c , Confocal microscopy analysis of UTF1 intensity in WT and Cdc14 DKO ESCs at time 0 ( T0 ) and after 1 day ( T1 ) in differentiation media . The quantification of UTF1 fluorescence mean intensity ( FMI ) per cell is shown in the plot to the right . Figures and horizontal bars indicate mean ± S . E . M . ( n = 1269 WT T0 cells , n = 2726 WT T1 , n = 974 Cdc14 DKO T0 , n = 1173 Cdc14 DKO T1 ) . * * , p < 0 . 01 ; * * * * , p < 0 . 0001 ; Student ' s t - test with Welch ' s correction .

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c, Confocal microscopy analysis of UTF1 intensity in WT and Cdc14 DKO ESCs at time 0 (T0) and after 1 day (T1) in differentiation media. The quantification of UTF1 fluorescence mean intensity (FMI) per cell is shown in the plot to the right. Figures and horizontal bars indicate mean ± S.E.M. (n=1269 WT T0 cells, n=2726 WT T1, n=974 Cdc14 DKO T0, n=1173 Cdc14 DKO T1). **, p<0.01; ****, p<0.0001; Student's t-test with Welch's correction.


d , Western blot analysis of UTF1 expression in WT and Cdc14 DKO ESCs at time 0 and after 3 days in differentiation media .

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d, Western blot analysis of UTF1 expression in WT and Cdc14 DKO ESCs at time 0 and after 3 days in differentiation media.


e , Western blot analysis of UTF1 expression in WT and Cdc14 DKO ESCs in absence or presence of the kinase inhibitors roscovitine ( R ) or trametinib ( T ) .

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e, Western blot analysis of UTF1 expression in WT and Cdc14 DKO ESCs in absence or presence of the kinase inhibitors roscovitine (R) or trametinib (T).


f , Immunoblot analysis of UTF1 protein stability after transfection of HeLa cells with UTF1 and GFP or CDC14B - GFP . Cells were treated with cycloheximide ( CHX ) and total proteins were detected at different time points as indicated . Cyclin B1 and β - catenin are used as controls .

O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O

f, Immunoblot analysis of UTF1 protein stability after transfection of HeLa cells with UTF1 and GFP or CDC14B-GFP. Cells were treated with cycloheximide (CHX) and total proteins were detected at different time points as indicated. Cyclin B1 and β-catenin are used as controls.


g , Western blot analysis of UTF1 WT or mutants in three ( UTF1 - 3KR ) or five ( UTF1 - 5KR ) lysines in absence or presence of trametinib ( T ) or roscovitine ( R ) .

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g, Western blot analysis of UTF1 WT or mutants in three (UTF1-3KR) or five (UTF1-5KR) lysines in absence or presence of trametinib (T) or roscovitine (R).


h , Immunoblot of UTF1 mutants in SPOP , SIAH or SPOP + SIAH potential binding sites , as well as S48D and S54D phosphorylation sites . Actin was used as a loading control .

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h, Immunoblot of UTF1 mutants in SPOP, SIAH or SPOP+SIAH potential binding sites, as well as S48D and S54D phosphorylation sites. Actin was used as a loading control.


( A ) Representative whole mount images of WT and Cep63T / T brains at P60 . Scale bar = 0 . 2 cm . ( B ) Telencephalon area of WT and Cep63T / T brains . WT littermates N = 4 , Cep63T / T N = 7 ; two - tailed Mann - Whitney t - test .

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(A) Representative whole mount images of WT and Cep63T/T brains at P60. Scale bar = 0.2 cm. (B) Telencephalon area of WT and Cep63T/T brains. WT littermates N = 4, Cep63T/T N = 7; two-tailed Mann-Whitney t-test.


( C ) Representative whole mount images of WT and Sas4cKO brains at P14 . Scale bar : 0 . 2 cm . ( D ) Telencephalon area of WT and Sas4cKO brains . WT littermates N = 8 , Sas4cKO N = 7 ; two - tailed Mann - Whitney t - test . Black circles represent Nestin - Cre + animals .

