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Structure of the zebrafish fasciclin I-related extracellular matrix protein (βig-h3) and its characteristic expression during embryogenesis
Gene Expression Patterns 3 (2003) 331–336 www.elsevier.com/locate/modgep
Structure of the zebrafish fasciclin I-related extracellular matrix protein (big-h3) and its characteristic expression during embryogenesis Yoshikazu Hiratea,b, Hitoshi Okamotob, Kyo Yamasua,* b
a Department of Regulation Biology, Faculty of Science, Saitama University, Saitama 338-8570, Japan Laboratory for Developmental Gene Regulation, RIKEN Brain Science Institute, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
Received 12 September 2002; received in revised form 17 January 2003; accepted 28 January 2003
Abstract big-h3, which is structurally related to the insect fasciclin I, is assumed to act as a cell adhesion molecule through binding to cell-surface integrins. In this study, we obtained cDNA clones for the zebrafish orthologue of big-h3 and examined the expression of the gene (big-h3) in zebrafish embryos using in situ hybridization. Expression is first seen at the bud stage in the presomitic mesoderm. Throughout the somitogenesis stage, big-h3 is expressed in all the segmented somites, as well as in the presomitic mesoderm (S0 and S-I). High expression is observed in the dorsolateral part of the somite until the mid-somitogenesis stage. At late somitogenesis stages, the big-h3 expression in the dorsolateral somite fades away, while expression is upregulated in the ventromedial part of the somite that corresponds to the sclerotome. In embryos after completion of somitogenesis and fry after hatching, big-h3 continues to be expressed in the sclerotome. In addition, new expression starts in the mesenchyme cells in the head, pharyngeal arches, and pectoral fins. In the embryonic brain, expression is observed along the anterior and postoptic commissures, as well as along the optic nerve. q 2003 Elsevier Science B.V. All rights reserved. Keywords: big-h3; Extracellular matrix protein; Fasciclin I; Fasciculation; Osteogenesis; RGD-CAP; Somitogenesis; Zebrafish
1. Results and discussion big-h3 was first identified in mammals as an extracellular matrix (ECM) protein induced by transforming growth factor-b (Skonier et al., 1992, 1994). big-h3 consists of four repeated domains related to those found in the insect neuronal adhesion molecule fasciclin I (fasciclin I-like repeat; Zinn et al., 1988). Proteins related to fasciclin I have been identified in a variety of organisms, including vertebrates (Skonier et al., 1992; Takeshita et al., 1993), sea urchins (Di Carlo et al., 1990; Brennan and Robinson, 1994; Hirate et al., 1999), and algae (Huber and Sumper, 1994), and most of these proteins are assumed to regulate cell – cell or cell – ECM interaction. Recent reports have revealed the roles played by big-h3 in cell adhesion using cell culture systems (Ohno et al., 1999; Kim et al., 2000b; Billings et al., 2002) as well as its involvement in cornea formation (Munier et al., 1997) and osteogenesis (Kim et al., 2000a), although its expression and function during early * Corresponding author. Tel.: þ 81-48-858-3417; fax: þ81-48-858-3698. E-mail address: [email protected] (K. Yamasu).
vertebrate development have not yet been addressed. Here, we show the structure of zebrafish big-h3 deduced from cDNA and expression of the big-h3 gene during embryogenesis. We obtained cDNA clones for a protein with fasciclin Ilike repeats by screening a zebrafish embryo cDNA library (Inoue et al., 1994) using as a probe the partial cDNA fragment for a fasciclin I-related protein obtained by RT – PCR using degenerate primers. The longest cDNA obtained, which is 3046 bp in length, encodes a putative protein of 677 amino acids (aa) with high similarity to big-h3 (overall identities to human and chick proteins; 62.7 and 61.4%, respectively; Fig. 1A). This result along with the phylogenetic tree (Fig. 1C) places the deduced protein in the big-h3 subgroup of fasciclin I-related proteins. Like related proteins in other animals, zebrafish big-h3 consists of four fasciclin I-like repeats, each of which contains two highly conserved sequences (H1 and H2; Fig. 1A,B). The distance between the two conserved sequences in the fasciclin I-like repeats is moderately conserved (66 –91 aa). Zebrafish bigh3 lacks the RGD sequence (Fig. 1A), which is seen in all reported big-h3 proteins. However, it has been shown that
1567-133X/03/$ - see front matter q 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S1567-133X(03)00035-8
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Fig. 1. Comparison of the zebrafish big-h3 protein with other fasciclin I-related proteins. (A) Alignment of the deduced amino acid sequences of zebrafish bigh3 and other vertebrate fasciclin I-related proteins. Red open boxes show amino acids shared by at least three proteins. Dashed lines show gaps introduced for the optimal sequence alignment. The positions of the four fasciclin I-like repeats (R1–4) are shown by the inward-pointing arrows at both ends of the respective repeats. Each repeat contains two highly conserved regions, shown by blue (H1 region) and green (H2 region) boxes. The RGD sequences shown by the gray box are seen only in human and chick big-h3. The solid red boxes in the human big-h3 sequence show peptides that are reported to mediate cell adhesion by binding to a1b3 integrin. (B) Comparison of the fasciclin I-like repeats in fasciclin I-related proteins. Colored boxes show amino acids shared by at least two proteins in regions H1 and H2. Although the amino acid sequences are not highly conserved between regions H1 and H2 (omitted, cf. A), the sizes of the linker regions between the two conserved regions are moderately conserved. (C) The phylogenetic tree of fasciclin I-related proteins. Multiple alignment and a phylogenetic tree were created with the program CLUSTAL W (Thompson et al., 1994). z, zebrafish; h, human; m, mouse; c, chick; D, Drosophila.
