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Expression of the FGF receptor 2 gene (fgfr2) during embryogenesis in the zebrafish Daniorerio
Mechanisms of Development 119S (2002) S173–S178 www.elsevier.com/locate/modo
Expression of the FGF receptor 2 gene ( fgfr2) during embryogenesis in the zebrafish Danio rerio Noriko Tonou-Fujimori a,1, Masayoshi Takahashi a,1, Hiroshi Onodera a, Hiroshi Kikuta a, Sumito Koshida b, Hiroyuki Takeda c, Kyo Yamasu a,* a
Department of Regulation Biology, Faculty of Science, Saitama University, 255 Shimo-Okubo, Saitama City, Saitama 338-8570, Japan b Center for Integrative Bioscience, Okazaki National Research Institutes, Nishigonaka 38, Myodaiji, Okazaki, Aichi 444-8585, Japan c Department of Biological Sciences, Graduate School of Science, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan Received 4 September 2002; received in revised form 21 October 2002; accepted 31 October 2002
Abstract We isolated a full-length cDNA clone for the zebrafish homologue of fibroblast growth factor receptor (FGFR) 2. The deduced protein sequence is typical of vertebrate FGFRs in that it has three Ig-like domains in the extracellular region. The expression of fgfr2 is initiated during epiboly in the paraxial mesoderm. During early somitogenesis, fgfr2 expression was noted in the anterior neural plate as well as in newly formed somites. Whereas fgfr2 expression in somites is transient, it increases in the central nervous system (CNS), i.e. in the ventral telencephalon, anterior diencephalon, midbrain, and respective rhombomeres of the hindbrain, from the mid-somitogenesis stage. The dorsal telencephalon and the region around the midbrain-hindbrain boundary are devoid of fgfr2 expression. Essentially the same expression pattern is observed until 48 h post-fertilization in the CNS, although rhombomeric expression in the hindbrain is progressively confined to narrower stripes. After somitogenesis, fgfr2 expression was also observed in the lens, hypochord, endoderm, and fin mesenchyme. We compared the expression of the four fgfr genes ( fgfr1–4) in the CNS of zebrafish embryos and show that fgfr1 is the only fgfr gene that is expressed in the dorsal telencephalon and isthmic region from which expression of fgfr2–4 is absent. q 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Brain development; Fibroblast growth factor; FGF receptor; fgfr2; Isthmus; Zebrafish
1. Results and discussion 1.1. Cloning and structural analysis of the cDNA for the zebrafish fgfr2 gene FGFRs are high-affinity receptors for fibroblast growth factors (FGFs) that constitute a family of more than 20 polypeptides. FGF signaling via FGFRs is implicated in diverse biological processes including embryogenesis and adult homeostasis (Ornitz and Itoh, 2001). Four vertebrate FGFR genes are known, which have distinct but overlapping patterns of expression during development (Green et al., 1996). Mutations in the genes for FGFR1, FGFR2, and FGFR3 have recently been implicated in a number of human craniosynostosis and dwarfism syndromes, and the molecular consequences of these mutations are beginning to be understood (McIntosh et al., 2000). However, despite the accumulation of data on this topic, the roles of the respective * Corresponding author. Tel.: 181-48-858-3417; fax: 181-48-858-3698. E-mail address: [email protected] (K. Yamasu). 1 These authors contributed equally to this work.
fgfr genes in vertebrate development are poorly understood. In zebrafish, expression of only two fgfr genes, fgfr3 and fgfr4, has been reported to date (Thisse et al., 1995; Sleptsova-Friedrich et al., 2001). Expression of fgfr2 has been examined in the embryos of several vertebrate species, such as amphibians and mice, although expression during early embryogenesis was not fully defined (Orr-Urtreger et al., 1991; Friesel and Brown, 1992; Peters et al., 1992; Shi et al., 1994; Golub et al., 2000). In this study we isolated a partial cDNA clone for the zebrafish fgfr2 gene using nested RT–PCR with degenerate primers. Using this cDNA as a probe, we screened a cDNA library that was constructed from zebrafish embryos at 25– 26 h post-fertilization (hpf). A protein of 815 amino acids (aa), which was deduced from the sequence of the longest cDNA clone, encoded (in order from the N-terminus) a signal sequence, three Ig-like domains, a transmembrane domain, and a split tyrosine kinase domain, which are typical features of FGFRs (Fig. 1). A BLAST search of the database showed that the deduced protein corresponds to the vertebrate FGFR2c, which is one of the two splice variants of FGFR2. Actually, we also obtained a cDNA
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Fig. 1. Comparison of the zebrafish FGFR2 sequence with those of other vertebrates. The deduced amino acid sequence of the zebrafish FGFR2 is aligned with the human, chick, and Xenopus FGFR2c sequences. Residues marked with asterisks are conserved among the FGFR2 proteins. The cysteine residues that are conserved in all the FGFR2s are marked with solid circles. Hyphens represent gaps that were introduced to maximize similarities. The signal sequence, three Ig-like domains (Ig I–III), acid box, transmembrane domain (TM), and split tyrosine kinase domain (PTK I, II) are marked with light-blue, green, red, darkblue, and orange boxes, respectively. Sequence identities to the zebrafish FGFR2 are shown at the ends of the respective sequences. The sequence that is specific to FGFR2c and substituted with a different sequence in putative zebrafish FGFR2b is shown by underlines (see text).
