RT-PCR) and immunofluorescence methods after 24 hours. The supernatant interleukin-10 (IL-10) and secretory type TGF-β1 (sTGF-β1) were detected by enzyme-linked immunosorbent assay (ELISA). The apoptotic level was detected by immunofluorescence. The demethylation of Foxp3-Tregs-specific demethylated region (Foxp-3-TSDR) was detected by methylation specific PCR (MSP) in order to reflect the cellular stability of CD4+CD25+ Tregs. DNA binding activity of NF-κB signaling pathway was determined by electrophoretic mobility shift assay (EMSA), and activity of NF-κB signaling pathway was determined by Western blotting. Compared with control group, LPS could increase the cellular stability, including an increase in the mRNA and protein expressions of Foxp-3, CTLA-4 and TGF-β1m+ and secretion of IL-10 and sTGF-β1, decrease in the level of apoptosis and increase in the methylation of Foxp-3-TSDR. At the same time, LPS increased DNA binding activity of NF-κB signaling pathway and phosphorylation levels of the major molecules of NF-κB, including inhibitory protein kinaseβ (IKKβ) and p65, suggesting that the mechanism of enhancing cellular stability by LPS was related to the NF-κB signaling pathway. Compared with LPS group, PBS had no effect on cellular stability and NF-κB signaling pathway. However, administration of rSema3A further promoted cellular stability and activated NF-κB signaling pathway. Compared with LPS + PBS + rSema3A group, PDTC inhibited the function of rSema3A to increase cellular stability, as the mRNA and protein expressions of Foxp-3, CTLA-4 and TGF-β1m+ were significantly down-regulated in the LPS + PDTC + rSema3A group [Foxp-3 mRNA (2-ΔΔCt): 8.092±1.117 vs. 18.509±1.068, Foxp-3 protein (relative fluorescence intensity): 1.224±0.033 vs. 1.826±0.181; CTLA-4 mRNA (2-ΔΔCt): 3.254±0.760 vs. 11.840±0.827, CTLA-4 protein (relative fluorescence intensity): 1.305±0.058 vs. 1.842±0.111; TGF-β1m+ mRNA (2-ΔΔCt): 3.589±1.180 vs. 8.509±0.472, TGF-β1m+ protein (relative fluorescence intensity): 1.319±0.033 vs. 1.822±0.063, all P < 0.01], the secretion of IL-10 and sTGF-β1 were significantly decreased [IL-10 (ng/L): 445.33±54.08 vs. 992.67±83.10, sTGF-β1 (ng/L): 1 116.67±65.25 vs. 1 494.67±94.45, both P < 0.01], the apoptosis was significantly increased (fluorescence intensity: 0.398±0.031 vs. 0.268±0.046, P < 0.01), the methylation of Foxp-3-TSDR was significantly decreased (grey value: 0.467±0.048 vs. 1.780±0.119, P < 0.01), the DNA binding activity of NF-κB signaling pathway was significantly inhibited (grey value: 1.23±0.02 vs. 3.95±0.06, P < 0.01), and the phosphorylation levels of IKKβ and p65 were significantly decreased [p-IKKβ (p-IKKβ/IKKβ): 0.97±0.07 vs. 1.97±0.04, p-p65 (p-p65/p65): 0.95±0.08 vs. 1.93±0.06, both P < 0.01]. LPS increases the cellular stability of CD4+CD25+ Tregs through the NF-κB signaling pathway, and Sema3A further increases the cellular stability of CD4+CD25+ Tregs, and is related to the NF-κB signaling pathway.

Semaphorin 3A Contributes to Secondary Blood-Brain Barrier Damage After Traumatic Brain Injury.