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(C) Representative whole mount images of WT and Sas4cKO brains at P14. Scale bar: 0.2 cm. (D) Telencephalon area of WT and Sas4cKO brains. WT littermates N = 8, Sas4cKO N = 7; two-tailed Mann-Whitney t-test. Black circles represent Nestin-Cre+ animals.


( E ) Representative images of E14 . 5 WT , Cep63T / T and Sas4cKO cortices stained with antibodies against α - Tubulin ( green ) , γ - Tubulin ( red ) and DAPI ( blue ) . Magenta arrows indicate dead cells ; white and yellow arrows indicate cells with bipolar and monopolar spindles , respectively . Insets showing zoomed in view of 2 representative cells . Dashed lines showing the orientation of the cleavage plane relative to the ventricular surface . Scale bar = 25 μm . ( F ) Graph showing the percentage of bipolar , monopolar and multipolar cells at the ventricular surface of E14 . 5 cortices . WT n = 201 cells , N = 3 embryos ; Cep63T / T n = 312 cells , N = 3 embryos ; Sas4cKO n = 296 cells , N = 3 embryos ; # , chi - square test with * , post - hoc analysis , comparisons are made to WT .

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(E) Representative images of E14.5 WT, Cep63T/T and Sas4cKO cortices stained with antibodies against α-Tubulin (green), γ-Tubulin (red) and DAPI (blue). Magenta arrows indicate dead cells; white and yellow arrows indicate cells with bipolar and monopolar spindles, respectively. Insets showing zoomed in view of 2 representative cells. Dashed lines showing the orientation of the cleavage plane relative to the ventricular surface. Scale bar = 25 μm. (F) Graph showing the percentage of bipolar, monopolar and multipolar cells at the ventricular surface of E14.5 cortices. WT n = 201 cells, N = 3 embryos; Cep63T/T n = 312 cells, N = 3 embryos; Sas4cKO n = 296 cells, N = 3 embryos; #, chi-square test with *, post-hoc analysis, comparisons are made to WT.


( E ) Representative images of E14 . 5 WT , Cep63T / T and Sas4cKO cortices stained with antibodies against α - Tubulin ( green ) , γ - Tubulin ( red ) and DAPI ( blue ) . Magenta arrows indicate dead cells ; white and yellow arrows indicate cells with bipolar and monopolar spindles , respectively . Insets showing zoomed in view of 2 representative cells . Dashed lines showing the orientation of the cleavage plane relative to the ventricular surface . Scale bar = 25 μm .

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(E) Representative images of E14.5 WT, Cep63T/T and Sas4cKO cortices stained with antibodies against α-Tubulin (green), γ-Tubulin (red) and DAPI (blue). Magenta arrows indicate dead cells; white and yellow arrows indicate cells with bipolar and monopolar spindles, respectively. Insets showing zoomed in view of 2 representative cells. Dashed lines showing the orientation of the cleavage plane relative to the ventricular surface. Scale bar = 25 μm.


( G ) E14 . 5 cortices stained with antibodies against Ki67 ( red ) and phosphorylated - Histone H3 ( PH3 , yellow ) . Insets showing zoomed in view of cells in the ventricular zone ( VZ ) and sub - ventricular zone ( SVZ ) . Scale bar = 100 μm .

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(G) E14.5 cortices stained with antibodies against Ki67 (red) and phosphorylated-Histone H3 (PH3, yellow). Insets showing zoomed in view of cells in the ventricular zone (VZ) and sub-ventricular zone (SVZ). Scale bar = 100 μm.


( H ) Ratio of mitotic ( PH3 + ) to cycling ( Ki67 + ) cells in E14 . 5 cortices . WT N = 5 , Cep63T / T N = 3 , Sas4cKO N = 3 ; two - tailed Mann - Whitney t - test .