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clone for another form of FGFR2 whose C-terminal half of the third Ig-like domain (IgIII) shows a sequence of 45 aa instead of 47 aa for the FGFR2c described above (Fig. 1), which is more similar to the equivalent regions of FGFR2b of other vertebrates (unpublished data). Besides the isoform-specific region, this cDNA clone showed the same sequence, suggesting that the second form is derived from a splicing variant of fgfr2 that encodes the zebrafish FGFR2b. 1.2. Expression of fgfr2 in zebrafish embryos Northern blot analysis showed little maternal expression of fgfr2, while zygotic expression started at 6–10 hpf (data not shown). In order to know the expression of fgfr2 in embryos, we conducted whole-mount in situ hybridization (WMISH) using the full-length cDNA for FGFR2c. We also used the probes specific to the two FGFR2s (FGFR2b and FGFR2c), though we failed to obtain reliable staining and discriminate the expressions of the two splice variants probably because the probes were too short for detection of the transcripts (134 and 140 bp, respectively). fgfr2 expression is first detected at 90% epiboly by WMISH in the paraxial mesoderm (Fig. 2A). At the bud stage, fgfr2 is expressed in the anterior brain as well as in the paraxial mesoderm (Fig. 2B). fgfr2 expression continues at early stages of somitogenesis in the paraxial mesoderm, which includes the newly formed somites. At this stage, fgfr2 is also expressed in the mesenchyme underlying the fgfr2-expressing somites, which might correspond to the developing gonads (Fig. 2C,H,I; Weidinger et al., 2002). Besides the mesoderm, fgfr2 expression is also observed in the central nervous system (CNS), which include the forebrain plus the optic vesicles, the midbrain, and three transverse stripes in the hindbrain (Fig. 2C). Somitic fgfr2 expression is restricted to the most anterior 5–6 somites during early somitogenesis, as is shown by the somitic expression of fgfr2 at the 10-somite (Fig. 2I). Somitic expression of fgfr2 is transient, since it disappears by the 15-somite stage and is not detected later during development, while fgfr2 expression in the underlying mesenchyme persists at the late somitogenesis stage (data not shown). The expression of fgfr2 in the brain is essentially unchanged from the late somitogenesis (18 hpf) through the early pharyngula (24 hpf) stages (Figs. 2D–F,J and 3B,F). In the anterior region, fgfr2 is expressed in the ventral telencephalon as well as in the anterior/ventral diencephalon, while the posterior/dorsal diencephalon displays low levels of fgfr2 expression. In the midbrain, fgfr2 expression is strong in the anterior half, but weaker in the posterior region. Up to seven stripes are seen in the hindbrain at 24 hpf. The first, fourth, and sixth stripes are the most strongly stained, and probably correspond to the three stripes that are observed at the earlier stages (Fig. 2C,D). Two-color WMISH with krox20, which is expressed specifically in rhombomeres 3 and 5 (r3 and r5; Oxtoby and Jowett,
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1993), showed that the fgfr2 stripes correspond to the respective rhombomeres (Fig. 2K). The fgfr2 gene is also expressed in the ventral ventricular zone of the hindbrain and anterior spinal cord (Fig. 2M,N). Importantly, the isthmic region lacks fgfr2 expression. Expression of fgfr2 is also seen in the CNS in a similar way at 36 hpf (data not shown) and 48 hpf (Fig. 2G), although it gradually becomes confined to sharper stripes in the hindbrain and to the ventricular zones in the forebrain and midbrain. In addition to the CNS, expression appears in the lens by 24 hpf (Fig. 2E,F, see also Fig. 3F), the prospective gut endoderm (Fig. 2L,N), and the hypochord (Fig. 2L,N). At this stage, when the lens is spherical but no histological differentiation is apparent (Easter and Nicola, 1996), fgfr2 is expressed in the entire lens structure (Fig. 2O). Lensspecific expression disappears by 48 hpf, when the lens is characterized by a core surrounded by surface epithelial cells. Meanwhile, by 48 hpf, fgfr2 expression has appeared at the bottom of the pectoral fin (Fig. 2G). 