Figure 7 ( A ) CSCs transfected with or without Notch1 ICD were analyzed for AMP / ATP ratio plus LKB1 phosphorylation . Mean ± SEM from 6 patients . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 . ( B ) CSCs treated with or without DAPT ( 10 μM ) were analyzed for AMP / ATP ratio plus LKB1 phosphorylation . Mean ± SEM from 6 patients . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 . ( C ) CSCs treated with or without CpG ODN ( 10 μg / ml ) were analysed for interactions between Notch1 and AMPK . Representative and mean ± SEM from 6 patients . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 . ( D ) CSCs transfected with LKB1 shRNA （ right ） or the control ( left ) were treated with CpG ODN ( 10 μg / ml ) and analysed for AMPK phosphorylation . Mean ± SEM from 6 patients . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 . ( E ) CSCs transfected with TAK1 shRNA or the control were treated with CpG ODN ( 10 μg / ml ) and analysed for AMPK phosphorylation . Mean ± SEM from 6 patients . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 . ( F ) CSCs transfected with LAMTOR1 shRNA or the control were treated with CpG ODN ( 10 μg / ml ) and analysed for AMPK phosphorylation . Mean ± SEM from 6 patients . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 . ( G ) CSCs transfected with AXIN1 shRNA or the control were treated with CpG ODN ( 10 μg / ml ) and analysed for AMPK phosphorylation . Mean ± SEM from 6 patients . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

O O B-PANEL_START 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 B-PANEL_START 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 B-PANEL_START 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 B-PANEL_START 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 B-PANEL_START 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 B-PANEL_START 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 B-PANEL_START 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

Figure 7(A) CSCs transfected with or without Notch1 ICD were analyzed for AMP/ATP ratio plus LKB1 phosphorylation. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.(B) CSCs treated with or without DAPT (10 μM) were analyzed for AMP/ATP ratio plus LKB1 phosphorylation. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.(C) CSCs treated with or without CpG ODN (10 μg/ml) were analysed for interactions between Notch1 and AMPK. Representative and mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.(D) CSCs transfected with LKB1 shRNA（right）or the control (left) were treated with CpG ODN (10 μg/ml) and analysed for AMPK phosphorylation. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.(E) CSCs transfected with TAK1 shRNA or the control were treated with CpG ODN (10 μg/ml) and analysed for AMPK phosphorylation. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.(F) CSCs transfected with LAMTOR1 shRNA or the control were treated with CpG ODN (10 μg/ml) and analysed for AMPK phosphorylation. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.(G) CSCs transfected with AXIN1 shRNA or the control were treated with CpG ODN (10 μg/ml) and analysed for AMPK phosphorylation. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


Figure 8 ( A ) NSCLC PDX models treated with or without chemotherapy for the first 2 weeks were analyzed for tumor growth at the indicated time . Mean ± SEM from 6 experiments . Data information : Paired t - test with Bonferroni method . * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . ( B ) Frequencies of CD133 - expressing CSCs from NSCLC xenografts treated with or without chemotherapy were determined after 4 weeks . Mean ± SEM from 6 experiments . Data information : Paired t - test with Bonferroni method . * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . ( C - E ) Flow cytometric analyses of protein expressions of TLR9 , activated Notch1 , and phosphor - AMPKα in cancer cells from NSCLC xenografts treated with or without chemotherapy after 4 weeks . Mean ± SEM from 6 experiments . Data information : Paired t - test with Bonferroni method . * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . ( F ) NSCLC PDX models were treated with chemotherapy alone or together with the indicated inhibitors , respectively , for the first 2 weeks . Tumor volume was determined at the indicated time . Mean ± SEM from 6 experiments . ( G ) Frequencies of CD133 - expressing CSCs from NSCLC xenografts treated with chemotherapy alone or with the indicated inhibitors after 4 weeks . Mean ± SEM from 6 experiments . Data information : ANOVA test with Turkey ' s method . * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

O O B-PANEL_START 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 B-PANEL_START 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 B-PANEL_START 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 B-PANEL_START 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

Figure 8(A) NSCLC PDX models treated with or without chemotherapy for the first 2 weeks were analyzed for tumor growth at the indicated time. Mean±SEM from 6 experiments. Data information: Paired t-test with Bonferroni method. **p < 0.01, ***p < 0.001, ****p < 0.0001.(B) Frequencies of CD133-expressing CSCs from NSCLC xenografts treated with or without chemotherapy were determined after 4 weeks. Mean±SEM from 6 experiments. Data information: Paired t-test with Bonferroni method. **p < 0.01, ***p < 0.001, ****p < 0.0001.(C-E) Flow cytometric analyses of protein expressions of TLR9, activated Notch1, and phosphor-AMPKα in cancer cells from NSCLC xenografts treated with or without chemotherapy after 4 weeks. Mean±SEM from 6 experiments. Data information: Paired t-test with Bonferroni method. **p < 0.01, ***p < 0.001, ****p < 0.0001.(F) NSCLC PDX models were treated with chemotherapy alone or together with the indicated inhibitors, respectively, for the first 2 weeks. Tumor volume was determined at the indicated time. Mean±SEM from 6 experiments. (G) Frequencies of CD133-expressing CSCs from NSCLC xenografts treated with chemotherapy alone or with the indicated inhibitors after 4 weeks. Mean±SEM from 6 experiments. Data information: ANOVA test with Turkey's method. **p < 0.01, ***p < 0.001, ****p < 0.0001.