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(H) Ratio of mitotic (PH3+) to cycling (Ki67+) cells in E14.5 cortices. WT N = 5, Cep63T/T N = 3, Sas4cKO N = 3; two-tailed Mann-Whitney t-test.


( J ) Representative images from live - cell movies of Nuclear - mCherry + WT , Cep63T / T and Sas4cKO NPCs . The timing of mitosis began at nuclear envelope breakdown ( NEBD ) and finished at nuclear envelope reformation . White arrows track the mitotic events of the mother cell from NEBD to the generation of daughter cells following cytokinesis . ( K ) Plot showing the duration of mitosis in WT , Cep63T / T and Sas4cKO NPCs . Triangles represent individual cells ; triangles of the same color represent cells derived from the same embryo . Circles represent the average mitotic duration of cells from each embryo . WT n = 202 cells , N = 4 embryos ; Cep63T / T n = 141 cells , N = 3 embryos ; Sas4cKO n = 138 cells , N = 3 embryos ; two - tailed Welch ' s t - test .

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(J) Representative images from live-cell movies of Nuclear-mCherry+ WT, Cep63T/T and Sas4cKO NPCs. The timing of mitosis began at nuclear envelope breakdown (NEBD) and finished at nuclear envelope reformation. White arrows track the mitotic events of the mother cell from NEBD to the generation of daughter cells following cytokinesis. (K) Plot showing the duration of mitosis in WT, Cep63T/T and Sas4cKO NPCs. Triangles represent individual cells; triangles of the same color represent cells derived from the same embryo. Circles represent the average mitotic duration of cells from each embryo. WT n = 202 cells, N = 4 embryos; Cep63T/T n = 141 cells, N = 3 embryos; Sas4cKO n = 138 cells, N = 3 embryos; two-tailed Welch's t-test.


( A - C ) Graph showing the mitotic duration of NPCs and the fate of their progeny in primary cultures generated from E14 . 5 embryos . Each bar represents one cell ; its height represents the amount of time the mother cell spent in mitosis ; bar color represents the fate of the progeny . Dashed line is set at 30 mins . WT n = 330 cells , N = 4 embryos ; Cep63T / T n = 260 cells , N = 3 embryos ; Sas4cKO n = 223 cells , N = 3 embryos . ( D - F ) Percentage of proliferating , arrested and apoptotic progeny within each group of the indicated mitotic duration , calculated from data shown in A - C ; groups with n < 20 cells were excluded from this quantification ; # , chi - square test with * , post - hoc analysis , within each genotype , comparisons are made to the 0 - 30min group .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A-C) Graph showing the mitotic duration of NPCs and the fate of their progeny in primary cultures generated from E14.5 embryos. Each bar represents one cell; its height represents the amount of time the mother cell spent in mitosis; bar color represents the fate of the progeny. Dashed line is set at 30 mins. WT n = 330 cells, N = 4 embryos; Cep63T/T n = 260 cells, N = 3 embryos; Sas4cKO n = 223 cells, N = 3 embryos. (D-F) Percentage of proliferating, arrested and apoptotic progeny within each group of the indicated mitotic duration, calculated from data shown in A-C; groups with n < 20 cells were excluded from this quantification; #, chi-square test with *, post-hoc analysis, within each genotype, comparisons are made to the 0-30min group.


( G ) Genome - wide copy number plots of single cells sequenced from dissociated dorsolateral telencephalons of E14 . 5 embryos . Individual cells are represented in rows with copy number states indicated by colors ; arrows on the zoomed in view ( right ) indicate aneuploid cells . WT n = 38 cells , N = 2 embryos ; Cep63T / T n = 40 cells , N = 2 embryos ; Sas4cKO n = 42 cells , N = 2 embryos . ( H ) Percentage of aneuploid cells from the single - cell sequencing data shown in G .

