Taqman RT-PCR for detection of Gli1 transcript. Ptc1 transcript was downregulated and Gli1 upregulated (C) in the Ptc1Lox/Lox;NestinCre VZ compared wildtype E14.5 Ptc1Lox/Lox VZ, as a result of Cre-mediated recombination. The data for Ptc1 and Gli1 was normalised based on the expression of Hprt and Gapdh respectively, and is presented as two independent pooled samples of each genotype. Found at: doi:10.1371/journal.pone.0014680.s001 (0.94 MB TIF) Figure S2 The irregularities in the thickness within the Ptc1Lox/Lox; NestinCre neocortex do not change in severity depending on the rostral/caudal positioning. E14.5 Haematoxylin and Eosin stained coronal sections at rostral (A, B), intermediate rostral-caudal (C, D), and caudal levels (E, F) of the Ptc1Lox/Lox;NestinCre neocortex. Scale bar (A–F), 500 mm. Found at: doi:10.1371/journal.pone.0014680.s002 (2.85 MB TIF) Figure S3 Shortening of cell cycle and increased re-entry in Ptc1Lox/Lox;NestinCre neocortex. Co-immunofluorescence analysis of BrdU and Ki67 expression in coronal sections of E14.5 neocortices. In the mutant neocortex, there is an increase in progenitor cells (Ki67+) undergoing S-phase (BrdU+) compared to the wildtype neocortex (A). Quantification of the percentage of progenitor cells (Ki67+, red) co-labelled with BrdU (green) after a 2 hour pulse label (B). Rate of cell division is increased in E14.5 Ptc1Lox/Lox;NestinCre neocortex (n = 3; 8361.4; p,0.0001) as compared to Ptc1Lox/Lox wild type littermates (n = 3; 6162.0) indicating mutant cells cycle faster. Cells re-entering the cell cycle are BrdU + /Ki67+ whereas cells no longer dividing and withdrawn from cell cycle are BrdU + /Ki672 after a single pulse label of BrdU 24 hours prior to being killed (C). Approximately twice as many wild type progenitors leave the cell cycle, as compared to Ptc1Lox/Lox;NestinCre progenitors (n = 3; p,0.0001) (D). DNA is stained with DAPI. Scale bar (A), 200 mm; (C), 50 mm. Co- immunofluorescence of GLAST (red) and Tbr2 (green) (E, F) on neocortical progenitors isolated from E14.5 Ptc1Lox/Lox;NestinCre and Ptc1Lox/Lox neocortex. GLAST+ radial glial cells from Ptc1Lox/Lox; NestinCre neocortex rarely differentiate into Tbr2+ basal progenitors after 24 hours (F, G). Arrowhead (in E) indicates Tbr2+ cells. Quantitative analysis for GLAST and Tbr2 (G) revealed that the percentage of RG cells undergoing symmetric differentiative divisions (1RG = 2 Basal progenitor (BP) cells) was significantly reduced in the Ptc1Lox/Lox;NestinCre neocortex. In addition, reduc- tion in the percentage of RG cells undergoing asymmetric differentiative (1RG = RG+BP) divisions was significant (G). Bars represent standard errors. **p,0.05. Scale bar (E, F), 10 mm. Found at: doi:10.1371/journal.pone.0014680.s003 (4.99 MB TIF) Figure S4 The radial glial progenitor population is expanded in Ptc1Lox/Lox;NestinCre neocortex (B). Nestin expression, which is a ubiquitous marker for neural progenitors, is expanded at E14.5 in the Ptc1Lox/Lox;NestinCre VZ (B) as compared to Ptc1Lox/Lox neocortex (A). Assessment of neuronal differentiation in the wild type and mutant neocortex. At E12.5 a layer of MAP2+ neurons emerges References 1. Huttner WB, Kosodo Y (2005) Symmetric versus asymmetric cell division during neurogenesis in the developing vertebrate central nervous system. Curr Opin Cell Biol 17: 648–657. 2. Gotz M, Barde YA (2005) Radial glial cells defined and major intermediates between embryonic stem cells and CNS neurons. Neuron 46: 369–372. 3. Miyata T, Kawaguchi A, Saito K, Kawano M, Muto T, et al. (2004) Asymmetric production of surface-dividing and non-surface-dividing cortical progenitor cells. Development 131: 3133–3145. 4. Miyata T, Kawaguchi A, Okano H, Ogawa M (2001) Asymmetric inheritance of radial glial fibers by cortical neurons. Neuron 31: 727–741. 