Volume 20, Issue 5, Pages 1148-1160 (August 2017) Loss of Brap Results in Premature G1/S Phase Transition and Impeded Neural Progenitor Differentiation  Alison A. Lanctot, Yan Guo, Yicong Le, Brittany M. Edens, Richard S. Nowakowski, Yuanyi Feng  Cell Reports  Volume 20, Issue 5, Pages 1148-1160 (August 2017) DOI: 10.1016/j.celrep.2017.07.018 Copyright © 2017 The Author(s) Terms and Conditions

Cell Reports 2017 20, 1148-1160DOI: (10.1016/j.celrep.2017.07.018) Copyright © 2017 The Author(s) Terms and Conditions

Figure 1 Loss of Brap Results in Increased BrdU LI without Changing NPC Structure and Population (A and B) Representative immunohistological (IH) images of transiently BrdU-labeled (30-min pulse) Pax6+ (A) or Tbr2+ (B) cortical NPCs at E12.5. (C) Representative IH images of phospho-vimentin 4A4-labeled M phase cortical NPCs co-labeled with Pax6 (RGCs) at E12.5. (D) Quantification of Pax6+ RGC and Tbr2+ INP populations in wild-type and BrapKO cortices normalized to 100 μm of apical surface. (E) BrdU pulse labeling indexes (LIs) of wild-type and BrapKO RGCs (Pax6+). Numbers represent BrdU+ cells/100 μm of apical surface. Mean ± SD, ∗p < 0.001. (F) BrdU pulse LIs of wild-type and BrapKO INPs (Tbr2+). Numbers represent BrdU+ cells/100 μm of apical surface. Mean ± SD. (G) Mitotic index of 4A4+ RGCs in wild-type and BrapKO cortices. Numbers represent 4A4+Pax6+ cells/100 μm of apical surface. Mean ± SD, p = 0.07. Nuclear DNA was stained with Hoechst 33342. Scale bars, 100 μm. CP, cortical plate; SVZ, subventricular zone; VZ, ventricular zone. See also Figure S1. Cell Reports 2017 20, 1148-1160DOI: (10.1016/j.celrep.2017.07.018) Copyright © 2017 The Author(s) Terms and Conditions

Figure 2 Prolonged DNA Replication and G2 Phase Arrest in BrapKO NPCs at E12.5 (A) Diagram of the experimental scheme of the S phase sequential labeling analysis. (B) Representative IH images of IdU (B44), CldU (BU1/75), and PH3 triple-stained cortical sections of wild-type and BrapKO embryos sequentially labeled by IdU and CldU. White and red arrowheads indicate cells stalled in S phase; blue arrows indicate M phase cells with low IdU incorporation in the mutant. (C) S phase duration (TS, hours) of wild-type and BrapKO NPCs. Mean ± SD, ∗p < 0.001. (D) Fraction of wild-type and BrapKO NPCs stalled in S phase (percentage of total S-phase NPCs). Mean ± SD, ∗p < 0.001. (E) Diagram illustrating the cell cycle-dependent interkinetic nuclear migration (INM) of cortical NECs/RGCs. (F) Representative IH images of PH3+ (G2/M phase) and 4A4+ (M phase) co-stained cortical sections of wild-type and BrapKO embryos. Arrowheads denote PH3+4A4− NPCs in the VZ of the BrapKO cortex. (G) Selected high magnification images of PH3+4A4− NPCs from (F), showing their lack of DNA condensation. (H) Fractions (percent) of PH3+ cells localized at the apical surface, in the VZ, or in the SVZ. (I and J) Representative immunoblotting (IB) images (I) and quantitative analysis (J) of Cdc25a and its phosphatase substrate pCdk1 (Tyr15) in cortical lysates of wild-type and BrapKO embryos. Mean ± SD, ∗p < 0.001 for Cdc25a, p < 0.01 for pCdk1. See also Figures S2 and S3. Cell Reports 2017 20, 1148-1160DOI: (10.1016/j.celrep.2017.07.018) Copyright © 2017 The Author(s) Terms and Conditions

