Sustained Hedgehog signaling in VZ progenitors promotes symmetric proliferative divisions of radial glial cells In vivo and in vitro loss of function studies have shown that Hh signaling is required for the maintenance of stem cells in the developing neocortex [9,11,12,21,34]. To directly address the consequences of sustained Hh signaling in neocortical precursors, the neurosphere culture assay was utilised to test for stem and progenitor cell properties [29]. We observed a substantial (approximately 8-fold) increase in sphere forming potential of cells derived from the Ptc1Lox/Lox;NestinCre neocortex (2.1546 0.267%) compared to Ptc1Lox/Lox neocortex (0.2760.047%, p,0.0001,) (Fig. 2A) indicating that Hh pathway activation in vivo can regulate the total number of stem and progenitor cells. Next, the neurospheres were passaged multiple times to select against lineage-committed progenitor cells not capable of self- renewal. The rate of clonal expansion of the neurospheres is represented as the slope of the line. There was a clear difference in the rate of fold increase between Ptc1Lox/Lox and Ptc1Lox/Lox;NestinCre neocortical samples (clonal expansion of 0.706060.03302 vs. 0.887760.06774, p,0.05) (Fig. 2B) indicating that Hh pathway activation is mitogenic for cells that have a capacity to self-renew over an extended period of time. We also investigated whether the enlargement of the neocortical VZ progenitor population was a result of changes in cell cycle kinetics due to Ptc1 inactivation. If enlargement of the progenitor pool is due to shortening of the cell cycle, the proportion of progenitor cells labelled by a single 2-hour pulse of BrdU in the total population of cycling progenitors (Ki67+) would increase. Indeed, a significant shortening of the cell cycle in the mutant E14.5 neocortex was detected confirming that Ptc1 inactivated progenitor cells divide significantly faster than wild type progenitors (Fig. S3B). In the Ptc1Lox/Lox;NestinCre neocortex, the proportion of VZ progenitors co-labelled with BrdU/Ki67 was approximately two-fold greater when compared to wild type littermates (Fig. S3D). Altogether, our results suggest that Hh pathway activation can regulate neocortical stem and/or progen- itor cell divisions and the rate of cell division in vivo. The expansion of the stem/progenitor population in the Ptc1 inactivated neocortex suggested a possible perturbation of number and types of differentiative divisions that radial glial cells undergo. In order to investigate this we utilized the clonal pair cell assay [30,31] which was performed on cells derived from the E14.5 neocortex of Ptc1Lox/Lox;NestinCre and Ptc1Lox/Lox mice. Immunoflu- orescence studies performed using GLAST (radial glial cells) and TuJ1 (post-mitotic neurons) antibodies revealed a marked reduction in the number of neurogenic divisions in cells from the Ptc1Lox/Lox;NestinCre neocortex compared to the wild type littermates (Fig. 2C, 2D, 2G). GLAST-positive neocortical cells divide either to generate two GLAST-positive radial glial cells (symmetric proliferative), a GLAST-positive radial glial cell plus a TuJ1-positive neuron (asymmetric neurogenic) or two TuJ1- positive neurons (symmetric neurogenic). The majority of the Ptc1Lox/Lox;NestinCre paired cells divided to generate two GLAST- positive radial glial cells and rarely differentiated into TuJ1- positive neurons (Fig. 2D). Whereas, Ptc1Lox/Lox neocortical cells were routinely GLAST+/TuJ1+ suggesting that these were early neurons that had recently differentiated from a radial glial cell (Fig. 2C). Quantification of the mode of cell division of paired cells stained with GLAST and TuJ1 revealed an approximate 4-fold increase (p,0.05) in the number of symmetric proliferative cell divisions and a subsequent 1.8 fold decrease (p,0.05) in the number of cells undergoing symmetric neurogenic cell divisions in the mutant Ptc1Lox/Lox;NestinCre neocortices (Fig. 2G). These data were further supported by immunocytochemical analysis of Control of Neural