Supporting InformationPao et al. 10.1073/pnas.1400783111SI Materials and MethodsAnimals. Animals strains used in this study are Nestin-Cre mice[B6.Cg-Tg (Nes-cre)1Kln/J; The Jackson Laboratory], BRCA1-null allele mice [STOCKBrca1tm1Cxd,Mouse Models of HumanCancer (MMHC) Mouse Repository at the National CancerInstitute (NCI) (1)], BRCA1 floxed allele mice [FVB;129-Brca1tm2Brn, MMHC Mouse Repository at NCI (2)], ATM KOmice (3), and p53 KO mice (4).

Immunohistochemistry. Primary antibodies used include the fol-lowing: rat anti-BrdU (Accurate), rabbit anti-GFP (MolecularProbes), rabbit anti-GFAP (Lab Vision/Neomarkers), rabbit anti-Bhlhb5 [kindly provided by Sarah E. Ross, University of Pittsburgh,Pittsburgh (5)], mouse anti-p73 (Lab Vision/Neomarkers), goatanti-Sox2 (Santa Cruz Biotechnology), mouse anti-Pax6 (Hy-bridoma Bank), mouse anti-Nestin (BD Pharmingen), mouseanti-ATM (Cell Signaling), mouse anti-proliferating cell nuclearantigen (Proliferating Cell Nuclear Antigen, Neomarkers), rab-bit anti-Caspase3 (Cell Signaling), mouse anti-Reelin (Reln;Chemicon), mouse anti-Tuj1 (Covance), rabbit anti-Calbindin(CB; Swant), rabbit anti-mouse p53 (Vector), and mouse anti-Map2(a+b) (Sigma).

Proliferation Assays. Short (2 hours) and long pulses (E15.5) ofBrdU were performed by injecting 200 μL of BrdU i.p. inpregnant females (E12.5 and E15.5). Image J software (NationalInstitutes of Health) was used for cell counting.

Cell Death Assay: TUNEL. TUNEL assays were carried out with theApopTag Apoptosis Detection Kit (Chemicon) as recommendedby the manufacturer.

Microarray Analysis. Brain total RNA was prepared using TRIzolreagent (Invitrogen). Total RNA was processed and hybridized withthe Affymetrix GeneChip Mouse Genome 430 2.0 Arrays (Affy-metrix Core Facility, The Salk Institute). The gene expression profilewas analyzed using the Bullfrog software package.

Cell Culture and Virus Infection. Primary neural progenitor cellswere isolated from adult mice containing one breast cancer sus-ceptibility gene 1 (BRCA1) null allele and one floxed BRCA1allele as described (6). The cells were cultured in DMEM/F12(1:1) medium with N2 supplement (Invitrogen), 20 ng/mL ofhuman fibroblast growth factor 2 (Peprotech), and 20 ng/mLhuman epithelial growth factor (Peprotech). Embryonic neuralprogenitors cells were isolated and cultured as described pre-viously (7). Cultured cells were retrovirally infected and in-cubated for a 12 hour period with an MOI of 5 and wereharvested at different times. To study cell proliferation, 10 μM ofBrdU (Sigma-Aldrich) or 5-ethynyl-2′deoxyuridine (Invitrogen)was pulsed for 30 min before fixation. Reconstitution experi-ments were performed on passage 2 embryonic neural pro-genitor cells and infected with either retrovirus expressing Cre-GFP or GFP alone. Lentiviral human BRCA1 expressed underthe control of the CAG promoter or control GFP was coinfectedwith the retroviral constructs. Approximately 72 hours after in-fection, cells were harvested for immunofluorescence staining.

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5. Ross SE, et al. (2012) Bhlhb5 and Prdm8 form a repressor complex involved in neuronalcircuit assembly. Neuron 73(2):292–303.

