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An essential role of a FoxD gene in notochord induction in Ciona embryos

Department of Zoology, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan

View larger version (45K): [in a new window]   Fig. 1. Expression of Cs-FoxD genes in early Ciona savignyi embryos revealed by whole-mount in situ hybridization. (A-F) Lineage of endodermal cells and notochord cells at the eight-cell (A), 16-cell (B), 32-cell (C), 64-cell (D), 110-cell (E) and tailbud (F) stages. Lateral views of the eight-cell embryo and tailbud embryo are shown, and early embryos at the 16- to 110-cell stages are viewed from the vegetal pole; anterior is upwards and posterior is downwards. Blastomeres are named according to Conklin (Conklin, 1905). Blastomeres shown in orange are presumptive notochord cells, while those in yellow are primordial endodermal cells whose fates are restricted to the endoderm. Light green dots in B and C show the expression of Cs-FoxD in endodermal cells. (G-N) Expression of Cs-FoxD at the eight-cell (G), 16-cell (H), 32-cell (I), 64-cell (J), 110-cell (K), gastrula (L), neurula (M) and tailbud (N) stages. Arrowheads in N indicate the five expression domains. In ascidians, in situ signals for zygotic gene expression are first detected in the nuclei of embryonic cells. Scale bar: 100 µm.

View larger version (51K): [in a new window]   Fig. 2. Gene product of Cs-FoxD. (A) Nucleotide and deduced amino acid sequence of Cs-FoxD clone. The 1915 bp insert includes a single open reading frame that encodes a polypeptide of 506 amino acids. The termination codon is shown by an asterisk. The predicted forkhead domain is shown by bold letters. The nucleotide sequence of the 5' region used to prepare morpholino oligonucleotides is underlined. (B) Comparison of amino acid sequences of the forkhead domains of Cs-FoxA5, Cs-FoxD and zebrafish z-FoxD1. (C) Molecular phylogenetic analysis of relationships of Cs-FoxD with Fox gene members. The tree was constructed by comparison of the amino acid sequences of the forkhead domains using the neighbor-joining method. Human FoxC1 and FoxC2 were used as the outgroup. Branch lengths are proportional to the evolutionary distance corrected for multiple substitutions with the scale denoting 0.01 amino acid substitutions per site. The numbers indicate the relative robustness of each node as assessed by bootstrap analysis (100 replications). This analysis strongly suggests that Cs-FoxD is a member of the FoxD subclass.

View larger version (88K): [in a new window]   Fig. 3. Expression of Cs-FoxD gene in ß-catenin-overexpressing embryos (A) and in cadherin-overexpressing embryos (B) at the 110-cell stage. Vegetal pole view; anterior is upwards and posterior is downwards. Scale bar: 100 µm.

View larger version (39K): [in a new window]   Fig. 4. (A) Sequence of the 5' flanking region between nucleotide positions –1142 and –963 of Cs-FoxD, which contains three Tcf-binding motifs [TBE1-3: CTTTG(A/T)(A/T)]. (B) Relative levels of lacZ expression when various deletion constructs were injected. Deletion constructs are shown on the left and relative ratios of lacZ expression on the right. The relative expression level was calculated as described in the Materials and Methods. (C) Relative levels of lacZ expression in embryos injected with ß-catenin mRNA, cadherin mRNA or various mutation constructs derived from p(–1138)Cs-FoxD/lacZ. Mutation N is shown in A. (D) Expression of lacZ when control p(–1138)Cs-FoxD/lacZ was injected. lacZ was detected in progeny cells of A5.1, A5.2 and B5.2 (endoderm, notochord and nerve cord cells, respectively) presumably due to the stability of ß-gal protein. (E) Expression of lacZ was not detected when p(–1138/del TBE1/2/3)Cs-FoxD/lacZ was injected.

