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Identification and characterization of a novel zebrafish semaphorin
Neuroscience Letters 488 (2011) 215–220
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Identification and characterization of a novel zebrafish semaphorin Masahiko Taniguchi a,∗ , Tomoyuki Masuda b , Yoshinori Mikami c , Masafumi Kimura d , Tomoyuki Yoshida c , Masayoshi Mishina c , Takao Shimizu d a
Department of Biochemistry, Cancer Research Institute, Sapporo Medical University School of Medicine, S-1 W-17, Chuo-ku, Sapporo, Hokkaido 060-8556, Japan Department of Anatomy, Fukushima Medical University School of Medicine, Fukushima 960-1295, Japan c Department of Molecular Neurobiology and Pharmacology, Faculty of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan d Department of Biochemistry and Molecular Biology, Faculty of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan b
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Article history: Received 31 August 2010 Received in revised form 12 November 2010 Accepted 12 November 2010 Keywords: Semaphorin Plexin Axon guidance Brain Lens Repellent
a b s t r a c t The semaphorin gene family contains numerous secreted and transmembrane proteins. Some of them function as the repulsive and attractive axon guidance molecules during development. Herein, we report the cloning and characterization of a novel member of zebrafish semaphorin gene, semaphorin 6E (sema6E). Sema6E is expressed predominantly in the nervous system during embryogenesis. Results also show that Sema6E binds Plexin-A1, but not other Plexins. Sema6E chemorepels not only dorsal root ganglion axons but also sympathetic axons. Therefore, Sema6E might utilize Plexin-A1 as a receptor to repel axons of the specific types during development. © 2010 Elsevier Ireland Ltd. All rights reserved.
During embryogenesis, axons reach their specific targets correctly to form the complex neural network found in a mature functional nervous system. Several groups of axon guidance molecules such as semaphorins, ephrins, netrins, and slits have been reported to repel or attract the growing axons that express their cognate receptors [6]. Semaphorins are secreted and transmembrane proteins containing a conserved domain (Sema domain) of about 500 amino acids and found in both vertebrates and invertebrates [23,30]. To date, semaphorin genes of more than 20 kinds have been identified and classified into seven classes and a virus semaphorin [31]. Among them, semaphorin 3A (Sema3A) is the first identified semaphorin in vertebrates based on its ability to induce the collapse of growth cones of dorsal root ganglion (DRG) axons [18]. Sema3A-deficient mice showed a severe abnormality in the patterns of the axonal projection in the peripheral nervous system during embryogenesis [38]. Neuropilins are functional receptors for class 3 semaphorins and Plexins are also known as receptors
Abbreviations: aa, amino acids; AP, alkaline phosphatase; DRG, dorsal root ganglion; hpf, hours post fertilization; Kd, dissociation constants; ORF, open reading frame; PFA, paraformaldehyde; RACE, rapid amplification of cDNA ends; RGC, retinal ganglion cell; SEM, standard error of the mean; Sema, semaphorin. ∗ Corresponding author. Tel.: +81 11 611 2111; fax: +81 11 611 2299. E-mail address: [email protected] (M. Taniguchi). 0304-3940/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2010.11.032
for semaphorins of other types [28,42]. Semaphorins have functions in many important biological processes in addition to the development of the nervous system, including angiogenesis, vasculogenesis, tumor progression, osteogenesis and immune regulation [3,10,24,32,34]. Zebrafish (Danio rerio) is an excellent model organism to investigate the development and function of the vertebrate nervous system because transparent embryos and Morpholino Oligo techniques facilitate in vivo function analyses of the interesting genes. Here, we report the cloning and tissue distribution of zebrafish sema6E: a novel member of class 6 semaphorins. Results show that Sema6E repels the axons of DRG and sympathetic neurons. The zebrafish AB strain was used. The zebrafish was raised and kept under standard conditions at 28 ◦ C [43]. We searched the zebrafish genomic DNA database (http://www.ensembl.org/Danio rerio/index.html) and identified a novel zebrafish semaphorin cDNA, which is a member of class 6 semaphorins. To clone the novel zebrafish cDNA, we performed PCR on the adult zebrafish brain cDNA library using 5 -ATGCTGTGTGGCTGAATTCTATGC-3 and 5 CCGCTCGAGTCAGTAGTTGAACTTCCCTGCCT-3 primers. The PCR fragments digested by EcoRI and XhoI were inserted in EcoRI and XhoI sites of pBluescript II SK (−) (Agilent Technologies Inc., Santa Clara, CA). The clones were sequenced using an ABI PRISM 3100 Genetic Analyzer (Life Technologies, Carlsbad, CA). Then 5 -RACE methods were performed for the 5 non-coding sequence on the
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Fig. 1. Alignment of zebrafish Sema6E and Sema6D proteins.