the RGD sequence does not account for the adhesive property of big-h3 (Ohno et al., 1999). Instead, the dipeptide sequences (Asp-Ile) positioned just ahead of the amino-terminal ends of the H2 regions in the second and
fourth repeats contribute to the adhesive property of human big-h3 via an interaction with a3b1 integrin (Kim et al., 2000b). Aspartic acid is conserved at the corresponding sites of zebrafish big-h3 (Fig. 1A).
Y. Hirate et al. / Gene Expression Patterns 3 (2003) 331–336
Fig. 2. Profile of zebrafish big-h3 mRNA expression during development. Ten micrograms of total RNA from each stage were electrophoresed on a 1% denaturing agarose gel, transferred onto a nylon membrane, and hybridized with the big-h3 partial cDNA fragment obtained by RT –PCR (see Section 2). The 3.1 kb signal (arrowhead) was detected from the bud stage onward. The ethidium bromide-stained pattern of 28S and 18S ribosomal RNA is shown at the bottom as a loading control. Staging of embryos is according to Kimmel et al. (1995).
big-h3 transcripts are first detected by northern analysis at the bud stage and the amounts increase throughout embryogenesis (Fig. 2). With in situ hybridization, expression is first seen at the bud stage in the presomitic mesoderm, which later gives rise to the first somite (Fig. 3A). Throughout somitogenesis, big-h3 is expressed in all the segmented somites (Fig. 3B – E,I – M), as well as in the presomitic mesoderm immediately before segmentation (S0 and S-I; Fig. 3I). Until the 18-somite stage, big-h3 expression is detected evenly in somites along the anteroposterior axis. Meanwhile, big-h3 is expressed only in the lateral part of the somite; no expression is seen in the ventromedial part where the sclerotome is to be formed (Fig. 3K,L; Morin-Kensicki and Eisen, 1997). big-h3 transcripts are also seen in the anterior paraxial mesoderm ahead of the somites from mid-somitogenesis stages. This expression progressively extends anteriorly, reaching just caudal to the optic vesicles by the 18-somite stage (Fig. 3D). In the late-segmentation period, big-h3 expression in the lateral part of the somite is gradually downregulated except those in the dorsal-most subregion and around the horizontal myoseptum, while ventromedial expression starts by the 20somite stage (Fig. 3M,N). When viewed laterally, the ventromedial expression assumes an ‘L-shape’ pattern which is a characteristic feature of the zebrafish sclerotome (Fig. 3E; Morin-Kensicki and Eisen, 1997). Importantly, the ventromedial expression domain includes cells positive for pax9a expression, which is specific to the sclerotome (compare Fig. 3N,O; Nornes et al., 1996). The sclerotomal
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expression is maintained during late-embryogenesis stages and beyond the hatching period (Fig. 3F – H). Interestingly, big-h3 is transiently expressed around 25 h post fertilization (hpf) in the periphery of the notochord (Fig. 3N). In contrast, notochord lacks the big-h3 expression at earlier stages (Fig. 3L,M) or at 48 hpf (data not shown). At the pharyngula and hatching stages, big-h3 is also expressed in several mesenchymal regions; mesenchyme cells around the presumptive mouth opening (Fig. 4A,B), beneath the hindbrain (Fig. 4A,C), around the otic vesicles (Fig. 4A,C) where otic capsules are to be formed, and surrounding the eyes (Fig. 4A – C). At 48 hpf, strong expression of big-h3 is seen in the head mesenchyme that develops into the neurocranium and encases the embryonic brain (Fig. 4D). big-h3 expression was also seen in the pharyngeal arch mesenchyme (Fig. 4F) and pectoral fin mesenchyme (Fig. 4G –I). Interestingly, in contrast to being absent from the brain per se (Fig. 4D), big-h3 expression is seen along the anterior commissure (ac) and optic nerve (on) (Fig. 4E). Since expression of big-h3 was not detected in the somata of ac in the telencephalon and those in the retinal ganglion cells (data not shown), big-h3 may be expressed not in the neurons but in glial cells enveloping the ac and on. Besides, big-h3 is also expressed along the postoptic commissure (data not shown).
