Control of Neural Stem Cells Figure 4. Loss of Rbpj leads to disruption of neocortical patterning. Immunostaining of E14.5 neocortex using TuJ1 (red) (A–H); (green), (I– P) and Nestin (green) (A–H) or Sox2 (red) (I–P) antibodies. High magnification confocal images of the medial region of coronal sections from A–D, I– L are shown in E–H, M–P respectively. The zone of cells that are Sox2 and Nestin positive is expanded in Ptc1Lox/Lox;NestinCre and Ptc1Lox/Lox;RbpjLox/Lox; NestinCre samples. Sox2+ cells were even present in the CP (arrowheads in N). TuJ1+ cells were present throughout the RbpjLox/Lox;NestinCre neocortex, from the VZ to the pial surface. Abbreviations: CP, cortical plate; CP’, choroid plexus; VZ, ventricular zone. Scale bar (A–D, I–L), 100 mm; (E–H, M–P), 20 mm. doi:10.1371/journal.pone.0014680.g004 expected, but does not respond to Hedgehog pathway activation. On the whole, the data presented in Figure 5 suggest that the undoubted interaction observed at the developmental and stem cell level between Hedgehog and Notch signaling is reflected in part by an apparent Rbpj-dependent regulation by Hedgehog signaling of some, but not all, Notch effectors in the VZ. Discussion The processes that direct the neuroepithelium to generate cerebral cortex are clearly central to the functioning of higher mammals. The Hedgehog pathway has long been recognized to play a key role in the development of ventral aspects of the CNS and the maintenance of neural stem cells more broadly. Recently, Shh loss of function analyses have indicated that Hh signaling is required for the correct establishment of dorsal patterning in the forebrain, with the window of activity likely to extend at the least from E12.5–E15.5, a critical period for corticogenesis [12]. Similarly, regulation of Notch signaling is essential for neurogen- esis with activated Notch signaling being required for the maintenance of a progenitor fate in aspects of the CNS, including the VZ of the forebrain [13,14,19]. Here, we used a genetic approach to examine the interaction between Hedgehog and Notch signaling in the neocortex. Activity of the Hh pathway is PLoS ONE | www.plosone.org 9 February 2011 | Volume 6 | Issue 2 | e14680