M. Taniguchi et al. / Neuroscience Letters 488 (2011) 215–220
zebrafish 72 h post fertilization (hpf) cDNA using a SMART RACE cDNA Amplification Kit (Clontech Laboratories, Inc., Mountain View, CA). Consequently, a 3295 bp cDNA containing the open reading frame (ORF) was obtained. The ORF is 3192 bp (1064 aa). The accession number of sema6E is deposited as AB185147. In situ hybridization was done as described in an earlier report [15]. Briefly, embryos were fixed in 4% paraformaldehyde (PFA) at 4 ◦ C overnight. The 36, 48 and 72 hpf embryos were treated with 10 g/ml proteinase K. Hybridization was performed at 55 ◦ C for 12–16 h. For RNA probe of sema6E, the 799 bp fragment (2497–3295) of sema6E amplified using PCR was cloned in pCR II (Invitrogen Corp., Carlsbad, CA). The DIG-labeled sense and antisense RNA probes for zebrafish sem6E gene were synthesized using a DIG RNA labeling Kit (F. Hoffmann-La Roche Ltd., Basel, Switzerland). The embryos were reacted with anti-DIG antibody
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conjugated alkaline phosphatase (AP) (F. Hoffmann-La Roche Ltd.) and developed using BCIP/NBT solution (Sigma–Aldrich Corp., St. Louis, MO). The ORF of zebrafish sema6E cDNA (33–654 aa) was inserted in SfiI and BglII sites of pAPtag-5 expression vector containing AP gene (GenHunter Corp., Nashville, TN) (pZ6E-AP). The expression vectors of Plexin-As were transfected into COS-7 cells with Lipofectamine Plus (Invitrogen Corp.). The binding assay was performed as described previously [39]. Equilibrium dissociation constants (Kd) of Plexin-A1 with Sema6E–AP fusion proteins were measured as described [5]. The HEK 293-T cells were transfected with pZ6E-AP using Lipofectamine 2000 reagent (Invitrogen Corp.). The cell aggregates were prepared using the hanging drop method as described previously [14]. Then DRG explants were dissected from stage 26
Fig. 2. Sema6E expression pattern by in situ hybridization with the antisense riboprobe (A, C, E, G, H, J–L) and sense riboprobe (B, D, F, I, and M). A–D are from 24 hpf zebrafish, E–G are 36 hpf; H–J are 48 hpf; and K–M are 72 hpf. (A and C) Dorsal (A) and ventral (C) views of 24 hpf embryos showing that sema6E was expressed in the lens. (E and G) Lateral (E) and ventral (G) views of 36 hpf embryos showing that sema6E expression was detected in the lens and telencephalon. (H and J) Lateral (H) and dorsal (J) views of 48 hpf embryos showing that sema6E was detected in the lens. (K and L) Dorsal (K) and lateral (L) views of 72 hpf, showing expression in the diencephalon and rhombencephalon.
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chick embryos [13]. Sympathetic ganglion explants were dissected from stage 37 chick embryos. These explants were embedded in a collagen gel approximately 300–400 m distant from the aggregates of 293-T cells transfected with empty vector (mock) or pZ6E-AP. Cultures were incubated at 37 ◦ C for 24–36 h in DMEM containing 50 ng/ml 7S nerve growth factor (Chemicon International Inc., Temecula, CA). Cultures were fixed for several days with 4% PFA. Whole-mount immunohistochemistry of cultures and analyses of repulsive activities were performed as described [20]. Recently, we identified the zebrafish semaphorin 6D [15]. Regarding the cloning process by searching the zebrafish DNA database, we identified a novel zebrafish semaphorin cDNA. To obtain the novel zebrafish semaphorin cDNA, we performed PCR on the adult zebrafish brain cDNA library. The length of a novel semaphorin cDNA was 3295 bp and the ORF is 3192 bp (1064 aa) with predicted molecular weight of about 118 kDa. The predictable protein of a novel zebrafish semaphorin was most similar to that of mouse sema6D in mammalian semaphorin. It showed a 45.5% identity with mouse Sema6D. A novel zebrafish semaphorin protein showed approximately 40% identity with other mammalian class 6 semaphorins. Sema4E has no known orthologues identified in other species [12,41]. Sema4E protein shows approximately 40% identity with mammalian class 4 semaphorins. Zebrafish semphorin proteins of mammalian orthologues show approximately 70% identity. For example, zebrafish Sema6D protein shows a 70.8% identity with mouse Sema6D protein. Therefore, we considered that the novel cloned cDNA is not an orthologue of mammalian class 6 semaphorins, and designated it as semaphorin 6E (sema6E). Results show that Sema6E has 45.0% identity with zebrafish Sema6D (Fig. 1). Therefore, these genes are not paralogs. Comparison between sema6E cDNA and zebrafish genomic DNA sequences revealed that sema6E gene comprises at least 19 exons and spans about 150 kb of the genomic DNA.
