Control of Neural Stem Cells wild type control (slope: 21.33763.400e+038, p,0.0001 and slope: 20.0809960.04816, p value compared to control ,0.0001, respectively). Error bars indicate standard deviation (A, B). (C–F): Co-immunofluorescence of GLAST (red) and TuJ1 (green) on neocortical progenitors isolated from E14.5 Rbpj and Ptc1 mutant and wild types neocortices. Cells were counterstained with DAPI (blue). Arrows (in C, E, F) indicate TuJ1+ cells and arrowheads (D) indicate GLAST+ radial glial cells. RG cells from Ptc1Lox/Lox;NestinCre neocortex rarely differentiate into TuJ1+ neurons after 24 hours (D, G), whereas concomitant loss of the Rbpj and Ptc1 alleles results in an increased rate of neurogenesis in Ptc1Lox/Lox;RbpjLox/Lox;NestinCre neocortical cells (F, G). On the other hand, cells from RbpjLox/Lox;NestinCre neocortex mostly differentiate into TuJ1+ neurons after 24 hours. Also, (G): Quantitative analysis for GLAST and TuJ1 revealed that the percentage of RG cells undergoing symmetric neurogenic divisions was significantly increased (p,0.05) in the Ptc1Lox/Lox;RbpjLox/Lox;NestinCre compared to the Ptc1Lox/Lox;NestinCre neocortices. On the other hand, there was a reduction in the number of symmetric proliferative cell divisions in Ptc1Lox/Lox;RbpjLox/Lox;NestinCre compared to Ptc1Lox/Lox;NestinCre (p,0.05) samples. Bars represent standard errors. ***p,0.0001. Scale bar (C–F), 10 mm. doi:10.1371/journal.pone.0014680.g002 on neocortical neurogenesis at E14.5, we examined the expression of neuronal marker TuJ1 in conjunction with Nestin, which marks neuroepithelial and radial glial cells or Sox2. Interestingly, the zone of Nestin+ cells was expanded in Ptc1Lox/Lox;NestinCre and Ptc1Lox/Lox; RbpjLox/Lox;NestinCre neocortices compared to the wild type neocortex, with Nestin+ cells even present in the region of the cortical plate (Fig. 4F, 4H; Fig. S4B, S6B, S6D). On the other hand, RbpjLox/Lox; NestinCre neocortex had fewer Nestin+ cells (Fig. 4C, 4G). Also, as previously reported, TuJ1+ neurons were observed across the entire neocortex, from the ventricular surface to the pial surface in the RbpjLox/Lox;NestinCre forebrains (Fig. 4G). These results indicate that the delicate balance between proliferative and neurogenic divisions of neural precursors is disturbed as a result of over-activation of the Hh pathway and loss of Notch activity. As Sox2 expression has been associated with actively dividing undifferentiated neural progenitor/stem cells and loss of Sox2 has been shown to be responsible for premature neuronal differentiation [43,44,45], we examined whether Sox2 expression was altered in Hh- activated and Rbpj-deleted neocortices. Sox2 expression was not restricted to the ventricular zone of Ptc1Lox/Lox;NestinCre and Ptc1Lox/Lox;RbpjLox/Lox;NestinCre neocortices with Sox2+ cells present in the more differentiated cells of the cortical plate in Ptc1Lox/Lox;NestinCre neocortex (arrowheads, Fig. 4N), suggesting a failure of progenitor cells to exit cell cycle and differentiate before migration. In support of our observation for Nestin staining, RbpjLox/Lox;NestinCre neocortex showed a dramatic reduction in Sox2+ cells in the ventricular zone (Fig. 4O, Fig. S6). Overall, due to disruption of the neocortical structure as a result of Hh pathway activation and deletion of Rbpj in the Ptc1Lox/Lox;NestinCre, RbpjLox/Lox;NestinCre and Ptc1Lox/Lox; RbpjLox/Lox;NestinCre neocortex, the layer-specific expression of Nestin, Sox2 and TuJ1 was perturbed. However, a distinct expansion of the Sox2 and Nestin domain was noted in the Ptc1Lox/Lox;NestinCre and Ptc1Lox/Lox;RbpjLox/Lox;NestinCre samples. Disruption of Notch signaling promotes symmetric neurogenic divisions and results in loss of putative neural stem cells in the Ptc1Lox/Lox;RbpjLox/Lox;NestinCre neocortex To examine whether the imbalance in the number of neurons versus radial glia in the Ptc1-deleted and Rbpj-deleted neocortices was due to a shift in the number of symmetric proliferative divisions as a consequence of loss of Rbpj, the clonal pair