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(G) Genome-wide copy number plots of single cells sequenced from dissociated dorsolateral telencephalons of E14.5 embryos. Individual cells are represented in rows with copy number states indicated by colors; arrows on the zoomed in view (right) indicate aneuploid cells. WT n = 38 cells, N = 2 embryos; Cep63T/T n = 40 cells, N = 2 embryos; Sas4cKO n = 42 cells, N = 2 embryos. (H) Percentage of aneuploid cells from the single-cell sequencing data shown in G.


( A ) Representative whole mount images of WT , Sas4cKO , Sas4cKO ; Trp53bp1 - / - , Sas4cKO ; Usp28cKO animals at P14 . Arrows indicate the cerebellar hypoplasia resulting from the lack of primary cilia . Scale bar = 0 . 2 cm . ( B ) Telencephalon area of P14 brains of the indicated genotypes . WT littermates N = 8 , Sas4cKO N = 7 , Sas4cKO ; Trp53bp1 - / - N = 6 , Sas4cKO ; Usp28cKO N = 10 , Sas4cKO ; Trp53 - / - N = 5 ; one - way ANOVA with post - hoc analysis . WT and Sas4cKO data are from Figure 1D and shown alongside for comparison . Black circles represent Nestin - Cre + animals . ( C ) Cortical thickness of P14 brains of the indicated genotypes . WT littermates N = 4 , Sas4cKO N = 4 , Sas4cKO ; Trp53bp1 - / - N = 4 , Sas4cKO ; Usp28cKO N = 4 , Sas4cKO ; Trp53 - / - N = 4 ; one - way ANOVA with post - hoc analysis . WT and Sas4cKO data are from Figure EV1D and shown alongside for comparison . Black circles represent Nestin - Cre + animals .

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(A) Representative whole mount images of WT, Sas4cKO, Sas4cKO;Trp53bp1-/-, Sas4cKO;Usp28cKO animals at P14. Arrows indicate the cerebellar hypoplasia resulting from the lack of primary cilia. Scale bar = 0.2 cm. (B) Telencephalon area of P14 brains of the indicated genotypes. WT littermates N = 8, Sas4cKO N = 7, Sas4cKO;Trp53bp1-/- N = 6, Sas4cKO;Usp28cKO N = 10, Sas4cKO;Trp53-/- N = 5; one-way ANOVA with post-hoc analysis. WT and Sas4cKO data are from Figure 1D and shown alongside for comparison. Black circles represent Nestin-Cre+ animals. (C) Cortical thickness of P14 brains of the indicated genotypes. WT littermates N = 4, Sas4cKO N = 4, Sas4cKO;Trp53bp1-/- N = 4, Sas4cKO;Usp28cKO N = 4, Sas4cKO;Trp53-/- N = 4; one-way ANOVA with post-hoc analysis. WT and Sas4cKO data are from Figure EV1D and shown alongside for comparison. Black circles represent Nestin-Cre+ animals.


( D ) Representative whole mount images of WT , Cep63T / T , Cep63T / T ; Trp53bp1 - / - , Cep63T / T ; Usp28 - / - animals at P60 . Scale bar = 0 . 2 cm . ( E ) Telencephalon area of P60 brains of the indicated genotypes . WT littermates N = 4 , Cep63T / T N = 7 , Cep63T / T ; Trp53bp1 - / - N = 6 , Cep63T / T ; Usp28 - / - N = 5 ; one - way ANOVA with post - hoc analysis . WT and Cep63T / T data are from Figure 1B and shown alongside for comparison . ( F ) Cortical thickness of P60 brains of the indicated genotypes . WT littermates N = 4 , Cep63T / T N = 5 , Cep63T / T ; Trp53bp1 - / - N = 3 , Cep63T / T ; Usp28 - / - N = 3 ; one - way ANOVA with post - hoc analysis . WT and Cep63T / T data are from Figure EV1B and shown alongside for comparison .

