Supplementary Figure S1. Alterations in Fzr1( / );Nestin-Cre brains. (a) P10 Cdh1-deficient brains display low levels of Myelin basic protein (MBP)

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1 Supplementary Figure S1. Alterations in Fzr1( / );Nestin-Cre brains. (a) P10 Cdh1-deficient brains display low levels of Myelin basic protein (MBP) in the cortex (area 1 as defined in Fig. 2a), and corpus callosum dysgenesis (white arrows, area 2). (b) H&E staining of P15 mice showing dysgenesis of the corpus callosum (area 2), and the presence of oedema in the wall of the lateral ventricle. (c) H&E staining of P35 mutant mice with an aggressive hydrocephalus, vacuolized cortex and damage in the ependymal layer (arrow). Scale bars, 250 m (a) 200 m (b, c). 1

2 Supplementary Figure S2. Cdh1 expression in the developing brain. The expression of Cdh1 was detected by immunofluorescence (red signal) in three different areas of E14.5 brains. The localization of these areas in the brain section is shown in the top panel. DAPI (blue) was used to detect nuclei. (a-f) Wild-type E14.5 brains. d, e, f correspond to the insets indicated in a, b, c, respectively. (g-i) The specificity of the Cdh1 antibody was tested by comparing wild-type brains with Fzr1( / );Nestin-Cre brain sections. nctx, neocortex; lge,: lateral ganglionic eminence; lv, lateral ventricle; 3v, third ventricle. Dashed white lines mark the limit of ventricles. Scale bars, 100 m (a, b, c), 25 m (d, e, f), 75 m (g, h, i). 2

3 Supplementary Figure S3. Defective development of Fzr1( / );Sox2-Cre mice. (a) Representative micrographs of E18.5 Fzr1( / );Sox2-Cre embryos with detailed images of the bone and the brain cortex. Quantification of the thickness of the cortex is shown in the histogram (n=3). ***; p<0.001, Student t test. Error bars indicate SEM. (b) Survival curve of Fzr1( / );Sox2-Cre and control mice (left), and representative pictures of these mutant mice at postnatal day (P)10 indicating the weight of these animals (right). (c) Microscopic analysis of representative aberrations displayed by Fzr1( / );Sox2-Cre mice at P10. The majority of mutant tissues (including liver, lung, skin and hematopoietic tissues) present a significant increase in active caspase-3 staining (apoptosis). H&E, hematoxylin and eosin. Scale bars, 100 m. 3

4 Supplementary Figure S4. Analysis of Cdh1 substrates. (a) Quantification of Cdh1 substrates in E14.5 brains of the indicated genotypes.(n=3 brains per genotype) ***; p<0.001, Student t test. Error bars indicate SEM. (b) Reduction in the levels of Cdk inhibitory proteins in Cdh1-deficient primary and secondary neurospheres. Immunoblot of Fzr1( / );Nestin-Cre primary and secondary neurospheres showing decreased levels of p21 Cip1 and p27 Kip1. Phosphorylation of histone H3 (ph3) is increased in Fzr1( / );Nestin-Cre primary neurospheres (probably as a consequence of increase arrest in late G2; see main text) but decreases in secondary neurospheres suggesting lack of proliferation. Primary neurospheres also display accumulation of phosphorylated (p)rpa, a marker of DNA damage. *, non-specific band. 4

5 Supplementary Figure S5. Accumulation of p53 and apoptosis in the ventricular area of the embryonic brain in the absence of Cdh1. (a) Progressive increase in p53 and active caspase-3 (C3A) signal (brown) during embryonic development (E12.5-E16.5). ns, not significant (p>0.05), **, p<0.01; ***, p<0.001; Student t test (n=3 embryos per genotype). Error bars indicate SEM. (b) Increased proliferation and apoptosis in the brain cortex of E16.5 Fzr1( / ); Nestin-Cre embryos. (a) are sagital sections; (b) are coronal sections. Scale bars, 100 m. 5

6 Supplementary Figure S6. Accumulation of damage in Fzr1( / ); Sox2-Cre brains. The ventricular area of E14.5 brains from Fzr1( / ); Sox2-Cre mice was stained with antibodies against phosphorylated H2AX H2AX), p53 or activecaspase 3 (C3A). These data indicate that replicative stress, p53 induction and apoptotic cell death in Fzr1( / ); Sox2-Cre embryos is similar to that observed in Fzr1( / ); Nes-Cre embryos. Coronal sections. Scale bars, 100 m. 6

7 Supplementary Figure S7. Replicative stress in Cdh1-deficient neural progenitors is prevented by partial inhibition of Cdk activity or nucleosides addition. (a) Quantification of H2AX or EdU intensity in control (grey) or Cdh1-deficient (red) neural progenitors from primary neurospheres treated for 8h with the indicated dose of Roscovitine ( M) or Doxorubicin (0.5 g/ml), or the addition of nucleosides (upper panel). (b) Effect of similar treatments in wild-type controls. *, p<0.05; **, p<0.01; ***, p<0.001; Student t test. At least 400 cells per condition were analyzed. Data represent one representative experiment out of three replicates. 7

