Control of Neural Stem Cells Figure 5. Inactivation of Ptc1 results in regulation of downstream targets of Notch signaling at E14.5. (A, B, C): Taqman RT-PCR for detection of Hes1, Hes5 and Blbp transcript from microdissected VZ (n$3 for each genotype). Hes1 transcript was 65-fold upregulated in Ptc1Lox/Lox; NestinCre neocortex compared to Ptc1Lox/Lox VZ (A) and Blbp transcript was approximately two-fold upregulated compared to wild type VZ (p,0.05) (C). The expression of Hes1, Blbp and Hes5 was significantly downregulated following deletion of Rbpj compared to Ptc1 deletion alone (p,0.05). The data were normalised based on the expression of Gapdh. doi:10.1371/journal.pone.0014680.g005 dependent upon the interaction between Ptc1 and Smo, with the activity of Hedgehog ligands serving to disrupt that relationship. At the molecular level, Hh signaling is generated from unre- strained Smo, not the Hedgehog ligand per se. Ptc1 is therefore a negative regulator of Hh signaling and here we have demonstrated that loss of Ptc1 in Nestin+ neuroepithelial cells and progeny leads to sustained, cell autonomous signaling from E11.5/E12.5 (Fig. 1 and data not shown). By taking this approach we were able to model a high level of Hh signal activation over a time window reflecting the expression and activity of endogenous Shh ligand, which continues at least until E15.5 [12]. It is clear from our data that this model likely generates a level of Hedgehog signal and response in excess to that of the endogenous Shh ligand, which is present in the VZ, SVZ and cortical plate. This approach enabled us to effectively address two issues. First, although loss of function studies have suggested that Hh signaling is a required mitogen for stem cells of the neocortex (and tectum), and in vitro activation studies have suggested the same [10,11,12]. Our model enabled us for the first time to examine the consequences of Hh activation in vivo, particularly in the control of symmetric/asymmetric stem cell divisions of VZ progenitor cells. Secondly, loss of Hedgehog function in the developing dorsal CNS creates severe phenotypes which have made interpretation difficult when superimposing other genotypes, and the gain of Hh function phenotype we define here has enabled the consequences of concomitant loss of Notch function to be readily assessed. Notch signaling utilizes a membrane bound ligand that contacts an appropriate receptor leading to the release of a cytoplasmic fragment of the receptor which then forms a multi- unit complex capable of transcriptional regulation. In mammals, PLoS ONE | www.plosone.org 10 February 2011 | Volume 6 | Issue 2 | e14680