cell assay was performed on cells from E14.5 Ptc1Lox/Lox;RbpjLox/Lox;NestinCre and RbpjLox/Lox;NestinCre mice. Immunostaining performed using GLAST and TuJ1 indicated that the majority of GLAST+ cells from RbpjLox/Lox;NestinCre neocortex undergo symmetric neurogenic divisions to expand the pool of post-mitotic neurons (Fig. 2E, 2G), observed previously across the entire neocortex (Fig. 4C, 4G) in accordance with the published data [16]. The number of radial glial cells undergoing symmetric neurogenic divisions was approximately double in Ptc1Lox/Lox;RbpjLox/Lox;NestinCre neocortex compared to Ptc1Lox/Lox;NestinCre neocortex, while the number of symmetric proliferative divisions were three-fold reduced (p,0.05, Fig. 2G). Moreover, the numbers of both symmetric proliferative and neurogenic divisions in the Ptc1Lox/Lox;RbpjLox/Lox;NestinCre neocortex were comparable to the wild type. Overall, these results suggest that although the disorganisation of the neocortical structure was not completely rescued (Fig. 4), inactivation of the Notch signaling pathway in Hh-activated progenitors restores the balance between symmetric neurogenic and proliferative divisions in the mutants to wild type levels. In addition to the Hh pathway, the canonical Rbpj mediated Notch pathway has been shown to regulate neural stem cells [17]. To investigate the effect of loss of Notch signaling on Hh-activated neocortical progenitor cells, we utilised the SFUA. As described previously, an 8-fold increase in sphere forming potential was observed between wild type and Ptc1Lox/Lox;NestinCre neocortex (Fig. 2A). Loss of both Rbpj and Ptc1 alleles in Nestin+ progenitors led to a 4.3-fold reduction (p = 0.0200) in the sphere forming capacity of neocortical cells (0.50060.094%) compared to Ptc1Lox/Lox;NestinCre neocortex (2.15460.267%). Moreover, these levels were comparable to the sphere forming potential of wild type neocortex. Interestingly, a 4.6-fold increase in sphere forming potential was observed in Ptc1Lox/Lox;RbpjLox/Lox;NestinCre neocortex compared to RbpjLox/Lox;NestinCre neocortex (p = 0.0427) suggesting that Hh signaling can compensate for loss of Notch signaling to support the sphere forming properties of neocortical progenitors (Fig. 2A). To further examine whether loss of Rbpj regulates Ptc1 deleted neocortical stem cells, the neurosphere assay was performed on neocortical samples. As detailed previously, a significant increase in the rate of clonal expansion of Ptc1Lox/Lox; NestinCre neocortex stem cells was observed compared to the Ptc1Lox/Lox neocortex (0.887760.06774 vs. 0.706060.03302, p,0.05; Fig. 2B). In contrast, stem cells from RbpjLox/Lox;NestinCre neocortex failed to expand clonally in the neurosphere assay post passage 1 (n = 3), although live cells were observed in the culture up to passage 4 (Fig. 2B, Fig. S7B). The rate of clonal expansion of RbpjLox/Lox;NestinCre neocortex was significantly reduced (21.33763.400e+038, p,0.0001) compared to Ptc1Lox/Lox wild type neocortex (Fig. 2B). Homozygous loss of Rbpj in addition to loss of Ptc1 led to a significant reduction in the number of stem cells (20.0809960.04816) compared to wild type samples. Moreover, an apparent reduction in neurosphere size and altered morphology in comparison to neurosphere cultures derived from the wild type neocortex was noted (Fig. S7B), possibly due to failure of stem cells to survive. Taken together, these data suggest that homozygous loss of Rbpj, irrespective of the presence or absence of Ptc1, ultimately results in loss of clonal expansion of neural stem cells in vitro. Some Notch effectors are upregulated in Hedgehog activated ventricular zone cells Clearly, our data indicate that Hedgehog pathway and Notch pathway activity can co-operate to regulate neurogenic divisions of neocortical progenitors. Therefore, we next examined whether the expansion of progenitor cells and the concomitant depletion of neurons in the VZ of E14.5 Ptc1Lox/Lox;NestinCre mice was PLoS ONE | www.plosone.org 7 February 2011 | Volume 6 | Issue 2 | e14680