To clarify the distribution of sema6E, in situ hybridization was performed (Fig. 2). At 12 hpf, no sema6E expression signal was detected (data not shown). At 24 hpf, sema6E expression signals were detected in the lens, diencephalon, and telencephalon (Fig. 2A and C). At 36 hpf, strong hybridization signals for sema6E were detected in the lens and weak signals were detected in the telencephalon, diencephalon, and hindbrain (Fig. 2E and G). Results show that Sema6E had been expressed in the lens, telencephalon, diencephalons, mesencephalon, and rhombencephalon by 48 hpf (Fig. 2H and J). Similarly, sema6E was found to be expressed in the telencephalon, diencephalons, mesencephalon, and rhombencephalon by 72 hpf and its expression of the lens was decreased (Fig. 2K and L). Plexins are known to be semaphorin receptors [28,42]. PlexinAs are class 6 semaphorin receptors [42]. For example, Plexin-A1 is a Sema6D receptor [15,40]. To clarify whether or not zebrafish Sema6E binds Plexin-As, Sema6E–AP fusion protein was reacted with COS-7 cells expressing various Plexin-As. Sema6E bound Plexin-A1-expressing cells, but not mock-transfected, Plexin-A3, or Plexin-A4-expressing cells (Fig. 3). Western blot analyses confirmed that the cells used in this study expressed Plexin-As (data not shown). We also performed binding assay using mouse Plexins of seven kinds (Plexin-A1–4, -B1, -C and -D). Zebrafish Sema6E bound mouse Plexin-A1 only (data not shown). We estimated the binding affinity of Sema6E–AP fusion proteins to cells expressing Plexin-A1 in equilibrium binding experiments. The estimated Kd was 6.2 ± 2.9 nM (SD; n = 6). To study whether Sema6E exhibits a significant biological activity, we performed a repulsive assay with chick DRG and sympathetic neurons (Fig. 4). Previous studies revealed that Plexin-A1 is expressed in DRG and sympathetic neurons [33,35]. Chick stage 26 DRG and stage 37 sympathetic explants were co-cultured with Sema6E-, or mock-transfected HEK 293-T cell aggregates in a collagen gel. For quantitative analyses, DRG and sympathetic axonal
Fig. 3. Sema6E binds Plexin-A1, but not Plexin-A3 and Plexin-A4. Supernatants from HEK 293-T cells transfected with pZ6E-AP were reacted with COS-7 cells expressing Plexin-As. (A, C and D) Sema6E–AP fusion protein did not bind mock-transfected COS-7 cells (A), or Plexin-A3 and Plexin-A4-expressing cells (C and D). (B) Sema6E–AP fusion protein bound Plexin-A1-expressing cells.
M. Taniguchi et al. / Neuroscience Letters 488 (2011) 215–220
Fig. 4. DRG and sympathetic axons are repelled by Sema6E. (A) Sema6E-, or mocktransfected HEK 293-T cell aggregates were co-cultured with chick stage 26 DRG explants (DRG) and chick stage 37 sympathetic ganglion explants (SYM). The cells secreting Sema6E chemorepelled DRG and sympathetic axons. Scale bars: 300 m. (B) Quantification of chemorepulsive activities of Sema6E. The axonal growth ratio p/d is, respectively, a measure of repulsive activity, with the ratios of 0 and 1 indicating complete and no repulsion. The bars represent mean + SEM (*P < 0.01, **P < 0.001) and the number of co-cultures is shown in the bar.