1.3. Expression of the fgfr genes in the CNS of zebrafish embryos Since FGF signaling plays an important role in the establishment of the midbrain-hindbrain boundary (MHB; Shamim et al., 1999) as well as in the organizer activity of the MHB/isthmic region (Crossley et al., 1996), we examined the expression of the fgfr genes around the MHB region at 24 hpf. Expression of fgfr2 is detected in most regions of the midbrain and in the r1 of the hindbrain as described above, but is absent from the posterior-most midbrain and the anterior-most r1 (Fig. 3B,F). This pattern of expression is complementary to that of pax2.1 (Fig. 3J), which confirms that the pax2.1-positive MHB region (Krauss et al., 1991) is devoid of fgfr2. Both fgfr3 and fgfr4 are expressed in the posterior end of r1 (Fig. 3C,D), as was reported previously (Thisse et al., 1995; SleptsovaFriedrich et al., 2001), and show a relatively wide negative r1 region posterior to the pax2.1-positive MHB region (Fig. 3K,L). This finding was confirmed by two-color WMISH for fgfr2 and fgfr3 or for fgfr2 and fgfr4 (Fig. 3G,H). Despite its rather ubiquitous expression in the CNS, fgfr1 is expressed at relatively high levels in the MHB region (Fig. 3A,E). The fgfr1 stripe at the MHB is similar to that seen for pax2.1 expression, although it is slightly displaced towards the posterior region (Fig. 3I). Taken together, fgfr1 is the only member of the zebrafish fgfr gene family that is expressed in the isthmic region. In addition, fgfr1 is expressed in the entire telencephalon in contrast to fgfr2, fgfr3, and fgfr4; fgfr3 is not expressed in the telencephalon (Sleptsova-Friedrich et al., 2001, see also Fig. 3C), while fgfr2 (this study) and fgfr4 (Thisse et al., 1995; see also Fig. 3D) are expressed exclusively in the ventral telencephalon. Thus, fgfr1 is the only fgfr that can transmit the FGF signal in the dorsal telencephalon. In the midbrain, fgfr3 expression is negative, and fgfr4 is
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Fig. 2. Analysis of fgfr2 expression in zebrafish embryos using in situ hybridization. (A–E,G,I) Dorsal views with the animal pole at the top (A) or anterior to the left (B–E,G,I). (Inset of A,F, and J–L) Lateral views with anterior to the left and dorsal to the top. (A) At 90% epiboly, fgfr2 expression is seen in the paraxial mesoderm (arrow) but not in the ectoderm (inset). (B) At the bud stage, fgfr2 is expressed in the anterior brain (arrowhead) and paraxial mesoderm where somitogenesis is initiated (arrow). The expression of pax2.1 at the MHB is visualized with fluorescein probe (red). (C) At the 8-somite stage, fgfr2 is expressed in the forebrain, optic vesicles, and midbrain. In the hindbrain, fgfr2 expression is visualized as three transverse stripes. fgfr2 is also expressed in the newly formed somites (arrow) and underlying mesenchyme cells (arrowhead). (D) At 18 hpf, fgfr2 is expressed strongly in the anterior forebrain, midbrain, and hindbrain. The expression in the hindbrain consists of three transverse stripes. (E,F) At 28 hpf, fgfr2 is expressed in the ventral telencephalon, anterior diencephalon, midbrain, hindbrain, and lens (arrowheads). In the hindbrain the expression is seen in respective rhombomeres, among which r1, r4 and r6 show most striking expression. Meanwhile, fgfr2 expression is absent from the MHB region. (G) The pattern of fgfr2 expression is essentially the same in the CNS at 48 hpf, although expression is confined to the ventricular zone in the forebrain and midbrain, and to the sharper stripes in the hindbrain. In addition to the CNS, fgfr2 is expressed in the mesenchyme that flanks the entire brain (arrowheads) and at the base of the pectoral fins (arrow), while expression is absent from the lens. (H) Cross section of the 8-somite-stage embryo at the anterior trunk showing expression in the newly formed somites (open arrow) and underlying mesenchyme cells (open arrowhead). (I) At the 10-somite stage, fgfr2 is expressed in the anterior-most 5–6 somites (arrow) but not in the posterior somites. Respective somites are delimited with oblique lines, and the first somite is marked with a solid dot. The fgfr2 gene is also expressed in the mesenchyme cells underlying the anterior somites (arrowhead). (J) The anterior brain of the 18-hpf embryo shown in (D) is visualized laterally. fgfr2 is expressed in the ventral telencephalon and the adjoining diencephalon. Open arrowheads mark the ventricle separating the telencephalon and diencephalon. (K) Comparisons with the expression of krox20 in r3 and r5 (red) at 24 hpf show that the stripes of fgfr2 expression in the hindbrain correspond to the rhombomeres. (L) At 24 hpf, fgfr2 is also expressed in the hypochord (arrowhead), gut endoderm, and anus (arrows). (M,N) Cross sections of 24-hpf embryos at the level of the r5 of the hindbrain (M) or at the level of the posterior hindbrain (N). Positive signals are seen in the ventral ventricular zone (open arrows; compare with the Developmental Atlas in the Zebrafish Information Network, http://zfin.org/zf_info/anatomy/24hrs/24hrs.html), hypochord (open arrowhead), endoderm (solid arrow), and paraxial mesoderm (solid arrowhead). (O) Horizontal section of the eye at 24 hpf with anterior to the top. F, forebrain; T, telencephalon; D, diencephalon; M, midbrain; H, hindbrain; OV, otic vesicle. The MHB is shown with asterisks. Scale bars: 100 mm (A–G,I–L); 50 mm (H,M–O).
