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¹ Department of Biochemistry and Molecular Biology and ² Department of Molecular Neurobiology and Pharmacology, Faculty of Medicine, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
³ Department of Anatomy, School of Medicine, Fukushima Medical University, Hikarigaoka, Fukushima 960-1295, Japan
⁴ Department of Anatomy, Hokkaido University School of Medicine, Sapporo, Hokkaido 060-8638, Japan

	Abstract

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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.

	Introduction

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During embryogenesis, axons reach their specific targets correctly to form the complex neural network found in the mature functional nervous system. Several groups of axon guidance molecules such as semaphorins, ephrins, netrins, and Slits have been reported to repel or attract growing axons that express their cognate receptors (Chisholm & Tessier-Lavigne 1999).

Semaphorins are secreted type or transmembrane type proteins with a conserved domain of about 500 amino acids (aa), sema domain, and found in both vertebrates and invertebrates (Nakamura et al. 2000; Raper 2000). So far, more than 20 kinds of semaphorin genes have been identified and classified into seven classes and a virus semaphorin (Semaphorin Nomenclature Committee 1999). Among them, semaphorin 3A (Sema3A) is the first identified semaphorin in vertebrates on the basis of its ability to induce the collapse of axonal growth cones of the dorsal root ganglion (DRG) (Luo et al. 1993). We have developed Sema3A-deficient mice, and they showed a severe abnormality in the axonal projection pattern in the peripheral nervous system during embryogenesis (Taniguchi et al. 1997), and also showed the distortion of the odor maps in the olfactory bulb (Taniguchi et al. 2003). Neuropilins (NPs) are functional receptors for class 3 semaphorins and plexin-As are co-receptors for class 3 semaphorins (Chen et al. 1997, 2000; He & Tessier-Lavigne 1997; Kitsukawa et al. 1997; Kolodkin et al. 1997; Giger et al. 1998, 2000; Takahashi et al. 1999; Cheng et al. 2001; Sasaki et al. 2002). Plexins are also known as receptors for other types of semaphorins (Winberg et al. 1998; Tamagnone et al. 1999). Semaphorin 4D/CD100 functions in the immune system and semaphorin 3C (Sema3C) functions during cardiac development (Shi et al. 2000; Feiner et al. 2001). Thus, semaphorin family genes perform a variety of important biological functions besides the axon guidance.

We cloned a novel secreted type of mouse semaphorin gene and termed semaphorin 3G (Sema3G). The expression pattern and functional activities of Sema3G suggest its important roles in the brain development and functions.

	Results

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Cloning of human and mouse semaphorin 3G genes

We identified one novel human secreted semaphorin cDNA, FLJ00014 Sequence analysis revealed that FLJ00014contained a predictable partial ORF and was a novel member of class 3 semaphorins. It encodes a 725 amino acids protein, although it did not contain the full length ORF. Then, mouse semaphorin was cloned, and termed semaphorin 3G (Sema3G) according to the rule of Semaphorin Nomenclature Committee (Semaphorin Nomenclature Committee 1999). The length of Sema3G cDNA was 3686 bp and the ORF is 2340 bp (780 amino acids) with a predicted molecular weight of about 87 kDa. Mouse Sema3G protein showed an 86.3% identity with human SEMA3G protein (encoded by FLJ00014, and similarity to other class 3 semaphorins was as follows: Sema3A (46.0% identity) and Sema3F (44.5% identity). Sema3G cDNA consists of at least 16 exons and is mapped on chromosome 14.