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Fig. S1. BRCA1 mRNA expression pattern and confirmation of the disruption of the BRCA1 gene by PCR. (A) At E12.5, BRCA1 mRNA is located in the uppermost part of the neocortical ventricular zone (VZ) and in the ventricular mantle of the ganglionic eminence (GE). High magnifications are shown in a–c pointingto same locations in A. (B) PCR detection of deletion of exons 5–13. Groups 1–7: DNA from cerebellum (1), cortex (2), hippocampus (3), olfactory bulbs (4),brainstem (5), spleen (6), and tail (7). In each group, PCR products of the LoxP sites on the 5′ (left lane) and 3′ (right lane) sites are shown. (C) PCR productspanning the deletion. No PCR products were detected in control (CTRL) samples or BRCA1 KO spleens. There was deletion in the tail DNA due to the presenceof CNS cells. In both cases (B and C), the samples were extracted from P10 BRCA1 KO (KO) mice and littermate controls. Ncx, neocortex; PrP, preplate. (Scale bar:A, 125 μm; a–c, 75 μm.)

Fig. S2. Transcription factors Dlx5 and Ngn2 are severely reduced in the BRCA1 KO brains. (A–D) E15.5 embryonic brain slices showing the expression of Dlx5(A and B) and Ngn2 (C and D). (A and B) Dlx5 expression is severely reduced in a rudimentary ganglionic eminence (GE) in the BRCA1 KO vs. control (CTRL).Arrowheads indicate Dlx5 expression in the ganglionic eminences. (C and D) Ngn2 expression is very diminished in the neocortical ventricular zone (VZ) of theBRCA1 KO vs. control. Choroid plexus (ChP, arrows) are hypertrophic in the BRCA1 KO (B and D). Ncx, neocortex. (Scale bars: A–D, 125 μm.)

Fig. S3. BrdU pulses confirm that upper layer neurons are not generated in the BRCA1 KO mice. (A–J) Long pulses of BrdU were performed in BRCA1 KO (F–J)and control (CTRL; A–E) animals. Injection day was at E15.5 and harvesting day at P8. Colabeling with upper layer neurons marker Bhlhb5 (green), Brdu labelingneurons generated at E15.5 (red), and DAPI as counterstaining were used. Arrowheads point to upper layers neurons colabeled with BrdU and Bhlhb5 incontrols and the same region in the BRCA1 KOmice with almost absence of Bhlhb5 and BrdU-positive neurons in the upper layers. Arrowhead in J points to onecell positive for Bhlhb5 and BrdU in the BRCA1 KO. White bars point to the Bhlhb5-positive cortical plate (CP) in the control (B) and BRCA1 KO (G). MZ, marginalzone; VZ, ventricular zone. (Scale bars: A–C, 150 μm; D and E, 100 μm; F–H, 150 μm; I and J, 100 μm.)

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Fig. S4. Reelin expression unmask lamination defects associated to the loss of BRCA1. Reelin (Reln) expression in cerebellum (A and B; Cb), olfactory bulb(C and D; Ob), and hippocampus (E and F; Hc) of the BRCA1 KO and control (CTRL) at P7. (A and B) Reelin-positive granular cells located in the internal granularlayer (igl) are severely diminished in the BRCA1 KO vs. control. (C and D) Reelin-positive mitral cells are reduced in cellular density with an abnormal laminationpattern in the BRCA1 KO. (E and F) Reelin immunoreactive interneuronal population (arrows) is dramatically diminished in the BRCA1 KO compared withcontrol. DG, dentate gyrus; Mcl, mitral cell layer. (Scale bars: A–D, 500 μm; E and F, 100 μm.)

Fig. S5. Defects on glial markers Nestin and GFAP associated to the loss of BRCA1. (A–D) At E15.5, Nestin-positive radial glia are severely diminished, pre-senting misaligned and shorten glial fibers in the BRCA1 KO compared with control (CTRL). (E and F) At P7, GFAP labeling shows an increase in reactive as-trocytes throughout the entire cortical wall in the BRCA1 KO mice versus CTRL. CP, cortical plate; LV, lateral ventricle; pCP, partial cortical plate; VZ, ventricularzone. (Scale bars: A and B, 125 μm; C and D, 75 μm; E and F, 100 μm.)