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Fig. 3. Expression of fgfr genes in the brain of 24-hpf zebrafish embryos. Lateral views (A–D) and dorsal views (E–L) with the anterior region to the left. (A–D) Optic vesicles were removed for visualization of gene expression in the anterior brains. (A,E) fgfr1 expression is rather ubiquitous, and particularly strong in the anterior brain and at the MHB. (B,F) fgfr2 expression is seen in the ventral telencephalon, anterior diencephalon, midbrain, and hindbrain. The expression in the hindbrain is seen in respective rhombomeres, among which the first, fourth, and sixth stripes are more strongly stained than the others are. (C) fgfr3 is expressed mainly in the diencephalon and posterior r1. (D) fgfr4 is expressed in the ventral telencephalon, diencephalon, midbrain, and posterior r1. (G–L) Two-color WMISH was performed using digoxygenin-labeled (blue) and fluorescein-labeled (red) probes. (G,H) fgfr2 is expressed in the midbrain and r1, except in the vicinity of the MHB, whereas the expression of fgfr3 and fgfr4 is seen at the posterior end of r1, but is absent (fgfr3) or at a reduced level (fgfr4) in the anterior tectum. (I) The fgfr1 domain overlaps with the pax2.1 domain at the MHB, although it is slightly displaced caudally. (J) fgfr2 expression and the pax2.1 domain are complementary at the MHB. (K,L) The anterior and central domains of r1 are negative for fgfr3/4 expression. MHBs are shown with asterisks. Strong expression of the fgfr genes in r1, r4, and/or r6 is also marked (r1, r4, r6). T, telencephalon; D, diencephalon; M, midbrain; H, hindbrain. Scale bars: 100 mm.
expressed weakly in the anterior tectum, showing that fgfr1 and fgfr2 are the main FGF receptor genes that are expressed in the midbrain. 2. Experimental procedures 2.1. Animals Zebrafish embryos were raised at 28.5 8C until they reached appropriate stages. Morphological features and the number of hours post-fertilization (hpf) were used to determine the stage of the embryos (Kimmel et al., 1995). 2.2. Reverse transcription–polymerase chain reaction (RT– PCR) and cDNA cloning The cDNA was prepared by reverse-transcribing total RNA from 12-hpf embryos with an oligo(dT)17 primer and M-MLV reverse transcriptase (Gibco BRL). Amplification of fgfr cDNA was performed by nested PCR using degen-
erate primers that were designed based on the three highly conserved amino acid sequences among vertebrate FGFRs (i.e. HKNIIN, EYLDL, and EGHRMD). Screening of the cDNA library from zebrafish 25–26-hpf embryos was performed by hybridization as described previously (Church and Gilbert, 1984) using the partial fgfr cDNA, which was obtained by RT–PCR. The nucleotide sequences of the cDNAs for FGFR2b and FGFR2c were deposited with the accession numbers AB094118 and AB084105, respectively, in the DDBJ/EMBL/GenBank databases. Full-length or partial cDNA clones for fgfr1, fgfr3, and fgfr4 were also obtained, which was confirmed by comparison with the sequences deposited in the database ( fgfr1, AF389400; fgfr3, AF157560; fgfr4, U23839), and used for in situ analyses. 2.3. Whole-mount in situ hybridization Whole-mount in situ hybridization was performed essentially as described previously (Schulte-Merker et al., 1992).
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Stained embryos were embedded in JB-4 (Polysciences Inc.) and sectioned to a thickness of 10–20 mm. Acknowledgements We wish to thank Dr. T. Jowett for providing the krox20 cDNA. We are also grateful to Dr. H. Okamoto for providing us with fish strains and the cDNA library, and for general advice on fish biology. This work was partially supported by Grants-in-Aid to K.Y. (No. 09780677 and No. 10220203) from the Ministry of Education, Science, Sports and Culture, of Japan. References Church, G.M., Gilbert, W., 1984. Genomic sequencing. Proc. Natl. Acad. Sci. USA 81, 1991–1995. Crossley, P.H., Martinez, S., Martin, G.R., 1996. Midbrain development induced by FGF8 in the chick embryo. Nature 380, 66–68. Easter Jr, S.S., Nicola, G.N., 1996. The development of vision in the zebrafish (Danio rerio). Dev. Biol. 180, 646–663. Friesel, R., Brown, S.A.N., 1992. Spatially restricted expression of fibroblast growth factor receptor-2 during Xenopus development. Development 116, 1051–1058. Golub, R., Adelman, Z., Clementi, J., Weiss, R., Bonasera, J., Servetnick, M., 2000. Evolutionarily conserved and divergent expression of members of the FGF receptor family among vertebrate embryos, as revealed by FGFR expression patterns in Xenopus. Dev. Genes Evol. 210, 345–357. Green, P.J., Walsh, F.S., Doherty, P., 1996. Promiscuity of fibroblast growth factor receptors. BioEssays 18, 639–646. 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.
