A Maternal System Initiating the Zygotic Developmental Program through Combinatorial Repression in the Ascidian Embryo
Fig 3
Tcf7-binding sites are critical for expression in the vegetal hemisphere domain (VD).
(A) Analysis of a regulatory region of Fgf9/16/20. Illustrations on the left depict the constructs. Green boxes indicate the Gfp gene and SV40 polyadenylation signal. The numbers indicate the relative nucleotide positions from the transcription start site of Fgf9/16/20. Mutant Tcf7-binding sites are indicated by X. The graphs show the percentage of blastomeres expressing the reporter in the anterior vegetal blastomeres, posterior vegetal blastomeres, and animal blastomeres. Note that not all cells or embryos could express the reporter because of mosaic incorporation of the electroporated plasmid. (B, C) Images showing Gfp expression, which was revealed by in situ hybridization, in embryos electroporated with the fourth and eighth constructs shown in (A). Scale bar, 100 μm. (D) Mapping of the Tcf7 ChIP data onto genomic regions consisting of the exons and upstream region of Fgf9/16/20. ChIP-chip data are shown in bars. ChIP-seq data are shown as a magenta line. Each graph shows the fold enrichment (y-axis) for the chromosomal region over Fgf9/16/20 (x-axis). A green box indicates the region essential for specific expression, which was revealed by reporter gene assays. This region overlapped peaks identified by the peak caller programs for ChIP-seq and ChIP-chip. (E) Gel-shift analysis showing that Tcf7-binding site b did not bind the GST protein but bound the Tcf7-GST fusion protein. The shifted band was greatly reduced by incubation with a specific competitor, but not a competitor with a mutant Tcf7-binding site b.