2. Experimental procedures Degenerate primers designed for the conserved amino acid sequences of big-h3 proteins (NEAFEK in the H1 region of the second repeat and TNGV(V/I)H in the H2 region of the fourth repeat) were used to amplify a partial cDNA fragment by RT –PCR. The partial cDNA fragment, which corresponds to the region from þ 847 to þ 1880 of the cDNA nucleotide sequence deposited in the DDBJ/EMBL/GenBank (AB086407), was used to synthesize probes for screening a cDNA library of 18– 20-hpf zebrafish embryos as well as for in situ hybridization. cDNA screening followed the standard protocol (Sambrook et al., 1989). Whole-mount in situ hybridization was performed as described previously (Westerfield, 1995). When necessary, two-color in situ hybridization was conducted using digoxygenin and fluorescein probes.
Acknowledgements We thank Drs. Hiroyuki Takeda and Terje Johansen for kindly providing us with the cDNAs for myoD and pax9a, respectively. We are grateful to Dr. Takashi Suyemitsu for his support of our research. This work was supported in part by Grants-in-Aid to K.Y. (Nos. 09780677 and 10220203)
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Fig. 3. big-h3 expression in the somite and sclerotome. Dorsal (A–C,I), ventral (J), and lateral (inset of A,D–H) views of zebrafish embryos with anterior to the left. Arrows show big-h3 expression in the unsegmented paraxial mesoderm (A), anterior-most somites (B–D), or sclerotomes (E– H). big-h3 is expressed in the paraxial mesoderm at the bud stage (A) and in the somitic mesoderm at the 4-somite stage (B), 8-somite stage (flat-mount, C), and 18-somite stage (D). At late somitogenesis stages, big-h3 is also expressed in the ventral head mesoderm (arrowhead, D). From the late-somitogenesis stage onward, big-h3 is expressed mainly in the ventral portion of respective somites (E–H). By 22-somite stage (E), the expression in the somite is downregulated to leave an ‘Lshape’ pattern similar to the zebrafish sclerotome at this stage. Besides, the expression is also retained in the dorsal and lateral subregions in the somite (for symbols see the legend to M,N). (I,J) Two-color in situ hybridization was conducted for big-h3 (digoxigenin probe, blue) and myoD (fluorescein probe, red) in a 16-somite stage embryo (flat-mounted, I) or for big-h3 (fluorescein probe, red) and pax9a (digoxigenin probe, blue) in a 22-somite stage embryo (J). (I) SI shows the nascent somite, and other somites are marked relative to the SI somite (SII, S0, and S-I; Pourquie´ and Tam, 2001). big-h3 is first expressed during somitogenesis (blue) in the unsegmented presomitic mesoderm (S-I) together with myoD (red). (J) big-h3-expressing somites include the pax9a-expressing cells in their ventromedial portions. (K) Parasagittal section of a 5-somite stage embryo counterstained with toluidine blue. (L –O) Cross sections with dorsal to the top. Embryos stained for big-h3 (L– N) or pax9a (O) were hand-cut with thicknesses of 30–40 mm. (L) At 18-somite stage, big-h3 expression is seen in the dorsolateral portion of the somite. (M,N) By 20-somite stage, the expression is downregulated besides the dorsal (thin arrows) and lateral portions (arrowheads) of the somite, while expression newly appears in the ventromedial portion and becomes conspicuous by 25 hpf (thick arrows) where pax9a-expressing cells are also observed (O). At 25 hpf, big-h3 is transiently expressed in the notochord, though this expression is downregulated at later stages (data not shown). Scale bars: 120 mm (A –D), 60 mm (E–H), 30 mm (I –O).