Krauss, S., Johansen, T., Korzh, V., Fjose, A., 1991. Expression of the zebrafish paired box gene pax[zf-b] during early neurogenesis. Development 113, 1193–1206. McIntosh, I., Bellus, G.A., Jab, E.W., 2000. The pleiotropic effects of fibroblast growth factor receptors in mammalian development. Cell Struct. Funct. 25, 85–96. Ornitz, D.M., Itoh, N., 2001. Fibroblast growth factors. Genome Biol. 2, reviews3005.1–3005.12. Orr-Urtreger, A., Givol, D., Yayon, A., Yarden, Y., Lonai, P., 1991. Developmental expression of two murine fibroblast growth factor receptors, flg and bek. Development 113, 1419–1434. Oxtoby, E., Jowett, T., 1993. Cloning of the zebrafish krox-20 gene (krx-20) and its expression during hindbrain development. Nucleic Acids Res. 21, 1087–1095. Peters, K.G., Werner, S., Chen, G., Williams, L.T., 1992. Two FGF receptor genes are differentially expressed in epithelial and mesenchymal tissues during limb formation and organogenesis in the mouse. Development 114, 233–243. Schulte-Merker, S., Ho, R.K., Herrmann, B.G., Nusslein-Volhard, C., 1992. The protein product of the zebrafish homologue of the mouse T gene is expressed in nuclei of the germ ring and the notochord of the early embryo. Development 116, 1021–1032. Shamim, H., Mahmood, R., Logan, C., Doherty, P., Lumsden, A., Mason, I., 1999. Sequential roles for Fgf4, En1 and Fgf8 in specification and regionalization of the midbrain. Development 126, 945–959. Shi, D.L., Launay, C., Fromentoux, V., Feige, J.J., Boucaut, J.C., 1994. Expression of fibroblast growth factor receptor-2 splice variants is developmentally and tissue-specifically regulated in the amphibian embryo. Dev. Biol. 164, 173–182. Sleptsova-Friedrich, I., Li, Y., Emelyanov, A., Ekker, M., Korzh, V., Ge, R., 2001. fgfr3 and regionalization of anterior neural tube in zebrafish. Mech. Dev. 102, 213–217. Thisse, B., Thisse, C., Weston, J.A., 1995. Novel FGF receptor (Z-FGFR4) is dynamically expressed in mesoderm and neurectoderm during early zebrafish embryogenesis. Dev. Dyn. 203, 377–391. Weidinger, G., Wolke, U., Ko¨ prunner, M., Thisse, C., Thisse, B., Raz, E., 2002. Regulation of zebrafish primordial germ cell migration by attraction towards an intermediate target. Development 129, 25–36.
Expression of the FGF receptor 2 gene ( fgfr2) during embryogenesis in the zebrafish Danio rerio Noriko Tonou-Fujimori a,1, Masayoshi Takahashi a,1, Hiroshi Onodera a, Hiroshi Kikuta a, Sumito Koshida b, Hiroyuki Takeda c, Kyo Yamasu a,* a
Department of Regulation Biology, Faculty of Science, Saitama University, 255 Shimo-Okubo, Saitama City, Saitama 338-8570, Japan b Center for Integrative Bioscience, Okazaki National Research Institutes, Nishigonaka 38, Myodaiji, Okazaki, Aichi 444-8585, Japan c Department of Biological Sciences, Graduate School of Science, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan Received 4 September 2002; received in revised form 21 October 2002; accepted 31 October 2002
Abstract We isolated a full-length cDNA clone for the zebrafish homologue of fibroblast growth factor receptor (FGFR) 2. The deduced protein sequence is typical of vertebrate FGFRs in that it has three Ig-like domains in the extracellular region. The expression of fgfr2 is initiated during epiboly in the paraxial mesoderm. During early somitogenesis, fgfr2 expression was noted in the anterior neural plate as well as in newly formed somites. Whereas fgfr2 expression in somites is transient, it increases in the central nervous system (CNS), i.e. in the ventral telencephalon, anterior diencephalon, midbrain, and respective rhombomeres of the hindbrain, from the mid-somitogenesis stage. The dorsal telencephalon and the region around the midbrain-hindbrain boundary are devoid of fgfr2 expression. Essentially the same expression pattern is observed until 48 h post-fertilization in the CNS, although rhombomeric expression in the hindbrain is progressively confined to narrower stripes. After somitogenesis, fgfr2 expression was also observed in the lens, hypochord, endoderm, and fin mesenchyme. We compared the expression of the four fgfr genes ( fgfr1–4) in the CNS of zebrafish embryos and show that fgfr1 is the only fgfr gene that is expressed in the dorsal telencephalon and isthmic region from which expression of fgfr2–4 is absent. q 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Brain development; Fibroblast growth factor; FGF receptor; fgfr2; Isthmus; Zebrafish
1. Results and discussion 1.1. Cloning and structural analysis of the cDNA for the zebrafish fgfr2 gene FGFRs are high-affinity receptors for fibroblast growth factors (FGFs) that constitute a family of more than 20 polypeptides. FGF signaling via FGFRs is implicated in diverse biological processes including embryogenesis and adult homeostasis (Ornitz and Itoh, 2001). Four vertebrate FGFR genes are known, which have distinct but overlapping patterns of expression during development (Green et al., 1996). Mutations in the genes for FGFR1, FGFR2, and FGFR3 have recently been implicated in a number of human craniosynostosis and dwarfism syndromes, and the molecular consequences of these mutations are beginning to be understood (McIntosh et al., 2000). However, despite the accumulation of data on this topic, the roles of the respective * Corresponding author. Tel.: 181-48-858-3417; fax: 181-48-858-3698. E-mail address: [email protected] (K. Yamasu). 1 These authors contributed equally to this work.
