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Developmental expression of the hemichordate otx ortholog
Mechanisms of Development 91 (2000) 337±339
Gene expression pattern
www.elsevier.com/locate/modo
Developmental expression of the hemichordate otx ortholog Yoshito Harada a,*, Noko Okai a, Shunsuke Taguchi a, Kunifumi Tagawa a,b, Tom Humphreys b, Nori Satoh a b
a Department of Zoology, Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan Kewalo Marine Laboratory, Paci®c Biomedical Research Center, University of Hawaii, 41 Ahui Street, Honolulu, HI 96813-5511, USA
Received 8 September 1999; received in revised form 15 October 1999; accepted 15 October 1999
Abstract The phylogenetic location of hemichordates is unique because they seem to ®ll an evolutionary gap between echinoderms and chordates. We report here characterization of Pf-otx, a hemichordate ortholog of otx, with its embryonic and larval expression pattern. Pf-otx is initially expressed in the vegetal plate of the blastula. Expression remains evident in the archenteron through gastrulation and then disappears. A new expression domain appears near the mouth along the preoral and postoral ciliated bands in the early tornaria larva. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Hemichordates; Otx; Vegetal plate; Ciliated band
1. Results Hemichordates occupy a critical phylogenetic location as they exhibit both echinoderm-like and chordate-like morphological characteristics (e.g. Nielsen, 1995). Therefore better understanding of the ontogeny of hemichordates will provide insight into the evolutionary nexus between the variety of deuterostome body plans. The larvae of echinoderms and hemichordates show considerable morphological similarity to each other, leading to the conceptualization of a hypothetical `dipleurula larva', as an archetype of these two phyla (Garstang, 1928; Nielsen, 1995). `Dipleurula larva' also has been considered as an archetype of chordates, and a relationship between the ciliated bands of `dipleurula larva' and the chordate neural tube has been pointed out (Garstang, 1928; Crowther and Whittaker, 1992; Nielsen, 1999). The homeobox gene, otx, has been demonstrated to play a highly conserved role in the head development in animals (for review see Finkelstein and Boncinelli, 1994). Among chordates, expression of the otx genes in early embryos can be summarized as showing two conserved domains, the anterior mesoendoderm and the anterior neurectoderm (Simeone et al., 1992, 1993; Wada et al., 1996; Williams and Holland, 1998). From non-chordate deuterostomes, only sea urchin otx genes have been reported (Li et al., * Corresponding author. Tel.: 181-75-753-4095; fax: 181-75-7051113. E-mail address: [email protected] (Y. Harada)
1997; Mitsunaga-Nakatsubo et al., 1998). Sea urchin otx mRNA does not show prominent spatial localization before gastrulation. As development proceeds, it begins to exhibit a relatively intense expression in archenteron and oral ectoderm of gastrula. In this study, we isolated a hemichordate otx ortholog and studied its embryonic expression pattern. We termed the gene Pf-otx (Ptychodera flava-otx). Its orthology to other otx homeobox genes was veri®ed by speci®c conserved amino acid residues within the encoded homeodomain (Fig. 1). Cleavage of P. ¯ava is holoblastic and radial with the embryo developing into a hollow blastula (Had®eld, 1975; Tagawa et al., 1998a). Pf-otx expression is observed prior to gastrulation. At the blastula stage, Pf-otx is expressed in the vegetal plate (Fig. 2A). This expression continues until the mid-gastrula stage with hybridization signal being detected in the entire archenteron (Fig. 2B±D). As gastrulation proceeds, the region of positive signal becomes narrower, and by the late gastrula stage, the signal is restricted to an equatorial band of the archenteron (Fig. 2E, arrowhead). This vegetal plate-originated zone of expression disappears by the early tornaria stage. A new expression domain appears in the ectoderm near the mouth and becomes more intense as the tornaria larva develops. Fig. 2F shows an oral view of an early tornaria larva with two latitudinal bands of positive hybridization signal (arrowheads) on both the animal and the vegetal sides of the mouth. Later, Pf-otx expression appears as
0925-4773/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0925-477 3(99)00279-8
338
Y. Harada et al. / Mechanisms of Development 91 (2000) 337±339
Fig. 1. Alignment of amino acids of the homeobox region and hexa-peptide in the C-terminus of Pf-Otx with those of other Otx orthologs. Asterisks indicate identities of amino acids among all members. Dots indicate similarities of amino acids.