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Fig. 4. Spatial pattern of big-h3 expression during organogenesis. Lateral (A,F,G) or ventral (B,C,E) views of embryos with anterior to the left. (A –C) At 36 hpf, big-h3 is expressed in the mesenchyme cells in the head such as those surrounding the eye (ey; solid arrowheads), surrounding the otic vesicles (ov; white arrowheads), underlying the hindbrain (white open arrows), and around the presumptive mouth opening (black open arrows). Expression is also seen in the pectoral fin bud (pf). Black arrow marks the sclerotomal expression. (D) Parasagittal section of an embryo with anterior to the left. The big-h3 expression in the frame is enlarged in the inset. big-h3 is expressed in the head mesenchyme (hm) that encases the developing brain. (E) At 48 hpf, big-h3 expression is seen along the anterior commissure (ac) and optic nerve (on). The staining along the optic nerve is overlapped with the staining in the mesenchyme. The expression around the eye is marked with an arrowhead. (F) big-h3 is expressed in the pharyngeal arch mesenchyme. The staining at the top left is due to big-h3 expression in the head mesenchyme. ch, ceratohyal; P3 –P7, pharyngeal arches 3– 7. (G –I) big-h3 expression is seen in the pectoral fin bud from 36 hpf to 120 hpf. (H) Section of a pectoral fin bud of a 72-hpf embryo along the proximo-distal axis with anterior to the left. The expression is seen in the mesenchyme cells in the fin bud, while it is absent from the chondroblasts (solid arrowhead). (I) Developing pectoral fin taken from a 120-hpf embryo with distal to the right and dorsal to the top. big-h3 expression is seen in the dorsal mesenchyme cells. Scale bars: 120 mm (A), 60 mm (B–D,I), 30 mm (E–H).
from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
References Billings, P.C., Whitbeck, J.C., Adams, C.S., Abrams, W.R., Cohen, A.J., Engelsberg, B.N., Howard, P.S., Rosenbloom, W.R., 2002. The transforming growth factor-b-inducible matrix protein big-h3 interacts with fibronectin. J. Biol. Chem. 277, 28003–28009. Brennan, C., Robinson, J.J., 1994. Cloning and characterization of HLC-32, a 32-kDa protein component of the sea urchin extraembryonic matrix, the hyaline layer. Dev. Biol. 165, 556 –565. Di Carlo, M., Montana, G., Bonura, A., 1990. Analysis of the sequence and expression during sea urchin development of two members of a multigenic family, coding for butanol-extractable proteins. Mol. Reprod. Dev. 25, 28–36. Hirate, Y., Tomita, K., Yamamoto, S., Kobari, K., Uemura, I., Yamasu, K., Suyemitsu, T., 1999. Association of the sea urchin EGF-related peptide, EGIP-D, with fasciclin I-related ECM proteins from the sea urchin Anthocidaris crassispina. Dev. Growth Differ. 41, 483 –494. Huber, O., Sumper, M., 1994. Algal-CAMs: isoforms of a cell adhesion
molecule in embryos of the alga Volvox with homology to Drosophila fasciclin I. EMBO J. 13, 4212– 4222. Inoue, A., Takahashi, M., Hatta, K., Hotta, Y., Okamoto, H., 1994. Developmental regulation of islet-1 mRNA expression during neuronal differentiation in embryonic zebrafish. Dev. Dyn. 199, 1–11. Kim, J.-E., Kim, E.-H., Han, E.H., Park, R.-W., Park, I.-H., Jun, S.-H., Kim, J.-C., Young, M.-F., Kim, I.-S., 2000a. A TGF-b-inducible cell adhesion molecule, big-h3, is downregulated in melorheostosis and involved in osteogenesis. J. Cell. Biochem. 77, 169 –178. Kim, J.-E., Kim, S.-J., Lee, B.-H., Park, R.-W., Kim, K.-S., Kim, I.-S., 2000b. Identification of motifs for cell adhesion within the repeated domains of transforming growth factor-b-induced gene, big-h3. J. Biol. Chem. 275, 30907–30915. Kimmel, C.B., Ballard, W.W., Kimmel, S.R., Ullmann, B., Schilling, T.F., 1995. Stages of embryonic development of the zebrafish. Dev. Dyn. 203, 253–310. Morin-Kensicki, E.M., Eisen, J.S., 1997. Sclerotome development and peripheral nervous system segmentation in embryonic zebrafish. Development 124, 159–167. Munier, F.L., Korvatska, E., Djema, I¨., A, ., Le Paslier, D., Zografos, L., Pescia, G., Schorderet, D.F., 1997. Kerato-epithelin mutations in four 5q31-linked corneal dystrophies. Nat. Genet. 15, 247–251. Nornes, S., Mikkola, I., Krauss, S., Delghandi, M., Perander, M., Johansen, T., 1996. Zebrafish Pax9 encodes two proteins with distinct