Figure 1Cortical malformations observed in six novel BBSOAS patients Patient ( P ) 1 shows abnormally elongated occipital convolutions ( arrowheads in A ) , whereas abnormal gyrification of supramarginal and angular gyri is present in five out of six individuals ( P2 - P6 B ' , C ' and C ' ' highlight the abnormally convoluted regions boxed in B and C . Cortical malformations observed in six novel BBSOAS patients Patient ( P ) 1 shows abnormal gyrification of supramarginal and angular gyri is present in five out of six individuals ( P2 - P6 ; ( H , H ' ) TBR2 ( green in H ) , SOX2 ( red in H ) and NR2F1 ( yellow in H ' ) immunofluorescence ( IF ) of a GW11 section of human neocortex Data information : Nuclei ( blue ) were stained with DAPI . Scale bars : 50µm . CP : cortical plate ; IZ : intermediate zone ; oSVZ : outer subventricular zone ; SVZ : subventricular zone ; VZ : ventricular zone . ( I ) NR2F1 expression ( quantified by pixel intensity ) at different L - M levels and in different regions along the A - P extent of the cortex , as indicated . n ≥ 3 sections from n = 1 fetal brain . Data information the number of positive cells was quantified in 100µm - width boxes , randomly placed across the cortex . In graphs , data are represented as means ± SEM . 2 - way ANOVA ( I ) ( * P < 0 . 05 , * * * P < 0 . 001 ) . ( J - L ' ) NR2F1 ( red ) IF of human GW14 neocortex , showing expression levels in the CP ( K , L ) and VZ ( K ' , L ' ) around primary convolutions in the posterior - most cortex High NR2F1 is detected in the neurogenic area of a gyrus ( L ' ) . Data information : Nuclei ( blue ) were stained with DAPI . In Scale bars : 50µm . CP : cortical plate ; IZ : intermediate zone ; oSVZ : outer subventricular zone ; SVZ : subventricular zone ; VZ : ventricular zone . ( M , M ' ) NR2F1 level as measured on single cells in the VZ ( M ) and CP ( M ' ) of sulci and gyri n ≥ 4 sections from n = 1 fetal brain . ( N - O ) NR2F1 ( red ) and TBR2 ( green ) immunostaining NR2F1 level as measured in single TBR2 + IPs by pixel intensity is shown in graph ( O ) . n ≥ 4 convolutions from n = 1 fetal brain ( n = 2 sections ) . Data information In graphs , data are represented as means ± SEM . Student t - test ( M , M ' , O ( * P < 0 . 05 , * * * P < 0 . 001 ) . ( P - R ) NR2F1 ( red ) and HOPX ( green ) immunostaining Virtually all HOPX + bRGs are also NR2F1 + ( magnification in P ' ' ) and their number is greatly increased in gyri ( P ' ' ) compared to sulci ( P ' ) , as quantified in ( R ) . At a single cell level , NR2F1 pixel intensity is higher in oSVZ HOPX + cells in gyri compared to sulci ( Q ) . n ≥ 4 sections from n = 1 fetal brain . Data information : Nuclei ( blue ) were stained with DAPI . the number of positive cells was quantified in 100µm - width boxes , randomly placed across the cortex . In graphs , data are represented as means ± SEM . Student t - test Q , R ) ( * P < 0 . 05 , * * * P < 0 . 001 ) . oSVZ : outer subventricular

O B-PANEL_START 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 B-PANEL_START 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 B-PANEL_START 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 B-PANEL_START 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 B-PANEL_START 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 B-PANEL_START 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 B-PANEL_START 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

Figure 1Cortical malformations observed in six novel BBSOAS patients Patient (P)1 shows abnormally elongated occipital convolutions (arrowheads in A), whereas abnormal gyrification of supramarginal and angular gyri is present in five out of six individuals (P2-P6 B', C' and C'' highlight the abnormally convoluted regions boxed in B and C.Cortical malformations observed in six novel BBSOAS patients Patient (P)1 shows abnormal gyrification of supramarginal and angular gyri is present in five out of six individuals (P2-P6;(H,H') TBR2 (green in H), SOX2 (red in H) and NR2F1 (yellow in H') immunofluorescence (IF) of a GW11 section of human neocortex Data information: Nuclei (blue) were stained with DAPI. Scale bars: 50µm. CP: cortical plate; IZ: intermediate zone; oSVZ: outer subventricular zone; SVZ: subventricular zone; VZ: ventricular zone.(I) NR2F1 expression (quantified by pixel intensity) at different L-M levels and in different regions along the A-P extent of the cortex, as indicated. n ≥ 3 sections from n = 1 fetal brain. Data information the number of positive cells was quantified in 100µm-width boxes, randomly placed across the cortex. In graphs, data are represented as means ± SEM. 2-way ANOVA (I) (*P<0.05, ***P<0.001).(J-L') NR2F1 (red) IF of human GW14 neocortex, showing expression levels in the CP (K,L) and VZ (K',L') around primary convolutions in the posterior-most cortex High NR2F1 is detected in the neurogenic area of a gyrus (L'). Data information: Nuclei (blue) were stained with DAPI. In Scale bars: 50µm. CP: cortical plate; IZ: intermediate zone; oSVZ: outer subventricular zone; SVZ: subventricular zone; VZ: ventricular zone.(M,M') NR2F1 level as measured on single cells in the VZ (M) and CP (M') of sulci and gyri n ≥ 4 sections from n = 1 fetal brain. (N-O) NR2F1 (red) and TBR2 (green) immunostaining NR2F1 level as measured in single TBR2+ IPs by pixel intensity is shown in graph (O). n ≥ 4 convolutions from n = 1 fetal brain (n = 2 sections). Data information In graphs, data are represented as means ± SEM. Student t-test (M,M',O (*P<0.05, ***P<0.001).(P-R) NR2F1 (red) and HOPX (green) immunostaining Virtually all HOPX+ bRGs are also NR2F1+ (magnification in P'') and their number is greatly increased in gyri (P'') compared to sulci (P'), as quantified in (R). At a single cell level, NR2F1 pixel intensity is higher in oSVZ HOPX+ cells in gyri compared to sulci (Q). n ≥ 4 sections from n = 1 fetal brain. Data information: Nuclei (blue) were stained with DAPI. the number of positive cells was quantified in 100µm-width boxes, randomly placed across the cortex. In graphs, data are represented as means ± SEM. Student t-test Q,R) (*P<0.05, ***P<0.001). oSVZ: outer subventricular zone


