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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.
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