lengths in the proximal quadrant (p), toward the cell aggregates, were compared to those in the distal quadrant (d), away from the cells. The axonal outgrowth ratio p/d value is, respectively, a measure of the repulsive activity, with ratios of 0 and 1 indicating complete and no repulsion (see Fig. 4B). Aggregates of control HEK 293-T cells (mock-transfected cells) co-cultured with DRG and sympathetic explants did not chemorepel these axons (Fig. 4A and B). However, cells secreting Sema6E chemorepelled both DRG and sympathetic axons (Fig. 4A and B). This study identified and cloned a novel zebrafish semaphorin, sema6E. The predictable protein of Sema6E is 1064 aa. Sema6E has no known orthologues identified in other species. Sema4E is also identified only in zebrafish [12,41]. Sema4E functions as a repulsive guidance molecule for facial and gill motor axons within the pharyngeal arches [41]. In an earlier study, we cloned a zebrafish sema6D and identified a sema6D isoform [15]. This isoform was generated by alternative splicing. All isoforms of class 6 semaphorins were identified in mammals [7,16,29,37]. These isoform expressions are controlled in a tissue-dependent and stage-dependent manner, although functional differences between the isoforms have not been reported. However, we did not identify a sema6E isoform.
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Sema6E was expressed predominantly in the nervous system during embryogenesis (Fig. 2). Characteristic expression of sema6E is in the lens (Fig. 2). Actually, Sema3A and sema3D are expressed in the lens during embryogenesis [1,11,25]. Sema3A produced by the lens functions as a chemorepellent during corneal nerve ring formation in chicks [19]. The embryonic lens produces a strong chemorepulsive activity for retinal ganglion cell (RGC) axons [25]. To form the functional topographic map, RGC axons must project to the correct target regions in the optic tectum/superior colliculus. RGC axons navigate out of the eye, across the optic chiasm, and dorsally through the optic tract to reach the optic tectum/superior colliculus [8]. The lens might be a source of guidance molecules that prevents aberrant RGC axons from growing toward it. In zebrafish, class 3 semaphorins are spatiotemporally placed to influence RGC axon growth [4]. Sema3D in the ventral tectum acts normally to inhibit ventral RGC axons from extending into the ventral tectum, ensuring their correct innervation of the dorsal tectum in zebrafish [17]. In sema6s, Sema6A was detected in the RGC layer in mice [26]. Sema6E expression in the lens was detected at 24 hpf (Fig. 2). The initial RGC axons had exited the eye by 32 hpf and projected toward the midline, which they crossed in the optic chiasm between 34 and 36 hpf in zebrafish [2]. Results show that Sema6E binds Plexin-A1, but not other Plexins (Fig. 3). Plexin-As are class 6 semaphorin receptors [42]. Plexin-A1 might be a Sema6E receptor. Sema6E exhibits chemorepulsive activity for chick DRG and sympathetic axons expressing Plexin-A1 (Fig. 4) [33,35]. In mice Plexin-A1 was detected in RGC axons during development [22]. These results suggest that Sema6E might function as a repulsive axon guidance molecule of RGC axons. Zebrafish sema6D expression in the lens was also detected at 24 hpf and Plexin-A1 is a Sema6D receptor [15,40]. Thus, Sema6E coordinated or competed with Sema6D might function in the axon guidance of RGC axons for functional topographic map formation. Plexin-A1 might be a Sema6E receptor and is a Sema6D receptor. Plexin-A1 is also a Sema3A co-receptor [33,35]. Plexin-A1 is required for Sema3A- and Sema6D-mediated repulsion of growing axon in mice [33,35,44]. In zebrafish Plexin-A3 acts as an essential component of the receptor for Sema3A signaling in the axonal pathfinding [9,27,36] and Plexin-A4 functions in axonal branching [21]. These results suggest that, in zebrafish, Plexin-As function in the axon guidance process. Although further studies are needed, Sema6E might utilize Plexin-A1 as a receptor to exert chemorepulsion. Taken together, Sema6E might function in the axon guidance as a repulsive guidance cue in the nervous system. We identified a novel zebrafish class 6 semaphorin, sema6E. Sema6E is expressed predominantly in the nervous system during embryogenesis. Sema6E binds Plexin-A1 and induces repulsion of DRG and sympathetic axons. Although further functional analyses of in vitro and in vivo are needed, these results suggest that Sema6E functions in the axon guidance as a repulsive guidance cue in the nervous system. Acknowledgments We are grateful to Dr. Hitoshi Okamoto (RIKEN, BSI) for the gift of zebrafish Plexin-A4 expression vector. This work was supported by Grants-in-Aid for Scientific Research (C) from the Japan Society for the Promotion of Science (JSPS), The Suhara Memorial Foundation, and The Akiyama Life Science Foundation. References [1] Z.Z. Bao, Z. Jin, Sema3D Sema7A have distinct expression patterns in chick embryonic development, Dev. Dyn. 235 (2006) 2282–2289. [2] J.D. Burrill, S.S. Easter Jr., Development of the retinofugal projections in the embryonic and larval zebrafish (Brachydanio rerio), J. Comp. Neurol. 346 (1994) 583–600.
