Redundant functions but temporal and regional regulation of two alternatively spliced isoforms of Semaphorin 3F in the nervous system

Molecular and Cellular Neuroscience 24 (2003) 409 – 418

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Redundant functions but temporal and regional regulation of two alternatively spliced isoforms of Semaphorin 3F in the nervous system Sophie Kusy,a,1 Lydiane Funkelstein,b,1 David Bourgais,b Harry Drabkin,c Genevie`ve Rougon,b Joe¨lle Roche,a and Vale´rie Castellanib,* a

Laboratoire de Ge´ne´tique Humaine, IBMIG, EA 2224, Universite´ de Poitiers, 40, Av. du recteur Pineau, 86022 Poitiers cedex, France Laboratoire de Neurogene`se et Morphogene`se dans le De´veloppement et chez l’Adulte, UMR CNRS 6156, Universite´ de la Me´diterrane´e, IBDM, Parc Scientifique de Luminy 13288 Marseille cedex 9, France c University of Colorado Health Sciences Center, Division of Medical Oncology, Box B171, 4200 East Ninth Avenue, Denver, CO 80262, USA b

Received 14 May 2003; accepted 20 May 2003

Abstract SEMA3F is a secreted semaphorin that affects axon and cell guidance in the developing nervous system, and is also thought to have anti-tumor activity. Two spliced forms of SEMA3F have been identified that differ by the insertion of 31 amino acids in the sema domain. Here, we investigated the bioactivity of these isoforms and show, using coculture and binding assays, that they share common axonal chemorepulsive properties and binding to neuropilin receptors. SEMA3F isoforms were also found to regulate endothelial cell morphology by remodeling lamellipodial protrusions. Although Sema3F expression globally decreased during mouse development, we noted an enrichment of the longest isoform at postnatal stages in some territories such as the brainstem and spinal cord. These results indicate that although functionally redundant in cell culture assays, Sema3F spliced forms are characterized in vivo by a temporal and regional specific regulation during maturation of the nervous system. © 2003 Elsevier Inc. All rights reserved.

Introduction Semaphorins are a large family of secreted and transmembrane molecules that play an important role in the formation of neuronal connectivity (He et al., 2002). Secreted semaphorins repel and collapse axonal growth cones by binding to receptors composed of neuropilin (NP) and plexin complexes (Tamagnone and Comoglio, 2000) and activating intracellular pathways that control actin dynamics (Castellani and Rougon, 2002). Several reports suggest that semaphorins and their receptors also regulate cell morphology and motility, and contribute to organogenesis in a variety of non neuronal tissues (Brown et al., 2001; Loes et al., 2001; Bismuth and Boumsell, 2002; Fujii et al., 2002; Ginzburg et al., 2002; Kagoshima et al., 2001; Neufeld et al., 2002; Takashima et al., * Corresponding author. Fax: ⫹33-4-91-26-97-48. E-mail address: [email protected] (V. Castellani). 1 Contributed equally. 1044-7431/03/$ – see front matter © 2003 Elsevier Inc. All rights reserved. doi:10.1016/S1044-7431(03)00197-0

2002). In addition, semaphorins have been implicated in tumorigenic processes. The human secreted semaphorins, SEMA3B and SEMA3F, were both isolated within a common 3p21 chromosomal region deleted in lung cancer (Roche et al., 1996; Xiang et al., 1996; Sekido et al., 1996). Introduction of SEMA3B or SEMA3F in a lung cancer cell line expressing predominantly the NP1 receptor led to growth inhibition (Tse et al., 2002; Xiang et al., 2002; Tomizawa et al., 2001), and SEMA3B was shown to induce apoptosis in this setting (Tomizawa et al., 2001). Although the functional properties of semaphorins responsible for these activities remain to be clarified, SEMA3F expression patterns and localization of the protein in cell protrusions are suggestive of a possible role in the control of adhesion or movement (Brambilla et al., 2000; Nasarre et al., 2003). Such functions were also suggested by the observation, based on differential display, that metastatic lung adenocarcinoma cells overexpress H-SemaE, now called SEMA3C (Martin-Satue and Blanco, 1999). The recent finding that SEMA3B gene expression is inducible by p53 raises the

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and the morphology of endothelial cells. In addition, their expression pattern in the mouse developing central nervous system was studied.

