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Multiple functions of a Zic-like gene in the differentiation of notochord, central nervous system and muscle in Ciona savignyi embryos

Kaoru S. Imai^*,, Yutaka Satou^* 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 (79K): [in a new window]   Fig. 1. Nucleotide and deduced amino acid sequences of cDNA for Cs-ZicL. The 1217-bp insert includes a single open reading frame that encodes a polypeptide of 355 amino acids. The termination codon is indicated by an asterisk, and polyadenylation signal sequences is underlined. Predicted zinc finger domains are shown by bold letters. The nucleotide sequence of the 5' region used to prepare the morpholino is boxed.

View larger version (51K): [in a new window]   Fig. 2. Molecular relationship of Cs-ZicL with Zic-related proteins. (A) Alignment of amino acid sequences of the zinc finger domains (boxed) of Cs-ZicL, Cs-macho1, macho-1, and mouse Zic3. Identical residues are shaded black, and similar residues are shaded gray. Cysteine and hystidine residues conserved in Zic-related proteins are indicated by asterisks. (B) Unrooted molecular phylogenetic tree constructed by the neighbor-joining method using the zinc-finger domain sequences (Saitou and Nei, 1987).

View larger version (77K): [in a new window]   Fig. 3. Zygotic expression of Cs-ZicL in Ciona savignyi embryos, as revealed by whole-mount in situ hybridization. A'-D' are drawings of A-D, respectively, to illustrate the Cs-ZicL expression. The dots indicate expression in the nucleus. (A) A 16-cell stage embryo, vegetal view with the anterior pole on top. Scale bar represents 100 µm for A-G. (B) A 32-cell stage embryo, vegetal view. Zygotic transcripts of Cs-ZicL appear in A6.2, A6.4, B6.2 and B6.4 cell-pairs. (C) A 64-cell stage embryo, vegetal view. Signals are evident in A7.3, A7.4, A7.7, A7.8, B7.3, B7.4, B7.7, B7.8 and B7.5 cell-pairs. (D) A 110-cell stage embryo, vegetal view. Zygotic transcripts are seen in a-line CNS cells and b-line CNS and muscle cells in addition to A-line nerve cord cells and B-line muscle cells. (E) A gastrula, vegetal view. Signals are evident in cells of the CNS. (F) A neurula, dorsal view, showing Cs-ZicL transcripts in a few anterior-most cells of the embryo (arrowhead). (G) An early tailbud embryos, lateral view. Signals have become undetectable.

View larger version (63K): [in a new window]   Fig. 4. Effects of suppression of Cs-ZicL function on differentiation of (A) endodermal cells, (B) epidermal cells, and (C,D) mesenchyme cells. (A,A') Histochemical detection of endoderm-specific alkaline phosphatase (AP) activity, and in situ hybridization with probe for (B,B’) epidermis-specific gene Cs-Epi1 and (C,C',D,D') mesenchyme-specific gene Cs-mech1. (A-C) Control embryos and (A'-C') embryos developed from eggs injected with Cs-ZicL morpholino. (D,D') Expression of Cs-mech1 in (D) control and (D') experimental embryos developed from eggs injected with Cs-ZicL morpholino, arrested at the 110-cell stage with cytochalasin B. Scale bar (in A) represents 100 µm for all panels.

View larger version (83K): [in a new window]   Fig. 5. Effects of suppression of Cs-ZicL function on differentiation of notochord cells. (A-A'') Expression of Cs-fibrn in (A) control embryos at the early-tailbud stage and (A') experimental embryos developed from eggs injected with Cs-ZicL morpholino. (A'') Expression of Cs-fibrn in experimental embryos developed from eggs co-injected with Cs-ZicL morpholino and Cs-ZicL mRNA. (B,B') Expression of Cs-fibrn in (B) control and (B') experimental embryos developed from eggs injected with Cs-ZicL morpholino, arrested at the 110-cell stage. Scale bar (in A) represents 100 µm for all panels.

View larger version (41K): [in a new window]   Fig. 6. Relationship between Cs-ZicL, ß-catenin, and Cs-FoxD. (A) Control embryos at the 32-cell stage showing Cs-ZicL expression in two pairs of A-line cells (arrowheads) and two pairs of B-line cells (arrows). Scale bar represents 100 µm. (B) Cadherin-overexpressing embryos at the 32-cell stage, showing Cs-ZicL expression in the two pairs of B-line cells (arrows), but not in the two pairs of A-line cells. (C) Cs-ZicL expression is found only in the two pairs of B-line cells in the 32-cell stage embryos developed from eggs injected with Cs-FoxD morpholino. Scale bar represents 100 µm. (D) Ectopic Cs-ZicL expression in many cells in the 32-cell stage embryos developed from eggs injected with Cs-FoxD mRNA.

View larger version (85K): [in a new window]   Fig. 7. Effects of suppression of Cs-ZicL function on differentiation of cells of the CNS. (A) Control embryos showing expression of CNS-specific Cs-ETR gene. Scale bar represents 100 µm. (A') Experimental embryos developed from eggs injected with Cs-ZicL morpholino.

View larger version (94K): [in a new window]   Fig. 8. Effects of functional suppression of Cs-ZicL and/or Cs-macho1 on the differentiation of muscle cells assessed by in situ hybridization with a probe for muscle actin gene Cs-MA1. (A-A'') Control embryos at (A) the early-gastrula stage, (A') mid-gastrula stage and (A'') early-tailbud stage. (B-B'') Cs-ZicL morpholino-injected embryos at (B) the early-gastrula stage, (B') mid-gastrula stage and (B'') early-tailbud stage. (C) Experimental embryos developed from eggs co-injected with Cs-ZicL morpholino and Cs-ZicL mRNA, showing recovery of Cs-MA1 expression in B8.7 and B8.8 cells (arrowheads). (D-D'') Cs-macho1 morpholino-injected embryos at (D) the early-gastrula stage, (D') mid-gastrula stage and (D'') early-tailbud stage. Arrowheads indicate the expression of Cs-MA1 in B6.2-derived muscle cells and arrows indicate the expression in B6.4-derived muscle cells. Scale bar (in A) represents 100 µm for all panels.

View larger version (47K): [in a new window]   Fig. 9. Relationship between Cs-macho1 and Cs-ZicL. (A) The 32-cell stage embryos developed from eggs injected with Cs-macho1 morpholino, showing that Cs-ZicL expression is not affected by functional inhibition of Cs-macho1. Scale bar represents 100 µm. (B,C) Expression of Cs-MA1 is suppressed in (B) early-gastrulae and (C) early-tailbud embryos, developed from eggs co-injected with Cs-macho1 morpholino and Cs-ZicL morpholino.