ligands include members of the Delta-like (DLL1, DLL3, DLL4) and Jagged (Jag1, Jag2) families, and receptors include Notch1-4. Of the receptors, at least Notch1-3 are expressed and functional in the developing forebrain and of the ligands, Jag1 and DLL1, 3 [49,50,51]. Although the potential combinatorial complexity of Notch ligand/receptor interaction in the developing forebrain is high, canonical Notch signaling requires the CSL/Rbpj (Rbpj) transcription co-factor. So in practice, inhibition of Rbpj function will abrogate Notch receptor signaling [52]. Accordingly, here we took the approach of attenuating Notch signaling by deleting Rbpj, which has proved to be a highly effective approach in the developing forebrain and other tissues [17,25,52]. In the absence of the Notch ICD the interaction of Rbpj with target promoters is thought to be transient and weak, nonetheless it is possible that deletion of Rbpj may itself lead to ‘‘de-repression’’ of some Notch target genes but the neocortical phenotype we observed in this study is consistent with data indicating a loss of Notch activity [39,40,53]. Following the loss of Ptc1 under the control of NestinCre, we observed a robust Hh response from E11.5 in the neocortex as indicated by the upregulation of Gli1 and Ptc1 itself. At the gross anatomical level this resulted in an irregular and folded neocortex by E14.5 and more detailed analysis indicated an expanded VZ/ SVZ region with a high mitotic index, with the neurosphere assay indicating this region contained an increased number of stem/ progenitor cells. It has been known for some time that neocortical stem/progenitor cells can be expanded in vitro by the addition of Shh [20,21] and here we have demonstrated that ligand- independent activation of the Hh pathway can produce a similar expansion in vivo. Clonal expansion studies of neurosphere cultures are one measure of the long-term proliferative potential of stem cells and our data indicate that the Hh pathway is mitogenic for such cells. The progenitor cell in the neocortex is the radial glial (RG) cell, which also acts physically as a migratory scaffold for differentiating cells to populate the dorsal layers with the majority of the neurons in the brain being derived directly or indirectly (through basal progenitors) from radial glial cells [2,54, 55,56,57,58]. Studies described here have demonstrated that TuJ1+ neurons were also positive for GLAST suggesting that majority of neurons originated from the radial glial progenitor cell. We quantified the consequences of Hh pathway activation in Nestin+ progenitors on the fate of RG cells using the pair cell assay and determined that at E14.5 there was an increase in the number of symmetric proliferative divisions at the expense of both neurogenic and differentiative divisions, with immunohistochem- ical data supporting those conclusions. Even though previous studies have suggested that Hh is a mitogen in vitro for neural stem cells, our data indicate that Hh activation can not only increase the number of stem/progenitor cells in vivo, but in doing so shifts the balance of RG cell neurogenic divisions. The mechanisms that underlie the switch from symmetric proliferative to symmetric/ asymmetric neurogenic divisions are poorly understood. Accord- ing to the cell cycle length hypothesis, lengthening of the cell cycle allows a neurogenic cell fate determinant sufficient time to induce a cell fate change resulting in differentiation [59]. Consistent with this hypothesis, here we demonstrate that Ptc1-inactivated neocortical VZ progenitors cycle more rapidly, and fail to exit the cell cycle, potentially accounting for the decrease in neurogenic divisions. During development it is likely the basal low level of Ptc1 expression in radial glial cells is sufficient to antagonise Hh pathway activity to preserve the balance between stem cell maintenance and neurogenesis. Several reports have suggested the importance of Notch signaling in gliogenesis [13,60] and in vivo and in vitro studies have Control of Neural Stem Cells established that Notch 1 and Notch 3 activation promotes radial glial cell fate [14,39], similar to our findings with Hh pathway activation. Indeed, in a number of tissues such as cerebellum and skin [26,61] there appears to be interaction between the Hh and Notch pathways. For example, in the cerebellum it has been demonstrated that Notch1 controls cerebellar granule-cell prolif- eration and differentiation [62], as does Sonic Hedgehog [63]. Furthermore, the elevation of Notch2 and Hes5 in Smoothened- induced medulloblastomas suggests that Hh pathway activation interacts with Notch signaling [64]. Apart from the brain, deregulation of Hh, Notch and Bmi pathways has been proposed to induce transformation of mammary stem cells [65]. Taken together, these reports provide evidence in favour of interplay between the Hh and Notch signaling pathways. The results presented here demonstrate that activation of the Hh signaling pathway by Ptc1 deletion increases the stemness of radial glial cells likely by upregulating the Notch signaling pathway. Further, concomitant inactivation of Rbpj in Hh-activated neural progen- itors restored the balance between symmetric proliferative and neurogenic divisions, thereby suggesting a crosstalk between the Hh and Notch signaling pathways during normal corticogenesis. The transcription factor Hes1 is possibly a key molecule in the Hh/Notch co-regulation of corticogenesis. Hes1 is generally regarded as a canonical Notch pathway effector and it is essential for maintaining neural stem cells through out the CNS, including the developing dorsal structures [66]. However, early in neuroep- ithelial development there appears to be instances where Hes1 expression is likely not dependent upon Notch signaling [67]. Furthermore, studies have shown that Hes1 may be regulated by Shh without the requirement for Notch signaling in cerebellar granule neurons and mesodermal cells [46,47]. In the retina, progenitor cell proliferation is under the control of Notch- independent Sonic hedgehog/Hes1 activity and the Hh signal effector Gli2 binds directly to the Hes1 promoter [48]. Here, our data in the neocortex similarly suggests that Hes1 is upregulated by Hh signaling but is not influenced by concomitant loss of Rbpj, consistent with our findings in the ventricular zone of the cerebellum [61]. We would therefore suggest that Hes1 is not only a critical regulator of neuronal cell fate in multiple niches but also uniquely represents a molecular convergence point between Hh and Notch signaling in those niches, and an important avenue for further study. Although there have been many studies conducted on the role of Notch signaling in radial glial cells, the crosstalk between the Notch and Hh signaling pathways in controlling progenitor cell expansion is still poorly understood. As radial glial cells play a central role in the cytoarchitecture of the neocortex acting both as migratory scaffolds and glial progenitors, the understanding of radial glial development is crucial for treatment of both developmental and degenerative disorders. Specifically, in vivo approaches have demonstrated that administration of Notch ligand to stimulate the neural stem cell niche has been of direct therapeutic benefit in animal models of ischaemic brain injury [68] and Parkinson’s disease [69]. On the basis of our data we suggest that agonists of the Hedgehog pathway alone or in combination with Notch pathway manipulation may also be a novel and useful therapeutic approach to stimulating neuronal cell niches in vivo as well as in vitro. Supporting Information Figure S1 Genotyping of microdissected VZ and Real Time PCR analysis. Genotyping of microdissected E14.5 VZ demonstrates that Ptc1 is inactivated (as indicated by the loss of the wild type 450 bp transcript) upon Nestin Cre-mediated recombination (A). (B, C): SYBR Green RT-PCR for detection of Ptc1 deleted transcript and PLoS ONE | www.plosone.org 11 February 2011 | Volume 6 | Issue 2 | e14680