Results and discussion SEMA3F isoforms bind to neuropilin-1 and neuropilin-2

Fig. 1. Binding of SEMA3F-GFP and SEMA3F⌬-GFP to NP-1- and NP-2-expressing cells. COS7 cells, transfected with expression constructs for NP-1 (A) and NP-2 (B), were incubated with SEMA3F-GFP and SEMA3F⌬-GFP. Using immunodetection of GFP, both SEMA3F isoforms were observed to bind to the cells. Thus, the extra amino acid sequences characteristic of the long isoform does not prevent interaction of SEMA3F with NP-1 or NP-2. Scale bar: 50 ␮m.

possibility for the semaphorin proteins to influence cell growth associated with tumor progression (Ochi et al., 2002). In mouse, Sema3F is expressed from early developmental stages in various neuronal and nonneuronal tissues (Eckhardt and Meyerhans, 1998; Xiang et al., 1996). In the nervous system, Sema3F was shown to inhibit neurite outgrowth, to collapse several populations of axons (Che´ dotal et al., 1998; de Castro et al., 1999), and to exert chemotactic attractive effect on oligodendrocyte precursor cell migration (Spassky et al., 2002). Intringuingly, two alternative spliced forms of human and mouse SEMA3F mRNA have been identified (Roche et al., 1996; Eckhardt and Meyerhans, 1998), one of which contains an extra exon encoding a 31-amino acid addition within the sema domain. No particular motif was found in these additional amino acids and its function, heretofore, has been largely uninvestigated except in a tumor suppression model where both spliced forms behaved similarly (Xiang et al., 2002). Several transcriptional variants exist for the class VI semaphorins, SEMA6C and SEMA6D, although their functional consequences are also unknown (Correa et al., 2001; Qu et al., 2002). An interesting feature concerning the SEMA3F isoforms is that their ratio strongly differs in various lung tumors (Roche et al., 1996), suggesting functional or regulatory differences. In the present study, we investigated the biological properties of these SEMA3F isoforms using a hippocampal axon repelling test,

We first examined whether the two SEMA3F isoforms differ in their capacity to interact with neuropilin-1 and -2, the ligand-binding proteins in the semaphorin receptor complexes (Tamagnone and Comoglio, 2000). To this end, COS7 cells transfected with NP-1 or NP-2 were incubated with supernatant containing SEMA3F-GFP (the longest form of SEMA3F) or SEMA3F⌬-GFP (the shortest form). To facilitate their visualization, the semaphorin proteins were preclustered with anti-GFP antibodies. Immunodetection of GFP indicated that both isoforms bind to NP-1 and NP-2, demonstrating that no apparent difference distinguishes SEMA3F from SEMA3F⌬ (Fig. 1). As controls, neither SEMA3F nor SEMA3F⌬ bound to untransfected cells, nor was binding observed with a supernatant of COS7 cells transfected with a control vector expressing only GFP (not shown). Although previous studies established that the sema domain confers specific binding features to different members of the secreted semaphorins (Koppel et al., 1997), constructs deleted for the sema domain were still observed to bind NP-1, indicating that the remaining Ig and basic domains are sufficient (Feiner et al., 1997). Thus, our observations that both forms of SEMA3F bind the two NP proteins are not unexpected, although they do not exclude the possibility that these isoforms differ in their affinity or avidity for NP receptors. Both SEMA3F isoforms repel hippocampal axons The sema domain is a particularly characteristic region of semaphorin proteins and is sufficient to confer biological activity (Koppel et al., 1997). Thus, the insertion of an extra exon in the sema domain could confer some functional difference. To explore this issue, we first used guidance assays to examine the influence of the two forms on growth cone trajectory. Neonatal hippocampal slices were cocultured with COS7 cell aggregates secreting SEMA3F-GFP, SEMA3F⌬-GFP, or control-GFP. Control cells exerted no effect on axon trajectory, as fibers extended radially from the hippocampal slices (Fig. 2A). This was also reflected by a guidance index close to zero (Fig. 2B). As expected from these qualitative observations, the number and length of axons growing away and toward the cell aggregates were equivalent (Figs. 2C and D). In contrast, COS7 cells secreting SEMA3F strongly deflected the axons, as illustrated in Fig. 2, in accordance with previous reports indicating a chemorepulsive effect of SEMA3F on hippocampal axons (Che´ dotal et al., 1998; Pozas et al., 2001). SEMA3F⌬ was