View larger version (61K): [in a new window]   Fig. 5. Effects of suppression of Cs-FoxD function with specific morpholino on the differentiation of endoderm. (A,B) Histochemical detection of endoderm-specific alkaline phosphatase (Cs-AP) activity. (C,D) In situ hybridization examining expression of an endoderm-specific gene for thyroid hormone-receptor (Cs-THR). (A-D) Normal embryos; (B,D) control embryos arrested at the 110-cell stage. (A',B',C',D') Injection of Cs-FoxD morpholino into fertilized eggs: (A',C') injected embryos at the tailbud stage and (B',D') injected embryos arrested at the 110-cell stage. Arrowheads in D' indicate the expression of Cs-THR in presumptive notochord cells in addition to endoderm cells. Scale bars: in A, 100 µm for A',C,C'; in B, 100 µm for B',D,D'.

View larger version (68K): [in a new window]   Fig. 6. Effects of suppression of Cs-FoxD function with morpholino on differentiation of (A,A') epidermal cells, (B,B') muscle cells, (C,C') mesenchyme cells and (D,D') the nervous system. In situ hybridization with probes for (A) epidermis-specific gene Cs-Epi1, (B) muscle-specific actin gene Cs-MA, (C) mesenchyme-specific gene Cs-mech1 or (D) nervous system-specific gene Cs-ETR. (A-D) Control embryos and (A'-D') embryos injected with Cs-FoxD morpholino. Scale bars: in A, 100 µm for A,A'; in D, 100 µm for B-D'.

View larger version (105K): [in a new window]   Fig. 7. Effects of suppression of Cs-FoxD function with morpholino on induction of notochord cells. (A-F) In situ hybridization to examine Cs-Bra expression. (A) Normal 110-cell embryo, (B) Cs-FoxD morpholino-injected embryo and (C) an embryo injected with both Cs-FoxD morpholino and synthetic Cs-FoxD mRNA. (D) A 110-cell stage embryo developed from two-cell embryo whose right AB2 was injected with Cs-FoxD morpholino; (E) 110-cell stage embryo developed from four-cell embryo whose right B3 was injected with Cs-FoxD morpholino, and (F) 110-cell stage embryo developed from four-cell embryo whose right A3 was injected with Cs-FoxD morpholino. Black arrowheads indicate Cs-Bra expression in A-line notochord cells. Black arrows indicate Cs-Bra expression in B-line notochord cells. White arrowheads and white arrows indicate the failure of Cs-Bra expression. (G-I) Expression of a notochord-specific gene, Cs-fibrn. (G) Normal tailbud embryo; (H) Cs-FoxD morpholino-injected embryo, and (I) an embryo injected with both Cs-FoxD morpholino and synthetic Cs-FoxD mRNA. Scale bars: in A, 100 µm for B,C,D,E,F; in G, 100 µm for H,I.

View larger version (34K): [in a new window]   Fig. 8. Semi-quantitative RT-PCR analysis of the amounts of Cs-THR and Cs-ETR mRNAs in control embryos and embryos injected with Cs-FoxD morpholino.

View larger version (66K): [in a new window]   Fig. 9. Expression of Cs-Notch gene. (A) The partial amino acid sequence of Cs-Notch encoded by the cDNA used. Although the sequence lacks the N-terminal half, it contains four EGF repeats, three DSL domains, the transmembrane domain and six ankyrin repeats. (B-F) Expression of Cs-Notch revealed by whole-mount in situ hybridization. (B) Fertilized egg, (C) eight-cell stage embryo, (D) 32-cell stage embryo, (E) neurula and (F) tailbud embryo. Scale bar: 100 µm.

View larger version (44K): [in a new window]   Fig. 10. Relationships of Cs-FoxD with Cs-FGF4/6/9 and Cs-Notch. Expression of a notochord-specific Cs-fibrn gene in (A) control embryos, (B) embryos developed from eggs injected with the constitutively active form of Notch mRNA, (C) embryos developed from eggs co-injected with Cs-FoxD morpholino and constitutively active form of Notch mRNA, and (D) embryos developed from eggs co-injected with Cs-FoxD morpholino and Cs-FGF4/6/9 mRNA.