Kinetics and yield of cDEX-E and cDEX-A uncaging at 365 nm as evidenced by HPLC. Time evolution of the composition of 3 μM cDEX-E (a) and 7 μM cDEX-A (b) solutions upon 365 nm illumination at 1.6×10−4 (a) and 1.1×10−4 (b) E.m−2.s−1. Markers: concentrations of the caged compounds cDEX-E or cDEX-A (squares) and photoreleased DEX (circles) as extracted from the peak areas in the HPLC chromatograms; Solid lines: Monoexponential fit with Eq. (S1) respectively yielding 800 and 904 s for the characteristic times of cDEX-E and cDEX-A, and 1061 s and 1434 s for DEX. Solvent: 10 mM PBS pH=7.4. T=293 K.

cDEX-A photoactivation in Ventx-GR-injected zebrafish and Xenopus embryos. Whereas the zebrafish (a) and Xenopus (d) embryos injected with Ventx-GR mRNA and incubated in the dark with 15 μM cDEX-A at 4 hpf (a,b; inset; scale bar: 100 μm) and blastula stage (d,e; inset; scale bar: 500 μm) exhibit normal phenotypes at 24 hpf and tailbud stage respectively, upon illumination at 365 nm for 600 s these zebrafish (b) and Xenopus (e) embryos reveal strong phenotypes at 24 hpf (b) and tailbud stage (e); c,f: Extent of Ventx-GR-induced phenotype determined at 24 hpf (c) and tailbud stage (f) in zebrafish (c) and Xenopus (f) embryos incubated (+) or not (−) at 4 hpf (c) and blastula stage (f) with 7 or 15 μM DEX or cDEX-A and illuminated (+) or not (−) at 4 hpf (c) and blastula stage (f) at 365 nm with 5.7×10−4 E.m−2.s−1 for 600 s (see Tables S3 and S4 for zebrafish and Xenopus embryos respectively). Bars: Extent of phenotypes C–I (dark cyan), C–II (orange), and C–III (dark red) (see Figures S3 and S4 and Tables S3 and S4). T=293 K.

Towards orthogonal light-driven phenotype generation from using caged dexamethasone and caged cyclofen. a-b: Evidence that photoreleased cyclofen CYC does not activate the GR receptor activated by photoreleased dexamethasone DEX in vivo. Representative phenotypes generated at 24 hpf from illuminating Ventx-GR-injected zebrafish embryos conditioned with 10 μM solution of cDEX-A (a) and cCYC (b) at 4 hfp and illuminated with 365 nm light to fully release DEX promoting GR activation (inset; scale bar: 100 μm). White and red arrow represent head and tail part of the zebrafish embryos respectively; c: Phenotype extents from a,b evaluated as reported in Figure S3 and Table S5; d-e: Evidence that DEX does not activate the ERT receptor targeted by photoreleased cyclofen CYC in vivo. d: Observed at 3 dpf, Tg(β−actin:loxP-EOS-stop-loxP-KRASG12V−T2A−H2B-mTFP; ubi:Cre-ERT; myl7:EGFP) zebrafish transgenic growing embryos ubiquitously express the EOS fluorescent protein in the cell cytoplasm in the absence of any CRE-ERT activation (no cCYC and UV); it results in the observation of cytoplasmic green fluorescence (top). In contrast, when these embryos have been further conditioned at 24 hfp with 6 μM cCYC and illuminated with 365 nm light to fully liberate CYC that promotes CRE-ERT activation, they express the H2B-mTFP fluorescent protein in their nuclei (bottom). e: Upon 10 μM DEX treatment at 24 hfp, the zebrafish transgenic growing embryos do not exhibit any CRE-ERT activation as evidenced by the observation of ubiquitous green fluorescence from EOS (top) and by the absence of any nuclear blue fluorescence from TFP (bottom). In d,e, the asterisk (*) indicates the eyes and the white arrow the heart, whereas a (anterior), p (posterior), d (dorsal), and v (ventral) indicate the body axes of the zebrafish transgenic growing embryos (inset; scale bar: 250 μm); f: Phenotype extents determined from d,e in the zebrafish transgenic growing embryos (see Table S6). T=301 K.