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Fig. 2. SEMA3F and SEMA3F⌬ repel hippocampal axons. (A) Cocultures of neonatal hippocampal slices with COS7 cell aggregates secreting SEMA3FGFP, SEMA3F⌬-GFP, or control-GFP. The microphotographs illustrate the chemorepulsive effect produced by SEMA3F and its isoforms on the trajectory of hippocampal fibers. (B) This repulsion was indicated by a guidance index that shifted from a value close to zero in the control to a negative value when cells were confronted to a source of semaphorins. (C, D) Quantitative analysis confirmed these observations, as the number and length of axons growing toward the aggregates were significantly reduced, compared with the outgrowth directed away. In the control experiments, the outgrowth from the side facing or opposed to the aggregates was equivalent. Statistical analysis was performed with ANOVA.* P ⬍ 0.001.

also observed to deflect the axons (Figs. 2B–D). Quantitative analysis of the number and length of fibers growing toward and away from the aggregates showed no statistical difference in the strength of the effects (Figs. 2C and D), indicating redundant functions of the two isoforms in axon chemorepulsion of hippocampal neurons. SEMA3F isoforms regulate the morphology of endothelial cells Another functional property assigned to semaphorins is the regulation of the cell morphology. For example, secreted semaphorins induce contraction of COS7 cells transfected to express functional receptors (Tamagnone and Comoglio, 2000). Interestingly, several recent studies have established

that endothelial cells are equipped with semaphorin receptors (Soker et al., 1998; Miao et al., 1999; reviewed by Neufeld et al., 2002), and Sema3A was observed to modulate the formation of endothelial cell protrusions (Miao et al., 1999). Such modifications of the cytoskeleton are thought to underlie changes in cell motility (Miao et al., 1999). We therefore used this latter model to investigate whether SEMA3F regulates endothelial cell morphology and also to determine whether the two isoforms are functionally equivalent. Endothelial cells derived from mouse forebrain were cultured for 24 h and then incubated for 1 h with Sema3A, SEMA3F, SEMA3F⌬, or control supernatants. To visualize morphological changes, cells were stained with Phalloidin–FITC that labels their actin cy-

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toskeletal organization. Two types of cytoskeletal extensions were scored in the quantitative analyses: the filopodial and lamellipodial elements (Fig. 3A, see Experimental Methods). Consistent with a previous report, Sema3A treatment resulted in a statistically significant 48.8% reduction in the number of lamellipodia. Similar effects were also seen for both SEMA3F isoforms when compared to control treatments (i.e., reduction by 53.2 and 60.5%, respectively, for SEMA3F and SEMA3F⌬, Fig. 3C). The disruption of lamellipodia was reflected by shrinkage of actin structures, an effect clearly visible with Phalloidin–FITC staining (Fig. 3B). Concomitant with the disruption of lamellipodia, we detected a twofold increase in the number of filopodial extensions (Fig. 3D). However, no statistical difference could be detected between the two SEMA3F isoforms in their efficiency to induce these dual morphological changes. These results indicate that SEMA3F, as previously observed with Sema3A (Miao et al., 1999), is able to modulate the cytoskeketal structures of endothelial cells. Although not statistically significant, we noticed a tendency for SEMA3F⌬ to exert more potent effects than SEMA3F. This is perhaps due to slight differences in the concentration of the proteins or their efficacy. The mechanism by which secreted semaphorins exert dual effects on the cell morphology, i.e., decreasing lamellipodia while increasing filopodia, was unclear. One possibility was that filopodia extension is a consequence of lamellipodia retraction. Such coupling has been described in the context of axon guidance with sprouting of lateral branches observed to occur following growth cone collapse (Davenport et al., 1999; Campbell et al., 2001). To test this idea, we examined in time-lapse video-microscopy the behavior of the cell protrusions following SEMA3F application. We observed that SEMA3F induced a reshaping of the cell surface (Fig. 4). In a range of time of 10 minutes, small lamellipodia almost totally retracted (Fig. 4A). Long lamellipodia became thinner, some of them then resembling filopodia (Fig. 4B). Spikes were also found to sprout from lamellipodia retraction, and became stabilized, as observed in recordings over 60 min (Fig. 4C). In contrast, the cell protrusions were remarkably stable under control conditions, as no obvious changing could be detected either in their shape or in their number (Fig. 4D). This analysis indicates that SEMA3F induces fast morphological changing of the endothelial cell protrusions. These reorganizations might reflect coordinated changes in the activity and subcellular localization of Rho-GTPases, a family of proteins that couples extracellular signals such as guidance cues to internal cytoskeletal dynamics (Dickson, 2001; Liu and Strittmatter, 2001). For instance, RhoA inhibition was shown to abolish growth cone collapse while increasing the length of induced lateral extensions, whereas Rac-1A or Cdc42 inhibition reduced both the number and length of these branches without affecting collapse (Thies and Davenport, 2003). Thus, changing the level of activation of a single Rho-GTPase could be the basis for the semaphorin-