Figure 3 G1 Progression Is Hastened in BrapKO NPCs at E12.5 (A) Cell cycle diagram illustrating TGF = TG2 + TM + TG1 = TC – TS. (B) Representative IH images of BrdU and Pax6 double-labeled RGCs in the cumulative BrdU labeling study, showing that both wild-type and BrapKO RGCs reach maximum labeling (TGF) in 9 hr. (C) BrdU cumulative LIs) (mean ± SD) for wild-type and BrapKO Pax6+ RGCs at E12.5. (D) Representative images of IB analysis of cyclins (D1, E, and B1) in cortical lysates of BrapKO and wild-type littermates at E12.5. (E) Representative images of IB analysis of G1/S phase CKIs p21Cip1 and p27Kip1 and cyclin A in cortical lysates of BrapKO and wild-type littermates at E12.5. (F) Quantification of the IB analyses of (D) and (E); fold changes of protein levels in BrapKO over wild-type littermates are presented. Mean ± SD, ∗p < 0.05. (G) Representative images of IB analysis of whole-cell lysates of cultured NPCs derived from wild-type and BrapKO cortices at E12.5. Cell Reports 2017 20, 1148-1160DOI: (10.1016/j.celrep.2017.07.018) Copyright © 2017 The Author(s) Terms and Conditions

Figure 4 Brap-Skp2 Interaction and Its Mediated G1/S Phase Regulation (A) The interaction of Brap with Skp2 demonstrated by a GST pull-down study. The diagram represents the structure of Brap protein, including RING-H2 (RH), ubiquitin protease-like zinc finger (ZnF), and coiled-coil leucine heptad repeat (CC) domains. The previously identified Ras binding domain (Ras BD) and GST fusion constructs used are also depicted. (B) Co-immunoprecipitation (coIP) analysis of Brap or the BrapC263A mutant with myc-SKP2 co-expressed in 293FT cells. Cells were incubated with the proteasome inhibitor MG132 for 24 hr before IP analysis using the anti-myc 9E10 monoclonal antibody. (C) IP of SKP2 from 293FT cells transfected with full-length Brap, BrapC263A, or vector, followed by 24-hr incubation with the proteasome inhibitor MG132. The IP products were analyzed by ubiquitin (Ub) and SKP2 IB, respectively. (D) Representative IB images and quantitative analyses of SKP2 and p21Cip1 levels in 293 cells transfected with Brap, BrapC263A, or vector. Data are presented as mean ± SD relative to the vector control. ∗p < 0.05. (E) The nuclear localization of SKP2 in 293FT cells 24 hr after GFP or GFP-Brap transfection. Fractions (percent) of nuclear SKP2 were scored from over 200 randomly photographed GFP+ cells. (F) IH analysis of Skp2 in cerebral cortical NPCs labeled sequentially by IdU and CldU at E12.5. NPCs in G1 phase were identified by the lack of IdU and CldU immunosignals, whereas NPCs in S, G2, and M phases were identified by their nuclear location and IdU and CldU labeling pattern. Note the high nuclear Skp2 immunosignals in S, G2, and M phases and the weak cytoplasmic Skp2 immunosignals in G1 phase. (G) IB analysis of Skp2 and its substrate c-Myc in wild-type and BrapKO cortical lysates at E12.5. Fold change of BrapKO over wild-type littermates is presented. Mean ± SD, ∗p < 0.05. (H) Representative IB images and quantitative analysis of primary cortical NPCs isolated from wild-type or BrapKO littermates and cultured with or without the presence of the Skp2 inhibitor SKPin C1. Fold change of BrapKO over wild-type NPCs is presented. Mean ± SD, ∗p < 0.05. (I) A representative IB image and quantitative analysis of Dcx levels in cortical NPCs isolated from wild-type or BrapKO littermates and cultured with or without the presence of the Skp2 inhibitor SKPin C1. Fold change of BrapKO relative to wild-type NPCs is presented. Mean ± SD, ∗p < 0.05. See also Figure S4. Cell Reports 2017 20, 1148-1160DOI: (10.1016/j.celrep.2017.07.018) Copyright © 2017 The Author(s) Terms and Conditions