Semaphorin 3A (SEMA3A) is a member of the Semaphorins family, a class of membrane-associated protein that participates in the construction of nerve networks. SEMA3A has been reported to affect vascular permeability previously, but its influence in traumatic brain injury (TBI) is still unknown. To investigate the effects of SEMA3A, we used a mouse TBI model with a controlled cortical impact (CCI) device and a blood–brain barrier (BBB) injury model in vitro with oxygen-glucose deprivation (OGD). We tested post-TBI changes in SEMA3A, and its related receptors (Nrp-1 and plexin-A1) expression and distribution through western blotting and double-immunofluorescence staining, respectively. Neurological outcomes were evaluated by modified neurological severity scores (mNSSs) and beam-walking test. We examined BBB damage through Evans Blue dye extravasation, brain water content, and western blotting for VE-cadherin and p-VE-cadherin in vivo, and we examined the endothelial cell barrier through hopping probe ion conductance microscopy (HPICM), transwell leakage, and western blotting for VE-cadherin and p-VE-cadherin in vitro. Changes in miR-30b-5p were assessed by RT-PCR. Finally, the neuroprotective function of miR-30b-5p is measured by brain water content, mNSSs and beam-walking test. SEMA3A expression varied following TBI and peaked on the third day which expressed approximate fourfold increase compared with sham group, with the protein concentrated at the lesion boundary. SEMA3A contributed to neurological function deficits and secondary BBB damage in vivo. Our results demonstrated that SEMA3A level following OGD injury almost doubled than control group, and the negative effects of OGD injury can be improved by blocking SEMA3A expression. Furthermore, the expression of miR-30b-5p decreased approximate 40% at the third day and 60% at the seventh day post-CCI. OGD injury also exhibited an effect to approximately decrease 50% of miR-30b-5p expression. Additionally, the expression of SEMA3A post-TBI is regulated by miR-30b-5p, and miR-30b-5p could improve neurological outcomes post-TBI efficiently. Our results demonstrate that SEMA3A is a significant factor in secondary BBB damage after TBI and can be abolished by miR-30b-5p, which represents a potential therapeutic target.

Semaphorin 3A Negatively Affects Proliferation of Mouse Thymus Epithelial Cells In Vitro.

We studied effects of semaphorin 3A, keratinocyte growth factor (KGF), hepatocyte growth factor (HGF), and their combinations on the proliferative activity of cortical (cTEC1-2) and medullary (mTEC3-10) thymus epithelium cell lines. Semaphorin 3A inhibited the proliferative activity of epithelial cells, while HGF and KGF, in contrast, exerted a stimulating effect. The effect of KGF and semaphorin 3A on different cell lines depended on the expression of receptors for these two factors. When the combination of two factors was used, semaphorin 3A was able to neutralize the stimulating effect of HGF and KGF. It can be assumed that semaphorin 3A synthesized in the thymus stroma, can act as a functional antagonist of HGF and KGF and have an inhibitory effect when these drugs are administered into the body for the therapeutic purpose of restoring thymus functions.

Assaying the Action of Secreted Semaphorins on Vascular Permeability.

Blood vessels compartmentalize blood flow and irrigate tissues, while allowing bidirectional exchanges. Vascular permeability permits fluids, small molecules and cells to cross the endothelial barrier. This process can be modulated, among others, by the guidance molecules from the semaphorin family. Here, we describe both in vitro and in vivo assays to evaluate the effects of Semaphorin 3A on vascular permeability. We also focused this chapter on methods to preclude semaphorin effects validating its pro-permeability action. These approaches could then be applied to other potential semaphorins and molecules modulating permeability.

The effect of semaphorin 3A on fracture healing in osteoporotic rats

BackgroundsSemaphorin 3A (Sema3A) was demonstrated to exert an osteoprotective effect by both inhibiting osteoclastic bone resorption and promoting osteoblastic bone formation. The effect of Sema3A on fracture healing of osteoporotic rats was investigated in this study. MethodsTwelve weeks after bilateral ovariectomy, all animals underwent unilateral transverse osteotomy on the proximal tibiae, and were then randomly divided into two groups. Rats received vehicle (control) or weekly local injection of Sema3A (500μg/kg) into the injury site (group Sema3A) after fracture surgery until sacrifice at 4 and 8weeks. Specimens were harvested and examined by radiography, iDXA, histology, micro-CT, and three-point bending test. ResultsCompared to control, Sema3A treatment significantly increased bone mineral density, percent bone volume and biomechanical strength of the callus at 4 and 8weeks post-fracture. At 8weeks after fracture, the bone volume of callus showed no difference between groups, while the average cross-sectional area of callus in the control group was 43.8% higher than that of Sema3A group. Histological images showed increased callus formation at 4weeks post-fracture and better callus ossification in the Sema3A group, while callus remodeling in the control group seemed to be delayed and not well bridged. ConclusionsResults in this study indicated that Sema3A treatment increased callus volume and density at 4weeks post-fracture, and induced promoted callus ossification and remodeling at 8weeks post-fracture compared to control.