induced dual effects. Which Rho-GTPase(s) is involved in the transduction of the SEMA3F signal is unclear, although it was shown in cancer cell lines that SEMA3F collapsing effects are accompanied by a relocalization of Rac-1 (Nasarre et al., 2003). Endothelial cells could provide an alternative model of neuronal growth cones for addressing this question. NP-2 but not NP-1 is required for the regulation of filopodial and lamellipodial extensions We asked whether the regulation of cell protrusions was triggered by activation of a receptor complex requiring NP-2, as was the case for the SEMA3F-induced axon chemorepulsion, or whether some of the observed effects could be mediated by NP-1 which also binds SEMA3F albeit with 10-fold less affinity than NP-2 (Chen et al., 1997). To address these questions, we blocked NP-1 with antibodies and NP-2 with a secreted NP-2-Fc chimeric construct, and examined the SEMA3F-induced changes in endothelial cell morphology. The endothelial cells endogenously expressed NP-1 and NP-2 at the cell membrane, as indicated by immunochemistry (Fig. 5A). Application of anti-NP-1 antibodies had no detectable effects on the number of extensions, indicating that NP-1 is dispensable for the two SEMA3F isoforms to regulate endothelial cell morphology (Figs. 5B and C). In contrast, NP-2-Fc significantly reduced the effects induced by SEMA3F isoforms on filopodial and lamellipodial protrusions (Figs. 5B and C), suggesting that both types of regulation require NP-2. Because NP-2-Fc might exert its effect by titration of the semaphorin ligand rather than by blocking the endogenous NP-2 receptor, we confirmed this conclusion by the use of anti-NP-2 antibodies that were found to almost totally prevent SEMA3F from regulating the lamellipodial and filopodial formation (Figs. 5B and C). From the initial finding that NP-1 is expressed by endothelial cells and is a receptor for vascular endothelial growth factor (VEGF), a potent mitogenic, chemotactic, and angiogenic signal, an increasing number of reports have described functions for semaphorins and their receptors during vasculogenesis and angiogenesis (reviewed by Neufeld et al., 2002). Consistent with NP-1 and NP-2 expression patterns (Herzog et al., 2001; Yuan et al., 2002), in vivo manipulations in zebrafish and loss-of-function studies in mice indicate that these ligand–receptor families regulate development of the cardiovascular system (Lee et al., 2002; Takashima et al., 2002; Yuan et al., 2002; Feiner et al., 2001). Interestingly, NP-2 expression is restricted to veins and lymphatic vessels, and phenotypic defects associated with NP-2 loss-of-function suggest that it is required for the formation of small vessels and capillaries (Yuan et al., 2002). Indeed, the proliferation of NP-2-/- lymphatic endothelial cells appeared to be decreased and some vessels were abnormally positioned, pointing to alterations in proliferative and cell migration events (Yuan et al., 2002). The

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present finding that SEMA3F modulates EC morphology through interaction with NP-2 therefore suggests that SEMA3F might participate in some of the events controlling the appropriate positioning of endothelial cells during lymphangiogenesis.

in the nervous system, particularly by addressing the possibility of cell-type specific expressions of the different mRNAs, should bring insights into the regulatory mechanisms of semaphorin expression in both physiological and pathological situations.

