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Fig. 2. FoxF is an immediate target of the FGF/MAPK/Ets pathway.
(A, top) Map of the FoxF gene (red) and 5' upstream
region on chromosome arm 3q. (A, middle) VISTAplot showing sequence
conservation (50-100%) between two Ciona species
(http://genome.lbl.gov/vista/index.shtml).
(A, bottom) Summary diagram of trunk ventral cell (TVC) and epidermis
expression with various 5' enhancers fused to the lacZ
reporter. (B) Embryo expressing the -3052 to +1 enhancer attached to
the lacZ reporter, showing TVC (arrow) and epidermal expression.
(C) The FoxF minimal TVC enhancer sequence (-1135 to -840) is
highly conserved between C. intestinalis and C. savignyi.
Boxes indicate the E-box (red) and the three Ets1/2-binding sites (green).
(D) A 295 bp (-1135 to -840 bp) genomic DNA fragment, fused to the
Forkhead (FoxA-a) basal promoter, drives lacZ
expression specifically in the TVCs (arrow). (E) Mutational analysis.
The histogram displays the proportions of embryos showing TVC staining
(n=total embryos). Diagrams depict the wild-type -1135 to -840
fragment and the indicated mutations. (F) The -3052 to +1
cis-regulatory region with deleted E-box motif drives expression in epidermis
only. (G) The -1135 to -840 (
E-box) does not drive lacZ
expression in TVCs (arrow). (H) Cis-trans complementation test.
Ets:VP16 can restore enhancer activity of the -1135 to -840 (E-box)
construct. The cis-trans complementation is abolished when the three Ets1/2
sites are mutated (errors bars indicate standard deviation). (I) The
-1135 to -840 (E-box) enhancer co-electroporated with Mesp>Ets:VP16.
Ets:VP16 causes all four B7.5 cells to migrate into the trunk and causes the
mutated enhancer to drive lacZ expression in all cells. Arrows in
B,D,F,G,I point to the TVCs (not stained in F and G).
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