The effect of semaphorin 3A in the process of apoptosis in oxygen induced retinopathy in rats

To observe the change of semaphorin 3A (SEMA3A) expression in the retina of oxygen induced retinopathy (OIR) in rats and to investigate its influence on retinal degeneration in OIR. Experimental study. Forty-eight newborn pups were randomly classified into four groups and only one eye was examined in each pup. Thirty-six pups was induced into OIR model through aspirating 50% and 10% oxygen every 24 hours alternatively while the control group (12 pups) was raised under normal atmosphere. OIR+SEMA3Aab group accepted intravitreous injection of rabbit anti-rat SEMA3A antibody (Abcam Co.LTD, 40 mg/L, 2 µl) while OIR+IgG group was injected the same amount of non-active rabbit IgG at postnatal day 7. And the OIR group had no injections. All the pups were executed at postnatal day 18. Histological changes of retinas were examined through HE stain while Immunoflurecence staining and TUNEL procedure were used to detect focal expression of SEMA3A and apoptosis respectively. Total tissue protein was extracted and the expression of SEMA3A and Caspase-3 p17subunit were examined by Western blot. The data including retinal thickness, cell counting of retinal ganglion cells layers (RGCL) , endothelia outside inner limiting membrane, retinal apoptosis index (AI), the relative expression of SEMA3A and Caspase3 p17 subunit were analyzed by One-way ANOVA and followed LSD-t test to compare group differences. There were significant differences of retinal thickness, cell counting of RGCL and endothelia outside inner limiting membrane among each groups respectively (F = 13.222, F = 22.537, F = 14.478;P < 0.01) . The retinal thickness was (117.07 ± 8.13) µm in normal control group, (70.93 ± 5.68) µm in OIR group, (91.28 ± 4.58) µm in OIR+SEMA3Aab group, and (67.27 ± 10.15) µm in OIR+IgG group; the cell counting of RGCL was 42.7 ± 3.6 in normal control group, 24.3 ± 3.1 in OIR group, 35.0 ± 6.2 in OIR+SEMA3Aab group, and 22.8 ± 4.3 in OIR+IgG group while the endothelia outside inner limiting membrane was 1.0 ± 0.3 in normal control group, 14.2 ± 3.2 in OIR group, 9.6 ± 1.1 in OIR+SEMA3Aab group, and 10.8 ± 1.6 in OIR+IgG group. The retinal thickness and cell counting of RGCL in OIR+SEMA3Aab group were significantly lower than those in the normal control group (P < 0.01) , but were apparently higher than the data of OIR group and OIR+IgG group respectively (P < 0.01) . There were no more endothelia on the vitreal side of the inner limiting membrane in OIR+SEMA3Aab group than OIR group or OIR+IgG group (P > 0.05) although these three groups had much more endothelia than it in the normal control group (P < 0.01). The retinal AI detected by TUNEL staining was 27.67 ± 2.51 in normal control, 58.33 ± 8.50 in OIR group, 37.33 ± 5.03 in OIR+SEMA3Aab group and 61.67 ± 6.65 in OIR+IgG group. There was significant difference of the retinal AI among the four groups (F = 19.250, P = 0.001) . Apoptotic cells were significantly reduced in OIR+SEMA3Aab group compared with OIR + IgG group or OIR group (P < 0.01). The difference of SEMA3A protein expression among the groups detected by Western blot was significant (F = 38.59, P = 0.000) . SEMA3A expression in OIR group was 0.97 ± 0.05, which was significantly upregulated compared with the normal control group (0.64 ± 0.03) ( P < 0.01) . And its expression was successfully neutralized in OIR + SEMA3Aab group (0.41 ± 0.02) with comparison with OIR+IgG group (1.03 ± 0.15) through Western blot (P < 0.01) . SEMA3A was detected apparently in the photoreceptors layer in the normal control while its fluorescence was stronger and much more scattered in the whole retina. However, anti-SEMA3Aab intravitreous injection successfully reduced its fluorescence compared with the IgG injection. The cleavage of Caspase-3 was not detected in the normal control while the relative expression of Caspase-3 p17 subunit was significantly different in the other three groups (F = 304.619, P < 0.01). OIR + SEMA3Aab group had much less Caspase-3 p17 subunit (0.12 ± 0.01) than OIR group (0.30 ± 0.02) or OIR +IgG group (0.27 ± 0.02) (P < 0.01) . The over regulation of SEMA3A probably enhances the aggravation of apoptosis in OIR rats. Inhibition of SEMA3A is helpful to protect neuroretina.