Figure 2 ( A ) Representative images of wild - type ( WT ) and mutant ( KO ) neurospheres obtained from E15 . 5 neocortices and cultured in vitro . The culture passage ( " step " ) corresponds to 3 days of culture after dissociation and re - plating . KO aggregates are more numerous and larger in size . Data information : Scale bars : 50µm . ( B ) Graph showing the number of neurospheres per P100 cell plate at different steps , as indicated . Nr2f1KO neurospheres proliferate for a longer time ( up to 30 passages tested ) , whereas WT cells exhaust around step 9 . n ≥ 3 culture wells from n = 2 batches . Data information : data are represented as means ± SEM . 2 - way ANOVA ( B ) ( * * P < 0 . 01 , * * * P < 0 . 001 ) . ( C ) Panel of WT and KO isolated neurospheres grown from day 1 to day 10 . Blue and red lines represent the diameter of WT and KO neurospheres , respectively . Data information Scale bars : 50µm . ( D , E ) PH3 ( green ; dividing cells ) IF on cross - sections of day 4 neurospheres . White arrowheads point at mitotic figures , which are quantified in ( E ) as percentage of dividing cells over DAPI - stained nuclei ( blue ) . n ≥ 3 neurospheres from n = 2 batches . Data information : data are represented as means ± SEM . Student t - test ( E ) ( * * P < 0 . 01 , * * * P < 0 . 001 ) . Scale bars : 50µm .

O O B-PANEL_START 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 B-PANEL_START 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 B-PANEL_START 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 B-PANEL_START 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

Figure 2(A) Representative images of wild-type (WT) and mutant (KO) neurospheres obtained from E15.5 neocortices and cultured in vitro. The culture passage ("step") corresponds to 3 days of culture after dissociation and re-plating. KO aggregates are more numerous and larger in size. Data information: Scale bars: 50µm.(B) Graph showing the number of neurospheres per P100 cell plate at different steps, as indicated. Nr2f1KO neurospheres proliferate for a longer time (up to 30 passages tested), whereas WT cells exhaust around step 9. n ≥ 3 culture wells from n = 2 batches. Data information: data are represented as means ± SEM. 2-way ANOVA (B) (**P<0.01, ***P<0.001).(C) Panel of WT and KO isolated neurospheres grown from day 1 to day 10. Blue and red lines represent the diameter of WT and KO neurospheres, respectively. Data information Scale bars: 50µm.(D,E) PH3 (green; dividing cells) IF on cross-sections of day 4 neurospheres. White arrowheads point at mitotic figures, which are quantified in (E) as percentage of dividing cells over DAPI-stained nuclei (blue). n ≥ 3 neurospheres from n = 2 batches. Data information: data are represented as means ± SEM. Student t-test (E) (**P<0.01, ***P<0.001). Scale bars: 50µm.


Figure 3 ( A - D ) Ki67 ( green ; mitotically active progenitors ) and Tuj1 ( red ; post - mitotic neurons ) IF at E14 . 5 ( A , A ' ) , E16 . 5 ( B , B ' ) and E18 . 5 ( C , C ' ) of lateral pallia of wild - type ( WT ) versus mutant ( KO ) brains . Ki67 + proliferating cells ( quantified in D ) accumulate in mutant cortices . n ≥ 3 brains . Data information : Nuclei ( blue ) were stained with DAPI . the number of positive cells was quantified in 100µm - width boxes , randomly placed across the LP . 2 - way ANOVA ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) . Scale bars : 50µm . CP : cortical plate ; IZ : intermediate zone ; SVZ : subventricular zone ; VZ : ventricular zone . ( E - F ' ) Tbr2 ( green ; IPs ) and Pax6 ( red ; radial glia cells ) IF of E13 . 5 ( E , E ' ) and E17 . 5 ( F , F ' ) lateral pallia of WT and KO brains . Data information : Nuclei ( blue ) were stained with DAPI . Scale bars : 50µm . CP : cortical plate ; IZ : intermediate zone ; SVZ : subventricular zone ; VZ : ventricular zone . Quantification of different classes of cortical progenitors : apical radial glia cells ( aRGs ) as Pax6 + NPs in VZ ( G ) , basal intermediate progenitors ( IPs ) as Tbr2 + cells in SVZ ( H ) n ≥ 3 brains . Data information : the number of positive cells was quantified in 100µm - width boxes , randomly placed across the LP . 2 - way ANOVA ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) . Quantification of different classes of cortical progenitors : , basal RGs as Pax6 + cells in outer SVZ ( I ) . n ≥ 3 brains . Data information : the number of positive cells was quantified in 100µm - width boxes , randomly placed across the LP . 2 - way ANOVA ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) . ( J - L ) E12 . 5 dorsal brain views ( J , J ' ) and representative sections of posterior hemispheres ( K , K ' ) , showing a vesicle enlargement in mutant brains ( K ' ) . The extension of the ventricular surface was quantified along the A - P axis ( L ) ; red color code represents Nr2f1 gradient in WT brains . 12µm - thick sections were collected on series of 10 slides ; consecutive measurement levels are 120µm apart from each other . n ≥ 3 brains . Data information : In graphs , data are represented as means ± SEM . 2 - way ANOVA ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) . ( N - P ) GFP ( green ; expressed under Sox2 promoter ) and RFP ( red ; expressed under Tis21 promoter ) IF of E13 . 5 lateral pallia , 18 - hours after IUE . The proportion of single or double positive NPs is shown in pie charts in ( P ) . n = 3 electroporated brains . Data information : Nuclei ( blue ) were stained with DAPI . 2 - way ANOVA ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) . ( Q - R ' ) Ki67 ( green ; progenitors ) and EdU ( red ) IF of E13 . 5 WT ( Q , Q ' ) and KO ( R , R ' ) embryos , injected with EdU at E12 . 5 . Differentiating cells ( EdU + Ki67 - ) are located in the IZ / CP ; percentages shown in ( Q ' , R ' ) were obtained from n ≥ 4 sections of n ≥ 2 brains . Data information : Nuclei ( blue ) were stained with DAPI . Student t - test ( Q ' , R ' ; E13 . 5 , EdU injected at E12 . 5 ; WT / KO : * = 0 . 03967 ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) . Scale bars : 50µm . CP : cortical plate ; IZ : intermediate zone ; SVZ : subventricular zone ; VZ : ventricular zone .

