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First published online March 1, 2004
doi: 10.1242/10.1242/dev.01011

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A zinc finger transcription factor, ZicL, is a direct activator of Brachyury in the notochord specification of Ciona intestinalis

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

View larger version (58K): [in a new window]   Fig. 1. (A) Alignment of the amino acid residues of the central region of Ci-ZicL with those of Cs-ZicL. The five zinc finger motifs are boxed, and conserved CCHH are indicated by asterisks. This region was used to produce a GST/ZicL(ZF) fusion protein. (B-D) Zygotic expression of Ci-ZicL, as revealed by whole-mount in-situ hybridization. (B) A 32-cell, (C) a 64-cell and (D) a 110-cell stage embryo, vegetal view. (E,F) The effect of suppression of Ci-ZicL function on expression of Ci-Bra, in (E) a control and (F) a Ci-ZicL-suppressed embyro at the 110-cell stage. (G-J) Schematic representations of the expression of FoxD, ZicL, Snail and Brachyury genes. Embryos at (G) the 16-cell, (H) 32-cell, (I) 64-cell and (J) tailbud stage, viewed from (G-I) the vegetal pole and (J) the lateral side. Blastomeres are named according to the nomenclature of Conklin (1905). Blastomeres whose names are colored green in (H) and (I) are those that give rise to notochord cells.

View larger version (65K): [in a new window]   Fig. 3. ZicL-binding sequences in the cis-regulatory region of the Ci-Bra gene. (A) DNA sequence of the minimal 483-bp Ci-Bra enhancer (see Corbo et al., 1997; Fujiwara et al., 1998). Solid unfilled boxes, dotted unfilled boxes, filled boxes and the dotted line indicate Ci-Snail-binding sites, Su(H)-binding sites, E box sequences and a TATA element, respectively. The arrow represents the presumptive transcription start site. Solid lines represent elements with sequence similarity to the consensus ZicL-binding sequence. (B,C) A gel shift assay of the binding of GST/Ci-ZicL(ZF) to oligonucleotides corresponding to various locations in the Ci-Bra transcription regulatory region. Oligonucleotide sequences shown in (B) were examined in a gel-shift assay (C). The boxed letters of ZicL-b indicate the consensus ZicL-binding sequence. The shaded letters in Bra-123 to Bra-1996 indicate nucleotides identical to those in the consensus ZicL-binding sequence. The binding seemed specific because it was not detected under conditions of zinc-removed incubation (lane 1), pre-incubation with x100 molar excess of unlabeled competitor DNA or replacement by GST protein (data not shown). (D) Comparison of the Brachyury upstream sequences between Ciona intestinalis (upper) and C. savignyi (lower). Boxes in the C. intestinalis sequence indicate presumptive ZicL-binding sites, and solid lines in the C. savignyi sequence indicate sequences that resemble the consensus ZicL-binding sequence.

View larger version (80K): [in a new window]   Fig. 4. Requirement of potential ZicL-binding sites for Ci-Bra expression in notochord cells. Reporter gene expression in embryos microinjected with (A-C) p(-483)Ci-Bra/lacZ and (D-F) p(-3.5k)Ci-Bra/lacZ, revealed by whole-mount in-situ hybridization. (A,B,D,E) Constructs without mutation or (C,F) ZicL-b1/ZicL-b2 double-mutations were introduced and examined at the (A,D) 64-cell stage or (B,C,E,F) 110-cell stage. (G) The frequency of the tailbud stage embryos with the reporter gene expression, revealed by histochemical detection of ß-galactosidase activity. The electroporated Ci-Bra/lacZ fusion genes with the mutated ZicL-binding sites are listed on the left. Reporter gene expression in embryos electroporated with (H-K) p(-483)Ci-Bra/lacZ and (L-O) p(-3.5k)Ci-Bra/lacZ. (H,L) Constructs without mutation, (I,M) ZicL-b1 mutated, (J,N) ZicL-b2 mutated or (K,O) ZicL-b1/ZicL-b2 double-mutated. High-magnification images of the embryos are shown in insets.

View larger version (70K): [in a new window]   Fig. 2. Characterization of nucleotide sequences recognized by GST/ZicL(ZF) fusion protein. (A) SDS-PAGE of purified GST/Ci-ZicL(ZF) fusion protein (lane 2) and GST protein (lane 3). The molecular mass relative to the Marker (lane 1) shows the successful production of the fusion protein. (B) Compilation of the GST/Ci-ZicL(ZF) and GST/Cs-ZicL(ZF) binding sequences. DNA sequences that bound to the fusion proteins were selected and determined as described in Materials and methods. Cloned random sequences, after eight or 10 rounds of selection for GST/Ci-ZicL(ZF) and eight rounds for GST/Cs-ZicL(ZF), were aligned. The bottom sequence in each table represents the compiled most favored sequence at each nucleotide position. The underlined ggatc is identical to the 3' end of primer F1. (C,D) A mutation analysis of the binding sequence of GST/Ci-ZicL(ZF). Eleven types of oligonucleotides (C) were examined by a gel-shift assay (D). The boxed uppercase letters of the ZicL-b oligonucleotides indicate the consensus ZicL-binding sequence. The shaded lowercase letters in the µA-µJ oligonucleotides indicate mutated nucleotides in each mutant oligonucleotide. The binding seemed specific because it was not detected under conditions of zinc-removed incubation (lane 1), pre-incubation with x100 molar excess of unlabeled competitor DNA or replacement with GST protein (data not shown).

View larger version (85K): [in a new window]   Fig. 5. Misexpression of Ci-ZicL promotes ectopic lacZ expression in blastomeres of non-notochord lineages, while suppression of Ci-ZicL does not activate lacZ expression. (A) p(-3.5k)Ci-Bra/lacZ and Ci-ZicL mRNA, (B) p(-3.5k)Ci-Bra/lacZ with ZicL-b1/ZicL-b2 double-mutation and Ci-ZicL mRNA, or (C) p(-3.5k)Ci-Bra/lacZ and Ci-ZicL morpholino antisense oligonucleotides were injected into fertilized eggs, and the reporter expression was examined at the 110-cell stage by whole-mount in-situ hybridization. (D,E) p(-3.5k)Ci-Bra/lacZ, (F) p(-3.5k)Ci-Bra/lacZ and Ci-ZicL mRNA, or (G) p(-3.5k)Ci-Bra/lacZ with ZicL-b1/ZicL-b2 double-mutation and Ci-ZicL mRNA were injected into fertilized eggs, embryos were allowed to develop to the 110-cell stage and cleavage was arrested for about 6 hours (equivalent to the early tailbud stage), and then the reporter gene expression was examined by histochemical detection of ß-galactosidase activity.

View larger version (25K): [in a new window]   Fig. 6. Schematic representation of the transcriptional control of Ci-Bra. See the text for details.