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Fig. S6. Cell cycle profiles of cultured neural progenitor cells. FACS analysis of BRCA1 KO NPCs (Cre-GFP) showing an increase in both S and G2/M phases. Thesedata indicate an arrest of the cell cycle in S and G2/M phases in the BRCA1-deficient NPCs compared with Mock and GFP controls. G1 and subG1, cell cyclephases G1 and subG1.

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Fig. S7. Diagram summarizing the neocortical defects associated with the loss of BRCA1 and the rescue of the phenotype by concomitant deletion of p53. Innormal conditions [control (CTRL)], early embryonic neurogenesis begins with symmetric divisions of the neuroepithelial cell (NEC) pool, expanding theneurogenic progenitor niche in the ventricular zone (VZ) (1). Around E12, NECs can differentiate into radial glia cells (RGs) that have the potential to divideasymmetrically generating neurons for the lower cortical layers (V and VI) and either another RG cell or a basal progenitor cell (BP) (1). Around E13.5, a secondprogenitor compartment named the subventricular zone (SVZ) starts to develop by the migration of the BP cells generated in the VZ, which simultaneouslystart to divide (R) in the SVZ, populating and expanding the pool of BP cells in the SVZ (2–4). BP cells start to generate upper layer neurons (II–IV) that migrateradially to their correspondent cortical layer (3, 5–8). The generation and migration of the cortical neurons is restrictedly controlled according to an inside-outgradient, in which layer VI neurons are the first to be generated, and then layer V neurons, and so on until layers II–III are formed (9, 10). At around E17, RGsstart to decline and a second gliogenesis period develops as GFAP astrocytes start to appear in the proliferative compartments (1, 11). Around birth, VZ is nolonger proliferative and transforms into an ependymal layer (E), whereas the SVZ niche still maintains proliferative capabilities throughout adult life (11). In theBRCA1 KO (B1KO), neurogenesis defects start to be evident after E11.5, which is the onset of the recombination for the Nestin-Cre driver (12). At around E12,earlier progenitors generate layer VI neurons that start to migrate forming a disorganized and reduced layer VI. Layer V is barely developed and severelydepleted of layer V neurons. We do not detect any layer above layer V, neither morphologically nor using neuronal specific markers for upper layers (II–IV),suggesting that the neurogenesis was interrupted after layer V neuronal-committed progenitors were being generated. Due to the brain tissue damagecaused, we did observe an abruptly increase in reactive GFAP astrocytes all over the cortical wall. The concomitant deletion of p53 and BRCA1 rescues thelamination phenotype. Thus, cortical layers are reestablished, and upper layers (II–IV) are developed. However, the polarity of the pyramidal cells forming thecortical layers is still aberrant and lack the typical radial orientation observed in control, which indicates additional BRCA1 functions not related with p53-dependent apoptosis. Abbreviations are displayed as color keys in the figure.

1. Götz M, Huttner WB (2005) The cell biology of neurogenesis. Nat Rev Mol Cell Biol 6(10):777–788.2. Miyata T, et al. (2004) Asymmetric production of surface-dividing and non-surface-dividing cortical progenitor cells. Development 131(13):3133–3145.3. Molyneaux BJ, Arlotta P, Menezes JR, Macklis JD (2007) Neuronal subtype specification in the cerebral cortex. Nat Rev Neurosci 8(6):427–437.4. Noctor SC, Martínez-Cerdeño V, Ivic L, Kriegstein AR (2004) Cortical neurons arise in symmetric and asymmetric division zones and migrate through specific phases. Nat Neurosci 7(2):

136–144.5. Nieto M, et al. (2004) Expression of Cux-1 and Cux-2 in the subventricular zone and upper layers II-IV of the cerebral cortex. J Comp Neurol 479(2):168–180.6. Smart IH, McSherry GM (1982) Growth patterns in the lateral wall of the mouse telencephalon. II. Histological changes during and subsequent to the period of isocortical neuron

