Dose-responsiveness and specificity of retinoic acid signaling sentinels. Dose-responsiveness and specificity of retinoic acid signaling sentinels. (A)

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Dose-responsiveness and specificity of retinoic acid signaling sentinels. Dose-responsiveness and specificity of retinoic acid signaling sentinels. (A) ES lines containing the DR5 RA response elements substituted for deletions of the Rosa26 promoter to −60, −228, −1217 and −3000 bp, visualized by phase contrast microscopy and for CFP fluorescence, in the presence and absence of 10 μM all-trans RA for 24 hours. The −60 construct had evident background fluorescence and exhibited increased visual fluorescence with RA treatment. The −228 construct had no background fluorescence and lower induced fluorescence with RA. The −1217 and −3000 constructs exhibited little or no fluorescence under either condition. Separate TA-CFP constructs without DR5 elements, inserted at −60 and −228, exhibited no fluorescence (data not shown). An experiment conducted with a 4.5-hour induction time is shown in supplementary material Fig. S1. (B) Higher magnification views of induced panels in A. (C) Dose-response curves where the percentage of CFP fluorescence-positive cells above a threshold defined in supplementary material Fig. S2 are quantified by FACS over a range of RA concentrations, with and without 10−5 M antagonist (AGN193109). Individual FACS profiles for each experimental point are shown in supplementary material Fig. S2, indicating that the cell induction profile distributions appear similar at each concentration of RA. (D) We tested a range of different ligands: RA, 10−6 M; activin A (ActA), 100 ng/ml; BMP4, 10 ng/ml; Fgf2, 50 ng/ml; Shh, 15 nM; and Wnt3a, 100 ng/ml. The data show that the −60 construct is sensitive to lower concentrations of RA than the −228 construct (C), and that both constructs exhibit a high degree of specificity of response to RA versus the other pathway agonists tested (D). The extracts in D were assayed after freezing at length, explaining why the overall %CFP+ cells is lower than in C. One-tailed t-tests of drug-treated samples compared with DMSO controls; *P<0.05. Palle Serup et al. Dis. Model. Mech. 2012;5:956-966 © 2012. Published by The Company of Biologists Ltd