Importantly, the phenotype can also be rescued by a nonphosphorylatable form of Ndel1 (Ndel1SA) in which three of the key PP4c target residues have been mutated to Alanine. However, the phosphomimetic form

of Ndel1 (Ndel1SE) LY2157299 cell line cannot rescue the spindle orientation defect ( Figures 5B and 5C). In addition, we observed similar spindle orientation defects when plasmids expressing Cre driven by a CAG promoter were electroporated into PP4cfl/fl embryonic brains, indicating that the phenotype is specific to the loss of PP4c. Ndel1SA, but not Ndel1SE, could rescue the spindle orientation defects caused by the loss of PP4c ( Figures 5B and 5C). To examine whether the nonphosphorylatable form of Ndel1 can also rescue the lineage defects at the onset of neurogenesis, we performed in utero electroporation at E11.5. Downregulation of PP4c leads to an increase of neuronal differentiation with the depletion of the progenitor pool, which is consistent with what we observed in PP4cfl/fl;Emx1Cre brains ( Figures S6A, S6B, S6D, and S6E). This phenotype was again rescued by coelectroporation of Ndel1SA ( Figures S6C, S6D, and S6E). Thus, our data suggest that excessive phosphorylation of Ndel1 results in disruption of the Ndel1/Lis1 complex in PP4c mutant mice and is responsible

for the spindle orientation defect. In the future, FG-4592 purchase it will be interesting to examine which regulatory subunit forms the complex with PP4c to regulate spindle orientation. To address how cell fates might be affected by the spindle

orientation defects, we analyzed the Notch signaling pathway. Notch signaling plays an essential role in regulating neural progenitor proliferation and differentiation (Pierfelice et al., 2011) and has been proposed to be an important downstream mediator of the asymmetric cell division machinery in the mammalian epidermis (Williams et al., 2011). To determine Notch activity, we used a Notch reporter (CBFRE-EGFP) that carries a CBF1-reponse element upstream of EGFP. The intensity of EGFP in the cell reflects endogenous Notch activity (Mizutani et al., 2007 and Bultje et al., 2009). The CBFRE-EGFP construct was coelectroporated with either a PP4c or a scrambled shRNA into E13.5 mouse brains. The resulting downregulation of PP4c by shRNA or genetic removal of PP4c Rolziracetam via Cre expression in PP4cfl/fl background caused a significant reduction in EGFP fluorescence when compared to controls ( Figures 6A and 6F). Counting the numbers of highly GFP-positive cells in which the Notch pathway is active showed that this effect is highly significant ( Figure 6I). The defect is specific as the decreased Notch activity can be restored by coelectroporation of an RNAi-resistant PP4c construct ( Figures 6C and 6I). We then asked whether overexpression of Ndel1SA could rescue the Notch signaling defect caused by the downregulation of PP4c.