Rosa26 promoter analysis and design of deletions for signaling sentinel substitution. (A) Scheme depicts the Rosa26 locus; arrow, transcribed region. Boxes above depict sequence identities over 95% and 80% for mouse and rat (mo:ra) and mouse and human (mo:hu), respectively. Red bars within the boxes indicate conserved domains, largely extending to −3 kb of the start site of transcription. (B) VISTA plot of transcription factor motif distribution across the proximal 3 kb of the mouse Rosa26 promoter region. Vertical dotted line indicates a marked increase in motif density at −1.2 kb. (C) Comparison of the mouse and human Rosa26 promoter regions. A natural break occurs at −228 bp and a CCAAT box is evident downstream of −60 bp. (D) RMCE at the Rosa26 locus (upper scheme) results in substitutions that replace the regions from −4 kb to +1 kb with recombinant sequences where signaling response sentinels (green boxes) are inserted between deletion endpoints −60, −228, −1217 and −3000 and a TATA box fused to a coding sequence for CFP.

RMCE at the Rosa26 locus to generate signaling sentinel ES cells. (A) Upper scheme depicts the natural Rosa26 locus; lower scheme, Rosa26 with an acceptor cassette substitution generated by homologous recombination in ES cells. Orange boxes indicate heterologous lox target sites, allowing unidirectional insertion of donor cassette in step B. The puroR sequence allows positive selection for recombination. (B) Upper scheme indicates signaling sentinel delivery plasmid transfected into acceptor ES cells. When co-transfected with a plasmid expressing cre recombinase, the plasmid undergoes unidirectional insertion into the acceptor locus (lower scheme). The appropriately structured ES lines are confirmed by PCR and used to generate chimeric mice; the mice are bred for germ line transmission of the recombined locus. (C) When the resulting mouse strain is crossed to a line expressing FRT recombinase in its germ lineage, the FRT recombination sites (blue) allow the subsequent excision of the selectable markers, yielding a mouse with the appropriate signaling sentinel at the Rosa26 locus.

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.

Retinoic acid sentinels in live embryos and responding to exogenous RA in half-embryo cultures. (A) Merged bright field and fluorescence images of live E8.25 and E9.5 embryos containing the retinoic response sentinel DR5-TA-CFP at the −60 and −228 sites of the endogenous Rosa26 promoter. Composite images were generated with Photoshop. Fluorescent domains are noted. (B) Half-embryo fragments from E8.5 embryos of the designated genotypes were cultured overnight with 1 μM RA or DMSO carrier (control). Images are shown before (left) and after (right) culture, revealing an anterior-ward extension of the fluorescent signal upon RA treatment for each construct (white arrows). In supplementary material Fig. S3, data are shown indicating that only RA extended the CFP expression domain anteriorly, and not the RA inhibitor AGN193109, BMP4, noggin, a TGFβ inhibitor or a MAPK inhibitor, demonstrating specificity.

Dose-responsiveness and specificity of BMP and activin A signaling sentinels. (A) ES cell signaling sentinel lines with response elements for BMP (IBRE-4-CFP) or activin A (AR8-TA-mCherry) inserted at the −60 and −228 sites of the endogenous Rosa26 locus. All cells exhibited responses to the respective agonists and not their antagonists (100 nM LDN193189 and 10 μM SB431542, respectively). Assays were for 48 hours. (B) Dose-response tests of the BMP and activin A (ActA) sentinels substituted at −60 and −228, demonstrating high sensitivity of both constructs for each reporter type. Individual FACS profiles are shown in supplementary material Fig. S4. (C) Specificity tests demonstrate high specificity of responsiveness, particularly illustrating that the BMP reporter responds to BMP4 and not activin A, whereas the activin A reporter responds to activin A and not BMP (doses as for Fig. 3). One-tailed t-tests of drug-treated samples compared with DMSO controls; **P<0.01.

BMP and activin A sentinels in live embryos. (A) Live E6.5 and E8.25 embryos containing the BMP sentinel IBRE4-TA-CFP substituted at −60 and −228 of the endogenous Rosa26 promoter. Fluorescence is in embryo domains as shown. (B) Live E9.5 embryos containing the BMP or activin A sentinels at −60 (BMP) or −60 and −228 (activin A). The heart, midgut and branchial arches constitute overlapping expression domains, whereas BMP signaling is more sharply revealed in the somatic regions and into portions of the head. Activin A signaling is reported more diffusely in the limb buds and elsewhere.

Specificity of Wnt signaling sentinels and activity in live embryos. (A) Cell signaling sentinel lines with response elements for Wnt (suTOP-TA-CFP) inserted at the −60 and −228 sites of the endogenous Rosa26 locus. All cells exhibited responses to medium containing LIF plus 2i, which contains 3 μM CHIR99021, a GSK3β antagonist that promotes Wnt signaling, and not to medium containing LIF plus BMP4, with the −60 construct exhibiting a stronger fluorescence signal. The cells harboring the −60 construct responded more weakly to Wnt3a added to the medium, whereas the −228 cells exhibited minimal induction. The presence of the Dkk1 inhibitor of Wnt signaling suppressed the Wnt3a-dependent response in the −60 cells. (B) Dose-responsive tests of the −60 and −228 reporters, as quantified from FACS profiles shown in supplementary material Fig. S5. The data show that the Wnt reporters do not respond to N2B27 medium, N2B27 medium with 1500 U/ml LIF (L) and 10 ng/ml BMP4 (B), 320 ng/ml Dkk1, 100 ng/ml activin A (ActA), 10 ng/ml BMP4, 50 ng/ml Fgf2, 10⁻⁶ M RA or 15 mM Shh. (C) Different live E9.5 embryos harboring the −60/SuTOP-TA-CGP construct, exhibiting fluorescence in designated areas. One-tailed t-tests of drug-treated samples compared with DMSO controls; *P<0.05.

Developing live cell sentinel for Notch signaling. Cell signaling sentinel lines with 12×CSL-TA-mOrange response elements for Notch were inserted at the −60 and −228 sites of the endogenous Rosa26 locus. The ES sentinel lines were transfected with an empty IRES-GFP vector; the vector encoding a constitutively active NICD, followed by the IRES-GFP; the vector encoding the membrane-bound NEXT; and the vector with NEXT in the presence of the γ-secretase inhibitor Compound XVIII (1 μM). Transfected cells were quantified by GFP fluorescence, which was then used to normalize for mOrange fluorescence (see supplementary material Fig. S6). The NICD induced mOrange significantly, the NEXT fragment more weakly so, and the γ-secretase inhibitor blocked such signaling for both the −60 and −228 constructs.