fgfr genes in vertebrate development are poorly understood. In zebrafish, expression of only two fgfr genes, fgfr3 and fgfr4, has been reported to date (Thisse et al., 1995; Sleptsova-Friedrich et al., 2001). Expression of fgfr2 has been examined in the embryos of several vertebrate species, such as amphibians and mice, although expression during early embryogenesis was not fully defined (Orr-Urtreger et al., 1991; Friesel and Brown, 1992; Peters et al., 1992; Shi et al., 1994; Golub et al., 2000). In this study we isolated a partial cDNA clone for the zebrafish fgfr2 gene using nested RT–PCR with degenerate primers. Using this cDNA as a probe, we screened a cDNA library that was constructed from zebrafish embryos at 25– 26 h post-fertilization (hpf). A protein of 815 amino acids (aa), which was deduced from the sequence of the longest cDNA clone, encoded (in order from the N-terminus) a signal sequence, three Ig-like domains, a transmembrane domain, and a split tyrosine kinase domain, which are typical features of FGFRs (Fig. 1). A BLAST search of the database showed that the deduced protein corresponds to the vertebrate FGFR2c, which is one of the two splice variants of FGFR2. Actually, we also obtained a cDNA
0925-4773/03/$ - see front matter q 2003 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0925-477 3(03)00112-6
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Fig. 1. Comparison of the zebrafish FGFR2 sequence with those of other vertebrates. The deduced amino acid sequence of the zebrafish FGFR2 is aligned with the human, chick, and Xenopus FGFR2c sequences. Residues marked with asterisks are conserved among the FGFR2 proteins. The cysteine residues that are conserved in all the FGFR2s are marked with solid circles. Hyphens represent gaps that were introduced to maximize similarities. The signal sequence, three Ig-like domains (Ig I–III), acid box, transmembrane domain (TM), and split tyrosine kinase domain (PTK I, II) are marked with light-blue, green, red, darkblue, and orange boxes, respectively. Sequence identities to the zebrafish FGFR2 are shown at the ends of the respective sequences. The sequence that is specific to FGFR2c and substituted with a different sequence in putative zebrafish FGFR2b is shown by underlines (see text).
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clone for another form of FGFR2 whose C-terminal half of the third Ig-like domain (IgIII) shows a sequence of 45 aa instead of 47 aa for the FGFR2c described above (Fig. 1), which is more similar to the equivalent regions of FGFR2b of other vertebrates (unpublished data). Besides the isoform-specific region, this cDNA clone showed the same sequence, suggesting that the second form is derived from a splicing variant of fgfr2 that encodes the zebrafish FGFR2b. 1.2. Expression of fgfr2 in zebrafish embryos Northern blot analysis showed little maternal expression of fgfr2, while zygotic expression started at 6–10 hpf (data not shown). In order to know the expression of fgfr2 in embryos, we conducted whole-mount in situ hybridization (WMISH) using the full-length cDNA for FGFR2c. We also used the probes specific to the two FGFR2s (FGFR2b and FGFR2c), though we failed to obtain reliable staining and discriminate the expressions of the two splice variants probably because the probes were too short for detection of the transcripts (134 and 140 bp, respectively). fgfr2 expression is first detected at 90% epiboly by WMISH in the paraxial mesoderm (Fig. 2A). At the bud stage, fgfr2 is expressed in the anterior brain as well as in the paraxial mesoderm (Fig. 2B). fgfr2 expression continues at early stages of somitogenesis in the paraxial mesoderm, which includes the newly formed somites. At this stage, fgfr2 is also expressed in the mesenchyme underlying the fgfr2-expressing somites, which might correspond to the developing gonads (Fig. 2C,H,I; Weidinger et al., 2002). Besides the mesoderm, fgfr2 expression is also observed in the central nervous system (CNS), which include the forebrain plus the optic vesicles, the midbrain, and three transverse stripes in the hindbrain (Fig. 2C). Somitic fgfr2 expression is restricted to the most anterior 5–6 somites during early somitogenesis, as is shown by the somitic expression of fgfr2 at the 10-somite (Fig. 2I). Somitic expression of fgfr2 is transient, since it disappears by the 15-somite stage and is not detected later during development, while fgfr2 expression in the underlying mesenchyme persists at the late somitogenesis stage (data not shown). The expression of fgfr2 in the brain is essentially unchanged from the late somitogenesis (18 hpf) through the early pharyngula (24 hpf) stages (Figs. 2D–F,J and 3B,F). In the anterior region, fgfr2 is expressed in the ventral telencephalon as well as in the anterior/ventral diencephalon, while the posterior/dorsal diencephalon displays low levels of fgfr2 expression. In the midbrain, fgfr2 expression is strong in the anterior half, but weaker in the posterior region. Up to seven stripes are seen in the hindbrain at 24 hpf. The first, fourth, and sixth stripes are the most strongly stained, and probably correspond to the three stripes that are observed at the earlier stages (Fig. 2C,D). Two-color WMISH with krox20, which is expressed specifically in rhombomeres 3 and 5 (r3 and r5; Oxtoby and Jowett,