Distinct temporal regulation of Sema3F and Sema3F⌬ mRNAs in the mouse spinal cord

Experimental methods

Together, these experiments indicate that the two SEMA3F isoforms are functionally redundant, at least in the repertoire of responses examined here. Another possibility was that the two SEMA3F isoforms could exhibit expression differences in the nervous system. This was examined in the developing CNS by using quantitative real-time RTPCR. First, Sema3F mRNA was globally assessed at embryonic stage E12 where it was detected in the brainstem and spinal cord at 3.5 higher levels than in the telencephalon (Fig. 6A). Sema3F expression was then examined in several regions of the Postnatal Day 1 CNS. The mRNA was particularly more abundant in the cerebral cortex and striatum than in the hippocampus, cerebellum, or olfactory bulb. Finally, it was only moderately expressed in the rachidian bulb and spinal cord (Fig. 6B). Between P1 and P3, the global level of Sema3F expression sharply decreased in all regions of the forebrain and midbrain examined (Fig. 6C). These findings are consistent with previous studies that demonstrated a decrease of semaphorin expression concomitant with birth in many regions of the CNS with highly dynamic regulation occurring in a short range of time (Wright et al., 1995; Giger et al., 1996; Skaliora et al., 1998). Next, we examined whether specific temporal regulation could distinguish the two mRNA forms. To this end, the isoform ratio was determined during the exponential phase of PCR amplification at both P1 and P3 stages (Figs. 7A and B). In most regions, the two mRNA forms were detected with a ratio close to 1, indicating that they were present in similar proportion (Fig. 7C). However, the long isoform of Sema3F mRNA was expressed at a higher ratio at P1 and P3 in the brainstem and spinal cord, respectively (Fig. 7C). To further examine this difference, we measured the ratio in the embryonic spinal cord at a stage where high semaphorin expression was reported (Wright et al., 1995). In contrast to postnatal stages, at E12 both mRNAs were present at the same level in the spinal cord. Together, these findings indicate that Sema3F expression is down-regulated with maturation and particularly in regions of the central nervous system such as the spinal cord. This decrease is accompanied by an enrichment of the long Sema3F isoform. These results demonstrate that differential regulation of the mRNAs is a physiological feature of SEMA3F expression and is therefore not specific to cancer cells. Rather, differences in the ratio between the two forms might reflect heterogeneity in the cell population. Further investigation of the significance of the region and time-dependent regulation

Plasmid construction SEMA3F or SEMA3F⌬ was PCR-amplified (excluding the stop codon) and cloned into the EcoRI site of pEGFPN3 (Clontech) to yield SEMA3F-GFP and SEMA3F⌬-GFP, respectively. The forward and reverse primers were 5⬘ GCG AAT TCT CTT GTC GCC GGT CTT CTT 3⬘ and 5⬘ C GGA ATT CGA TGT GTC CGG AGG GTG GTG CCG 3⬘. The constructs fused GFP to the 3⬘ end of SEMA3F. Binding assays COS7 cells were transiently transfected using lipofection method with expression vectors encoding myc-tagged NP-1 or HA-tagged NP-2. After two days in vitro, cells were incubated with supernatants containing SEMA3F-GFP or SEMA3F⌬-GFP for 1 h at 37°C. Prior to the binding, the SEMA3F forms were preclustered with rabbit anti-GFP antibodies (1:20, InVitrogen) for 1 h at 37°C. Following incubation, the cells were fixed with 4% PFA and processed for immunofluorescence detection of the GFP with FITCconjugated anti-rabbit antibodies. After secondary antibody incubation, the cells were washed with PBS and mounted in Mowiol (Calbiochem). Coculture experiments The hippocampus of neonatal mice was removed and cut into 200-␮m-thick slices with a McIlwain tissue chopper. Explants containing the CA1 and CA3 regions were prepared from individual slices. COS-7 cells were transfected with the expression vector coding for SEMA3F-GFP or SEMA3F⌬-GFP, or with the vector alone, as a control, using a lipofection method (Lipofectamine, InVitrogen). The cells were aggregated and cocultured with hippocampal explants in a three-dimensional plasma clot for 24 to 48 h. Cultures were then fixed and analyzed with a phase-contrast microscope (Axiovert 35-M, Zeiss), as described (Castellani et al., 2000). A qualitative guidance index from ⫺2 to ⫹2 was given to the cocultures. Quantitative analysis was performed on the number and length of axons growing toward and away from the cell aggregates using a computer analysis software (Visiolab 2000, Biocom). For immunofluorescence labeling of axons, cultures were incubated first with the antiphosphorylated neurofilament antibody SM131 (1:1000, Sternberger and Meyer, Inc.), and second with Texas red-conjugated secondary antibody.