Expression pattern of Sema3G

To clarify the regional expression of Sema3G, Northern blot hybridization was performed (Fig. 1). Sema3G transcript was about 4.5 kb in size, which was comparable with the size of the cloned Sema3G cDNA. In the adult tissues, it was expressed predominantly in the lung and kidney, moderately in the heart and placenta, and only slightly in the brain (Fig. 1A). We then analyzed its regional distribution in the adult rat brain (Fig. 1B). Notably, Sema3G was expressed only in the cerebellum. To further study the expression patterns in the rat brain, in situ hybridization was performed (Fig. 2). Although strong Sema3G signals were detected in the dorsal root ganglion (DRG) and kidney at E17, few signals were found in the brain (Fig. 2A). In the central nervous system, Sema3G signals appeared in the olfactory bulb and cerebellum at P2 (Fig. 2B). Its signals in the olfactory bulb disappeared at P10 (Fig. 2C), whereas the signals in the cerebellum increased toward P20 (Fig. 2D) and adult (data not shown). The strong Sema3G signals in the cerebellum at P10 were present in the meningeal cell layer attaching the cerebellum, whereas moderate and weak Sema3G signals were detected in the differentiating external granular and internal granular layers, respectively (Fig. 2E). Moreover, few Sema3G signals at P10 were detected in the mitotic external granular layer (Fig. 2E). At P20, the expression of Sema3G was restricted to the granular layer in the cerebellum (Fig. 2D,F). The expression pattern of Sema3G suggests that the cerebellar granule cells express Sema3G from the differentiating and migrating stages of the granule cells through the adult stage.

View larger version (63K): [in this window] [in a new window]   Figure 1  Analysis of Sema3G expression by Northern blotting. Each lane contained 20 µg of total RNA. (A) The distribution of Sema3G in adult mouse tissues. Sema3G was expressed predominantly in the lung, and a little in the brain. (B) Regional distribution of Sema3G in adult rat brain. Sema3G was expressed highly in the cerebellum. The lines show the location of murine 28S and 18S ribosomal RNAs. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression patterns are also shown as references.

View larger version (165K): [in this window] [in a new window]   Figure 2  Sema3G expression pattern by in situ hybridization. (A–D) X-ray film macroautoradiographs of parasagittal sections showed the distribution of Sema3G mRNA in rat brains at E17 (A), P2 (B), P10 (C), and P20 (D). (A) Few Sema3G signals were detected in the nervous system at E17, except for the DRG. (D) Sema3G expression at P20 was restricted to the granular layer in the cerebellum. (E, F) Bright-field micrographs of the cerebellar cortex at P10 (E) and P20 (F). Note strong Sema3G signals in the meningeal cell layer (MCL) at P10 (E) and granular layer (GL) at P20 (F). Asterisks indicate Purkinje cell perikarya. Cb, cerebellum; Cx, cerebral cortex; DRG, dorsal root ganglion; dEGL, differentiating external granular layer; GL, granular layer; IGL, internal granular layer; Ki, kidney; Mb, mibdrain; mEGL, mitotic external granular layer; ML, molecular layer; MO, medulla oblongata; OB, olfactory bulb. Scale bars: 1 mm (A–D); 50 µm (E, F).

NPs are known to bind class 3 semaphorins (Nakamura et al. 2000). It is reported that NP-1 is a functional Sema3A receptor, and NP-2 is a functional Sema3F receptor (Chen et al. 1997, 2000; He & Tessier-Lavigne 1997; Kitsukawa et al. 1997; Kolodkin et al. 1997; Giger et al. 1998, 2000). To clarify whether or not newly identified Sema3G binds NPs, Sema3G-alkaline phosphatase (AP) fusion protein was reacted with COS-7 cells expressing either NP-1 or NP-2 (Fig. 3). Sema3G bound NP-2-expressing cells, but not mock-transfected or NP-1-expressing cells (Fig. 3A,B,D). Sema3A-AP fusion protein bound NP-1-expressing cells as reported previously (Fig. 3C). Western blot analysis confirmed that the cells used in this study (Fig. 3B–D) expressed NPs (data not shown). We estimated the binding affinity of Sema3G-AP fusion proteins to cells expressing NP-2 in equilibrium binding experiments. The estimated Kd was 58 ± 17 pM (SD; n = 6).

View larger version (135K): [in this window] [in a new window]   Figure 3  Sema3G binds NP-2, but not NP-1. The supernatants from HEK 293-T cells transfected with p3G-AP or p3A-AP were reacted with COS-7 cells expressing NPs. (A, B) Sema3G-AP fusion protein did not bind mock-transfected COS-7 cells (A) and NP-1-expressing cells (B). (C) Sema3A-AP fusion protein bound NP-1-expressing cells. (D) Sema3G-AP fusion protein bound NP-2-expressing cells.