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9. Lambert de Rouvroit C, Goffinet AM (1998) The reeler mouse as a model of brain development. Adv Anat Embryol Cell Biol 150:1–106.10. Perez-Garcia CG, Tissir F, Goffinet AM, Meyer G (2004) Reelin receptors in developing laminated brain structures of mouse and human. Eur J Neurosci 20(10):2827–2832.11. Alvarez-Buylla A, Garcia-Verdugo JM (2002) Neurogenesis in adult subventricular zone. J Neurosci 22(3):629–634.12. Chou SJ, Perez-Garcia CG, Kroll TT, O’Leary DD (2009) Lhx2 specifies regional fate in Emx1 lineage of telencephalic progenitors generating cerebral cortex. Nat Neurosci 12(11):

1381–1389.

Fig. S8. A hypomorph KO of BRCA1 in the brain showed a much milder phenotype. (A) P10 animals of a hypomorph BRCA1 conditional KO did not showgrowth retardation. (B) P28 animals had a slight brain volume reduction but are externally indistinguishable from the control brains.

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Table S1. Neurogenesis and cortical differentiation

Genes Fold change

Endothelial differentiation, lysophosphatidic acid Gprotein-coupled receptor, 2

2.66

Chemokine (C-X-C motif) ligand 12 4.92Doublecortin −2.13Ectonucleotide pyrophosphatase/phosphodiesterase 1 2.66Tachykinin 2 −4.42Forkhead box C1 2.99Sialyltransferase 8 (alpha-2, 8-sialyltransferase) B −1.74Neuronal pentraxin 2 1.82Synaptoporin −1.65Disabled homolog 2 (Drosophila) 2Vasoactive intestinal polypeptide −6.06Distal-less homeobox 2 −2.74Distal-less homeobox 1 −3.73Distal-less homeobox 5 −2.13Aristaless related homeobox gene (Drosophila) −2.21T-box18 6.02Vimentin 1.8NK2 transcription factor related, locus 2 (Drosophila) −1.68Membrane associated guanylate kinase

interacting protein-like 1−2.28

G protein–coupled receptor GPR17 −4.16Endothelial differentiation, sphingolipid G

protein-coupled receptor, 32.16

SRY-box containing gene 10 −2.62Neurofascin −1.9

Table S2. Oligodendrocyte myelination

Genes Fold change

Peripheral myelin protein, 22 kda 1.71Myelin basic protein −9.05Myelin-associated oligodendrocytic basic protein −7.24Proteolipid protein (myelin) −3.25Proteolipid protein (myelin) −5Myelin basic protein −8.84Myelin-associated oligodendrocytic basic protein −13.2Myelin basic protein −8.77Sialyltransferase 8 (alpha-2, 8-sialyltransferase) B −2.02Sphingosine kinase 1 2.32Proteolipid protein (myelin) −6.86Myelin basic protein −6.35Myelin basic protein −5.36Myelin-associated glycoprotein −2.7Myelin and lymphocyte protein, T-cell differentiation protein −6.81Myelin-associated oligodendrocytic basic protein −33.77SRY-box containing gene 10 −2.62Neurofascin −1.9

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Table S3. Oxidative response

Genes Fold change

Thioredoxin interacting protein 1.91Lysyl oxidase 2.05GST, alpha 4 1.74Stress induced protein 1.5Dicarbonyl L-xylulose reductase 2.06S100 calcium binding protein A6 (calcyclin) 2.44Epoxide hydrolase 1, microsomal 2.6Metallothionein 2 1.77Cytochrome b-245, alpha polypeptide 2