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C-terminal transactivating domains of different potency negatively regulated by adjacent N-terminal sequences. J. Biol. Chem. 271, 26914–26923. Ohno, S., Noshiro, M., Makihira, S., Kawamoto, T., Shen, M., Yan, W., Kawashima-Ohya, Y., Fujimoto, K., Tanne, K., Kato, Y., 1999. RGDCAP (big-h3) enhances the spreading of chondrocytes and fibroblasts via integrin a1b1. Biochim. Biophys. Acta 1451, 196–205. Pourquie´ , O., Tam, P.P.L., 2001. A nomenclature for prospective somites and phases of cyclic gene expression in the presomitic mesoderm. Dev. Cell 1, 619–620. Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Skonier, J., Neubauer, M., Madisen, L., Bennett, K., Plowman, G.D., Purchio, A.F., 1992. cDNA cloning and sequence analysis of big-h3, a novel gene induced in a human adenocarcinoma cell line after treatment with transforming growth factor-b. DNA Cell Biol. 11, 511–522. Skonier, J., Bennett, K., Rothwell, V., Kosowski, S., Plowman, G.,
Wallace, P., Edelhoff, S., Disteche, C., Neubauer, M., Marquardt, H., Rodgers, J., Purchio, A.F., 1994. big-h3: a transforming growth factorb-responsive gene encoding a secreted protein that inhibits cell attachment in vitro and suppresses the growth of CHO cells in nude mice. DNA Cell Biol. 13, 571–584. Takeshita, S., Kikuno, R., Tezuka, K., Amann, E., 1993. Osteoblast-specific factor 2: cloning of a putative bone adhesion protein with homology with the insect protein fasciclin I. Biochem. J. 294, 271–278. Thompson, J.D., Higgins, D.G., Gibson, T.J., 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673–4680. Westerfield, M., 1995. The Zebrafish Book – A Guide for the Laboratory Use of Zebrafish (Danio rerio), 3rd Edition, University of Oregon Press, Eugene, OR. Zinn, K., McAllister, L., Goodman, C.S., 1988. Sequence analysis and neuronal expression of fasciclin I in grasshopper and Drosophila. Cell 53, 577–587.
Structure of the zebrafish fasciclin I-related extracellular matrix protein (big-h3) and its characteristic expression during embryogenesis Yoshikazu Hiratea,b, Hitoshi Okamotob, Kyo Yamasua,* b
a Department of Regulation Biology, Faculty of Science, Saitama University, Saitama 338-8570, Japan Laboratory for Developmental Gene Regulation, RIKEN Brain Science Institute, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
Received 12 September 2002; received in revised form 17 January 2003; accepted 28 January 2003
Abstract big-h3, which is structurally related to the insect fasciclin I, is assumed to act as a cell adhesion molecule through binding to cell-surface integrins. In this study, we obtained cDNA clones for the zebrafish orthologue of big-h3 and examined the expression of the gene (big-h3) in zebrafish embryos using in situ hybridization. Expression is first seen at the bud stage in the presomitic mesoderm. Throughout the somitogenesis stage, big-h3 is expressed in all the segmented somites, as well as in the presomitic mesoderm (S0 and S-I). High expression is observed in the dorsolateral part of the somite until the mid-somitogenesis stage. At late somitogenesis stages, the big-h3 expression in the dorsolateral somite fades away, while expression is upregulated in the ventromedial part of the somite that corresponds to the sclerotome. In embryos after completion of somitogenesis and fry after hatching, big-h3 continues to be expressed in the sclerotome. In addition, new expression starts in the mesenchyme cells in the head, pharyngeal arches, and pectoral fins. In the embryonic brain, expression is observed along the anterior and postoptic commissures, as well as along the optic nerve. q 2003 Elsevier Science B.V. All rights reserved. Keywords: big-h3; Extracellular matrix protein; Fasciclin I; Fasciculation; Osteogenesis; RGD-CAP; Somitogenesis; Zebrafish
1. Results and discussion big-h3 was first identified in mammals as an extracellular matrix (ECM) protein induced by transforming growth factor-b (Skonier et al., 1992, 1994). big-h3 consists of four repeated domains related to those found in the insect neuronal adhesion molecule fasciclin I (fasciclin I-like repeat; Zinn et al., 1988). Proteins related to fasciclin I have been identified in a variety of organisms, including vertebrates (Skonier et al., 1992; Takeshita et al., 1993), sea urchins (Di Carlo et al., 1990; Brennan and Robinson, 1994; Hirate et al., 1999), and algae (Huber and Sumper, 1994), and most of these proteins are assumed to regulate cell – cell or cell – ECM interaction. Recent reports have revealed the roles played by big-h3 in cell adhesion using cell culture systems (Ohno et al., 1999; Kim et al., 2000b; Billings et al., 2002) as well as its involvement in cornea formation (Munier et al., 1997) and osteogenesis (Kim et al., 2000a), although its expression and function during early * Corresponding author. Tel.: þ 81-48-858-3417; fax: þ81-48-858-3698. E-mail address: [email protected] (K. Yamasu).