O O B-PANEL_START 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 O O O O B-PANEL_START 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 B-PANEL_START 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 B-PANEL_START 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 B-PANEL_START 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 B-PANEL_START 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 B-PANEL_START 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 O O O O O O O O

Figure 3(A-D) Ki67 (green; mitotically active progenitors) and Tuj1 (red; post-mitotic neurons) IF at E14.5 (A,A'), E16.5 (B,B') and E18.5 (C,C') of lateral pallia of wild-type (WT) versus mutant (KO) brains. Ki67+ proliferating cells (quantified in D) accumulate in mutant cortices. n ≥ 3 brains. Data information: Nuclei (blue) were stained with DAPI. the number of positive cells was quantified in 100µm-width boxes, randomly placed across the LP. 2-way ANOVA (*P<0.05, **P<0.01, ***P<0.001). Scale bars: 50µm. CP: cortical plate; IZ: intermediate zone; SVZ: subventricular zone; VZ: ventricular zone.(E-F') Tbr2 (green; IPs) and Pax6 (red; radial glia cells) IF of E13.5 (E,E') and E17.5 (F,F') lateral pallia of WT and KO brains. Data information: Nuclei (blue) were stained with DAPI. Scale bars: 50µm. CP: cortical plate; IZ: intermediate zone; SVZ: subventricular zone; VZ: ventricular zone.Quantification of different classes of cortical progenitors: apical radial glia cells (aRGs) as Pax6+ NPs in VZ (G), basal intermediate progenitors (IPs) as Tbr2+ cells in SVZ (H) n ≥ 3 brains. Data information: the number of positive cells was quantified in 100µm-width boxes, randomly placed across the LP. 2-way ANOVA (*P<0.05, **P<0.01, ***P<0.001).Quantification of different classes of cortical progenitors: , basal RGs as Pax6+ cells in outer SVZ (I). n ≥ 3 brains. Data information: the number of positive cells was quantified in 100µm-width boxes, randomly placed across the LP. 2-way ANOVA (*P<0.05, **P<0.01, ***P<0.001).(J-L) E12.5 dorsal brain views (J,J') and representative sections of posterior hemispheres (K,K'), showing a vesicle enlargement in mutant brains (K'). The extension of the ventricular surface was quantified along the A-P axis (L); red color code represents Nr2f1 gradient in WT brains. 12µm-thick sections were collected on series of 10 slides; consecutive measurement levels are 120µm apart from each other. n ≥ 3 brains. Data information: In graphs, data are represented as means ± SEM. 2-way ANOVA (*P<0.05, **P<0.01, ***P<0.001).(N-P) GFP (green; expressed under Sox2 promoter) and RFP (red; expressed under Tis21 promoter) IF of E13.5 lateral pallia, 18-hours after IUE. The proportion of single or double positive NPs is shown in pie charts in (P). n = 3 electroporated brains. Data information: Nuclei (blue) were stained with DAPI. 2-way ANOVA (*P<0.05, **P<0.01, ***P<0.001).(Q-R') Ki67 (green; progenitors) and EdU (red) IF of E13.5 WT (Q,Q') and KO (R,R') embryos, injected with EdU at E12.5. Differentiating cells (EdU+Ki67-) are located in the IZ/CP; percentages shown in (Q',R') were obtained from n ≥ 4 sections of n ≥ 2 brains. Data information: Nuclei (blue) were stained with DAPI. Student t-test (Q',R'; E13.5, EdU injected at E12.5; WT/KO: *=0.03967 (*P<0.05, **P<0.01, ***P<0.001). Scale bars: 50µm. CP: cortical plate; IZ: intermediate zone; SVZ: subventricular zone; VZ: ventricular zone.