5. Noctor SC, Flint AC, Weissman TA, Dammerman RS, Kriegstein AR (2001) Neurons derived from radial glial cells establish radial units in neocortex. Nature 409: 714–720. Control of Neural Stem Cells beneath the pial surface in both the Ptc1Lox/Lox neocortex (C) and Ptc1Lox/Lox;NestinCre neocortex (D). BrdU-birthdating of neurons was performed by injecting pregnant females with BrdU at E11.5 (E, F) or E12.5 (G, H) and sacrificing at E14.5. BrdU immunostaining revealed that radial distribution of neurons born at E11.5 (E, F) or E12.5 (G, H) is indistinguishable at E14.5 between the Ptc1Lox/Lox neocortex (E, G) and Ptc1Lox/Lox;NestinCre neocortex (F, H). Abbreviations: CP, cortical plate; IZ, interme- diate zone; MZ, marginal zone; PP, pre-plate; SP, sub-plate; SVZ, sub-ventricular zone; VZ, ventricular zone. Scale bar (A–B, E–H), 20 mm; (C, D), 10 mm. Found at: doi:10.1371/journal.pone.0014680.s004 (6.05 MB TIF) Figure S5 Disruption of neocortical structure in the RbpjLox/Lox; NestinCre mice. (A, B): Haematoxylin and Eosin stained coronal sections of E14.5 neocortex. (B): shows a higher magnification of the mutant neocortex in (A). The neocortex is highly disorganised, lacks diencephalon and contains blood clots (arrowheads) within the neocortex and the LV. (C): Nissl-stained coronal sections of E14.5 neocortices. Scale bar, 100 mm. Abbreviations: LV, lateral ventricle; NC, neocortex. Found at: doi:10.1371/journal.pone.0014680.s005 (6.60 MB TIF) Figure S6 Loss of Rbpj leads to disruption of the neocortical patterning. Immunostainingof E14.5 neocortex using TuJ1 (red) (A–D); (green) (E–H) and Nestin (green) (A–D) or Sox2 (red) (E–H) antibodies and counterstained with DAPI (blue). Confocal images of medial region of the coronal sections are shown. Abbreviations: CP, cortical plate; VZ, ventricular zone. Scale bar (A–H), 20 mm. Found at: doi:10.1371/journal.pone.0014680.s006 (9.19 MB TIF) Figure S7 Neurosphere assay of mutant neocortices compared to wild type spheres. (A–D): Bright field microscope images of passage 7 neurosphere cultures (A, C, D) and passage 1 (B). Inset images for (A–D) reveal microspikes on Ptc1Lox/Lox neurospheres while homozygous loss of Rbpj results in no sphere generation (C, D). Scale bar (A–D), 10 mm. Found at: doi:10.1371/journal.pone.0014680.s007 (2.60 MB TIF) Acknowledgments The authors would like to thank members of the Wainwright lab especially Lena Constantin for critical reading of the manuscript and technical assistance, Rob Wechsler-Reya, Carol Wicking, Veronica Palma and Linda Richards for helpful discussions and Stacey Cole for technical assistance. We thank T.Sudo and R. Kageyama for generously providing antibodies. Author Contributions Conceived and designed the experiments: RKD TE BJW. Performed the experiments: RKD TE MCT JPR CA. Analyzed the data: RKD TE MCT JPR CA HMC BAR BJW. Contributed reagents/materials/analysis tools: EJ PFB HMC. Wrote the paper: RKD TE BJW. 6. Noctor SC, Martinez-Cerdeno V, Ivic L, Kriegstein AR (2004) Cortical neurons arise in symmetric and asymmetric division zones and migrate through specific phases. Nat Neurosci 7: 136–144. 7. Briscoe J, Sussel L, Serup P, Hartigan-O’Connor D, Jessell TM, et al. (1999) Homeobox gene Nkx2.2 and specification of neuronal identity by graded Sonic hedgehog signaling. Nature 398: 622–627. 8. Ericson J, Briscoe J, Rashbass P, van Heyningen V, Jessell TM (1997) Graded sonic hedgehog signaling and the specification of cell fate in the ventral neural tube. Cold Spring Harb Symp Quant Biol 62: 451–466. 9. Blaess S, Corrales JD, Joyner AL (2006) Sonic hedgehog regulates Gli activator and repressor functions with spatial and temporal precision in the mid/ hindbrain region. Development 133: 1799–1809. PLoS ONE | www.plosone.org 12 February 2011 | Volume 6 | Issue 2 | e14680