vertebrate development have not yet been addressed. Here, we show the structure of zebrafish big-h3 deduced from cDNA and expression of the big-h3 gene during embryogenesis. We obtained cDNA clones for a protein with fasciclin Ilike repeats by screening a zebrafish embryo cDNA library (Inoue et al., 1994) using as a probe the partial cDNA fragment for a fasciclin I-related protein obtained by RT – PCR using degenerate primers. The longest cDNA obtained, which is 3046 bp in length, encodes a putative protein of 677 amino acids (aa) with high similarity to big-h3 (overall identities to human and chick proteins; 62.7 and 61.4%, respectively; Fig. 1A). This result along with the phylogenetic tree (Fig. 1C) places the deduced protein in the big-h3 subgroup of fasciclin I-related proteins. Like related proteins in other animals, zebrafish big-h3 consists of four fasciclin I-like repeats, each of which contains two highly conserved sequences (H1 and H2; Fig. 1A,B). The distance between the two conserved sequences in the fasciclin I-like repeats is moderately conserved (66 –91 aa). Zebrafish bigh3 lacks the RGD sequence (Fig. 1A), which is seen in all reported big-h3 proteins. However, it has been shown that
1567-133X/03/$ - see front matter q 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S1567-133X(03)00035-8
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Fig. 1. Comparison of the zebrafish big-h3 protein with other fasciclin I-related proteins. (A) Alignment of the deduced amino acid sequences of zebrafish bigh3 and other vertebrate fasciclin I-related proteins. Red open boxes show amino acids shared by at least three proteins. Dashed lines show gaps introduced for the optimal sequence alignment. The positions of the four fasciclin I-like repeats (R1–4) are shown by the inward-pointing arrows at both ends of the respective repeats. Each repeat contains two highly conserved regions, shown by blue (H1 region) and green (H2 region) boxes. The RGD sequences shown by the gray box are seen only in human and chick big-h3. The solid red boxes in the human big-h3 sequence show peptides that are reported to mediate cell adhesion by binding to a1b3 integrin. (B) Comparison of the fasciclin I-like repeats in fasciclin I-related proteins. Colored boxes show amino acids shared by at least two proteins in regions H1 and H2. Although the amino acid sequences are not highly conserved between regions H1 and H2 (omitted, cf. A), the sizes of the linker regions between the two conserved regions are moderately conserved. (C) The phylogenetic tree of fasciclin I-related proteins. Multiple alignment and a phylogenetic tree were created with the program CLUSTAL W (Thompson et al., 1994). z, zebrafish; h, human; m, mouse; c, chick; D, Drosophila.
the RGD sequence does not account for the adhesive property of big-h3 (Ohno et al., 1999). Instead, the dipeptide sequences (Asp-Ile) positioned just ahead of the amino-terminal ends of the H2 regions in the second and
fourth repeats contribute to the adhesive property of human big-h3 via an interaction with a3b1 integrin (Kim et al., 2000b). Aspartic acid is conserved at the corresponding sites of zebrafish big-h3 (Fig. 1A).