four latitudinal bands on the tornaria larva (Fig. 2G,H, arrowheads). In Fig. 2G,H, the upper two bands of signal near the mouth run parallel to the preoral ciliated band,
while the lower two bands of signal are in parallel along the postoral ciliated band. The Pf-otx hybridization signal was mainly observed in the oral side of tornaria; however, a
Fig. 2. Spatial expression of Pf-otx. (A) A blastula. (B) An early gastrula. (C,D) A mid-gastrula; (C) lateral view, (D) vegetal view. (E) A late gastrula. The signal is restricted to an equatorial region of inner layer of the embryo (arrowhead). (F) Oral view of an early tornaria. Two latitudinal lines with signals are observed (arrowheads). (G,H) An early tornaria larva, which is at slightly later stage than shown in F. Four latitudinal lines with signals are observed (arrowheads). The upper two correspond to pre-oral/postoral ciliated bands respectively, while lower two correspond to circumanal ciliated bands. (G) oral view, (H) lateral view. (I) Lateral view of an early tornaria at the same stage shown in (G) or (H). Focused on the lateral ectoderm. Some cells along the postoral ciliated band are observed with faint positive signals (arrowheads). ar, archenteron; m, mouth; vp, vegetal plate.
Y. Harada et al. / Mechanisms of Development 91 (2000) 337±339
less intense signal was observed along the postoral ciliated band on the lateral sides (Fig. 2I, arrowheads). 2. Methods PCR cloning was carried out using a P. ¯ava gastrula cDNA library (Tagawa et al., 1998b). The primer sequences were as follows; forward: 5 0 -ACNMGNTAYCCNGAYATHTTYATG-3 0 , reverse: 5 0 -YTTNGCNCKNCKRTTYTTRAACCA-3 0 (where H not G, K G or T, M A or C, N any, R A or G, and Y C or T). The nucleotide sequences and amino acid sequences of Pfotx are available on database (accession number: AB028220). Whole-mount specimens were obtained as described previously (Tagawa et al., 1998a), and hybridized in situ basically as described by Tagawa et al. (1998b). Acknowledgements We thank A. Nishino for useful discussions. Y.H. and K.T. were supported by postdoctoral fellowships from the Japan Society for the Promotion of Science for Japanese Junior Scientists with Research Grants #8910 and #03725. This research was also supported by a Grant-in-Aid for Specially Promoted Research (No. 07102012) from the Monbusho, Japan to N.S. References Crowther, R.J., Whittaker, J.R., 1992. Structure of the caudal neural tube in
339
an ascidian larva: vestiges of its possible evolutionary origin from a ciliated band. J. Neurobiol. 23, 280±292. Finkelstein, R., Boncinelli, E., 1994. From ¯y head to mammalian forebrain: the story of otd and Otx. Trends Genet. 10, 310±315. Garstang, W., 1928. The morphology of the tunicata, and its bearing on the phylogeny of the chordata. Q. J. Microsc. Sci. 72, 58±187. Had®eld, M., 1975. In: Giese, A., Pearse, J. (Eds.). Reproduction of Marine Invertebrates, Academic Press, New York, pp. 185±240. Li, X., Chuang, C.K., Mao, C.A., Angerer, L.M., Klein, W.H., 1997. Two Otx proteins generated from multiple transcripts of a single gene in Strongylocentrotus purpuratus. Dev. Biol. 187, 253±266. Mitsunaga-Nakatsubo, K., Akasaka, K., Sakamoto, N., Takata, K., Matsumura, Y., Kitajima, T., Kusunoki, S., Shimada, H., 1998. Differential expression of sea urchin Otx isoform (HpOtxE and HpOtxL) mRNAs during early development. Int. J. Dev. Biol. 42, 645±651. Nielsen, C., 1995. Animal Evolution, Oxford University Press, Oxford. Nielsen, C., 1999. Origin of the