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Fig. 3. SEMA3F isoforms modulate the protrusions of endothelial cells. Endothelial cell cultures were incubated with Sema3A, SEMA3F isoforms, or control supernatants. The number of filopodial and lamellipodial extensions formed by the cells was determined in phase contrast or immunofluorescent observations of actin labeling with Phalloidin treatment. (A) The microphotographs illustrate the morphology of endothelial cells in control and SEMA3F-treated conditions. The arrows show the lamellipodia, and the asterisks the filopodia. Note that SEMA3F reduced the lamellipodia but increased the filopodia. (B) These effects were also clearly visible with Phalloidin–FITC staining showing that SEMA3F treatment induced a contraction of actin structures, as the labeling is packed in the cell membranes, a phenomenon that was accompanied by the formation of filopodia. (C, D) Histograms representing the proportion of protrusions normalized to the control situation. Sema3A and SEMA3F isoforms significantly reduced the lamellipodial protrusions, whereas they increased the number of filopodia formed by the cells. The slighly different proportion of extensions exhibited by cells treated with SEMA3F and SEMA3F⌬ was not statistically different.* P ⬍ 0.001. Scale bar: 50 ␮m.

Endothelial cell cultures Cells derived from mouse forebrain (bEnd3 cell line) were cultured in DMEM and fetal calf serum (FCS) 10% on poly-D-lysin-coated glass coverslips. The cultures were incubated for 1 h with Sema3A-AP, SEMA3F-GFP, SEMA3F⌬-GFP, or control supernatants diluted in serumfree OptiMEM medium (InVitrogen). In some experiments, anti-NP-2 antibodies (gift of A. Kolodkin), anti-NP-1 anti-

bodies (gift of Z. He), or NP-2-Fc chimera (gift of M. Tessier-Lavigne) were added in the SEMA3F-treated cultures. Cells were rapidly rinsed with OptiMEM, fixed with PFA 4%, and processed for immunocytochemistry. Cells were permeabilized with cold acetone for 30 s to 1 min, incubated with Phalloidin–FITC (1:1000) for 1 h, rinsed with PBS, and mounted in Mowiol. Analysis of the cell morphology was performed using an inverted phase-contrast microscope (Axiovert 35-M, Zeiss) according to the

Fig. 4. Time-lapse imaging showing the SEMA3F-induced reshaping of the endothelial cell protrusions. (A) The behavior of the lamellipodia was examined through recordings every 15 or 30 s. Images selected here show a retraction of a short lamellipodia. (B) In this sequence of 10 min, the large lamellipodia (left) became thinner, and the cytoplasmic content retracted from the right lamellipodia, which looked like a filopodia at the end of the recording. (C) Progressive collapse of a lamellipodia and the concomitant formation of a spike at the location of the retraction that became stabilized. (D) In control experiments, the cytomembranar extensions are stable, as illustrated the images selected from recordings over 60 min. Scale bar: 30 ␮m.

following criteria: 20 to 40 individual cells per coverslip were randomly selected and the number of extensions with filopodia and lamellipodia morphology was counted. The analysis was performed on three independent experiments. The proportion of extensions was normalized to the control condition. Phalloidin–FITC staining was examined with a confocal microscope (CARV, Zeiss). Statistical analysis was done with ANOVA test. For time-lapse video-microscopy, endothelial cells were treated with SEMA3F and viewed using phase-contrast microscopy (Axiovert 35-M, Zeiss) and were heated on the microscope stage at 37°C. Images were collected with a video camera (Cool View,