Y. Hirate et al. / Gene Expression Patterns 3 (2003) 331–336
Fig. 2. Profile of zebrafish big-h3 mRNA expression during development. Ten micrograms of total RNA from each stage were electrophoresed on a 1% denaturing agarose gel, transferred onto a nylon membrane, and hybridized with the big-h3 partial cDNA fragment obtained by RT –PCR (see Section 2). The 3.1 kb signal (arrowhead) was detected from the bud stage onward. The ethidium bromide-stained pattern of 28S and 18S ribosomal RNA is shown at the bottom as a loading control. Staging of embryos is according to Kimmel et al. (1995).
big-h3 transcripts are first detected by northern analysis at the bud stage and the amounts increase throughout embryogenesis (Fig. 2). With in situ hybridization, expression is first seen at the bud stage in the presomitic mesoderm, which later gives rise to the first somite (Fig. 3A). Throughout somitogenesis, big-h3 is expressed in all the segmented somites (Fig. 3B – E,I – M), as well as in the presomitic mesoderm immediately before segmentation (S0 and S-I; Fig. 3I). Until the 18-somite stage, big-h3 expression is detected evenly in somites along the anteroposterior axis. Meanwhile, big-h3 is expressed only in the lateral part of the somite; no expression is seen in the ventromedial part where the sclerotome is to be formed (Fig. 3K,L; Morin-Kensicki and Eisen, 1997). big-h3 transcripts are also seen in the anterior paraxial mesoderm ahead of the somites from mid-somitogenesis stages. This expression progressively extends anteriorly, reaching just caudal to the optic vesicles by the 18-somite stage (Fig. 3D). In the late-segmentation period, big-h3 expression in the lateral part of the somite is gradually downregulated except those in the dorsal-most subregion and around the horizontal myoseptum, while ventromedial expression starts by the 20somite stage (Fig. 3M,N). When viewed laterally, the ventromedial expression assumes an ‘L-shape’ pattern which is a characteristic feature of the zebrafish sclerotome (Fig. 3E; Morin-Kensicki and Eisen, 1997). Importantly, the ventromedial expression domain includes cells positive for pax9a expression, which is specific to the sclerotome (compare Fig. 3N,O; Nornes et al., 1996). The sclerotomal
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expression is maintained during late-embryogenesis stages and beyond the hatching period (Fig. 3F – H). Interestingly, big-h3 is transiently expressed around 25 h post fertilization (hpf) in the periphery of the notochord (Fig. 3N). In contrast, notochord lacks the big-h3 expression at earlier stages (Fig. 3L,M) or at 48 hpf (data not shown). At the pharyngula and hatching stages, big-h3 is also expressed in several mesenchymal regions; mesenchyme cells around the presumptive mouth opening (Fig. 4A,B), beneath the hindbrain (Fig. 4A,C), around the otic vesicles (Fig. 4A,C) where otic capsules are to be formed, and surrounding the eyes (Fig. 4A – C). At 48 hpf, strong expression of big-h3 is seen in the head mesenchyme that develops into the neurocranium and encases the embryonic brain (Fig. 4D). big-h3 expression was also seen in the pharyngeal arch mesenchyme (Fig. 4F) and pectoral fin mesenchyme (Fig. 4G –I). Interestingly, in contrast to being absent from the brain per se (Fig. 4D), big-h3 expression is seen along the anterior commissure (ac) and optic nerve (on) (Fig. 4E). Since expression of big-h3 was not detected in the somata of ac in the telencephalon and those in the retinal ganglion cells (data not shown), big-h3 may be expressed not in the neurons but in glial cells enveloping the ac and on. Besides, big-h3 is also expressed along the postoptic commissure (data not shown).
2. Experimental procedures Degenerate primers designed for the conserved amino acid sequences of big-h3 proteins (NEAFEK in the H1 region of the second repeat and TNGV(V/I)H in the H2 region of the fourth repeat) were used to amplify a partial cDNA fragment by RT –PCR. The partial cDNA fragment, which corresponds to the region from þ 847 to þ 1880 of the cDNA nucleotide sequence deposited in the DDBJ/EMBL/GenBank (AB086407), was used to synthesize probes for screening a cDNA library of 18– 20-hpf zebrafish embryos as well as for in situ hybridization. cDNA screening followed the standard protocol (Sambrook et al., 1989). Whole-mount in situ hybridization was performed as described previously (Westerfield, 1995). When necessary, two-color in situ hybridization was conducted using digoxygenin and fluorescein probes.