chordate central nervous system ± and the origin of chordates. Dev. Genes Evol. 209, 198±205. Simeone, A., Acampora, D., Gulisano, M., Stornaiuolo, A., Boncinelli, E., 1992. Nested expression domains of four homeobox genes in developing rostral brain. Nature 358, 687±690. Simeone, A., Acampora, D., Mallamaci, A., Stornaiuolo, A., D'Apice, M.R., Nigro, V., Boncinelli, E., 1993. A vertebrate gene related to orthodenticle contains a homeodomain of the bicoid class and demarcates anterior neuroectoderm in the gastrulating mouse embryo. EMBO J. 12, 2735±2747. Tagawa, K., Nishino, A., Humphreys, T., Satoh, N., 1998a. The spawning and early development of the Hawaiian acorn worm (Hemichordate), Ptychodera ¯ava. Zool. Sci. 15, 85±91. Tagawa, K., Humphreys, T., Satoh, N., 1998b. Novel pattern of Brachyury gene expression in hemichordate embryos. Mech. Dev. 75, 151±155. Wada, S., Katsuyama, Y., Sato, Y., Itoh, C., Saiga, H., 1996. Hroth an orthodenticle-related homeobox gene of the ascidian, Halocynthia roretzi: its expression and putative roles in the axis formation during embryogenesis. Mech. Dev. 60, 59±71. Williams, N.A., Holland, P.W.H., 1998. Molecular evolution of the brain of chordates. Brain Behav. Evol. 52, 177±185.
Gene expression pattern
www.elsevier.com/locate/modo
Developmental expression of the hemichordate otx ortholog Yoshito Harada a,*, Noko Okai a, Shunsuke Taguchi a, Kunifumi Tagawa a,b, Tom Humphreys b, Nori Satoh a b
a Department of Zoology, Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan Kewalo Marine Laboratory, Paci®c Biomedical Research Center, University of Hawaii, 41 Ahui Street, Honolulu, HI 96813-5511, USA
Received 8 September 1999; received in revised form 15 October 1999; accepted 15 October 1999
Abstract The phylogenetic location of hemichordates is unique because they seem to ®ll an evolutionary gap between echinoderms and chordates. We report here characterization of Pf-otx, a hemichordate ortholog of otx, with its embryonic and larval expression pattern. Pf-otx is initially expressed in the vegetal plate of the blastula. Expression remains evident in the archenteron through gastrulation and then disappears. A new expression domain appears near the mouth along the preoral and postoral ciliated bands in the early tornaria larva. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Hemichordates; Otx; Vegetal plate; Ciliated band
1. Results Hemichordates occupy a critical phylogenetic location as they exhibit both echinoderm-like and chordate-like morphological characteristics (e.g. Nielsen, 1995). Therefore better understanding of the ontogeny of hemichordates will provide insight into the evolutionary nexus between the variety of deuterostome body plans. The larvae of echinoderms and hemichordates show considerable morphological similarity to each other, leading to the conceptualization of a hypothetical `dipleurula larva', as an archetype of these two phyla (Garstang, 1928; Nielsen, 1995). `Dipleurula larva' also has been considered as an archetype of chordates, and a relationship between the ciliated bands of `dipleurula larva' and the chordate neural tube has been pointed out (Garstang, 1928; Crowther and Whittaker, 1992; Nielsen, 1999). The homeobox gene, otx, has been demonstrated to play a highly conserved role in the head development in animals (for review see Finkelstein and Boncinelli, 1994). Among chordates, expression of the otx genes in early embryos can be summarized as showing two conserved domains, the anterior mesoendoderm and the anterior neurectoderm (Simeone et al., 1992, 1993; Wada et al., 1996; Williams and Holland, 1998). From non-chordate deuterostomes, only sea urchin otx genes have been reported (Li et al., * Corresponding author. Tel.: 181-75-753-4095; fax: 181-75-7051113. E-mail address: [email protected] (Y. Harada)