Figure 4 ( A - D ) Graphs showing the average number of PH3 + mitotic figures in the posterior lateral pallium of WT and mutant cortices . The total number of mitoses ( A ) as well as the number of dividing cells in VZ ( Pax6 + Sox2 + aRGs ; B ) , SVZ ( Tbr2 + IPs ; C ) and outer ( basal - most ) region of SVZ ( oSVZ / IZ ; Pax6 + oRGs ; D ) are shown . For graphs ( B - D ) , statistical analysis by 2 - way ANOVA n ≥ 3 brains per age / genotype . Data information : the number of positive cells was quantified in 100µm - width boxes , randomly placed across the lateral pallium . In graphs , data are represented as means ± SEM . 2 - way ANOVA * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) . SVZ : subventricular zone ; VZ : ventricular zone . ( E ) Cell cycle duration at different embryonic ages ( from E10 . 5 to E16 . 5 ) in WT ( blue bars ) or KO embryos ( orange line ) , as quantified upon a double EdU / BrdU injection protocol in posterior - most LP n ≥ 3 brains per age / genotype . Data information : the number of positive cells was quantified in 100µm - width boxes , randomly placed across the lateral pallium . In graphs , data are represented as means ± SEM . 2 - way ANOVA * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) . ( F - G ' ) Ki67 ( green ; NPs ) and EdU ( red ) IF in WT ( F , F ' ) and KO ( G , G ' ) after 6 hours consecutive EdU incorporation . Note the higher number of labelled NPs in KO cortices ( G ' ) suggesting faster cell cycle progression and / or longer S - phase . Data information : Nuclei ( blue ) were stained with DAPI . ( H ) Best linear fit of EdU + Ki67 + NP percentage after 8 hours EdU cumulative labelling in WT ( blue line ) and KO ( orange line ) brains . The x - and y - intercept are proportional to the S - phase length and to the number of cycling cells , respectively , while the steepness of the line is proportional to cell cycle duration . WT : y = 11 . 66x + 24 . 62 ; KO : y = 13 . 54x + 32 . 96 ) . n ≥ 2 brains per time point / genotype . Data information : the number of positive cells was quantified in 100µm - width boxes , randomly placed across the lateral pallium . In graphs , data are represented as means ± SEM . 2 - way ANOVA * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) . ( I - K ) PH3 ( green ; dividing NPs ) and Pax6 ( red ; aRGs ) IF of E13 . 5 WT ( I ) and KO ( J ) lateral pallia . Strong and weak / punctate PH3 patterns show M - phase and G2 - phase cells , respectively . G2 - phase NP percentage is quantified in ( K ) . As a result of shorter G1 - phase and globally shorter cell cycle time , the percentage of G2 - phase cells is increased in the KO NP pool . n ≥ 2 brains . Data information : Nuclei ( blue ) were stained with DAPI . the number of positive cells was quantified in 100µm - width boxes , randomly placed across the lateral pallium . In graphs , data are represented as means ± SEM . Student t - test ( K ; * P < 0 . 05 ) Scale bars : 50µm . SVZ : subventricular zone ; VZ : ventricular zone . ( L ) Cell cycle time of E13 . 5 NPs in the lateral pallia of WT ( upper column ) and KO cells ( lower column ) , as calculated by EdU cumulative labelling and PH3 staining KO NPs have a 16 . 1 % faster cell cycle time , due to a 30 . 9 % shorter G1 - phase compensating a 14 . 3 % longer S - phase . n ≥ 3 brains . In graphs , data are represented as means ± SEM . 2 - way ANOVA ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) .

O O B-PANEL_START 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 O O O O O O O O O O O B-PANEL_START 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 B-PANEL_START 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 B-PANEL_START 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 B-PANEL_START 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 O O B-PANEL_START 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

Figure 4(A-D) Graphs showing the average number of PH3+ mitotic figures in the posterior lateral pallium of WT and mutant cortices. The total number of mitoses (A) as well as the number of dividing cells in VZ (Pax6+Sox2+ aRGs; B), SVZ (Tbr2+ IPs; C) and outer (basal-most) region of SVZ (oSVZ/IZ; Pax6+ oRGs; D) are shown. For graphs (B-D), statistical analysis by 2-way ANOVA n ≥ 3 brains per age/genotype. Data information: the number of positive cells was quantified in 100µm-width boxes, randomly placed across the lateral pallium. In graphs, data are represented as means ± SEM. 2-way ANOVA *P<0.05, **P<0.01, ***P<0.001). SVZ: subventricular zone; VZ: ventricular zone.(E) Cell cycle duration at different embryonic ages (from E10.5 to E16.5) in WT (blue bars) or KO embryos (orange line), as quantified upon a double EdU/BrdU injection protocol in posterior-most LP n ≥ 3 brains per age/genotype. Data information: the number of positive cells was quantified in 100µm-width boxes, randomly placed across the lateral pallium. In graphs, data are represented as means ± SEM. 2-way ANOVA *P<0.05, **P<0.01, ***P<0.001).(F-G') Ki67 (green; NPs) and EdU (red) IF in WT (F,F') and KO (G,G') after 6 hours consecutive EdU incorporation. Note the higher number of labelled NPs in KO cortices (G') suggesting faster cell cycle progression and/or longer S-phase. Data information: Nuclei (blue) were stained with DAPI.(H) Best linear fit of EdU+Ki67+ NP percentage after 8 hours EdU cumulative labelling in WT (blue line) and KO (orange line) brains. The x- and y-intercept are proportional to the S-phase length and to the number of cycling cells, respectively, while the steepness of the line is proportional to cell cycle duration. WT: y = 11.66x +24.62; KO: y = 13.54x +32.96). n ≥ 2 brains per time point/genotype. Data information: the number of positive cells was quantified in 100µm-width boxes, randomly placed across the lateral pallium. In graphs, data are represented as means ± SEM. 2-way ANOVA *P<0.05, **P<0.01, ***P<0.001).(I-K) PH3 (green; dividing NPs) and Pax6 (red; aRGs) IF of E13.5 WT (I) and KO (J) lateral pallia. Strong and weak/punctate PH3 patterns show M-phase and G2-phase cells, respectively. G2-phase NP percentage is quantified in (K). As a result of shorter G1-phase and globally shorter cell cycle time, the percentage of G2-phase cells is increased in the KO NP pool. n ≥ 2 brains. Data information: Nuclei (blue) were stained with DAPI. the number of positive cells was quantified in 100µm-width boxes, randomly placed across the lateral pallium. In graphs, data are represented as means ± SEM. Student t-test (K; *P<0.05) Scale bars: 50µm. SVZ: subventricular zone; VZ: ventricular zone.(L) Cell cycle time of E13.5 NPs in the lateral pallia of WT (upper column) and KO cells (lower column), as calculated by EdU cumulative labelling and PH3 staining KO NPs have a 16.1% faster cell cycle time, due to a 30.9% shorter G1-phase compensating a 14.3% longer S-phase. n ≥ 3 brains. In graphs, data are represented as means ± SEM. 2-way ANOVA (*P<0.05, **P<0.01, ***P<0.001).