Acknowledgements We thank Drs. Hiroyuki Takeda and Terje Johansen for kindly providing us with the cDNAs for myoD and pax9a, respectively. We are grateful to Dr. Takashi Suyemitsu for his support of our research. This work was supported in part by Grants-in-Aid to K.Y. (Nos. 09780677 and 10220203)
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Fig. 3. big-h3 expression in the somite and sclerotome. Dorsal (A–C,I), ventral (J), and lateral (inset of A,D–H) views of zebrafish embryos with anterior to the left. Arrows show big-h3 expression in the unsegmented paraxial mesoderm (A), anterior-most somites (B–D), or sclerotomes (E– H). big-h3 is expressed in the paraxial mesoderm at the bud stage (A) and in the somitic mesoderm at the 4-somite stage (B), 8-somite stage (flat-mount, C), and 18-somite stage (D). At late somitogenesis stages, big-h3 is also expressed in the ventral head mesoderm (arrowhead, D). From the late-somitogenesis stage onward, big-h3 is expressed mainly in the ventral portion of respective somites (E–H). By 22-somite stage (E), the expression in the somite is downregulated to leave an ‘Lshape’ pattern similar to the zebrafish sclerotome at this stage. Besides, the expression is also retained in the dorsal and lateral subregions in the somite (for symbols see the legend to M,N). (I,J) Two-color in situ hybridization was conducted for big-h3 (digoxigenin probe, blue) and myoD (fluorescein probe, red) in a 16-somite stage embryo (flat-mounted, I) or for big-h3 (fluorescein probe, red) and pax9a (digoxigenin probe, blue) in a 22-somite stage embryo (J). (I) SI shows the nascent somite, and other somites are marked relative to the SI somite (SII, S0, and S-I; Pourquie´ and Tam, 2001). big-h3 is first expressed during somitogenesis (blue) in the unsegmented presomitic mesoderm (S-I) together with myoD (red). (J) big-h3-expressing somites include the pax9a-expressing cells in their ventromedial portions. (K) Parasagittal section of a 5-somite stage embryo counterstained with toluidine blue. (L –O) Cross sections with dorsal to the top. Embryos stained for big-h3 (L– N) or pax9a (O) were hand-cut with thicknesses of 30–40 mm. (L) At 18-somite stage, big-h3 expression is seen in the dorsolateral portion of the somite. (M,N) By 20-somite stage, the expression is downregulated besides the dorsal (thin arrows) and lateral portions (arrowheads) of the somite, while expression newly appears in the ventromedial portion and becomes conspicuous by 25 hpf (thick arrows) where pax9a-expressing cells are also observed (O). At 25 hpf, big-h3 is transiently expressed in the notochord, though this expression is downregulated at later stages (data not shown). Scale bars: 120 mm (A –D), 60 mm (E–H), 30 mm (I –O).
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Fig. 4. Spatial pattern of big-h3 expression during organogenesis. Lateral (A,F,G) or ventral (B,C,E) views of embryos with anterior to the left. (A –C) At 36 hpf, big-h3 is expressed in the mesenchyme cells in the head such as those surrounding the eye (ey; solid arrowheads), surrounding the otic vesicles (ov; white arrowheads), underlying the hindbrain (white open arrows), and around the presumptive mouth opening (black open arrows). Expression is also seen in the pectoral fin bud (pf). Black arrow marks the sclerotomal expression. (D) Parasagittal section of an embryo with anterior to the left. The big-h3 expression in the frame is enlarged in the inset. big-h3 is expressed in the head mesenchyme (hm) that encases the developing brain. (E) At 48 hpf, big-h3 expression is seen along the anterior commissure (ac) and optic nerve (on). The staining along the optic nerve is overlapped with the staining in the mesenchyme. The expression around the eye is marked with an arrowhead. (F) big-h3 is expressed in the pharyngeal arch mesenchyme. The staining at the top left is due to big-h3 expression in the head mesenchyme. ch, ceratohyal; P3 –P7, pharyngeal arches 3– 7. (G –I) big-h3 expression is seen in the pectoral fin bud from 36 hpf to 120 hpf. (H) Section of a pectoral fin bud of a 72-hpf embryo along the proximo-distal axis with anterior to the left. The expression is seen in the mesenchyme cells in the fin bud, while it is absent from the chondroblasts (solid arrowhead). (I) Developing pectoral fin taken from a 120-hpf embryo with distal to the right and dorsal to the top. big-h3 expression is seen in the dorsal mesenchyme cells. Scale bars: 120 mm (A), 60 mm (B–D,I), 30 mm (E–H).
from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
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