1997; Mitsunaga-Nakatsubo et al., 1998). Sea urchin otx mRNA does not show prominent spatial localization before gastrulation. As development proceeds, it begins to exhibit a relatively intense expression in archenteron and oral ectoderm of gastrula. In this study, we isolated a hemichordate otx ortholog and studied its embryonic expression pattern. We termed the gene Pf-otx (Ptychodera flava-otx). Its orthology to other otx homeobox genes was veri®ed by speci®c conserved amino acid residues within the encoded homeodomain (Fig. 1). Cleavage of P. ¯ava is holoblastic and radial with the embryo developing into a hollow blastula (Had®eld, 1975; Tagawa et al., 1998a). Pf-otx expression is observed prior to gastrulation. At the blastula stage, Pf-otx is expressed in the vegetal plate (Fig. 2A). This expression continues until the mid-gastrula stage with hybridization signal being detected in the entire archenteron (Fig. 2B±D). As gastrulation proceeds, the region of positive signal becomes narrower, and by the late gastrula stage, the signal is restricted to an equatorial band of the archenteron (Fig. 2E, arrowhead). This vegetal plate-originated zone of expression disappears by the early tornaria stage. A new expression domain appears in the ectoderm near the mouth and becomes more intense as the tornaria larva develops. Fig. 2F shows an oral view of an early tornaria larva with two latitudinal bands of positive hybridization signal (arrowheads) on both the animal and the vegetal sides of the mouth. Later, Pf-otx expression appears as
0925-4773/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0925-477 3(99)00279-8
338
Y. Harada et al. / Mechanisms of Development 91 (2000) 337±339
Fig. 1. Alignment of amino acids of the homeobox region and hexa-peptide in the C-terminus of Pf-Otx with those of other Otx orthologs. Asterisks indicate identities of amino acids among all members. Dots indicate similarities of amino acids.
four latitudinal bands on the tornaria larva (Fig. 2G,H, arrowheads). In Fig. 2G,H, the upper two bands of signal near the mouth run parallel to the preoral ciliated band,
while the lower two bands of signal are in parallel along the postoral ciliated band. The Pf-otx hybridization signal was mainly observed in the oral side of tornaria; however, a
Fig. 2. Spatial expression of Pf-otx. (A) A blastula. (B) An early gastrula. (C,D) A mid-gastrula; (C) lateral view, (D) vegetal view. (E) A late gastrula. The signal is restricted to an equatorial region of inner layer of the embryo (arrowhead). (F) Oral view of an early tornaria. Two latitudinal lines with signals are observed (arrowheads). (G,H) An early tornaria larva, which is at slightly later stage than shown in F. Four latitudinal lines with signals are observed (arrowheads). The upper two correspond to pre-oral/postoral ciliated bands respectively, while lower two correspond to circumanal ciliated bands. (G) oral view, (H) lateral view. (I) Lateral view of an early tornaria at the same stage shown in (G) or (H). Focused on the lateral ectoderm. Some cells along the postoral ciliated band are observed with faint positive signals (arrowheads). ar, archenteron; m, mouth; vp, vegetal plate.
