TY - JOUR TI - A Nestin-cre transgenic mouse is insufficient for recombination in early embryonic neural progenitors AU - Liang, H. AU - Hippenmeyer, S. AU - Ghashghaei, H. T. T2 - Biology Open AB - Summary Nestin-cre transgenic mice have been widely used to direct recombination to neural stem cells (NSCs) and intermediate neural progenitor cells (NPCs). Here we report that a readily utilized, and the only commercially available, Nestin-cre line is insufficient for directing recombination in early embryonic NSCs and NPCs. Analysis of recombination efficiency in multiple cre-dependent reporters and a genetic mosaic line revealed consistent temporal and spatial patterns of recombination in NSCs and NPCs. For comparison we utilized a knock-in Emx1cre line and found robust recombination in NSCs and NPCs in ventricular and subventricular zones of the cerebral cortices as early as embryonic day 12.5. In addition we found that the rate of Nestin-cre driven recombination only reaches sufficiently high levels in NSCs and NPCs during late embryonic and early postnatal periods. These findings are important when commercially available cre lines are considered for directing recombination to embryonic NSCs and NPCs. DA - 2012/9/27/ PY - 2012/9/27/ DO - 10.1242/bio.20122287 VL - 1 IS - 12 SP - 1200-1203 J2 - Biology Open LA - en OP - SN - 2046-6390 UR - http://dx.doi.org/10.1242/bio.20122287 DB - Crossref ER - TY - JOUR TI - A photoactivatable small-molecule inhibitor for light-controlled spatiotemporal regulation of Rho kinase in live embryos AU - Morckel, Allison R. AU - Lusic, Hrvoje AU - Farzana, Laila AU - Yoder, Jeffrey A. AU - Deiters, Alexander AU - Nascone-Yoder, Nanette M. T2 - DEVELOPMENT AB - 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. DA - 2012/1/15/ PY - 2012/1/15/ DO - 10.1242/dev.072165 VL - 139 IS - 2 SP - 437-442 SN - 0950-1991 KW - Small-molecule inhibitor KW - Rho kinase KW - Photocaging KW - Xenopus ER -