8 Supplementary Figure S8. The choroid plexus in Cdh1-deficient and control embryos. Sections correspond to E14.5 embryos of the indicated genotypes. H&E, hematoxylin and eosin. H2AX is only positive in ependymal cells. Scale bars, 100 m. 8

9 Supplementary Fig. S9. Massive hemorrhages in brains of Fzr1( / ); Nestin-Cre; Tp53( / ) mice. Red areas (arrows) indicate accumulation of red blood cells in the cortex (left) or near the ventricles (right panels). Images correspond to H&E staining of coronal sections. Scale bars, 200 m. 9

10 Supplementary Fig. S10. Increased DNA damage in Cdh1; p53 double deficient embryos. Samples from the third ventricle (top panels) or the lateral ventricle (bottom panels) from E14.5 embryos of the indicated genotypes were stained with antibodies against phosphorylated histone H3 (ph3) or phosphorylated H2AX ( H2AX). These data show that the accumulation of damaged cells affects all ventricular areas in double mutant brains. Scale bars, 25 m. 10

11 Supplementary Fig. S11. Accumulation of the proliferation marker Ki67 in the hippocampus, cortex and cerebellum of P10 Cdh1-deficient and double Cdh1; p53 mutant mice. The concomitant depletion of p53 leads to further accumulation of Ki67-positive cells (arrowheads). Cdh1-null and double Cdh1; p53-null mice display a similar reduction in the thickness of the cortex (arrows). Coronal sections. Scale bars, 100 m. 11

12 Supplementary Fig. S12. Accumulation of cells with a G2-like pattern in the phosphorylation of histone H3 (ph3) cells in the brain of P10 Cdh1-deficient and double Cdh1; p53 mutant mice. The concomitant depletion of p53 leads to further accumulation of Ki67- positive cells (arrowheads). Cdh1-null and double Cdh1; p53-null mice display a similar reduction in the thickness of the cortex (arrows). Coronal sections. Scale bars, 100 m. 12

13 Supplementary Figure S13. Full scans of blots shown in Fig. 3b (a), 3e (b) and Supplementary Fig. S4 (c). 13

14 Supplementary Table S1. Antibodies used in this work. Antigen Source Ig Source Dilution Catalogue# Use* Actin Mouse Sigma-Aldrich 1:2000 A4700 WB AurkA Mouse Becton Dickinson (BD) 1: IHC AurkA Mouse Abcam 1:500 ab13824 WB AurkB Rabbit Abcam 1:100 ab2254 IHC III-tubulin Mouse Sigma-Aldrich 1:500 T 8660 IF Bromo-deoxyuridine Mouse GE Healthcare 1:100 RPN202 IHC (BrdU) Caspase 3 Active (C3A) Rabbit RYD Systems 1:400 AF835 IHC Cdc6 Mouse J. Méndez (CNIO, Madrid) 1:1000 WB Cdh1 Rabbit J.-M. Peters (IMP, Vienna) 1:2000 IF Cdh1 Mouse Neomarkers 1:200 MS-1116-P WB Ctip2 Rat Abcam 1:500 Ab18465 IF Cyclin B1 Mouse Millipore 1:175 MAB3684 IHC Cyclin B Mouse Chemicon 1:1000 MAB3684 WB -Tubuliln Mouse Sigma-Aldrich 1:200-1:700 T6557 IF Geminin Rabbit Santa Cruz Biotech. 1:150-1:400 sc IHC, WB Glial Fibrillary Acidic Rabbit Dako 1:500 Z0334 IHC Protein (GFAP) Ki67 Rabbit Master Diagnostica Prediluted QD IHC Nestin Mouse Developmental Studies 1:10 IF Hybridoma Bank (DSHB) p21 Rabbit Santa Cruz Biotechnology 1:200 sc-397 WB p27 Mouse BD Transduction Labs. 1: WB p53 Rabbit Leica 1:300 NCL-p53-CM5p IHC PCNA Mouse Merck 1:1000 NA03 IHC Phospho-Histone H2A.X (Ser139) Mouse Millipore 1:1000-1: IHC, IF Phospho-Histone H3 Rabbit Millipore 1:200-1: IHC, IF (Ser10) Phospho-RPA32 Rabbit Bethyl 1:500 A A WB S100 Rabbit Dako 1:100-1:1000 Z0311 IHC, IF Satb2 Mouse Abcam 1:50 ab51502 IF Sox2 Rabbit Millipore 1:200-1:1000 AB5603 IHC, IF, WB Sox9 Rabbit Millipore 1:200-1:300 AB5535 IHC, IF Tbr1 Rabbit Abcam 1:200 ab31940 IF Tbr2 Rabbit Abcam 1:200 ab23345 IF Tpx2 Rabbit Lifespan Biosciences 1:250-1:1000 LS-B146 IHC, WB Tubulin, acetylated Mouse Sigma 1:700 T7451 IF Vinculin Mouse Sigma-Aldrich 1:3000 V9131 WB * IF, immunofluorescence; IHC, immunohistochemistry; WB, immunoblot. 14