The minimal promoter of cxcr4b. (A and B) transient expression of RFP under the control of the 139-bp cxcr4b promoter fragment in a migrating primordium (A) and in a deposited neuromast (B) in cldnb:gfp embryos. (Left) RFP fluorescence, (Right) RFP in red, merged with GFP in green. (C) Stable expression of RFP driven by the 139-bp promoter in a line doubly transgenic for cxcr4b:rfp and cldnb:gfp. (D) Coexpression of RFP (red) and GFP (green) in the same embryo. (C and D) Assembled from four consecutive frames of a Z-stack; the slight mismatch between green and red, in some cells, is due to migration during the imaging process. In this and all other figures anterior is to the left, and the primordium is migrating to the right. (Scale bars: 50 μm.) (E) Low-scale view of an embryo doubly transgenic for cxcr4b:rfp and cldnb:gfp, illustrating the complete absence of RFP expression in anterior lateral line neuromasts (ALL) and in the infraorbital anterior lateral line primordium (ALL io) as well as in the otic system (arrowheads). Faint RFP fluorescence can be detected in the PLL ganglion (PLLg). (F) Comparison of the sequences upstream of the TATA box in Danio cxcr4b, Homo cxcr4, and Danio cxcr4a promoters. The 139-bp cxcr4b minimal promoter defined by us begins one base upstream of the ESR1 putative binding site and includes the initiation ATG; the 157-bp minimal promoter defined in Homo (15) begins 10 bases upstream of the ESR1 binding site and does not include the initiation ATG. The corresponding sequence in the promoter of cxcr4a is also shown. Putative binding sites according the TESS software are colored. The ESR1 binding sites in cxcr4b and human cxcr4 (red) are both followed by a C, consistent with the presence of a pyrimidine at this position in the consensus binding site. The binding sites for SP1 and TATA Binding Protein have been shown to be instrumental for cxcr4 expression in the minimal human promoter (26).
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