Stem Cells Ptc1Lox/Lox;NestinCre neocortex using antibodies against Nestin, MAP2 and TuJ1 (Fig. S4A–D, Fig. S6A–D). Nestin is expressed in both neuroepithelial and radial glial cells but not basal progenitors [35,36], and its expression was found to be expanded and more intense in the Ptc1Lox/Lox;NestinCre neocortex compared to the Ptc1Lox/Lox neocortex. The expansion of the Nestin domain is consistent with the observed increase in proliferation. A population of MAP2+ neurons were observed in the E12.5 Ptc1Lox/Lox;NestinCre neocortex, indicating that prior to Ptc1 inactivation at E11.5/ E12.5 (although the expression of Nestin is turned on at E10.5, it takes 24–48 hours to result in Patched1 inactivation), neuronal differentiation commenced normally (also see Fig. S4E–H). Radial glial cells can also differentiate into a Tbr2+ basal progenitor cell through asymmetric/symmetric differentiative divisions, reviewed in [37], which then undergoes one symmetric division to yield two neurons. The number of Tbr2+ cells were markedly reduced in the Ptc1Lox/Lox;NestinCre neocortex compared to wild type (Fig. S3E–G), further supporting the notion that the decrease in neuronal differentiation is a result of both a failure of radial glial cells to generate neurons directly and indirectly through basal progenitors. Overall these data indicate that the expansion of the proliferative compartment in the Ptc1Lox/Lox;NestinCre neocortex is likely the result of an increase in the number of symmetrical divisions of radial glial cells at the expense of neurogenic divisions. Ablation of Notch signaling in Hh-activated neural progenitors results in disruption of the neocortical structure Several reports have suggested the importance of Notch signaling in controlling asymmetric/symmetric divisions in neural stem and progenitor cells (reviewed in [30,38]). Both in vivo and in vitro studies have established that Notch1 and Notch3 activation promote radial glial cell fate [14,39] and inactivation of Notch1 signaling leads to precocious neuronal differentiation [40]. Since our data indicate that Hedgehog pathway activation may play a similar role in the same cell types we next asked whether Notch and Hedgehog signaling interact in the regulation of homeostasis in the VZ. To examine whether the increase in the number of radial glial cells in Ptc1Lox/Lox;NestinCre neocortex required Notch signaling, inactivation of Rbpj and Ptc1 was carried out concomitantly in Nestin+ neural progenitors. Forebrains from the control genotypes (Ptc1Lox/Lox, Ptc1Lox/Lox;Rbpj+/Lox, Ptc1Lox/Lox;RbpjLox/Lox) were examined and demonstrated normal neocortical development, although only Ptc1Lox/Lox neocortex samples are presented as a control in Figures 3 and 4. Whole mounts of E14.5 embryos from the RbpjLox/Lox;NestinCre and Ptc1Lox/Lox;RbpjLox/Lox;NestinCre mice showed extensive hemorrhage in the neocortex and tectum (Fig. 3C, 3D, Fig. S5), possibly due to loss of Rbpj from Nestin+ endothelial cells, however this phenotype has not been reported for the Rbpj- conditional knockout mice generated using inducible Nestin-Cre [41]. Haematoxylin and Eosin staining of E14.5 forebrains clearly demonstrated a patterning defect in the Ptc1-inactivated, Rbpj-inactivated and the double mutants (Fig. 3E–H). The diencephalon was missing in the RbpjLox/Lox;NestinCre forebrains (Fig. 3G) and the lateral ventricles of the Ptc1Lox/Lox;RbpjLox/Lox; NestinCre forebrains were enlarged (Fig. 3H). The neocortex of double mutant mice was much thinner and the neocortical structure was disrupted in contrast to the wild type neocortex, however, the VZ/SVZ was reduced compared to the Ptc1-deleted neocortex (Fig. 3L, 3P). Precocious neuronal differentiation has been reported in mice with inactivated Notch signaling components [16,42]. Moreover, it has been well documented that Notch signaling is instrumental in promoting stem/glial cell fate. To assess the effect of loss of Rbpj PLoS ONE | www.plosone.org 5 February 2011 | Volume 6 | Issue 2 | e14680