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First published online 5 October 2005
doi: 10.1242/dev.02051

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Uncoupling heart cell specification and migration in the simple chordate Ciona intestinalis

Department of Molecular and Cellular Biology, Division of Genetics and Development, University of California, Berkeley, CA 94720, USA

View larger version (53K): [in a new window]   Fig. 1. T-box binding sites are essential components of the Ciona Mesp regulatory elements. (A) Diagram depicting the two lineages derived from the B7.5 cells (TVC, trunk ventral cells). (B) Transgenic, gastrulating embryo expressing the 110-bp Ci-Mesp-lacZ reporter gene, hybridized with a probe against lacZ mRNA. (C) Transgenic tailbud embryo expressing the 110-bp Ci-Mesp-lacZ reporter gene, stained with X-gal. (D) Transgenic tailbud embryo expressing the 107-bp Ci-Mesp-lacZ reporter gene, stained with X-gal. (E) Upstream sequences for Ci-Mesp and Cs-Mesp; numbers indicate the distance from the putative transcription start site. Black bars indicate minimal 110- and 105-bp enhancers. Red bars represent the distal deletions that rendered the minimal enhancers inactive. Putative T-box binding motifs are boxed and lettered; matches to the Ciona Tbx6-binding site are highlighted in green lettering. Gray boxed areas indicate the only stretches of conserved sequence between the two enhancers. (F) Summary of expression obtained with Ci-Mesp-lacZ fusion genes. Putative T-box binding motifs are indicated by lettered boxes. The motif sequences below show matches to the Tbx6 consensus-binding site (Yagi et al., 2005) in green; mutated nucleotides for disruption of the binding motif are shown in red, whereas those for enhancement of the motif are in blue. +++, strong, consistent staining of B7.5 lineages; +, weak, inconsistent staining; 0, no staining. All results are representative of at least two trials and were unambiguous for the hundreds of embryos observed in each trial.

View larger version (87K): [in a new window]   Fig. 2. Direct activation of Ciona Mesp by Tbx6c. (A-H) In situ hybridizations, all embryos are shown from the vegetal (future dorsal) side except for the one in G, which is shown from the ventral side; arrows in E-G indicate B7.5 lineage cells. (A) 32- to 64-cell stage embryo hybridized with a probe against Tbx6c (blue). (B) Mesp-lacZ transgenic embryo at the same stage hybridized with probe against lacZ (blue). (C) 110-cell embryo hybridized with probe against Tbx6c (blue). (D) Mesp-lacZ transgenic 110-cell embryo co-hybridized with probes against Tbx6c (blue) and lacZ mRNAs (red). Nuclear staining by the lacZ probe indicates nascent transcripts, whereas Tbx6c transcripts are detected in the cytoplasm owing to an earlier onset of expression. (E) Gastrulating embryo hybridized with a probe against Tbx6c (blue). (F) Mesp-lacZ transgenic embryo at the same stage hybridized with a probe against lacZ (blue). (G) Embryo following gastrulation hybridized with probe against Mesp (blue). The B7.5 lineage cells have now involuted to the ventral side, soon after this stage Mesp expression becomes undetectable. (H) Mesp-lacZ transgenic 110-cell embryo co-hybridized with probes against Tbx6b (red) and lacZ mRNA (green), such asymmetric left- or right-sided incorporation of the Mesp-lacZ reporter gene was a common occurrence. (I) Gel shift assays. The GST-Tbx6c fusion protein was incubated with radiolabeled sequences containing each of the putative Tbx6-binding sites from the Ci-Mesp 110-bp enhancer. The first lane for each probe demonstrates binding to the GST-Tbx6c fusion protein. In the second lane, unlabeled competitor inhibited binding. For site B, a fragment containing a mutated binding site (Ci-B Mut-1) fails to inhibit binding (lane 3). (J) Diagrams of conserved blocks of sequence from the 5' flanking regions of vertebrate Mesp2 and Mesp1 genes. The characterized mouse Mesp2 enhancer region (Haraguchi et al., 2001) is highly enriched for probable T-box binding motifs in two out of the three conserved sequence blocks. Alignment of zebrafish and Fugu Mesp1 flanking DNA reveals a small block of conserved proximal sequence highly enriched with putative T-box binding motifs. The characterized mouse Mesp1 enhancer region also contains numerous probable T-box binding sites (Haraguchi et al., 2001) (data not shown), but mouse and zebrafish sequences do not align. Conservation of the putative T-box binding sites is shown in green; in the motif sequence shown below, capital letters indicate conservation and green indicates a match to the consensus. Numbers indicate the distance in base pairs from the zebrafish translation start site.

View larger version (63K): [in a new window]   Fig. 3. Detailed visualization of heart cell migration. (A-H) Transgenic Mesp-GFP embryos. (A) Early tailbud embryo, 12 hours post-fertilization (hpf), ventral view. (B) Tailbud embryo (14 hpf), lateral view. (C) Ventrolateral view of a slightly more advanced embryo (15 hpf). Note that caudal siblings are elongated with punctate accumulations of GFP. (D) Late tailbud embryo (17 hpf), including magnified views of the two lineages. (E-G) High magnification ventral views of three sets of TVCs from progressively older embryos (17-18 hpf). (H) Ventrolateral view of an embryo at 18 hpf. All confocal images display GFP fluorescence in green and are shown with the anterior to the left. In A,C and H, the red channel displays phalloidin staining (Alexa-fluor 647, Molecular Probes). In B and D, the red channel displays auto-fluorescence.

View larger version (94K): [in a new window]   Fig. 4. Activator Mesp fusion protein blocks heart cell migration. (A-C) Transgenic Mesp-GFP embryos. (D-F) Embryos co-electroporated with Mesp-VP16. (A,D) Tailbud embryos (14 hpf) displayed laterally. (B,E) Late tailbud embryos (18 hpf) displayed laterally. (C,F) Late tailbud embryos (18 hpf) displayed ventrolaterally. Embryos are oriented with anterior to the left; GFP fluorescence is in green, phalloidin staining in red. (G,H) Still shots from the movies of transgenic Mesp-VP16 juveniles (Movies 1 and 2 in the supplementary material, respectively). Red arrowhead marks ectopic beating heart tissue; arrow marks the site of tail resorption. The site of typical heart formation is marked by a black arrowhead. E, endostyle; S, stomach. Lateral views with anterior to the left.

View larger version (59K): [in a new window]   Fig. 5. Activator and repressor Mesp fusion proteins alter the expression of B7.5 lineage markers. (A-C) Control, (D-F) Mesp-VP16. (A,D) Transgenic Mesp-lacZ embryos. (B,E) Embryos stained using a probe for Hand-like. (C,F) Embryos stained using a probe for Raldh2. All embryos are shown laterally, anterior to the left.

View larger version (18K): [in a new window]   Fig. 6. Model for heart specification in Ciona. Stages are shown on the top line. (A) At the 16- to 64-cell stages maternal Macho 1 directs the expression of Tbx6b and Tbx6c, whereas maternal ß-catenin directs the expression of an additional activator. At the 110-cell stage, Tbx6 and `Activator X' drive the expression of Mesp in the B7.5 cells. Mesp expression initiates conditional heart specification (represented by the red and yellow pattern). During gastrulation, the B7.5 cells have divided to form a cluster of four cells. An inductive `Signal X' then synergizes with Mesp to permit further heart differentiation in the anterior daughters, whereas the posterior daughters revert to an anterior tail muscle fate. (B) Targeted expression of the constitutively active MespVP16 bypasses the requirement for the inductive signal, leading to heart differentiation in the entire B7.5 lineage.

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