Fig. 5. NP-2 but not NP-1 is required by SEMA3F isoforms to modulate the morphology of endothelial cells. (A) Immunocytochemical labeling of NP-1 and NP-2 in endothelial cell cultures showing that both proteins are expressed by these cells. (B, C) The formation of lamellipodial and filopodial extensions was examined in conditions where function-blocking antibodies (anti-NP-1 antibodies), chimeric constructs antagonizing the endogenous receptors (NP2-Fc), or anti-NP-2 antibodies were applied combined with SEMA3F isoforms. As shown by the histograms, NP-2-Fc significantly reduced the effects exerted by SEMA3F and SEMA3F⌬. Application of anti-NP-2 antibodies confirmed the implication of this neuropilin, as it almost totally abrogated the modification of lamellipodial and filopodial structures induced by SEMA3F. In contrast, application of anti-NP-1 antibodies had no detectable consequence on the reduction of lamellipodia and increase of filopodia induced by SEMA3F isoforms. Thus, these modulatory effects are both mediated by NP-2 but not NP-1 in the semaphorin receptor complex. *P ⬍ 0.001 with ANOVA test; ns, not significant.

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and spinal cord). To isolate the striatum and the hippocampus, brains were cut into 800-␮m-thick slices with a tissue chopper and the regions dissected from the slices. Samples were recovered into RNAlater (Qiagen). Tissues from four mice were pooled for the experiment. Total RNA was prepared with the SV Total RNA isolation kit (Promega) and RT-PCR was performed with SuperscriptII reverse transcriptase (InVitrogen) using the procedure supplied by the manufacturer. Alternatively spliced 915- and 1008-bp fragments from the SEMA3F cDNA were amplified by PCR with the mouse-59H8 primer (5⬘-TTCAAC-TTT-CTG-CTC-AAC-3⬘) and the mouse-39G5 primer (5⬘-GAA-GAC-CAT-GCG-GAT-ATC-3⬘). Conditions of the PCR were standard buffer (Promega) with 1.5 mM MgCl2 using the following parameters; 20 or 25 cycles, 94°C for 1 min, 55°C for 1 min, 72°C for 1 min. Twelve microliters of a 1:100 dilution of the PCR reaction products was analyzed by Southern blot analysis with the SEMA3F probe labeled by random priming. Southern blot hybridizations were performed at 65°C using a nylon membrane (HYBOND-N⫹; Amersham Pharmacia Biotech) in the Rapid-hyb buffer (Amersham). Washings were performed two times for 15 min in 0.2⫻ SSC– 0.1% SDS. The signal was quantified by phosphorimaging. We assessed levels of global SEMA3F transcription, relative to GAPDH, by quantitative PCR carried out with

Fig. 6. Expression pattern of Sema3F in the mouse developing CNS. (A) The histogram depicts the amount of Sema3F isoforms contained in different territories of the CNS in the E12 mouse embryo. Sema3F was detected at high levels in the spinal cord and the brainstem, whereas expression was moderate in the telencephalon. (B) On the opposite at Postnatal Day 1 (P1), the highest expression level was observed in the telencephalon, and particularly in the cerebral cortex (Ctx) and the striatum (St). In the rachidian bulb (RB) and the spinal cord (SC), Sema3F expression is five fold lower than at embryonic stages. (C) Expression level sharply decreases at P3 in all regions examined.

