Department of Molecular Biomedical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC 27606, USA.
Development. 2012 Jan;139(2):437-42. doi: 10.1242/dev.072165.
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.
为了揭示胚胎发育的分子机制，理想的功能丧失策略应该能够以时间和空间精度靶向活胚胎的特定区域。为此，我们开发了一种新型药理学试剂，可以通过光激活来实现体内 Rho 激酶活性的时空有限抑制。将一种新的光不稳定笼状基团 6-硝基哌啶氧甲基 (NPOM) 安装在 Rho 激酶的小分子抑制剂 Rockout 上，生成“笼状 Rockout”衍生物。在哺乳动物细胞培养和非洲爪蟾胚胎中的互补生化、细胞、分子和形态发生测定验证了笼状化合物的抑制活性取决于暴露于光。方便的是，这种独特的试剂保留了传统小分子抑制剂的许多实际优势，包括在生长培养基中通过简单扩散传递和浓度依赖性可调性，但可以通过标准仪器的去笼化进行局部激活。将这种新型工具应用于胚胎左右不对称的空间异质性问题，揭示了原始肠管左侧和右侧的 Rho 信号传导存在差异需求，为产生不对称器官形态的分子机制提供了新的见解。由于许多芳香族/杂环小分子抑制剂都适合安装这种笼状基团，我们的结果表明光笼化学抑制剂可能是一种普遍适用的技术，可以产生方便的功能丧失试剂，在发育生物学中有广泛的应用潜力。
