1B). This could be caused by the use of different reporter genes (nuclear-targeted β-galactosidase

in the previous study vs. cytosolic EGFP in the current study) and the different mechanism by which genes were delivered to neurons. The efficiency of DNA entry into cells is also compromised in the IUE method, as a trade-off in preventing electroporation-induced damage to the embryo. Nevertheless, we found that transfected Purkinje LDE225 molecular weight cells could efficiently coexpress at least three transgenes (Figs 3 and 4). This situation is quite advantageous for electrophysiological analyses, because recordings from transfected and neighboring non-transfected (control) neurons can be easily compared. In addition, EGFP introduced at E11.5 remained highly expressed 1 month after birth (Fig. 2) and was maintained at least until P90 (data not shown). Immature Purkinje cells originally have a fusiform shape with a few dendrites. Purkinje cells lose these primitive dendrites almost completely Nutlin-3a datasheet by P3–P4 in rats (Sotelo & Dusart, 2009). As the virus-mediated overexpression of human RORα1 accelerates this process in wild-type and restores it in staggerer cerebellum organotypic slice cultures, RORα1 was proposed to play a crucial role in the regression of primitive dendritic branches (Boukhtouche et al., 2006). In the present study, we showed that the IUE-mediated overexpression of dominant-negative RORα1 in Purkinje cells in vivo could recapitulate the morphological

abnormalities observed in staggerer mice (Fig. 5). These results not only support but also extend the hypothesis that cell-autonomous activities of RORα1 in Purkinje cells are responsible for the process controlling the regression of primitive dendrites in vivo. Notably, because of the limited migration of Purkinje cells in organotypic slice cultures, the migration defect of staggerer Purkinje cells was not analysed previously (Boukhtouche et al., 2006), and it remains unclear whether the regressive phase begins during or after the migration of Purkinje cells to their final domains. We observed that some Purkinje cells expressing dominant-negative RORα1 did not reach the Purkinje cell

layer in vivo, indicating that RORα1 regulates not PAK5 only the regression of dendrites but also the migration process of Purkinje cells. It is unclear why the phenotypes of Purkinje cells expressing dominant-negative RORα1 were variable, but small differences in transgene expression levels and/or the developmental stage of the transfected Purkinje cell progenitors could have contributed to the variation. A more robust suppression of RORα1 gene expression by IUE-based RNA interference (Matsuda & Cepko, 2004) will help clarify the role of RORα1 in the early events during Purkinje-cell development. Future studies taking advantage of IUE to enable gene expression from the early postmitotic stage will facilitate studies on the mechanisms of Purkinje cell development and migration.