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Identification and characterization of a novel zebrafish semaphorin Masahiko Taniguchi a,∗ , Tomoyuki Masuda b , Yoshinori Mikami c , Masafumi Kimura d , Tomoyuki Yoshida c , Masayoshi Mishina c , Takao Shimizu d a
Department of Biochemistry, Cancer Research Institute, Sapporo Medical University School of Medicine, S-1 W-17, Chuo-ku, Sapporo, Hokkaido 060-8556, Japan Department of Anatomy, Fukushima Medical University School of Medicine, Fukushima 960-1295, Japan c Department of Molecular Neurobiology and Pharmacology, Faculty of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan d Department of Biochemistry and Molecular Biology, Faculty of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan b
a r t i c l e
i n f o
Article history: Received 31 August 2010 Received in revised form 12 November 2010 Accepted 12 November 2010 Keywords: Semaphorin Plexin Axon guidance Brain Lens Repellent
a b s t r a c t The semaphorin gene family contains numerous secreted and transmembrane proteins. Some of them function as the repulsive and attractive axon guidance molecules during development. Herein, we report the cloning and characterization of a novel member of zebrafish semaphorin gene, semaphorin 6E (sema6E). Sema6E is expressed predominantly in the nervous system during embryogenesis. Results also show that Sema6E binds Plexin-A1, but not other Plexins. Sema6E chemorepels not only dorsal root ganglion axons but also sympathetic axons. Therefore, Sema6E might utilize Plexin-A1 as a receptor to repel axons of the specific types during development. © 2010 Elsevier Ireland Ltd. All rights reserved.
During embryogenesis, axons reach their specific targets correctly to form the complex neural network found in a mature functional nervous system. Several groups of axon guidance molecules such as semaphorins, ephrins, netrins, and slits have been reported to repel or attract the growing axons that express their cognate receptors [6]. Semaphorins are secreted and transmembrane proteins containing a conserved domain (Sema domain) of about 500 amino acids and found in both vertebrates and invertebrates [23,30]. To date, semaphorin genes of more than 20 kinds have been identified and classified into seven classes and a virus semaphorin [31]. Among them, semaphorin 3A (Sema3A) is the first identified semaphorin in vertebrates based on its ability to induce the collapse of growth cones of dorsal root ganglion (DRG) axons [18]. Sema3A-deficient mice showed a severe abnormality in the patterns of the axonal projection in the peripheral nervous system during embryogenesis [38]. Neuropilins are functional receptors for class 3 semaphorins and Plexins are also known as receptors
Abbreviations: aa, amino acids; AP, alkaline phosphatase; DRG, dorsal root ganglion; hpf, hours post fertilization; Kd, dissociation constants; ORF, open reading frame; PFA, paraformaldehyde; RACE, rapid amplification of cDNA ends; RGC, retinal ganglion cell; SEM, standard error of the mean; Sema, semaphorin. ∗ Corresponding author. Tel.: +81 11 611 2111; fax: +81 11 611 2299. E-mail address: [email protected] (M. Taniguchi). 0304-3940/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2010.11.032
for semaphorins of other types [28,42]. Semaphorins have functions in many important biological processes in addition to the development of the nervous system, including angiogenesis, vasculogenesis, tumor progression, osteogenesis and immune regulation [3,10,24,32,34]. Zebrafish (Danio rerio) is an excellent model organism to investigate the development and function of the vertebrate nervous system because transparent embryos and Morpholino Oligo techniques facilitate in vivo function analyses of the interesting genes. Here, we report the cloning and tissue distribution of zebrafish sema6E: a novel member of class 6 semaphorins. Results show that Sema6E repels the axons of DRG and sympathetic neurons. The zebrafish AB strain was used. The zebrafish was raised and kept under standard conditions at 28 ◦ C [43]. We searched the zebrafish genomic DNA database (http://www.ensembl.org/Danio rerio/index.html) and identified a novel zebrafish semaphorin cDNA, which is a member of class 6 semaphorins. To clone the novel zebrafish cDNA, we performed PCR on the adult zebrafish brain cDNA library using 5 -ATGCTGTGTGGCTGAATTCTATGC-3 and 5 CCGCTCGAGTCAGTAGTTGAACTTCCCTGCCT-3 primers. The PCR fragments digested by EcoRI and XhoI were inserted in EcoRI and XhoI sites of pBluescript II SK (−) (Agilent Technologies Inc., Santa Clara, CA). The clones were sequenced using an ABI PRISM 3100 Genetic Analyzer (Life Technologies, Carlsbad, CA). Then 5 -RACE methods were performed for the 5 non-coding sequence on the
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Fig. 1. Alignment of zebrafish Sema6E and Sema6D proteins.