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1993), showed that the fgfr2 stripes correspond to the respective rhombomeres (Fig. 2K). The fgfr2 gene is also expressed in the ventral ventricular zone of the hindbrain and anterior spinal cord (Fig. 2M,N). Importantly, the isthmic region lacks fgfr2 expression. Expression of fgfr2 is also seen in the CNS in a similar way at 36 hpf (data not shown) and 48 hpf (Fig. 2G), although it gradually becomes confined to sharper stripes in the hindbrain and to the ventricular zones in the forebrain and midbrain. In addition to the CNS, expression appears in the lens by 24 hpf (Fig. 2E,F, see also Fig. 3F), the prospective gut endoderm (Fig. 2L,N), and the hypochord (Fig. 2L,N). At this stage, when the lens is spherical but no histological differentiation is apparent (Easter and Nicola, 1996), fgfr2 is expressed in the entire lens structure (Fig. 2O). Lensspecific expression disappears by 48 hpf, when the lens is characterized by a core surrounded by surface epithelial cells. Meanwhile, by 48 hpf, fgfr2 expression has appeared at the bottom of the pectoral fin (Fig. 2G). 1.3. Expression of the fgfr genes in the CNS of zebrafish embryos Since FGF signaling plays an important role in the establishment of the midbrain-hindbrain boundary (MHB; Shamim et al., 1999) as well as in the organizer activity of the MHB/isthmic region (Crossley et al., 1996), we examined the expression of the fgfr genes around the MHB region at 24 hpf. Expression of fgfr2 is detected in most regions of the midbrain and in the r1 of the hindbrain as described above, but is absent from the posterior-most midbrain and the anterior-most r1 (Fig. 3B,F). This pattern of expression is complementary to that of pax2.1 (Fig. 3J), which confirms that the pax2.1-positive MHB region (Krauss et al., 1991) is devoid of fgfr2. Both fgfr3 and fgfr4 are expressed in the posterior end of r1 (Fig. 3C,D), as was reported previously (Thisse et al., 1995; SleptsovaFriedrich et al., 2001), and show a relatively wide negative r1 region posterior to the pax2.1-positive MHB region (Fig. 3K,L). This finding was confirmed by two-color WMISH for fgfr2 and fgfr3 or for fgfr2 and fgfr4 (Fig. 3G,H). Despite its rather ubiquitous expression in the CNS, fgfr1 is expressed at relatively high levels in the MHB region (Fig. 3A,E). The fgfr1 stripe at the MHB is similar to that seen for pax2.1 expression, although it is slightly displaced towards the posterior region (Fig. 3I). Taken together, fgfr1 is the only member of the zebrafish fgfr gene family that is expressed in the isthmic region. In addition, fgfr1 is expressed in the entire telencephalon in contrast to fgfr2, fgfr3, and fgfr4; fgfr3 is not expressed in the telencephalon (Sleptsova-Friedrich et al., 2001, see also Fig. 3C), while fgfr2 (this study) and fgfr4 (Thisse et al., 1995; see also Fig. 3D) are expressed exclusively in the ventral telencephalon. Thus, fgfr1 is the only fgfr that can transmit the FGF signal in the dorsal telencephalon. In the midbrain, fgfr3 expression is negative, and fgfr4 is
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Fig. 2. Analysis of fgfr2 expression in zebrafish embryos using in situ hybridization. (A–E,G,I) Dorsal views with the animal pole at the top (A) or anterior to the left (B–E,G,I). (Inset of A,F, and J–L) Lateral views with anterior to the left and dorsal to the top. (A) At 90% epiboly, fgfr2 expression is seen in the paraxial mesoderm (arrow) but not in the ectoderm (inset). (B) At the bud stage, fgfr2 is expressed in the anterior brain (arrowhead) and paraxial mesoderm where somitogenesis is initiated (arrow). The expression of pax2.1 at the MHB is visualized with fluorescein probe (red). (C) At the 8-somite stage, fgfr2 is expressed in the forebrain, optic vesicles, and midbrain. In the hindbrain, fgfr2 expression is visualized as three transverse stripes. fgfr2 is also expressed in the newly formed somites (arrow) and underlying mesenchyme cells (arrowhead). (D) At 18 hpf, fgfr2 is expressed strongly in the anterior forebrain, midbrain, and hindbrain. The expression in the hindbrain consists of three transverse stripes. (E,F) At 28 hpf, fgfr2 is expressed in the ventral telencephalon, anterior diencephalon, midbrain, hindbrain, and lens (arrowheads). In the hindbrain the expression is seen in respective rhombomeres, among which r1, r4 and r6 show most striking expression. Meanwhile, fgfr2 expression is absent from the MHB region. (G) The pattern of fgfr2 expression is essentially the same in the CNS at 48 hpf, although expression is confined to the ventricular zone in the forebrain and midbrain, and to the sharper stripes in the hindbrain. In addition to the CNS, fgfr2 is expressed in the mesenchyme that flanks the entire brain (arrowheads) and