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(D) Representative whole mount images of WT, Cep63T/T, Cep63T/T;Trp53bp1-/-, Cep63T/T ;Usp28-/- animals at P60. Scale bar = 0.2 cm. (E) Telencephalon area of P60 brains of the indicated genotypes. WT littermates N = 4, Cep63T/T N = 7, Cep63T/T;Trp53bp1-/- N = 6, Cep63T/T;Usp28-/- N = 5; one-way ANOVA with post-hoc analysis. WT and Cep63T/T data are from Figure 1B and shown alongside for comparison. (F) Cortical thickness of P60 brains of the indicated genotypes. WT littermates N = 4, Cep63T/T N = 5, Cep63T/T;Trp53bp1-/- N = 3, Cep63T/T;Usp28-/- N = 3; one-way ANOVA with post-hoc analysis. WT and Cep63T/T data are from Figure EV1B and shown alongside for comparison.


( G ) Body weight of P7 animals of the indicated genotypes . WT littermates N = 18 , Cep63T / T N = 16 , Cep63T / T ; Trp53bp1 - / - N = 2 , Cep63T / T ; Usp28 - / - N = 10 ; one - way ANOVA with post - hoc analysis .

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(G) Body weight of P7 animals of the indicated genotypes. WT littermates N=18, Cep63T/T N=16, Cep63T/T;Trp53bp1-/- N=2, Cep63T/T;Usp28-/- N=10; one-way ANOVA with post-hoc analysis.


( H ) Quantification of total number of cells within a 500 μm - width column of cortices at P60 . WT littermates N = 4 , Cep63T / T N = 4 , Cep63T / T ; Trp53bp1 - / - N = 4 , Cep63T / T ; Usp28 - / - N = 3 ; one - way ANOVA with post - hoc analysis .

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(H) Quantification of total number of cells within a 500 μm-width column of cortices at P60. WT littermates N = 4, Cep63T/T N = 4, Cep63T/T;Trp53bp1-/- N = 4, Cep63T/T;Usp28-/- N = 3; one-way ANOVA with post-hoc analysis .


( I - J ) Quantification of the number of cells in the ( I ) superficial layer ( CUX1 + ) and ( J ) deep layer ( CTIP2 + ) within a 500 μm - width column of P60 cortices . WT littermates N = 3 , Cep63T / T N = 4 , Cep63T / T ; Trp53bp1 - / - N = 4 , Cep63T / T ; Usp28 - / - N = 3 ; one - way ANOVA with post - hoc analysis .

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(I-J) Quantification of the number of cells in the (I) superficial layer (CUX1+) and (J) deep layer (CTIP2+) within a 500 μm-width column of P60 cortices. WT littermates N = 3, Cep63T/T N = 4, Cep63T/T;Trp53bp1-/- N = 4, Cep63T/T;Usp28-/- N = 3; one-way ANOVA with post-hoc analysis .


( K ) P60 cortices of indicated genotypes stained with antibodies against the deep layer marker CTIP2 ( green ) , superficial layer marker CUX1 ( red ) and DAPI ( blue ) . Scale bar = 200 μm .

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(K) P60 cortices of indicated genotypes stained with antibodies against the deep layer marker CTIP2 (green), superficial layer marker CUX1 (red) and DAPI (blue). Scale bar = 200 μm.


( A ) Cortices from E14 . 5 brains stained with antibodies against the radial glial cell marker PAX6 ( yellow ) , intermediate progenitor marker TBR2 ( red ) and DAPI ( blue ) . Scale bar = 50 μm . ( B and C ) Quantification of the number of ( B ) radial glial cells ( PAX6 + ) and ( C ) intermediate progenitors ( TBR2 + ) within a 250 μm - width column of E14 . 5 cortices . WT N = 3 , Cep63T / T N = 3 , Cep63T / T ; Usp28 - / - N = 3 , Sas4cKO N = 3 , Sas4cKO ; Usp28cKO N = 3 ; one - way ANOVA with post - hoc analysis .