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zebrafish 72 h post fertilization (hpf) cDNA using a SMART RACE cDNA Amplification Kit (Clontech Laboratories, Inc., Mountain View, CA). Consequently, a 3295 bp cDNA containing the open reading frame (ORF) was obtained. The ORF is 3192 bp (1064 aa). The accession number of sema6E is deposited as AB185147. In situ hybridization was done as described in an earlier report [15]. Briefly, embryos were fixed in 4% paraformaldehyde (PFA) at 4 ◦ C overnight. The 36, 48 and 72 hpf embryos were treated with 10 g/ml proteinase K. Hybridization was performed at 55 ◦ C for 12–16 h. For RNA probe of sema6E, the 799 bp fragment (2497–3295) of sema6E amplified using PCR was cloned in pCR II (Invitrogen Corp., Carlsbad, CA). The DIG-labeled sense and antisense RNA probes for zebrafish sem6E gene were synthesized using a DIG RNA labeling Kit (F. Hoffmann-La Roche Ltd., Basel, Switzerland). The embryos were reacted with anti-DIG antibody
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conjugated alkaline phosphatase (AP) (F. Hoffmann-La Roche Ltd.) and developed using BCIP/NBT solution (Sigma–Aldrich Corp., St. Louis, MO). The ORF of zebrafish sema6E cDNA (33–654 aa) was inserted in SfiI and BglII sites of pAPtag-5 expression vector containing AP gene (GenHunter Corp., Nashville, TN) (pZ6E-AP). The expression vectors of Plexin-As were transfected into COS-7 cells with Lipofectamine Plus (Invitrogen Corp.). The binding assay was performed as described previously [39]. Equilibrium dissociation constants (Kd) of Plexin-A1 with Sema6E–AP fusion proteins were measured as described [5]. The HEK 293-T cells were transfected with pZ6E-AP using Lipofectamine 2000 reagent (Invitrogen Corp.). The cell aggregates were prepared using the hanging drop method as described previously [14]. Then DRG explants were dissected from stage 26
Fig. 2. Sema6E expression pattern by in situ hybridization with the antisense riboprobe (A, C, E, G, H, J–L) and sense riboprobe (B, D, F, I, and M). A–D are from 24 hpf zebrafish, E–G are 36 hpf; H–J are 48 hpf; and K–M are 72 hpf. (A and C) Dorsal (A) and ventral (C) views of 24 hpf embryos showing that sema6E was expressed in the lens. (E and G) Lateral (E) and ventral (G) views of 36 hpf embryos showing that sema6E expression was detected in the lens and telencephalon. (H and J) Lateral (H) and dorsal (J) views of 48 hpf embryos showing that sema6E was detected in the lens. (K and L) Dorsal (K) and lateral (L) views of 72 hpf, showing expression in the diencephalon and rhombencephalon.
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chick embryos [13]. Sympathetic ganglion explants were dissected from stage 37 chick embryos. These explants were embedded in a collagen gel approximately 300–400 m distant from the aggregates of 293-T cells transfected with empty vector (mock) or pZ6E-AP. Cultures were incubated at 37 ◦ C for 24–36 h in DMEM containing 50 ng/ml 7S nerve growth factor (Chemicon International Inc., Temecula, CA). Cultures were fixed for several days with 4% PFA. Whole-mount immunohistochemistry of cultures and analyses of repulsive activities were performed as described [20]. Recently, we identified the zebrafish semaphorin 6D [15]. Regarding the cloning process by searching the zebrafish DNA database, we identified a novel zebrafish semaphorin cDNA. To obtain the novel zebrafish semaphorin cDNA, we performed PCR on the adult zebrafish brain cDNA library. The length of a novel semaphorin cDNA was 3295 bp and the ORF is 3192 bp (1064 aa) with predicted molecular weight of about 118 kDa. The predictable protein of a novel zebrafish semaphorin was most similar to that of mouse sema6D in mammalian semaphorin. It showed a 45.5% identity with mouse Sema6D. A novel zebrafish semaphorin protein showed approximately 40% identity with other mammalian class 6 semaphorins. Sema4E has no known orthologues identified in other species [12,41]. Sema4E protein shows approximately 40% identity with mammalian class 4 semaphorins. Zebrafish semphorin proteins of mammalian orthologues show approximately 70% identity. For example, zebrafish Sema6D protein shows a 70.8% identity with mouse Sema6D protein. Therefore, we considered that the novel cloned cDNA is not an orthologue of mammalian class 6 semaphorins, and designated it as semaphorin 6E (sema6E). Results show that Sema6E has 45.0% identity with zebrafish Sema6D (Fig. 1). Therefore, these genes are not paralogs. Comparison between sema6E cDNA and zebrafish genomic DNA sequences revealed that sema6E gene comprises at least 19 exons and spans about 150 kb of the genomic DNA.