Figure 5 ( A - B ' ) Pax6 ( green ; aRGs ) and Nr2f1 ( red ) IF of E12 . 5 WT ( A , A ' ) and KO ( B , B ' ) lateral pallia . Data information : nuclei ( blue ) were stained with DAPI . Scale bars : 50µm . SVZ : subventricular zone ; VZ : ventricular zone . ( C ) Pax6 and Nr2f1 pixel intensity quantification at E12 . 5 and E14 . 5 indicating increased Pax6 levels upon Nr2f1 removal . n ≥ 3 brains . Data information : the pixel intensity was quantified in 100µm - width boxes , randomly placed across the lateral pallium . Data are represented as means ± SEM . 2 - way ANOVA ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) . ( D ) Real time RT - PCR quantification of Nr2f1 and Pax6 expression in E12 . 5 cortices . n ≥ 3 cortices . Data information : Data are represented as means ± SEM . 2 - way ANOVA ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) . ( E - G ' ' ) GFP ( green ; electroporated cells ) , Pax6 ( red ) and Nr2f1 ( blue ) IF of pCIG2 - Nr2f1 - IRES - GFP electroporated brains at E13 . 5 ( 24 - hours after IUE ) . VZ ( F - F ' ' ) and CP ( G - G ' ' ) regions are shown at high magnification . Arrowheads point to GFP + electroporated cells over - expressing Nr2f1 ( blue in F ' , G ' ) , down - regulating Pax6 ( F ' ' ) and rapidly migrating out of the VZ ( G - G ' ' ) . Scale bars : 50µm . SVZ : subventricular zone ; VZ : ventricular zone . ( H ) Scatter plot showing Pax6 and Nr2f1 pixel intensity of VZ electroporated progenitors , comparing Nr2f1 overexpressing cells ( orange dots ) with control pCIG2 - GFP electroporated cells ( blue dots ) . Average Nr2f1 and Pax6 pixel intensities of the 2 populations were compared by 2 - way ANOVA and resulted significantly different ( * * * = < 0 . 0001 ) . n = 2 electroporated brains . Data information Data are represented as means ± SEM . 2 - way ANOVA * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) . ( I ) Real time RT - PCR of cell cycle genes comparing WT ( blue line ) and KO ( orange line ) cortices . CyclinD1 and P21 transcripts are down - regulated whereas Dct is up - regulated in mutants . n = 3 cortices . Data information : Data are represented as means ± SEM . 2 - way ANOVA * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) . ( J - L ) GFP ( green ; electroporated cells ) and P21 ( red ) IF of E14 . 5 cortex electroporated 48 hours earlier with control PX458 plasmid ( J ) or CRISPR / Cas9 - expressing plasmid directed against Nr2f1 sequence ( PX458 - αNr2f1 ; K ) . Percentage of P21 / GFP double positive cells ( L ) . Arrowheads in ( J , K ) point to P21 + cells . n = 2 electroporated brains . Data information , nuclei ( blue ) were stained with DAPI . Student t - test ( L ; * P < 0 . 05 ( N ) Graph showing the number of differentially expressed genes ( DEG up - regulated ( UP ) or down - regulated ( DOWN ) in KO compared to WT , as detected by RNA - Seq of E15 . 5 neocortices . Data information : For statistical analysis of RNA - Seq data , see Methods . ( P ) Hierarchical clustering and heatmap of the expression level of DEGs belonging to the " neuron differentiation " Heat map color scale indicates normalized gene expression from high ( red ) to low ( white ) level . Data information : For statistical analysis of RNA - Seq data see Methods .

O O B-PANEL_START 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 B-PANEL_START 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 B-PANEL_START 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 B-PANEL_START 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 B-PANEL_START 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 B-PANEL_START 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 B-PANEL_START 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 B-PANEL_START 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 B-PANEL_START 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

Figure 5(A-B') Pax6 (green; aRGs) and Nr2f1 (red) IF of E12.5 WT (A,A') and KO (B,B') lateral pallia. Data information: nuclei (blue) were stained with DAPI. Scale bars: 50µm. SVZ: subventricular zone; VZ: ventricular zone.(C) Pax6 and Nr2f1 pixel intensity quantification at E12.5 and E14.5 indicating increased Pax6 levels upon Nr2f1 removal. n ≥ 3 brains. Data information: the pixel intensity was quantified in 100µm-width boxes, randomly placed across the lateral pallium. Data are represented as means ± SEM. 2-way ANOVA (*P<0.05, **P<0.01, ***P<0.001).(D) Real time RT-PCR quantification of Nr2f1 and Pax6 expression in E12.5 cortices. n ≥ 3 cortices. Data information: Data are represented as means ± SEM. 2-way ANOVA (*P<0.05, **P<0.01, ***P<0.001).(E-G'') GFP (green; electroporated cells), Pax6 (red) and Nr2f1 (blue) IF of pCIG2-Nr2f1-IRES-GFP electroporated brains at E13.5 (24-hours after IUE). VZ (F-F'') and CP (G-G'') regions are shown at high magnification. Arrowheads point to GFP+ electroporated cells over-expressing Nr2f1 (blue in F',G'), down-regulating Pax6 (F'') and rapidly migrating out of the VZ (G-G''). Scale bars: 50µm. SVZ: subventricular zone; VZ: ventricular zone.(H) Scatter plot showing Pax6 and Nr2f1 pixel intensity of VZ electroporated progenitors, comparing Nr2f1 overexpressing cells (orange dots) with control pCIG2-GFP electroporated cells (blue dots). Average Nr2f1 and Pax6 pixel intensities of the 2 populations were compared by 2-way ANOVA and resulted significantly different (***= <0.0001). n = 2 electroporated brains. Data information Data are represented as means ± SEM. 2-way ANOVA *P<0.05, **P<0.01, ***P<0.001).(I) Real time RT-PCR of cell cycle genes comparing WT (blue line) and KO (orange line) cortices. CyclinD1 and P21 transcripts are down-regulated whereas Dct is up-regulated in mutants. n = 3 cortices. Data information: Data are represented as means ± SEM. 2-way ANOVA *P<0.05, **P<0.01, ***P<0.001).(J-L) GFP (green; electroporated cells) and P21 (red) IF of E14.5 cortex electroporated 48 hours earlier with control PX458 plasmid (J) or CRISPR/Cas9-expressing plasmid directed against Nr2f1 sequence (PX458-αNr2f1; K). Percentage of P21/GFP double positive cells (L). Arrowheads in (J,K) point to P21+ cells. n = 2 electroporated brains. Data information , nuclei (blue) were stained with DAPI. Student t-test (L; *P<0.05(N) Graph showing the number of differentially expressed genes (DEG up-regulated (UP) or down-regulated (DOWN) in KO compared to WT, as detected by RNA-Seq of E15.5 neocortices. Data information: For statistical analysis of RNA-Seq data , see Methods.(P) Hierarchical clustering and heatmap of the expression level of DEGs belonging to the "neuron differentiation" Heat map color scale indicates normalized gene expression from high (red) to low (white) level. Data information: For statistical analysis of RNA-Seq data see Methods.