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(A) Cortices from E14.5 brains stained with antibodies against the radial glial cell marker PAX6 (yellow), intermediate progenitor marker TBR2 (red) and DAPI (blue). Scale bar = 50 μm. (B and C) Quantification of the number of (B) radial glial cells (PAX6+) and (C) intermediate progenitors (TBR2+) within a 250 μm-width column of E14.5 cortices. WT N=3, Cep63T/T N = 3, Cep63T/T;Usp28-/- N = 3, Sas4cKO N = 3, Sas4cKO;Usp28cKO N = 3; one-way ANOVA with post-hoc analysis.


( D ) Cortices from E14 . 5 embryos stained with antibodies against Cleaved - Caspase 3 ( CC3 , white ) , TP53 ( red ) and DAPI . Scale bar = 50 μm . ( E and F ) Quantification of the number of CC3 + apoptotic cells ( E ) and cells with TP53 activation ( F ) within a 250 μm - width column of E14 . 5 cortices . WT N = 5 , Cep63T / T N = 3 , Cep63T / T ; Usp28 - / - N = 3 , Sas4cKO N = 4 , Sas4cKO ; Usp28cKO N = 4 ; one - way ANOVA with post - hoc analysis .

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(D) Cortices from E14.5 embryos stained with antibodies against Cleaved-Caspase 3 (CC3, white), TP53 (red) and DAPI. Scale bar = 50 μm. (E and F) Quantification of the number of CC3+ apoptotic cells (E) and cells with TP53 activation (F) within a 250 μm-width column of E14.5 cortices. WT N = 5, Cep63T/T N = 3, Cep63T/T;Usp28-/- N = 3, Sas4cKO N = 4, Sas4cKO;Usp28cKO N = 4; one-way ANOVA with post-hoc analysis.


( A ) Representative images of E14 . 5 cortices stained with antibodies against Centrin ( green ) , γ - Tubulin ( red ) and DAPI ( blue ) . White and yellow arrows indicate cells with bipolar and monopolar spindles , respectively . Insets showing zoomed in view of 2 representative cells . Dashed lines showing the orientation of the cleavage plane relative to the ventricular surface . Scale bar = 10 μm .

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(A) Representative images of E14.5 cortices stained with antibodies against Centrin (green), γ-Tubulin (red) and DAPI (blue). White and yellow arrows indicate cells with bipolar and monopolar spindles, respectively. Insets showing zoomed in view of 2 representative cells. Dashed lines showing the orientation of the cleavage plane relative to the ventricular surface. Scale bar = 10 μm.


( B ) Percentage of bipolar , monopolar and multipolar cells at the ventricular surface of E14 . 5 cortices . WT n = 201 cells , N = 3 embryos ; Cep63T / T ; Usp28 - / - n = 205 cells , N = 3 embryos ; Sas4cKO ; Usp28cKO n = 308 cells , N = 3 embryos ; # , chi - square test with * , post - hoc analysis , comparisons are made to WT . WT data are from Figure 1F and shown alongside for comparison .

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(B) Percentage of bipolar, monopolar and multipolar cells at the ventricular surface of E14.5 cortices. WT n = 201 cells, N = 3 embryos; Cep63T/T;Usp28-/- n = 205 cells, N = 3 embryos; Sas4cKO;Usp28cKO n = 308 cells, N = 3 embryos; #, chi-square test with *, post-hoc analysis, comparisons are made to WT. WT data are from Figure 1F and shown alongside for comparison.


( C ) Ratio of mitotic ( PH3 + ) to cycling ( Ki67 + ) cells in E14 . 5 cortices . WT N = 5 , Cep63T / T ; Usp28 - / - N = 3 , Sas4cKO ; Usp28cKO N = 3 ; two - tailed Mann - Whitney t - test . 3 WT data points are from Figure 1H and shown alongside for comparison .

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(C) Ratio of mitotic (PH3+) to cycling (Ki67+) cells in E14.5 cortices. WT N = 5, Cep63T/T;Usp28-/- N = 3, Sas4cKO;Usp28cKO N = 3; two-tailed Mann-Whitney t-test. 3 WT data points are from Figure 1H and shown alongside for comparison.


