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Fig. 5. Ripply2 expression is regulated by the Notch and Wnt
pathways. (A) Comparative Vista plots of the mouse and human
Ripply2 loci identify highly conserved putative regulatory regions
(red) and coding exons (blue). Sequence identity (%) is indicated on the
right. lacZ reporter constructs driven by these Ripply2
regulatory elements are depicted below the Vista plot. Representative
transgenic founder embryos expressing the `enhancer/promoter' reporter (1),
the `enhancer' reporter (2), or the `promoter' reporter (3) are shown on the
right. The number of β-gal-positive transgenic embryos/total number of
transgenic founders is shown in brackets. The gradually diminishing reporter
expression in anterior somites is probably due to the perdurance of the stable
β-gal protein. (B) Luciferase reporter constructs are depicted on
the left. The enhancer and promoter elements are the same as in A. Graph
displaying activity of Ripply2 luciferase constructs in HEK293 cells
is shown on the right. Note that stabilized β-catenin expression had no
effect on Mesp2 or Tbx6 protein levels as assessed by western blot (not
shown). (C) Highly conserved region (33/34 bp identical) in the mouse
(m) and human (h) Ripply2 promoters contains an Ebox and
near-consensus Tbx6 binding site (BS). (D) An oligo containing the
putative Tbx6 BS bound specifically to FLAG-Tbx6 in EMSA assays. Binding of
Flag-Tbx6 protein to the wild-type oligo is specifically competed by adding
125x excess unlabeled oligo to the binding reaction.
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