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Early embryonic expression of a LIM-homeobox gene Cs-lhx3 is downstream of ß-catenin and responsible for the endoderm differentiation in Ciona savignyi embryos

Yutaka Satou^*,, Kaoru S. Imai^* and Nori Satoh

Department of Zoology, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan
^* These authors contributed equally to this work

View larger version (83K): [in a new window]   Fig. 1. Expression of genes downstream of ß-catenin in Ciona savignyi embryos. (A-E) Lineage of endodermal cells at the 16-cell stage (A), 32-cell stage (B), 64-cell stage (C), 110-cell stage (D) and tailbud stage (E). A-D, vegetal view; E, lateral view. Blastomeres shown by orange color are presumptive endodermal cells, while those in yellow are primordial endodermal cells whose fates are restricted to endoderm. (F-J) The expression of Cs-lhx3, (K-O) Cs-otx and (P-T) Cs-ttf1 gene, revealed by whole-mount in situ hybridization at the 16-cell stage (F,K,P), the 32-cell stage (G,L,Q), the 64-cell stage (H,M,R), the 110-cell stage (I,N,S) and the tailbud stage (J,O,T). Early embryos up to the 110-cell stage are viewed from the vegetal pole; anterior is upwards, posterior is downwards. The tailbud embryos in J and O are side views; T is a dorsal view. In ascidians, in situ signals for zygotic gene expression are first detected in nuclei of embryonic cells. Scale bar: 100 µm.

View larger version (39K): [in a new window]   Fig. 2. The Cs-lhx3 gene product of Ciona savignyi. (A) The predicted amino acid sequence. The LIM domains are shown by italic capitals and homeodomain is underlined. (B) Relationship of Cs-LHX3 with other LIM-homeodomain proteins. The phylogenetic tree of the homeodomains of the LIM-homeodomain proteins was constructed using the neighbor-joining method. The homeodomains of Cs-OTX and Cs-TTF1 were used as an outgroup. Branch length is proportional to the number of amino acid substitution; the scale bar indicates 0.1 amino acid substitution per position in the sequence.

View larger version (84K): [in a new window]   Fig. 3. Expression of Cs-lhx3 (A-D), Cs-otx (E-H) and Cs-ttf1 gene (I-L) in ß-catenin overexpressed embryos (A, C, E, G, I, K) or in cadherin overexpressed embryos (B, D, F, H, J, L), at the 32-cell stage (A, B, E, F, I, J) and at the 110-cell stage (C, D, G, H, K, L). Vegetal pole view; anterior is upwards, posterior is downwards. Scale bar: 100 µm.

View larger version (31K): [in a new window]   Fig. 4. The Cs-otx gene product. (A) The predicted amino acid sequence. The homeodomain is underlined. (B) Alignment of amino acid sequence of the homeodomain of Cs-OTX protein with those of Ci-OTX (Hudson and Lemaire, 2001) and Hr-OTX (Wada et al., 1996). Identities of the amino acid residues are shown by asterisks and similarities by dots.

View larger version (31K): [in a new window]   Fig. 5. The Cs-ttf1 gene product. (A) The predicted amino acid sequence. The homeodomain is underlined. (B) Alignment of amino acid sequence of the homeodomain of Cs-TTF1 protein with those of Ci-TTF1 (Ristoratore et al., 1999) and mouse TTF1 (Lazzaro et al., 1991). Identities of the amino acid residues are shown by asterisks and similarities by dots.

View larger version (99K): [in a new window]   Fig. 6. Effects of functional suppression with morpholino oligos of Cs-otx (D-F) and Cs-ttf1 (G-I) or overexpression of Cs-ttf1 (J-L) on the endodermal cell differentiation. (A-C) Larvae developed from eggs injected with lacZ morpholino (control). (A) Histochemical staining of AP in the endodermal cells (arrows) of the larval trunk. In Ciona larvae, AP is also expressed B-line notochord cells (arrowhead). (B,C) Larvae that show normal development of two sensory organs, otolith (arrow) and ocellus (arrowhead), which is evident by pigment cell development. (D-F) Larvae developed from eggs injected with Cs-otx morpholino. (D) Histochemical staining of AP. (E,F) Larvae that show a failure of development of two sensory organs. The development of the anterior head is also suppressed by Cs-otx morpholino, causing this region to appear smooth. (G-I) Larvae that developed from eggs injected with Cs-ttf1 morpholino. (G) Histochemical staining of AP activity. (H,I) Larvae that show normal development of two sensory organs, otolith (arrow) and ocellus (arrowhead). (J-L) Larvae developed from eggs injected with Cs-ttf1 mRNA. Histochemical staining of AP activity in tailbud embryos (J) and in 110-cell arrested embryo. Ventral view (K) and anterior view (L). Scale bars: in A, 100 µm for A,D,G,J; in B, 100 µm for B,E,H; in C, 100 µm for C,F,I; and in K, 100 µm for K,L.

View larger version (60K): [in a new window]   Fig. 7. Effects of functional suppression of Cs-lhx3 with morpholino oligos, rescue of the suppression by injection of synthetic Cs-lhx3 mRNA or overexpression of Ci-lhx3 on endodermal cell differentiation. (A,B) The embryos developed from eggs injected with morpholino against Cs-lhx3. Cs-lhx3 morpholino inhibits development of AP activity (A) and endoderm-specific thyroid hormone receptor gene expression (B). Insert in B shows the endoderm-specific expression of thyroid hormone receptor gene in control embryo (arrow). (C,D) Co-injection of Cs-lhx3 morpholino together with lacZ mRNA (C) or Cs-lhx3 mRNA (D). In control, AP activity did not develop (C), but embryos developed from eggs injected with Cs-lhx3 morpholino and synthetic mRNA showed AP activity (D). (E,F) Effects of Cs-lhx3 overexpression. AP expression in embryos developed from eggs injected with Cs-lhx3 mRNA or lacZ mRNA (control) and arrested at the 110-cell stage, vegetal view. (E) Control embryo showing AP activity in blastomeres of the endoderm lineage, while (F) Cs-lhx3 overexpressed embryos showing AP activity in non-endodermal blastomeres as well. Arrowheads indicate pigment cell development in the cleavage-arrested embryo. (G) AP expression in embryos developed from eggs injected with delE-Ci-cadherin mRNA and lacZ mRNA. (H) AP expression in embryos developed from eggs injected with delE-Ci-cadherin mRNA and Cs-lhx3 mRNA. Scale bars: in A, 100 µm for A-D; in E, 100 µm for E,F; in G, 100 µm for G,H.