Table S4. Proliferation

Genes Fold change

Transforming growth factor, beta induced, 68 kDa 5.53Inhibitor of DNA binding 3 1.82Connective tissue growth factor 2.08Jun oncogene 1.88Cyclin-dependent kinase inhibitor 1C (P57) 1.7Bone morphogenetic protein 7 2.76Cyclin-dependent kinase inhibitor 1A (P21) 6.75FBJ osteosarcoma oncogene 3.88Inhibitor of DNA binding 1 2.26Transforming growth factor, beta receptor II 2.03Smoothened homolog (Drosophila) 1.65Cysteine rich protein 61 3.91Transforming growth factor, beta induced, 68 kDa 7.18H19 fetal liver mrna 2.37Insulin-like growth factor 2 2.14T-box18 6.02Bone morphogenetic protein 6 2.66Insulin-like growth factor binding protein 2 1.71Sirtuin 2 (silent mating type information regulation 2,

homolog) 2 (S. cerevisiae)−1.95

LIM and senescent cell antigen like domains 2 −2.58Breast carcinoma amplified sequence 1 −4.25Kruppel-like factor 13 −1.87Histone 1, h2ad −2.19Ribosomal protein s15a 1.6

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Table S5. Extracellular matrix

Genes Fold change

Plasminogen activator, tissue 1.48Fibromodulin 1.87Matrix metalloproteinase 2 1.94Serine (or cysteine) proteinase inhibitor, clade F), member 1 2.54Cathepsin C 1.68Biglycan 2.35Elastin microfibril interface located protein 1 5.79Nidogen 1 1.92Epithelial membrane protein 3 2.24Microfibrillar-associated protein 2 1.6Laminin, alpha 1 4.29Procollagen, type XVIII, alpha 1 2.39Cathepsin H 1.74Ig superfamily containing leucine-rich repeat 2.83Annexin A2 1.85Lectin, galactose binding, soluble 1 2.79Vitronectin 2Procollagen, type IV, alpha 6 2.39Mannose receptor, C type 2 3.13Lectin, galactose binding, soluble 9 1.64Procollagen, type V, alpha 2 2.35Nidogen 2 2.48Lumican 3.35Procollagen, type I, alpha 1 3.13Laminin, gamma 1 1.88Procollagen, type VI, alpha 3 3.03Procollagen, type IV, alpha 5 3.4Laminin, alpha 2 1.98Procollagen, type IV, alpha 1 2.62Procollagen, type VI, alpha 2 1.95Procollagen, type XVIII, alpha 1 2.28Epidermal growth factor-containing fibulin-like

extracellular matrix protein 13.38

Procollagen, type III, alpha 1 3.79Cadherin 5 1.84Procollagen C-proteinase enhancer protein 3.48Transglutaminase 2, C polypeptide 1.92Fibromodulin 1.91Biglycan 2.56Matrix metalloproteinase 14 (membrane-inserted) 1.66Matrix gamma-carboxyglutamate (gla) protein 1.73Procollagen C-proteinase enhancer protein 2.21Thrombomodulin 2.64Procollagen, type VI, alpha 1 2.33Cathepsin S 2.03Procollagen, type XV 2.96Decorin 2.14Cathepsin K 2.41Serine (or cysteine) proteinase inhibitor, clade H, member 1 2.14Procollagen, type I, alpha 2 2.87Procollagen, type IV, alpha 1 2.32Procollagen, type VI, alpha 2 2.35Serine (or cysteine) proteinase inhibitor, clade F), member 1 2.41Vitronectin 2.14Lectin, galactose binding, soluble 1 2.68Supervillin 2.66Gelsolin −3.1Claudin 11 −3.7Plexin B3 −3.97A disintegrin-like and metalloprotease (reprolysin type)

with thrombospondin type 1 motif, 4−2.92

Fras1 related extracellular matrix protein 1 2.37Epidermal growth factor-containing fibulin-like

extracellular matrix protein 13.38

Procollagen, type III, alpha 1 3.79

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Movie S1. Ataxic phenotype observed in the BRCA1 KO animals. BRCA1 KO animals display overt signs of a severe deficiency in motor coordination includingataxia, tremors, and inability to maintain gait and balance during locomotion.

Movie S1

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