Next, to study whether newly identified Sema3G exhibits a significant biological activity, we performed a repulsive assay with DRG and sympathetic neurons (Fig. 4). Previous studies have revealed that NP-2 is highly expressed in sympathetic neurons (Chen et al. 1997). On the other hand, the expression of NP-2 is absent in mouse DRG neurons at later stages (E15.5; Chen et al. 1997). E15.5 mouse DRG and E10 chick or P2 mouse sympathetic explants were cocultured with Sema3G-, Sema3A-, or mock-transfected HEK 293-T cell aggregates in a collagen gel (Fig. 4A–F). For quantitative analyses, DRG and sympathetic axonal lengths in the proximal quadrant (p), toward the cell aggregates, was compared to those in the distal quadrant (d), away from cells. The axonal outgrowth ratio p/d-value is a measure of the repulsive activity, with ratios of 0 and 1 indicating complete and no repulsion, respectively. Aggregates of control HEK 293-T cells (mock-transfected cells) cocultured with DRG and sympathetic explants did not chemorepel these axons (Fig. 4G). However, cells secreting Sema3G chemorepelled mouse and chick sympathetic axons, but not mouse DRG axons (Fig. 4A–C,G). On the other hand, cells secreting Sema3A chemorepelled both DRG and sympathetic axons (Fig. 4D–G).

View larger version (36K): [in this window] [in a new window]   Figure 4  Sympathetic axons, but not DRG axons, are repelled by Sema3G. (A–F) Sema3G- (left sides of each panel), Sema3A- (right sides of each panel), or mock-transfected HEK 293-T cell aggregates were cocultured with mouse SCG explants (mSCG), chick SYM explants (cSYM), and mouse DRG explants (mDRG). (A–C) Cells secreting Sema3G chemorepelled mouse (A) and chick (B) sympathetic axons, but not mouse DRG axons (C). (D–F) Both sympathetic and DRG axons were chemorepelled by cells secreting Sema3A. Scale bars: 200 µm. (G) The quantification of chemorepulsive activities of Sema3G and Sema3A. The axonal growth ratio p/d is a measure of repulsive activity, with the ratios of 0 and 1 indicating complete and no repulsion, respectively. Mouse Sema3G repelled sympathetic axons (mSCG and cSYM). The bars represent mean ± SEM and the number of cocultures is shown in the bar.

	Discussion

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Sema3G was also expressed in the heart and placenta. Sema3A and Sema3C function during cardiac development (Behar et al. 1996; Feiner et al. 2001). Class 3 semaphorins control vascular morphogenesis (Serini et al. 2003). In fact, NP-1 and NP-2 are class 3 semaphorin receptors and also a co-receptor for vascular endothelial growth factor (VEGF) (Soker et al. 1998; Gluzman-Poltorak et al. 2000), functioning in vasculogenesis (Kawasaki et al. 1999; Yuan et al. 2002). The double NP-1/NP-2-deficient mice were killed at E8.5 and showed more severe abnormal vascular phenotypes than single deficient mice of NP-1 or NP-2 (Takashima et al. 2002). Plexin-A2 is a component of a Sema3A receptor complex with NP-1 (Takahashi et al. 1999; Sasaki et al. 2002) and may function during cardiac development (Brown et al. 2001). Semaphorins and their respective receptors play roles in vasculogenesis and angiogenesis besides the axon guidance. Human NP-2 is expressed in the heart and placenta (Rossignol et al. 2000). Thus, it is intriguing to determine if Sema3G and NP-2 may function in vasculogenesis and angiogenesis of the heart and placenta.