at the base of the pectoral fins (arrow), while expression is absent from the lens. (H) Cross section of the 8-somite-stage embryo at the anterior trunk showing expression in the newly formed somites (open arrow) and underlying mesenchyme cells (open arrowhead). (I) At the 10-somite stage, fgfr2 is expressed in the anterior-most 5–6 somites (arrow) but not in the posterior somites. Respective somites are delimited with oblique lines, and the first somite is marked with a solid dot. The fgfr2 gene is also expressed in the mesenchyme cells underlying the anterior somites (arrowhead). (J) The anterior brain of the 18-hpf embryo shown in (D) is visualized laterally. fgfr2 is expressed in the ventral telencephalon and the adjoining diencephalon. Open arrowheads mark the ventricle separating the telencephalon and diencephalon. (K) Comparisons with the expression of krox20 in r3 and r5 (red) at 24 hpf show that the stripes of fgfr2 expression in the hindbrain correspond to the rhombomeres. (L) At 24 hpf, fgfr2 is also expressed in the hypochord (arrowhead), gut endoderm, and anus (arrows). (M,N) Cross sections of 24-hpf embryos at the level of the r5 of the hindbrain (M) or at the level of the posterior hindbrain (N). Positive signals are seen in the ventral ventricular zone (open arrows; compare with the Developmental Atlas in the Zebrafish Information Network, http://zfin.org/zf_info/anatomy/24hrs/24hrs.html), hypochord (open arrowhead), endoderm (solid arrow), and paraxial mesoderm (solid arrowhead). (O) Horizontal section of the eye at 24 hpf with anterior to the top. F, forebrain; T, telencephalon; D, diencephalon; M, midbrain; H, hindbrain; OV, otic vesicle. The MHB is shown with asterisks. Scale bars: 100 mm (A–G,I–L); 50 mm (H,M–O).
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Fig. 3. Expression of fgfr genes in the brain of 24-hpf zebrafish embryos. Lateral views (A–D) and dorsal views (E–L) with the anterior region to the left. (A–D) Optic vesicles were removed for visualization of gene expression in the anterior brains. (A,E) fgfr1 expression is rather ubiquitous, and particularly strong in the anterior brain and at the MHB. (B,F) fgfr2 expression is seen in the ventral telencephalon, anterior diencephalon, midbrain, and hindbrain. The expression in the hindbrain is seen in respective rhombomeres, among which the first, fourth, and sixth stripes are more strongly stained than the others are. (C) fgfr3 is expressed mainly in the diencephalon and posterior r1. (D) fgfr4 is expressed in the ventral telencephalon, diencephalon, midbrain, and posterior r1. (G–L) Two-color WMISH was performed using digoxygenin-labeled (blue) and fluorescein-labeled (red) probes. (G,H) fgfr2 is expressed in the midbrain and r1, except in the vicinity of the MHB, whereas the expression of fgfr3 and fgfr4 is seen at the posterior end of r1, but is absent (fgfr3) or at a reduced level (fgfr4) in the anterior tectum. (I) The fgfr1 domain overlaps with the pax2.1 domain at the MHB, although it is slightly displaced caudally. (J) fgfr2 expression and the pax2.1 domain are complementary at the MHB. (K,L) The anterior and central domains of r1 are negative for fgfr3/4 expression. MHBs are shown with asterisks. Strong expression of the fgfr genes in r1, r4, and/or r6 is also marked (r1, r4, r6). T, telencephalon; D, diencephalon; M, midbrain; H, hindbrain. Scale bars: 100 mm.
expressed weakly in the anterior tectum, showing that fgfr1 and fgfr2 are the main FGF receptor genes that are expressed in the midbrain. 2. Experimental procedures 2.1. Animals Zebrafish embryos were raised at 28.5 8C until they reached appropriate stages. Morphological features and the number of hours post-fertilization (hpf) were used to determine the stage of the embryos (Kimmel et al., 1995). 2.2. Reverse transcription–polymerase chain reaction (RT– PCR) and cDNA cloning The cDNA was prepared by reverse-transcribing total RNA from 12-hpf embryos with an oligo(dT)17 primer and M-MLV reverse transcriptase (Gibco BRL). Amplification of fgfr cDNA was performed by nested PCR using degen-
erate primers that were designed based on the three highly conserved amino acid sequences among vertebrate FGFRs (i.e. HKNIIN, EYLDL, and EGHRMD). Screening of the cDNA library from zebrafish 25–26-hpf embryos was performed by hybridization as described previously (Church and Gilbert, 1984) using the partial fgfr cDNA, which was obtained by RT–PCR. The nucleotide sequences of the cDNAs for FGFR2b and FGFR2c were deposited with the accession numbers AB094118 and AB084105, respectively, in the DDBJ/EMBL/GenBank databases. Full-length or partial cDNA clones for fgfr1, fgfr3, and fgfr4 were also obtained, which was confirmed by comparison with the sequences deposited in the database ( fgfr1, AF389400; fgfr3, AF157560; fgfr4, U23839), and used for in situ analyses. 2.3. Whole-mount in situ hybridization Whole-mount in situ hybridization was performed essentially as described previously (Schulte-Merker et al., 1992).
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