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T-brain homologue (HpTb) is involved in the archenteron induction signals of micromere descendant cells in the sea urchin embryo

Takuya Fuchikami¹, Keiko Mitsunaga-Nakatsubo¹, Shonan Amemiya², Toshiya Hosomi¹, Takashi Watanabe¹, Daisuke Kurokawa^1,*, Miho Kataoka¹, Yoshito Harada³, Nori Satoh³, Shinichiro Kusunoki⁴, Kazuko Takata¹, Taishin Shimotori¹, Takashi Yamamoto¹, Naoaki Sakamoto¹, Hiraku Shimada¹ and Koji Akasaka^1,

¹ Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Higashi-Hiroshima 739-8526, Japan
² Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
³ Department of Zoology, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan
⁴ LSL, Nerima-ku, Tokyo 178-0061, Japan
^* Present address: Evolutionary Regeneration Biology Group, RIKEN Center for Developmental Biology, Kobe 650-0047, Japan

View larger version (109K): [in a new window]   Fig. 1. Nucleotide and deduced amino acid sequences of the cDNA clone for the sea urchin T-box gene, HpTb. The T-domain is underlined. The termination codon is shown with an asterisk, and the potential signal sequence for polyadenylation is double underlined. The position of 3' end of cDNAs isolated from cleavage stage embryos is depicted with arrowheads. DDBJ Accession Number for HpTb is AB048760.

View larger version (43K): [in a new window]   Fig. 2. Relationships of sea urchin T-domain proteins. (A) A phylogenetic tree of T domains. A molecular phylogenetic tree was constructed using the NJ method by a computer program (Genetyx, Software Development). The branch length is proportional to the number of amino acid substitutions. The number at each branch indicates the values of expected substitution of amino acid residues. (B) Alignment of the amino acid sequences of the T domain of T-brain subfamily members. Asterisks (*) indicate that all of the six T-brain proteins have an identical amino acid.

View larger version (90K): [in a new window]   Fig. 3. HpTb is activated zygotically and is expressed transiently during the blastula and gastrula stages. (A) Developmental northern blot. Two micrograms of total RNA extracted from sea urchin embryos at various developmental stages were run in parallel. The lower panel shows the rRNA bands in the ethidium bromide-stained gel. U, unfertilized egg; 16, 16-cell stage; M, morula; UB, unhatched blastula; HB, hatched blastula; MB, mesenchyme blastula; G, gastrula; Pr, prism; Pl, pluteus. (B-D) Whole-mount in situ hybridization showing spatial expression of HpTb transcript during embryogenesis. (B,C) Hatched blastulae, (B) lateral view and (C) vegetal view. (D) Early mesenchyme blastula, lateral view. PMCs migrating into the blastocoel show a positive signal. Scale bar: 50 µm.

View larger version (62K): [in a new window]   Fig. 4. HpTb protein is accumulated into PMC nucleus after blastula stage. (A) Developmental western blot. Thirty micrograms of protein prepared from embryos at various developmental stages were run in parallel. Detection procedure of HpTb protein is described in text. U, unfertilized egg; 16, 16-cell stage; M, morula; UB, unhatched blastula; HB, hatched blastula; MB, mesenchyme blastula; G, gastrula; Pr, prism; Pl, pluteus. (B,C) Immunostaining of embryos with antibodies to HpTb. (B) Morula; (C) mesenchyme blastula. Scale bar: 50 µm.

View larger version (41K): [in a new window]   Fig. 5. Effect of injection of HpTb morpholino antisense oligonucleotides on the embryogenesis. (A,C,E,G) Control embryos injected with lacZ morpholino (7x108 molecules/egg). (B) Embryo injected with low amounts of HpTb morpholino (1x108 molecules/egg). (D,F,H-J) Embryos injected with HpTb morpholino (7x108 molecules/egg). The repression of translation of HpTb was confirmed using indirect immunofluorescence with anti-HpTb antibodies on mesenchyme blastula, (I) bright-field view, (J) epifluorescence view. (K) Embryo co-injected with 1.5 pg of HpTb-mRNA and HpTb morpholino (7x108 molecules/egg). Embryos at 28 hours (C,D,I,J), 40 hours (E,F), 48 hours (A,B,K) and 72 hours (G,H). Scale bar: 50 µm.

View larger version (35K): [in a new window]   Fig. 6. RT-PCR analysis of various tissue specific genes in control and embryos injected with HpTb morpholino. Total RNA was isolated from 50 control embryos and 50 embryos injected with HpTb morpholino antisense oligonucleotides at 28 hours after fertilization. Southern blotting of RT-PCR products. (-) and (+) indicate uninjected and injected embryos. The genes analysed are discussed in the text.

View larger version (71K): [in a new window]   Fig. 7. Localization of PMC specific HpEts and oral ectoderm specific Hpoe with antibodies. (A,C,E) Control embryo and (B,D,F) embryo injected with HpTb morpholino. (A,B) Embryos stained with antibodies to HpEts at 23 hours after fertilization; (C,D) embryos stained with antibodies to Hpoe at 40 hours after fertilization; (E,F) phase image of C,D. Scale bar: 50 µm.

View larger version (51K): [in a new window]   Fig. 8. RT-PCR analysis of Ars in embryos derived from animal cap mesomeres and control embryos. The RNA was isolated from 50 embryos derived from animal cap mesomeres at 28 hours after fertilization when the control embryo had reached the late gastrula stage. Southern blotting of RT-PCR products. C, control embryos; AC, embryos derived from animal cap mesomeres.

View larger version (43K): [in a new window]   Fig. 9. Loss of organizing activity of micromeres by injecting with HpTb morpholino. (A,B) Chimera composed of animal cap mesomeres from normal embryo with the micromere quartet isolated from a 16-cell stage embryo developed from zygotes injected with 7x108 molecules of HpTb morpholino (A, vegetal view; B, lateral view). (C) Control embryo. (D,E) The micromeres were labelled with rhodamine-dextran. Chimera composed of micromere quartet from normal embryo with the animal cap mesomeres isolated from a 16-cell stage embryo developed from zygotes injected with 7x108 molecules of HpTb morpholino (D, lateral view; E, vegetal view). The animal cap mesomeres were labelled with rhodamine-dextran. Left, bright-field views; right, epifluorescence views. Scale bar: 50 µm.

View larger version (56K): [in a new window]   Fig. 10. RT-PCR analysis of HpTb and HpEts in embryos injected with intracellular domains of cadherin (LvG-cadherin) mRNA (+) and control (-) embryos. Total RNA was isolated from 50 control and 50 mRNA-injected embryos at 28 hours after fertilization. Southern blotting of RT-PCR products.

View larger version (91K): [in a new window]   Fig. 11. Effect of the ectopic expression of HpEts on the expression of HpTb and gastrulation. (A) RT-PCR analysis of HpTb in embryos injected with HpEts (+) mRNA and control (-) embryos. Total RNA was isolated from 50 control and 50 mRNA-injected embryos at 28 hours after fertilization. Southern blotting of RT-PCR products. (B) Control embryo and (C) embryo injected with HpEts-mRNA at 48 hours after fertilization. Scale bar: 50 µm.