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Development
2012 Jan 01;1392:437-42. doi: 10.1242/dev.072165.
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A photoactivatable small-molecule inhibitor for light-controlled spatiotemporal regulation of Rho kinase in live embryos.
Morckel AR
,
Lusic H
,
Farzana L
,
Yoder JA
,
Deiters A
,
Nascone-Yoder NM
.
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To uncover the molecular mechanisms of embryonic development, the ideal loss-of-function strategy would be capable of targeting specific regions of the living embryo with both temporal and spatial precision. To this end, we have developed a novel pharmacological agent that can be light activated to achieve spatiotemporally limited inhibition of Rho kinase activity in vivo. A new photolabile caging group, 6-nitropiperonyloxymethyl (NPOM), was installed on a small-molecule inhibitor of Rho kinase, Rockout, to generate a 'caged Rockout' derivative. Complementary biochemical, cellular, molecular and morphogenetic assays in both mammalian cell culture and Xenopus laevis embryos validate that the inhibitory activity of the caged compound is dependent on exposure to light. Conveniently, this unique reagent retains many of the practical advantages of conventional small-molecule inhibitors, including delivery by simple diffusion in the growth medium and concentration-dependent tuneability, but can be locally activated by decaging with standard instrumentation. Application of this novel tool to the spatially heterogeneous problem of embryonic left-right asymmetry revealed a differential requirement for Rho signaling on the left and right sides of the primitive gut tube, yielding new insight into the molecular mechanisms that generate asymmetric organ morphology. As many aromatic/heterocyclic small-molecule inhibitors are amenable to installation of this caging group, our results indicate that photocaging pharmacological inhibitors might be a generalizable technique for engendering convenient loss-of-function reagents with great potential for wide application in developmental biology.
Fig. 1. Synthesis of a photoactivatable Rho kinase inhibitor. (A) Caged Rockout (cRO) was generated by installing 6-nitropiperonyloxymethyl (NPOM) on the indole nitrogen of Rockout (RO). Exposure to UV light releases the NPOM caging group, restoring Rho kinase inhibitory activity. (B) Rho kinase activity was assayed in vitro using a Rho kinase assay, under standard conditions (---) or in the presence of DMSO (solvent control), 50 μM RO, or 50 μM cRO with (+ UV) and without (no UV) irradiation. The assay was also run without ATP as a negative control (no ATP). The data shown are representative of several independent trials; in the trial shown, the effect of cRO + UV is slightly greater than the effect of RO itself (P<0.05), but this result was not consistently observed. **, P<0.01 (one-way ANOVA); error bars indicate s.d. (C-H) NIH3T3 cells were untreated (C) or exposed to RO (D), DMSO (E,F) or cRO (G,H) and left in the dark (C-E,G) or exposed to UV irradiation (F,H). Blue, DAPI; green, phalloidin.
Fig. 2. In vivo efficacy of caged Rockout. (A-C) Stage 39 Xenopus embryos were exposed to 40 μM cRO for 2 hours, rinsed and individually irradiated on the right-hand side of the prospective gut (A); green-to-red photoconversion of EosFP indicates the decaged region (B, ventral view; C, right view). (D-I) Irradiated groups were then cultured in embryo medium (0.1× MMR) in the dark until the end of gut morphogenesis (stage 46). Embryos grown in the dark in 0.1× MMR (untreated, D), DMSO (F) or cRO (H) have long coiled guts, compared with those cultured in 30 μM RO (E), which have uniformly straight, un-elongated guts. Right side UV irradiation does not affect gut morphology in DMSO controls (G), but induces regions of defective elongation on the right side of the gut (arrowheads) in cRO-exposed embryos (I). (J-O) The proportion of normal (J), mild (K) and severe (L) gut elongation defects induced by decaging of cRO is dependent on the concentration of cRO to which the embryos are exposed (120 minutes uptake, 60 seconds irradiation; M), the uptake time (15 μM cRO, 60 seconds irradiation; N), and the length of UV irradiation (15 μM cRO, 120 minutes uptake; O). Control embryos may be exposed to UV for up to 2 minutes or cRO for up to 240 minutes (non-irradiated) without adverse effect (not shown).
Fig. 3. Caged Rockout locally affects gut epithelial morphogenesis in a light-dependent manner. (A-L) Stage 39 Xenopus embryos were exposed to cRO (A-D,I-L), rinsed and kept in the dark (A-D) or irradiated on the right-hand side (I-L), as in Fig. 2. Stage 46 embryos (A,E,I) were sectioned and stained for β-catenin (red) and smooth muscle actin (green); DAPI (blue) indicates nuclei. In contrast to the single-layer columnar epithelium in control (non-irradiated) embryos (B-D), epithelial architecture on the irradiated right side of cRO-treated embryos is highly stratified and disorganized (‘R’ in J-L), similar to that observed throughout the gut in embryos globally exposed to RO (F-H). In non-irradiated embryos, and on the non-irradiated left (‘L’) side of cRO-treated embryos, β-catenin is concentrated at the apical surface of the columnar epithelium (arrows in C,D,K,L). By contrast, very little β-catenin appears apically localized in RO guts (asterisks in H) or within the irradiated right side (‘R’) of cRO-treated guts (asterisks in L). B,F,J, 100×; C,G,K, 200×; D,H,L, 400×.
Fig. 4. Rho kinase plays different roles on contralateral sides of the gut. (A-C) Stage 38 Xenopus embryos were exposed to cRO, rinsed, and kept in the dark (A) or irradiated on the left (B) or right (C) side of the prospective gut, as in Fig. 2. The arc of the midgut curvature is indicated by the dashed line. Inset (C) shows a reversed curvature caused by ectopic concavity (arrow). (D) Average curvature for each condition (n≥10) was quantified as 1/radius of the best-fit circle superimposed on the greater curvature of the midgut. *, P<0.05; **, P<0.01 (one-way ANOVA); error bars indicate s.d.
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