( D ) Representative images from live - cell movies of Nuclear - mCherry + WT , Sas4cKO and Sas4cKO ; Usp28cKO NPCs . White arrows showing the nuclei of daughter cells following cytokinesis . ( E ) Plot showing quantification of mitotic duration of WT , Sas4cKO and Sas4cKO ; Usp28cKO NPCs . Triangles represent individual cells ; triangles of the same color represent cells derived from the same embryo . Circles represent the average mitotic duration of cells from each embryo . WT n = 202 cells , N = 4 embryos ; Sas4cKO n = 138 cells , N = 3 embryos ; Sas4cKO ; Usp28cKO n = 191 cells , N = 3 embryos ; two - tailed Welch ' s t - test . WT and Sas4cKO data are from Figure 1K and shown alongside for comparison . ( F ) Graph showing the mitotic duration of NPCs and the fate of their progeny in primary cultures generated from Sas4cKO ; Usp28cKO E14 . 5 embryos . Each bar represents one cell ; its height represents the amount of time the mother cell spent in mitosis ; bar color represents the fate of the progeny . Dashed line is set at 30 min . Sas4cKO ; Usp28cKO n = 271 cells , N = 3 embryos . ( G ) Percentage of proliferating , arrested and apoptotic progeny within each group of the indicated mitotic duration , calculated from data shown in F ; # , chi - square test with * , post - hoc analysis , comparisons are made to the 0 - 30min group .

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(D) Representative images from live-cell movies of Nuclear-mCherry+ WT, Sas4cKO and Sas4cKO;Usp28cKO NPCs. White arrows showing the nuclei of daughter cells following cytokinesis. (E) Plot showing quantification of mitotic duration of WT, Sas4cKO and Sas4cKO;Usp28cKO NPCs. Triangles represent individual cells; triangles of the same color represent cells derived from the same embryo. Circles represent the average mitotic duration of cells from each embryo. WT n = 202 cells, N = 4 embryos; Sas4cKO n = 138 cells, N = 3 embryos; Sas4cKO;Usp28cKO n = 191 cells, N = 3 embryos; two-tailed Welch's t-test. WT and Sas4cKO data are from Figure 1K and shown alongside for comparison. (F) Graph showing the mitotic duration of NPCs and the fate of their progeny in primary cultures generated from Sas4cKO;Usp28cKO E14.5 embryos. Each bar represents one cell; its height represents the amount of time the mother cell spent in mitosis; bar color represents the fate of the progeny. Dashed line is set at 30 min. Sas4cKO;Usp28cKO n = 271 cells, N = 3 embryos. (G) Percentage of proliferating, arrested and apoptotic progeny within each group of the indicated mitotic duration, calculated from data shown in F; #, chi-square test with *, post-hoc analysis, comparisons are made to the 0-30min group.


Graph showing the mitotic duration of NPCs and the fate of their progeny in primary cultures generated from Sas4cKO E14 . 5 embryos . Each bar represents one cell ; its height represents the amount of time the mother cell spent in mitosis ; bar color represents the fate of the progeny . Dashed line is set at 30 min . ( H and I ) Data from Figures 2C and 2F shown alongside for comparison with F and G .

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Graph showing the mitotic duration of NPCs and the fate of their progeny in primary cultures generated from Sas4cKO E14.5 embryos. Each bar represents one cell; its height represents the amount of time the mother cell spent in mitosis; bar color represents the fate of the progeny. Dashed line is set at 30 min. (H and I) Data from Figures 2C and 2F shown alongside for comparison with F and G.


( a ) WT and Smc5cKO cortices at E14 . 5 stained with antibodies against Centrin ( green ) , γ - Tubulin ( red ) and DAPI ( blue ) . Insets showing zoomed in view of 2 representative cells . Scale bar : 25 μm . ( B and C ) Quantification of the number of ( B ) Centrin foci and ( C ) γ - Tubulin foci in mitotic cells in the VZ of E14 . 5 cortices . WT n = 175 cells , N = 4 embryos ; Smc5cKO n = 134 cells , N = 3 embryos . # , chi - square test . WT data are from Figures EV1F and EV1G and shown alongside for comparison .