To clarify the distribution of sema6E, in situ hybridization was performed (Fig. 2). At 12 hpf, no sema6E expression signal was detected (data not shown). At 24 hpf, sema6E expression signals were detected in the lens, diencephalon, and telencephalon (Fig. 2A and C). At 36 hpf, strong hybridization signals for sema6E were detected in the lens and weak signals were detected in the telencephalon, diencephalon, and hindbrain (Fig. 2E and G). Results show that Sema6E had been expressed in the lens, telencephalon, diencephalons, mesencephalon, and rhombencephalon by 48 hpf (Fig. 2H and J). Similarly, sema6E was found to be expressed in the telencephalon, diencephalons, mesencephalon, and rhombencephalon by 72 hpf and its expression of the lens was decreased (Fig. 2K and L). Plexins are known to be semaphorin receptors [28,42]. PlexinAs are class 6 semaphorin receptors [42]. For example, Plexin-A1 is a Sema6D receptor [15,40]. To clarify whether or not zebrafish Sema6E binds Plexin-As, Sema6E–AP fusion protein was reacted with COS-7 cells expressing various Plexin-As. Sema6E bound Plexin-A1-expressing cells, but not mock-transfected, Plexin-A3, or Plexin-A4-expressing cells (Fig. 3). Western blot analyses confirmed that the cells used in this study expressed Plexin-As (data not shown). We also performed binding assay using mouse Plexins of seven kinds (Plexin-A1–4, -B1, -C and -D). Zebrafish Sema6E bound mouse Plexin-A1 only (data not shown). We estimated the binding affinity of Sema6E–AP fusion proteins to cells expressing Plexin-A1 in equilibrium binding experiments. The estimated Kd was 6.2 ± 2.9 nM (SD; n = 6). To study whether Sema6E exhibits a significant biological activity, we performed a repulsive assay with chick DRG and sympathetic neurons (Fig. 4). Previous studies revealed that Plexin-A1 is expressed in DRG and sympathetic neurons [33,35]. Chick stage 26 DRG and stage 37 sympathetic explants were co-cultured with Sema6E-, or mock-transfected HEK 293-T cell aggregates in a collagen gel. For quantitative analyses, DRG and sympathetic axonal
Fig. 3. Sema6E binds Plexin-A1, but not Plexin-A3 and Plexin-A4. Supernatants from HEK 293-T cells transfected with pZ6E-AP were reacted with COS-7 cells expressing Plexin-As. (A, C and D) Sema6E–AP fusion protein did not bind mock-transfected COS-7 cells (A), or Plexin-A3 and Plexin-A4-expressing cells (C and D). (B) Sema6E–AP fusion protein bound Plexin-A1-expressing cells.
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Fig. 4. DRG and sympathetic axons are repelled by Sema6E. (A) Sema6E-, or mocktransfected HEK 293-T cell aggregates were co-cultured with chick stage 26 DRG explants (DRG) and chick stage 37 sympathetic ganglion explants (SYM). The cells secreting Sema6E chemorepelled DRG and sympathetic axons. Scale bars: 300 m. (B) Quantification of chemorepulsive activities of Sema6E. The axonal growth ratio p/d is, respectively, a measure of repulsive activity, with the ratios of 0 and 1 indicating complete and no repulsion. The bars represent mean + SEM (*P < 0.01, **P < 0.001) and the number of co-cultures is shown in the bar.
lengths in the proximal quadrant (p), toward the cell aggregates, were compared to those in the distal quadrant (d), away from the cells. The axonal outgrowth ratio p/d value is, respectively, a measure of the repulsive activity, with ratios of 0 and 1 indicating complete and no repulsion (see Fig. 4B). Aggregates of control HEK 293-T cells (mock-transfected cells) co-cultured with DRG and sympathetic explants did not chemorepel these axons (Fig. 4A and B). However, cells secreting Sema6E chemorepelled both DRG and sympathetic axons (Fig. 4A and B). This study identified and cloned a novel zebrafish semaphorin, sema6E. The predictable protein of Sema6E is 1064 aa. Sema6E has no known orthologues identified in other species. Sema4E is also identified only in zebrafish [12,41]. Sema4E functions as a repulsive guidance molecule for facial and gill motor axons within the pharyngeal arches [41]. In an earlier study, we cloned a zebrafish sema6D and identified a sema6D isoform [15]. This isoform was generated by alternative splicing. All isoforms of class 6 semaphorins were identified in mammals [7,16,29,37]. These isoform expressions are controlled in a tissue-dependent and stage-dependent manner, although functional differences between the isoforms have not been reported. However, we did not identify a sema6E isoform.