Photonic Science) and digitized at intervals of 15 or 30 s using image processing software (Visiolab 2000, Biocom). The culture medium was recovered with mineral oil before microscope observation. Tissue analysis RT-PCR Tissues were collected from E12 embryos (telencephalon, trunk, and spinal cord) or P1 and P3 pups. For neonatal stages, the brain was removed, and different regions were isolated (olfactory bulb, rachidian bulb, cortex, cerebellum,

Fig. 7. Time- and region-specific regulation of Sema3F spliced forms. As illustrated by the presence of two bands in the electrophoresis, both Sema3F mRNAs were detected in the different regions of the postnatal CNS examined at P1 (A) and P3 (B), indicating a lack of regional specificity in the alternative splicing of sema3F mRNA. A ratio of 1 between the two Sema3F isoforms is found in most regions examined, except for the rachidian bulb at P1 and the spinal cord at P3 for which a 3 times enrichment of the longest form was found (C). This indicates that Sema3F mRNA synthesis is regulated during development and in a region-specific manner in the CNS. pBS␭5 and 3.6 are the controls for the shortest and the longest forms of Sema3F cDNA, respectively.

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the GeneAmp7000 (PE Biosystems) quantitative PCR system with SYBR Green chemistry. The cycle at which a particular sample reaches an arbitrary threshold fluorescent level (Ct) during exponential amplification is indicative of input quantity of the template. To adjust for variations in the amount of RNA, the Ct values for SEMA3F were normalized against the Ct values for the housekeeping gene GAPDH (⌬Ct ⫽ Ctsema3F ⫺ CtGAPDH). The results are displayed in terms of the relative expression (⫻1000) compared to GAPDH. The SEMA3F-7/21f primer 5⬘-AGC-AGA-CCC-AGGACA-TCA-G-3⬘ and the SEMA3F-7/21r primer 5⬘-AAGACC-ATG-CGG-ATA-TCA-GCC-3⬘ were used to amplify SEMA3F cDNA, giving a product of 114 bp. GAPDH cDNA was amplified with primers GAPDH For 5⬘-TGCACC-ACC-AAC-TGC-TTA-GC-3⬘ and GAPDH Rev 5⬘GGC-ATG-GAC-TGT-GGT-CAT-GAG-3⬘, giving an 87-bp product. The PCR was carried out in 20 ␮l consisting of 1⫻ PCR SYBR Green buffer, 0.125 ␮M primers, 200 ␮M each dNTP, and 0.025 units/␮l AmpliTaq Gold (PE). cDNA was amplified as follows: 50°C for 2 min, 95°C for 10 min followed by 35 cycles at 95°C ⫻ 15 s, 60°C ⫻ 1 min. Acknowledgments This work was supported by the CNRS for V.C. and G.R., the Ligue Nationale Contre le Cancer and ARC for S.K. and J.R., and NIH SPORE (CA58187) for H.D. We thank M. Tessier-Lavigne for the gift of NP-2-Fc construct, Z. He and A. Kolodkin for anti-neuropilin antibodies. We are grateful to P. Nasarre and V. Coronas for mouse brain dissection. References Bismuth, G., Boumsell, L., 2002. Controlling the immune system through semaphorins. Sci. STKE 128, RE4. Brambilla, E., Constantin, B., Drabkin, H., Roche, J., 2000. Semaphorin SEMA3F localization in malignant human lung and cell lines: a suggested role in cell adhesion and cell migration. Am. J. Pathol. 156, 939 –950. Brown, C.B., Feiner, L., Lu, M.M., Li, J., Ma, X., Webber, A.L., Jia, L., Raper, J.A., Epstein, J.A., 2001. PlexinA2 and semaphorin signaling during cardiac neural crest development. Development 128, 3071– 3080. Campbell, D.S., Regan, A.G., Lopez, J.S., Tannahill, D., Harris, W.A., Holt, C.E., 2001. Semaphorin 3A elicits stage-dependent collapse, turning, and branching in Xenopus retinal growth cones. J. Neurosci. 21, 8538 – 8547. Castellani, V., Che´ dotal, A., Schachner, M., Faivre-Sarrailh, C., Rougon, G., 2000. Analysis of the L1-deficient mouse phenotype reveals crosstalk between Sema3A and L1 signaling pathways in axonal guidance. Neuron 27, 237–249. Castellani, V., Rougon, G., 2002. Control of semaphorin signaling. Curr. Opin. Neurobiol. 12, 532–541. Che´ dotal, A., Del Rio, J.A., Ruiz, M., He, Z., Borrell, V., de Castro, F., Ezan, F., Goodman, C.S., Tessier-Lavigne, M., Sotelo, C., Soriano, E.,

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