A number of chemorepulsive molecules including semaphorins play an important role in the axon guidance in the central and peripheral nervous systems (reviewed in Masuda & Shiga 2005). We found that Sema3G can bind NP-2, but not NP-1 (Fig. 3). Consistently, Sema3G exhibits chemorepellent activity for sympathetic axons expressing NP-1 and NP-2, but not DRG axons at later stages which express NP-1 alone (Fig. 4; Chen et al. 1997). Although further studies are needed, these results suggest that Sema3G may utilize NP-2 as a receptor to exert chemorepulsion for sympathetic axons. The expression pattern of Sema3G in the brain is striking. Sema3G expression level in the cerebellum increased from P2 to P20 and its expression at P20 was detected only in the granular layer of the cerebellum (Figs 1, 2). At P10, the strong Sema3G signals were detected in the meningeal cell layer attaching the cerebellum and the weak signals in the migrating granule cells and internal granular layer (Fig. 2E). After the granule cells migrated to the granular layer, Sema3G signals were detected highly in the granular layer (Fig. 2F). Lack of ß1-class integrins in neurons and glia showed defectiveness of the development of cerebellar folia and the meninges in the mutants did not penetrate into the folia (Graus-Porta et al. 2001). Then, a large number of the granule cells formed ectopia along the fusion lines of adjacent folia and at the cerebellar surface underlying the meninges. The meningeal cells express extracellular matrix components and that are important for the assembly of the meningeal basement membrane (Sievers et al. 1994). Fusion of cerebellar folia are likely caused by defects in the basement membranes, which results in lack of expansion of the overlying meningeal cell layer into the folia. These results indicate that the meningeal cell layer may function in the migration of the granule cells. Based on the fact that the cerebellum and cultured cerebellar granule neurons express NP-2 (Chen et al. 1997; Moreno-Flores et al. 2003), our results suggest that Sema3G may play important roles in the granule cell differentiation, migration and the axon guidance of the granular layer of the cerebellum via NP-2.

In summary, we identified and characterized a novel semaphorin gene, Sema3G. We showed that Sema3G has unique expression patterns and chemorepulsive activities for sympathetic axons. Analyses of Sema3G-deficient mice would provide further understanding of the in vivo functions.

	Experimental procedures

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Identification and cloning of semaphorin 3G

We searched human EST databases and identified a novel human semaphorin cDNA, FLJ00014 which is a member of class 3 semaphorins and thus was named semaphorin 3G (SEMA3G) according to the rule of Semaphorin Nomenclature Committee (Semaphorin Nomenclature Committee 1999). To clone the mouse semaphorin 3G (Sema3G) cDNA, adult mouse (C57BL/6) brain cDNA library was screened with a 2.4 kb NheI fragment of human SEMA3G cDNA as a probe. Hybridization was performed in 6x SSC, 5x Denhardt solution, 0.2% SDS and 100 µg/mL herring sperm DNA at 55 °C overnight. The filters were washed with 2x SSC-0.2% SDS four times at 55 °C. The positive clones were sequenced with an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems). The accession number of Sema3G cDNA is deposited as AB127607.

Northern blot hybridization

Northern blot membranes were purchased from Seegene, Inc. Hybridization was performed in 50% formamide, 6x SSC, 5x Denhardt solution, 0.2% SDS and 100 µg/mL herring sperm DNA at 42 °C overnight. The filters were washed with 2x SSC-0.2% SDS three times and 0.2x SSC-0.2% SDS once at 65 °C. A 1.6 kb NcoI fragment of Sema3G cDNA was used as a probe.