Y. Harada et al. / Mechanisms of Development 91 (2000) 337±339
less intense signal was observed along the postoral ciliated band on the lateral sides (Fig. 2I, arrowheads). 2. Methods PCR cloning was carried out using a P. ¯ava gastrula cDNA library (Tagawa et al., 1998b). The primer sequences were as follows; forward: 5 0 -ACNMGNTAYCCNGAYATHTTYATG-3 0 , reverse: 5 0 -YTTNGCNCKNCKRTTYTTRAACCA-3 0 (where H not G, K G or T, M A or C, N any, R A or G, and Y C or T). The nucleotide sequences and amino acid sequences of Pfotx are available on database (accession number: AB028220). Whole-mount specimens were obtained as described previously (Tagawa et al., 1998a), and hybridized in situ basically as described by Tagawa et al. (1998b). Acknowledgements We thank A. Nishino for useful discussions. Y.H. and K.T. were supported by postdoctoral fellowships from the Japan Society for the Promotion of Science for Japanese Junior Scientists with Research Grants #8910 and #03725. This research was also supported by a Grant-in-Aid for Specially Promoted Research (No. 07102012) from the Monbusho, Japan to N.S. References Crowther, R.J., Whittaker, J.R., 1992. Structure of the caudal neural tube in
339
an ascidian larva: vestiges of its possible evolutionary origin from a ciliated band. J. Neurobiol. 23, 280±292. Finkelstein, R., Boncinelli, E., 1994. From ¯y head to mammalian forebrain: the story of otd and Otx. Trends Genet. 10, 310±315. Garstang, W., 1928. The morphology of the tunicata, and its bearing on the phylogeny of the chordata. Q. J. Microsc. Sci. 72, 58±187. Had®eld, M., 1975. In: Giese, A., Pearse, J. (Eds.). Reproduction of Marine Invertebrates, Academic Press, New York, pp. 185±240. Li, X., Chuang, C.K., Mao, C.A., Angerer, L.M., Klein, W.H., 1997. Two Otx proteins generated from multiple transcripts of a single gene in Strongylocentrotus purpuratus. Dev. Biol. 187, 253±266. Mitsunaga-Nakatsubo, K., Akasaka, K., Sakamoto, N., Takata, K., Matsumura, Y., Kitajima, T., Kusunoki, S., Shimada, H., 1998. Differential expression of sea urchin Otx isoform (HpOtxE and HpOtxL) mRNAs during early development. Int. J. Dev. Biol. 42, 645±651. Nielsen, C., 1995. Animal Evolution, Oxford University Press, Oxford. Nielsen, C., 1999. Origin of the chordate central nervous system ± and the origin of chordates. Dev. Genes Evol. 209, 198±205. Simeone, A., Acampora, D., Gulisano, M., Stornaiuolo, A., Boncinelli, E., 1992. Nested expression domains of four homeobox genes in developing rostral brain. Nature 358, 687±690. Simeone, A., Acampora, D., Mallamaci, A., Stornaiuolo, A., D'Apice, M.R., Nigro, V., Boncinelli, E., 1993. A vertebrate gene related to orthodenticle contains a homeodomain of the bicoid class and demarcates anterior neuroectoderm in the gastrulating mouse embryo. EMBO J. 12, 2735±2747. Tagawa, K., Nishino, A., Humphreys, T., Satoh, N., 1998a. The spawning and early development of the Hawaiian acorn worm (Hemichordate), Ptychodera ¯ava. Zool. Sci. 15, 85±91. Tagawa, K., Humphreys, T., Satoh, N., 1998b. Novel pattern of Brachyury gene expression in hemichordate embryos. Mech. Dev. 75, 151±155. Wada, S., Katsuyama, Y., Sato, Y., Itoh, C., Saiga, H., 1996. Hroth an orthodenticle-related homeobox gene of the ascidian, Halocynthia roretzi: its expression and putative roles in the axis formation during embryogenesis. Mech. Dev. 60, 59±71. Williams, N.A., Holland, P.W.H., 1998. Molecular evolution of the brain of chordates. Brain Behav. Evol. 52, 177±185.