Figure 6 ( A ) Real time RT - PCR analysis of Pax6 expression in neurospheres with different genotypes , as indicated . n = 3 cortices . N KO P HET : Nr2f1 KO , Pax6 HET . Data information : Data are represented as means ± SEM . 2 - way ANOVA ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) . ( B ) Representative images of Nr2f1 KO Pax6 HET neurospheres obtained from E15 . 5 neocortices and cultured in vitro . Scale bars : 50µm . ( C ) Graph showing the number of neurospheres per P100 cell plate , at different steps , as indicated . While Nr2f1 KO neurospheres ( orange line ) proliferate for a longer time compared to WT ones ( blue line ) , the loss of one Pax6 allele ( gray line ) almost restores normal proliferation rate and exhaustion time . Complete Pax6 loss is not compatible with stem cell renewal ( yellow line ) . n ≥ 3 culture wells from n = 2 independent batches . Data information : Data are represented as means ± SEM . 2 - way ANOVA ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) . ( D - E ) Paired cell analysis after neurosphere dissociation and 24 - hour culture at clonal density . Couples of dividing cells were identified by Map2 ( red ; N , neurons ) and BLBP ( green ; P , progenitors ) IF . Pie charts in ( E ) show the proportion of P - P ( proliferative ; D ) , P - N ( asymmetric differentiative ; D ' ) or N - N ( symmetric differentiative ; D ' ' ) couples in WT , Nr2f1KO and Nr2f1KO Pax6HET animals . n ≥ 3 samples from n = 2 culture batches . Data information : Nuclei ( blue ) were stained with DAPI . Data are represented as means ± SEM . 2 - way ANOVA ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 Scale bars : 50µm . ( F - I ) Pax6 ( green ) and Nr2f1 ( red ) IF in the posterior - most region of E12 . 5 cortex , showing Pax6 upregulation in Nr2f1 KO animals ( G , G ' ) compared to WT ( F ' , F ' ' ) . Normal Pax6 levels can be restored by loss of one Pax6 allele ( Nr2f1 KO , Pax6 HET ; H , H ' ) . n ≥ 3 brains . N KO : Nr2f1 KO ; P HET : Pax6 HET . Data information : Nuclei ( blue ) were stained with DAPI . Scale bars : 50µm . Pixel intensity quantification of Nr2f1 ( blue ) and Pax6 ( orange ) is shown in ( I ) . n ≥ 3 brains . N KO : Nr2f1 KO ; P HET : Pax6 HET . Data information : the pixel intensity or the number of positive cells was quantified in 100µm - width boxes , randomly placed across the LP . Data are represented as means ± SEM . 2 - way ANOVA ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) . ( J ) Cell cycle duration at E12 . 5 in different mutants as indicated , quantified in posterior regions with double EdU / BrdU injection protocol n ≥ 6 sections from n = 2 brains . Data information : the pixel intensity or the number of positive cells was quantified in 100µm - width boxes , randomly placed across the LP . Data are represented as means ± SEM . 2 - way ANOVA ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) . ( K ) Insets showing representative γ - Tubulin ( green ) and Phospho - Vimentin ( red ) IF of the cortical VZ surface to evaluate the orientation of the cleavage plane of dividing aRGs . Pie charts show the percentage of vertical ( green ) , oblique ( yellow ) and horizontal ( red ) division planes of mitotic figures in the E12 . 5 lateral pallium of animals with different genotypes , as indicated . n ≥ 2 brains per genotype . Data information : Nuclei ( blue ) were stained with DAPI . Data are represented as means ± SEM . 2 - way ANOVA ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) . cale bars : 50µm .

O O B-PANEL_START 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 B-PANEL_START 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-PANEL_START 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 B-PANEL_START 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 B-PANEL_START 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 B-PANEL_START 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 B-PANEL_START 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 B-PANEL_START 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