Figure 5 Impeded Neuronal Differentiation in BrapKO and BrapcKO Cortices (A) Representative IH images of BrdU and Tbr2 double-stained cortical sections from E12.5 wild-type and BrapKO embryos fixed 16 hr after BrdU pulse labeling. (B) Quantification of the fraction (percent) of BrdU-labeled NPCs differentiated into Tbr2+ INPs in wild-type and BrapKO embryos from E11.5 to E12.5. Numbers presented are relative to wild-type littermates; n = 3, mean ± SD, ∗p < 0.05. (C) Representative IH images of BrdU and Tbr2 double-stained cortical sections of E13.5 control and BrapcKO embryos fixed 16 hr after BrdU pulse labeling. (D) Quantification of BrdU-labeled NPCs that expressed Tbr2 (BrdU+Tbr2+) or BrdU-labeled NPCs that expressed neither Pax6 nor Tbr2 (BrdU+Pax6−Tbr2−) from E12.5 to E13.5. Numbers were from spatially matched control and BrapcKO littermates (n = 3), normalized to 100 μm of ventricular width, and are presented as mean ± SD; ∗p < 0.05. (E) Representative IH image of control and BrapcKO littermate cortices double-stained with the neuron marker Tbr1 and the subplate marker CSPG at E14.5. (F) Representative IH image of control and BrapcKO littermate cortices double-stained with the NPC marker Sox2 and the neuron marker Tuj1 at E16.5. (G) Representative images of Dcx, Tbr2, and Pax6 IB analysis of total protein extracts from embryonic cortical tissue of wild-type and BrapKO (E12.5) or control and BrapcKO (E14.5 and E16.5) littermates. (H) Quantitative analysis of Dcx, Tbr2, and Pax6 protein levels in cortical tissue from E12.5 to E16.5. Data from BrapKO or BrapcKO embryos are presented as fold change relative to wild-type (E12.5) or control (E14.5 and E16.5) littermates. Mean ± SD, ∗p < 0.05. (I) Quantification of BrdU+ neurons born at E12.5, E13.5, or E14.5 in control and BrapcKO cortices at E16.5. Numbers were normalized to 100 μm of ventricular surface. Mean ± SD, ∗p < 0.01. (J) Neuronal birth date analysis of the number and distribution of BrdU+ neurons at E16.5. Shown are representative images of E16.5 embryos pulse-labeled by BrdU at E12.5, E13.5, or E14.5. Nuclear DNA was stained with Hoechst 33342. Scale bars, 50 μm. IZ, intermediate zone. See also Figure S5. Cell Reports 2017 20, 1148-1160DOI: (10.1016/j.celrep.2017.07.018) Copyright © 2017 The Author(s) Terms and Conditions

Figure 6 Reduced Brain Weight and Superficial Cortical Neurons in BrapcKO Mice (A) H&E analysis of coronal brain sections from control and BrapcKO young adult littermates. (B) Total brain weight of BrapcKO (Brapf/KO Emx1 Cre+; n = 9) and control (n = 14) mice at weaning age (post-natal day 28 [P28]). Mean ± SD, ∗p < 0.001. (C) H&E analysis of coronal brain sections of control and BrapcKO mice at P28. Cortical layers I through VI are labeled. Note the unremarkable lamina organization of cortical neurons and preferential thinning of superficial layers in the mutant. (D) Representative IH image and quantitative analysis of Cux1+ cortical neurons (layers II/III) at P28. Cux1+ cell numbers were normalized to 100 μm of ventricular surface. Mean ± SD, p < 0.01. (E) Representative IH image and quantitative analysis of Foxp2+ cortical neurons (layer VI) at P28. Foxp2+ cell numbers were normalized to 100 μm of ventricular surface. Mean ± SD, p = 0.07. (F) Representative IH image and quantitative analysis of Foxp1+ cortical neurons (layers II–V) at P28. Foxp1+ cell numbers were normalized to 100 μm of ventricular surface. Mean ± SD, p < 0.01. (G) Representative IH image and quantitative analysis of Tbr1+ cortical neurons (layer II/III) at P28. Tbr1+ cell numbers were normalized to 100 μm of ventricular surface. Mean ± SD, p = 0.05. Nuclear DNA was stained with Hoechst 33342. Scale bars, 100 μm. See also Figure S6. Cell Reports 2017 20, 1148-1160DOI: (10.1016/j.celrep.2017.07.018) Copyright © 2017 The Author(s) Terms and Conditions