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Sema6E was expressed predominantly in the nervous system during embryogenesis (Fig. 2). Characteristic expression of sema6E is in the lens (Fig. 2). Actually, Sema3A and sema3D are expressed in the lens during embryogenesis [1,11,25]. Sema3A produced by the lens functions as a chemorepellent during corneal nerve ring formation in chicks [19]. The embryonic lens produces a strong chemorepulsive activity for retinal ganglion cell (RGC) axons [25]. To form the functional topographic map, RGC axons must project to the correct target regions in the optic tectum/superior colliculus. RGC axons navigate out of the eye, across the optic chiasm, and dorsally through the optic tract to reach the optic tectum/superior colliculus [8]. The lens might be a source of guidance molecules that prevents aberrant RGC axons from growing toward it. In zebrafish, class 3 semaphorins are spatiotemporally placed to influence RGC axon growth [4]. Sema3D in the ventral tectum acts normally to inhibit ventral RGC axons from extending into the ventral tectum, ensuring their correct innervation of the dorsal tectum in zebrafish [17]. In sema6s, Sema6A was detected in the RGC layer in mice [26]. Sema6E expression in the lens was detected at 24 hpf (Fig. 2). The initial RGC axons had exited the eye by 32 hpf and projected toward the midline, which they crossed in the optic chiasm between 34 and 36 hpf in zebrafish [2]. Results show that Sema6E binds Plexin-A1, but not other Plexins (Fig. 3). Plexin-As are class 6 semaphorin receptors [42]. Plexin-A1 might be a Sema6E receptor. Sema6E exhibits chemorepulsive activity for chick DRG and sympathetic axons expressing Plexin-A1 (Fig. 4) [33,35]. In mice Plexin-A1 was detected in RGC axons during development [22]. These results suggest that Sema6E might function as a repulsive axon guidance molecule of RGC axons. Zebrafish sema6D expression in the lens was also detected at 24 hpf and Plexin-A1 is a Sema6D receptor [15,40]. Thus, Sema6E coordinated or competed with Sema6D might function in the axon guidance of RGC axons for functional topographic map formation. Plexin-A1 might be a Sema6E receptor and is a Sema6D receptor. Plexin-A1 is also a Sema3A co-receptor [33,35]. Plexin-A1 is required for Sema3A- and Sema6D-mediated repulsion of growing axon in mice [33,35,44]. In zebrafish Plexin-A3 acts as an essential component of the receptor for Sema3A signaling in the axonal pathfinding [9,27,36] and Plexin-A4 functions in axonal branching [21]. These results suggest that, in zebrafish, Plexin-As function in the axon guidance process. Although further studies are needed, Sema6E might utilize Plexin-A1 as a receptor to exert chemorepulsion. Taken together, Sema6E might function in the axon guidance as a repulsive guidance cue in the nervous system. We identified a novel zebrafish class 6 semaphorin, sema6E. Sema6E is expressed predominantly in the nervous system during embryogenesis. Sema6E binds Plexin-A1 and induces repulsion of DRG and sympathetic axons. Although further functional analyses of in vitro and in vivo are needed, these results suggest that Sema6E functions in the axon guidance as a repulsive guidance cue in the nervous system. Acknowledgments We are grateful to Dr. Hitoshi Okamoto (RIKEN, BSI) for the gift of zebrafish Plexin-A4 expression vector. This work was supported by Grants-in-Aid for Scientific Research (C) from the Japan Society for the Promotion of Science (JSPS), The Suhara Memorial Foundation, and The Akiyama Life Science Foundation. References [1] Z.Z. Bao, Z. Jin, Sema3D Sema7A have distinct expression patterns in chick embryonic development, Dev. Dyn. 235 (2006) 2282–2289. [2] J.D. Burrill, S.S. Easter Jr., Development of the retinofugal projections in the embryonic and larval zebrafish (Brachydanio rerio), J. Comp. Neurol. 346 (1994) 583–600.
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