Control of Neural Stem Cells Figure 3. Hedgehog and Notch signaling are necessary for corticogenesis. Bright field microscopy images of whole mounts of E14.5 (A–D). Haematoxylin and Eosin stained coronal sections of E14.5 forebrains (E–L). (I–L): magnified views of (E–H). Nissl-stained coronal sections of E14.5 neocortices (M–P). In the Ptc1Lox/Lox;NestinCre neocortex, the VZ is expanded and the SVZ and CP cannot be identified (J, N) compared to the Ptc1Lox/Lox neocortex (I, M). The RbpjLox/Lox;NestinCre neocortex is highly disorganised, lacks diencephalon and contains blood clots (arrowheads) within the neocortex and the LV. (G, K, O). Ptc1Lox/Lox;RbpjLox/Lox;NestinCre neocortex (H, L, P) is thinner than the wild type neocortex, but the VZ and SVZ is comparable to the wild type. Abbreviations: CP, cortical plate; GE, ganglionic eminence; IZ, intermediate zone; LV, lateral ventricle; MZ, marginal zone; NC, neocortex; SVZ, sub- ventricular zone; T, tectum; VZ, ventricular zone. Scale bar (A–D), 2.0 mm; (E–H), 500 mm; (I–P), 50 mm. doi:10.1371/journal.pone.0014680.g003 potentially the result of the upregulation of Notch signaling. Figure 5A demonstrates that in wild type VZ Hes1 expression is low, and undetectable in the absence of Rbpj as expected. In the presence of Hedgehog pathway activation Hes1 activity is high but this activity is largely lost in the absence of Rbpj, suggesting that most of the Hedgehog-dependent Hes1 activity is dependent upon active canonical signaling. Interestingly, there is a minor component of Hh-dependent Hes1 activity that is not dependent upon Notch signaling, consistent with our results from the VZ of the cerebellum where there is also a Hedgehog and Notch- dependent Hes1 activity, and the data of others [46,47,48]. Overall, the Hes1 data indicate that in the developing VZ of the neocortex, Hedgehog pathway activation positively regulates Hes1 via Rbpj. Similarly, we analyzed Blbp expression (Figure 5C). Blbp has been shown to be a canonical Notch target in radial glial cells in the VZ [18]. Similar to Hes1, Blbp shows a distinct upregulation in response to activation of the Hedgehog pathway, and this activation is abrogated by loss of Rbpj. A third potential Notch effector in the VZ, Hes5 was also examined (Fig. 5B). From these data it can be observed that Hes5 expression requires Rbpj, as PLoS ONE | www.plosone.org 8 February 2011 | Volume 6 | Issue 2 | e14680