Abstract 379: Potential Protective Effects of Semaphorin3A on Macrophage Function

Semaphorin3A (SEMA3A) is a secreted member of the Semaphorin family, first found to play a role in axon guidance. Recently many studies revealed immune functions for this protein. However, how SEMA3A may regulate monocyte and macrophage functions associated with atherosclerosis is unknown. An early step in plaque formation is the adhesion of monocytes on activated endothelial cells expressing adhesion molecules. However, we found that 25-100 ng/ml SEMA 3A did not affect the adhesion of monocyes on ICAM-1- or VCAM-1-coated plates. Similarly, stimulation of mouse carotid arteries with SEMA3A in an ex-vivo perfusion system, either administered alone or in conjunction with TNF-α, did not increase adhesiveness of monocytes injected in the intraluminal compartment. Furthermore SEMA3A did not stimulate monocyte migration in a boyden chamber assay. However SEMA3A had significant effects on macrophages, the most abundant cells in the plaque. Inefficient apoptotic cell phagocytosis and macrophage retention in the artery wall are hallmarks of atherosclerosis progression. We found that SEMA3A increased the migration of M2 “wound healing” macrophages more than 4-fold (p&lt;0.05), but had no such effect on M1 “inflammatory” macrophages. Increased M2 migration was associated with phosphorylation of FAK Y397, and could be inhibited by RGD peptide that prevents integrin-matrix interaction and by a blocking antibody targeting the SEMA3A receptor Neuropilin-1. SEMA3A (100ng/ml) also significantly increased phagocytosis of apoptotic cells by M2 macrophages (1.8-fold, p&lt;0.01) but not M1 cells. Interestingly, Sema3A did not affect lipoprotein uptake by macrophages, which characterizes foam cell formation. Finally in an in vivo model of acute inflammation, SEMA3A plasmid pretreatment reduced redness, swelling, and macrophage infiltration associated with LPS injection.In summary, our data suggest that SEMA3A is unlikely to participate in atherosclerotic plaque formation. On the contrary, effects of SEMA3A on macrophage function in vitro and in vivo indicate a potential protective effect in the inflammatory setting.

Waiting axons shun semaphorins

The semaphorins are a large family of secreted and transmembrane axon guidance molecules that are conserved across phyla. In vertebrates, most attention has focused on semaphorin-3A (SEMA-3A), which mediates the chemorepulsion of a variety of axons, including those of sympathetic, sensory and olfactory neurons. Recent studies have shown that semaphorins signal via a receptor complex that includes neuropilins and plexins; neuropilin-1 (NP-1) in particular, binds SEMA-3A and is required for its function, but it also binds other semaphorins. Therefore, a promising strategy is to mis-express dominant-negative neuropilins in developing axons, and thus to explore the effect of blocking the function of one or more semaphorins. Previous in vitro studies had already shown that a truncated neuropilin-1 (dnNP-1) can act in a dominant-negative fashion, blocking the effects of SEMA-3A and SEMA-3C on sympathetic neurons.Now, Michael Renzi et al. 1xOlfactory sensory axons expressing a dominant-negative semaphorin receptor enter the CNS early and overshoot their target. Renzi, M.J et al. Neuron. 2000; 28: 437–447Abstract | Full Text | Full Text PDF | PubMedSee all References1 from Jonathan Raper's laboratory have used this dominant-negative approach to investigate the projection of olfactory sensory axon projections in the chick embryo. Olfactory projections extend towards the telencephalon, but pause for several days before they contact their target, the olfactory bulb. SEMA-3A expression was observed from embryonic days (E) 5–7 when axons first reach the telencephalon and undergo their waiting period, but expression declined at E9, coinciding with the timing of axonal invasion of the bulb. Because olfactory axons express NP-1 and are sensitive to SEMA-3A in vitro, this data implies that SEMA-3A mediates repulsion to control the duration of this olfactory ‘waiting period’.Renzi et al. electroporated constructs encoding dnNP-1, full-length neuropilin-1 (NP-1), and a control construct, into the olfactory placode of chick embryos in ovo, leading to expression of the construct by olfactory axons. In each case a construct encoding a tag was co-transfected in order to visualize transfected cells. Whereas axons that express the control or NP-1 molecules observed the normal waiting period within the telencephalon, a proportion of axons expressing dnNP-1 overshot their normal stopping point and projected onto the developing olfactory bulb and along the medial forebrain. Aberrant trajectories into the forebrain were also detected at later stages of development, during which dnNP-1-expressing axons were detected on the surface of the olfactory bulb, with some trajectories overshooting the border between the bulb and the forebrain to invade this latter territory.These data provide a nice example of the precise action of repellent axon guidance cues; the authors also speculate that the olfactory–axon waiting period might represent a sort of paralysis or growth-cone collapse, paralleling the in vitro phenomenon that can be induced by addition of semaphorins to responsive axons. This study also adds to the growing body of data that uses electroporation in the chick embryo to mis-express genes of interest, providing an alternative or complementary approach to transgenic mouse studies and to retrovirus-mediated gene transfer.