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(a) WT and Smc5cKO cortices at E14.5 stained with antibodies against Centrin (green), γ-Tubulin (red) and DAPI (blue). Insets showing zoomed in view of 2 representative cells. Scale bar: 25 μm. (B and C) Quantification of the number of (B) Centrin foci and (C) γ-Tubulin foci in mitotic cells in the VZ of E14.5 cortices. WT n = 175 cells, N = 4 embryos; Smc5cKO n = 134 cells, N = 3 embryos. #, chi-square test. WT data are from Figures EV1F and EV1G and shown alongside for comparison.


( D ) E14 . 5 cortices stained with antibodies against Ki67 ( red ) and phosphorylated - Histone H3 ( PH3 , yellow ) . Insets showing zoomed in view of cells in the VZ and SVZ . Scale bar = 100 μm .

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(D) E14.5 cortices stained with antibodies against Ki67 (red) and phosphorylated-Histone H3 (PH3, yellow). Insets showing zoomed in view of cells in the VZ and SVZ. Scale bar = 100 μm.


( E ) Ratio of mitotic ( PH3 + ) to cycling ( Ki67 + ) cells in E14 . 5 cortices . WT N = 5 , Smc5cKO N = 5 ; two - tailed Mann - Whitney t - test . WT data are from Figure 1H and shown alongside for comparison .

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(E) Ratio of mitotic (PH3+) to cycling (Ki67+) cells in E14.5 cortices. WT N = 5, Smc5cKO N = 5; two-tailed Mann-Whitney t-test. WT data are from Figure 1H and shown alongside for comparison.


( F Quantification of the number of ( F ) apoptotic cells ( CC3 + ) within a 250 μm - width column of E14 . 5 cortices . WT N = 5 , Smc5cKO N = 4 ; two - tailed Mann - Whitney t - test . WT data are from Figures 4E and 4G and shown alongside for comparison .

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(F Quantification of the number of (F) apoptotic cells (CC3+) within a 250 μm-width column of E14.5 cortices. WT N = 5, Smc5cKO N = 4; two-tailed Mann-Whitney t-test. WT data are from Figures 4E and 4G and shown alongside for comparison.


G ) Quantification of the number of ( G ) cells with TP53 activation within a 250 μm - width column of E14 . 5 cortices . WT N = 5 , Smc5cKO N = 4 ; two - tailed Mann - Whitney t - test . WT data are from Figures 4E and 4G and shown alongside for comparison .

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G) Quantification of the number of (G) cells with TP53 activation within a 250 μm-width column of E14.5 cortices. WT N = 5, Smc5cKO N = 4; two-tailed Mann-Whitney t-test. WT data are from Figures 4E and 4G and shown alongside for comparison.


( H ) Representative whole mount images of WT , Smc5cKO , Smc5cKO ; Usp28cKO , Smc5cKO ; Trp53 - / - brains at P21 . Scale bar = 0 . 2 cm . ( I ) Telencephalon area of P21 brains of the indicated genotypes . WT littermates N = 6 , Smc5cKO N = 5 , Smc5cKO ; Usp28cKO N = 4 , Smc5cKO ; Trp53 - / - N = 5 ; one - way ANOVA with post - hoc analysis .

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(H) Representative whole mount images of WT, Smc5cKO, Smc5cKO;Usp28cKO, Smc5cKO;Trp53-/- brains at P21. Scale bar = 0.2 cm. (I) Telencephalon area of P21 brains of the indicated genotypes. WT littermates N = 6, Smc5cKO N = 5, Smc5cKO;Usp28cKO N = 4, Smc5cKO;Trp53-/- N = 5; one-way ANOVA with post-hoc analysis.