In situ hybridization

In situ hybridization analysis was performed using specific oligonucleotide probes for rat Sema3G. Rat Sema3G cDNA was cloned by the suppression subtractive hybridization method as a cerebellum-specific gene (H. Kataoka, H. Mori and M. Mishina, unpublished observation, accession number AB190259). The sequences of synthetic oligonucleotide probes were 5'-aggggctgggttgccaccttctgtcttctcagggtctcctccacc-3' or 5'-taccaagtcttgggtgaggcagaagccacacttcgccaggctgag-3'. The brains were obtained from Wistar rats at embryonic day (E) 17, postnatal day (P) 2, P10, P20 and adult under the deep pentobarbital anesthesia and were frozen in powdered dry ice. The parasagittal brain sections (20 µm) were prepared using the cryostat (LEICA, CM1900) and mounted on the glass slides precoated with 3-aminopropyltriethoxysilane. The sections were treated at room temperature with following incubations; fixation with 4% paraformaldehyde for 10 min, and 2 mg/mL glycine in phosphate-buffered saline (PBS) for 10 min, acetylation with 0.25% acetic anhydride in 0.1 M triethanolamine-HCl (pH 8.0) for 10 min, and prehybridization for 1 h in a hybridization buffer (50% formamide, 50 mM Tris-HCl (pH 7.5), 0.02% Ficoll, 0.02% polyvinylpyrrolidone, 0.02% bovine serum albumin (BSA), 0.6 M NaCl, 0.25% SDS, 200 µg/mL tRNA, 1 mM EDTA, and 10% dextran sulfate). Hybridization was performed at 42 °C for 10 h in the hybridization buffer supplemented with 10 000 cpm/µL of ³³P-labeled oligonucleotide probes. The slides were washed twice at 55 °C for 40 min in 0.1x SSC containing 0.1% sarcosyl. Sections were exposed to BioMax films (Kodak) for three weeks or to nuclear track emulsion (NTB-2, Kodak) for five weeks. Emulsion-dipped sections were counterstained lightly with methyl green pyronine solution for bright-field microscopy. Specificity of the signals was confirmed by blank signals in the presence of excess unlabeled oligonucleotides.

Sema3G-alkaline phosphatase protein binding assay

The ORF of Sema3G cDNA (28–758 amino acids) was inserted in SfiI and HindIII sites of pAPtag-5 expression vector containing alkaline phosphatase (AP) gene (GenHunter Corp.) (p3G-AP). Like the creation of p3G-AP, p3A-AP vector was also created with the ORF of Sema3A and pAPtag-5 vector. The expression vectors of NPs were transfected into COS-7 cells with Lipofectamine Plus (Invitrogen Corp.). After 2 days, the cells were seeded into 6-well plates. Next day the supernatants from HEK 293-T cells tranfected with p3G-AP or p3A-AP were reacted with COS-7 cells expressing NPs for 1 h at room temperature. After washing with Hanks’ balanced salt solution (HBSS) with 0.05% BSA, 0.1% sodium azide and 20 mM HEPES (pH 7.0) four times, the cells were fixed with 4% paraformaldehyde solution for 2 min at room temperature. After washing with PBS, the cells were incubated for 1 h at 65 °C to inactivate endogenous AP. They were developed with NBT/BCIP in 100 mM Tris-HCl (pH 9.5), 100 mM NaCl and 5 mM MgCl₂. Equilibrium dissociation constants (Kd) of NP-2 with Sema3G-AP fusion proteins were measured as described (Cheng & Flanagan 1994).

Collagen gel cultures

HEK 293-T cells were transfected with p3G-AP or p3A-AP using Lipofectamine 2000 reagent (Invitrogen Corp.). The cell aggregates were prepared by the hanging drop method as previously described (Kennedy et al. 1994). DRG explants and superior cervical ganglion (SCG) explants were dissected from E15.5 mouse embryos and P2 mouse pups, respectively. Sympathetic ganglion (SYM) explants were dissected from stage 36 (E10) (Hamburger & Hamilton 1951) chick embryos. These explants were embedded in a collagen gel approximately 300 or 900 µm distant from the aggregates of the cells transfected with p3G-AP or p3A-AP. Cultures were incubated at 37 °C for 24–36 h in DMEM containing 50 ng/mL 7S nerve growth factor (NGF) (Chemicon). Cultures were fixed for several days with 4% paraformaldehyde. Whole-mount immunohistochemistry of cultures and analyses of repulsive activities were done as described (Masuda et al. 2003).

	Acknowledgements

 
We are grateful to Drs. T. Nagase (Kazusa DNA Res. Institute), H. Fujisawa (University Nagoya) and M. Tessier-Lavigne (Genentech Inc.) for gifts of FLJ00014cDNA, NP-1 cDNA and NP-2 expression vector, respectively, and H. Usui, M. Kimura, N. Murakami (University Tokyo) and F. Nakamura (University Yokohama City) for assistance. This work was supported by Grants-in-Aids from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan (M.T.).

	Footnotes

 
Communicated by: Hideyuki Okano

^* Correspondence: E-mail: taniguti{at}m.u-tokyo.ac.jp

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Received: 6 January 2005
Accepted: 25 April 2005

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