Figure 1(A) Haematoxylin and Eosin (HE) staining on sections of the neocortex from control (Co) and cyclin Y (Ccny) and cyclin Y-like 1 (Ccnyl1) double knockout (DKO) embryos at E13.5. Neocortex thickness (indicated by yellow bars) is reduced in DKO vs. control embryos while mediolateral neocortex length (indicated by pink arrows) does not display obvious differences. Scale bars 100 μm. (B) Hoechst (DNA) staining and IF for Tbr2 to calculate the thickness (white bars) of the VZ, SVZ and intermediate zone + cortical plate (IZ+CP) in control and DKO E13.5 neocortex. Scale bar 20 μm. (C) Quantification of VZ, SVZ and IZ+CP thickness in control and DKO E13.5 neocortex. Each zone is represented as percentage of total thickness (measured in µm) of the neocortex. Columns are means ± SEM (n=6 embryos, 4 litters). ( (D) IF for Pax6 and Tbr2 (not shown) in control and DKO E13.5 neocortex. White bars indicate thickness of Pax6+ zone (i.e. VZ). Scale bar 20 μm. (E) Quantification of the number of APs (Pax6+Tbr2-) from (D). Pax6+Tbr2+ cells were excluded from final counts. AP numbers are expressed as percentage of total cell number (sum of D, F and H). Columns are means ± SEM (n=4 embryos, 3 litters). (F) IF for Tbr2 in control and DKO E13.5 neocortex. White bars indicate thickness of Tbr2+ zone (i.e. SVZ). Scale bar 20 μm. (G) Quantification of the number of BPs from (F). Columns are means ± SEM (n=6 embryos, 3 litters). (H) IF for Tbr1 in control and DKO E13.5 neocortex. White bars indicate thickness of Tbr1+ zone (i.e. mostly CP). Scale bar 15 μm. (I) Quantification the number of deep-layer post-mitotic neurons from (H). Columns are means ± SEM (n=3 embryos, 3 litters). (J) IF for Ccny (a), Ccnyl1 (b) and LRP6 (c) (low-density lipoprotein receptor-related protein 6) phosphorylated at T1479 (pLRP6) in the neocortex of E13.5 control embryos. White arrowheads in (a,b) indicate apical membrane / cortex, shown at higher magnification in the insets. High levels of LRP6 phosphorylation are detected in mitotic APs (c, white arrowheads). Scale bar 50 μm. (K) IF for Ccny(a), Ccnyl1(b) and pLRP6(c), each together with IF for Tbr2, in the neocortex of E12.5 control embryos. White arrowheads in the insets of (a, b) depict Ccny/l1 puncta in Tbr2+ BPs. High levels of LRP6 phosphorylation are detected in mitotic BPs (c, inset, white arrowhead). DNA staining for mitotic pLRP6+ BP shown in bottom right inset (c, green arrowhead). Scale bar 20 μm. (L) IF for pLRP6 (T1479) in the neocortex of E13.5 control and DKO embryos. White arrowheads depict mitotic APs and purple arrowheads depict mitotic BPs. Scale bar 20 μm. (M) Quantification of relative pLRP6 staining intensity in mitotic APs and BPs from (L). Data are expressed as fold change vs. controls and are means ± SEM (n=3 embryos, 3 litters). (N) Immunoblot analysis with an antibody against LRP6 phosphorylated at S1490 (pLRP6 (S1490)) of protein lysates from E13.5 control and DKO forebrains. Numbers above blot indicate fold change vs. controls of phospho-LRP6 (S1490) protein levels normalized to total LRP6 (n=6 embryos, 3 litters). (O) Expression analysis of the β-catenin target gene Axin2 by RNAScope in situ hybridization on neocortex sections from E13.5 control and DKO embryos. Scale bar 20 μm. (P) Quantification of (O). Axin2+ area was calculated and normalized to total neocortex area. Data are expressed as fold change vs. controls and are means ± SEM (n=4 embryos, 3 litters). (Q) Axin2 and N-myc qPCR expression analysis on RNA extracted from E13.5 dorsal forebrains. Data are expressed as fold change vs. controls and are means ± SEM (n=3 embryos, 2 litters).