Figure 6(A) Real time RT-PCR analysis of Pax6 expression in neurospheres with different genotypes, as indicated. n = 3 cortices. N KO P HET: Nr2f1 KO, Pax6 HET. Data information: Data are represented as means ± SEM. 2-way ANOVA ; *P<0.05, **P<0.01, ***P<0.001).(B) Representative images of Nr2f1 KO Pax6 HET neurospheres obtained from E15.5 neocortices and cultured in vitro. Scale bars: 50µm.(C) Graph showing the number of neurospheres per P100 cell plate, at different steps, as indicated. While Nr2f1 KO neurospheres (orange line) proliferate for a longer time compared to WT ones (blue line), the loss of one Pax6 allele (gray line) almost restores normal proliferation rate and exhaustion time. Complete Pax6 loss is not compatible with stem cell renewal (yellow line). n ≥ 3 culture wells from n = 2 independent batches. Data information: Data are represented as means ± SEM. 2-way ANOVA ; *P<0.05, **P<0.01, ***P<0.001).(D-E) Paired cell analysis after neurosphere dissociation and 24-hour culture at clonal density. Couples of dividing cells were identified by Map2 (red; N, neurons) and BLBP (green; P, progenitors) IF. Pie charts in (E) show the proportion of P-P (proliferative; D), P-N (asymmetric differentiative; D') or N-N (symmetric differentiative; D'') couples in WT, Nr2f1KO and Nr2f1KO Pax6HET animals. n ≥ 3 samples from n = 2 culture batches. Data information: Nuclei (blue) were stained with DAPI. Data are represented as means ± SEM. 2-way ANOVA ; *P<0.05, **P<0.01, ***P<0.001 Scale bars: 50µm.(F-I) Pax6 (green) and Nr2f1 (red) IF in the posterior-most region of E12.5 cortex, showing Pax6 upregulation in Nr2f1 KO animals (G,G') compared to WT (F',F''). Normal Pax6 levels can be restored by loss of one Pax6 allele (Nr2f1 KO, Pax6 HET; H,H'). n ≥ 3 brains. N KO: Nr2f1 KO; P HET: Pax6 HET. Data information: Nuclei (blue) were stained with DAPI. Scale bars: 50µm.Pixel intensity quantification of Nr2f1 (blue) and Pax6 (orange) is shown in (I). n ≥ 3 brains. N KO: Nr2f1 KO; P HET: Pax6 HET. Data information: the pixel intensity or the number of positive cells was quantified in 100µm-width boxes, randomly placed across the LP. Data are represented as means ± SEM. 2-way ANOVA ; *P<0.05, **P<0.01, ***P<0.001).(J) Cell cycle duration at E12.5 in different mutants as indicated, quantified in posterior regions with double EdU/BrdU injection protocol n ≥ 6 sections from n = 2 brains. Data information: the pixel intensity or the number of positive cells was quantified in 100µm-width boxes, randomly placed across the LP. Data are represented as means ± SEM. 2-way ANOVA ; *P<0.05, **P<0.01, ***P<0.001).(K) Insets showing representative γ-Tubulin (green) and Phospho-Vimentin (red) IF of the cortical VZ surface to evaluate the orientation of the cleavage plane of dividing aRGs. Pie charts show the percentage of vertical (green), oblique (yellow) and horizontal (red) division planes of mitotic figures in the E12.5 lateral pallium of animals with different genotypes, as indicated. n ≥ 2 brains per genotype. Data information: Nuclei (blue) were stained with DAPI. Data are represented as means ± SEM. 2-way ANOVA ; *P<0.05, **P<0.01, ***P<0.001). cale bars: 50µm.


Figure 7 ( A - C ) NR2F1 expression in cerebral organoids during neural induction and differentiation in vitro ( day 40 and day 70 , respectively ) , together with the NP markers PAX6 ( green in A ) , SOX2 ( green in B , B ' ) and DCX as a neural marker ( blue in A , B , B ' ' ) . Quantification of NR2F1 levels in NPs is shown in ( C ) . n ≥ 4 organoids from n = 2 batches . Data information : the pixel intensity or the number of positive cells were quantified in 100µm - width boxes , randomly placed across the cortex / organoid neuroepithelia In graphs , data are represented as means ± SEM . 2 - way ANOVA test ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) . Scale bars : 50µm . ( D - G ) NR2F1 ( red ) and PAX6 ( green ) IF in day 40 organoids . Neuroepithelia with high NR2F1 levels show weak PAX6 expression ( E , E ' ) , and vice versa ( F , F ' ) . Quantification by pixel intensity analysis ( G ) . n ≥ 4 organoids from n = 2 batches . Data information : Nuclei ( blue stained with DAPI . the pixel intensity or the number of positive cells were quantified in 100µm - width boxes , randomly placed across the cortex / organoid neuroepithelia In graphs , data are represented as means ± SEM . 2 - way ANOVA test ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) . Scale bars : 50µm . ( I - J ) NR2F1 ( red ) and GFP ( green ) IF in day 40 human brain organoids upon electroporation of the PX458 - αNR2F1 plasmid . In ( I , I ' ) , GFP + NR2F1 - cells ( empty arrowheads ) and a GFP + NR2F1 + cell ( white arrowhead ) are shown ; quantification in ( J ) . n ≥ 6 organoids from n = 2 batches . Data information : Nuclei ( blue stained with DAPI . the pixel intensity or the number of positive cells were quantified in 100µm - width boxes , randomly placed across the cortex / organoid neuroepithelia In graphs , data are represented as means ± SEM . 2 - way ANOVA test ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) . Scale bars : 50µm . ( K - M ) TUJ1 ( red ; differentiating neurons ) and GFP ( green ) IF 7 days after electroporation of control ( K ) and PX458 - αNr2f1 plasmids ( L ) . Co - expression of GFP with SOX2 or TUJ1 distinguishes neural progenitors ( NPs ) from neurons ( Ns ) , as quantified in ( M ) . Arrowheads in ( K , L ) point to TUJ1 + GFP + cells . n ≥ 6 organoids from n = 2 batches . Data information : Nuclei ( blue stained with DAPI . the number of positive cells was normalized over the total number of GFP + cells . In graphs , data are represented as means ± SEM . 2 - way ANOVA test ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) . Scale bars : 50µm . ( N ) Red and green arrowheads in ( N ) point to a SOX2 + ( red ) progenitor and a TUJ1 + ( blue ) neuron , respectively . Scale bars : 50µm .

O O B-PANEL_START 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 B-PANEL_START 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 B-PANEL_START 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 B-PANEL_START 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 B-PANEL_START 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

Figure 7(A-C) NR2F1 expression in cerebral organoids during neural induction and differentiation in vitro (day 40 and day 70, respectively), together with the NP markers PAX6 (green in A), SOX2 (green in B,B') and DCX as a neural marker (blue in A,B,B''). Quantification of NR2F1 levels in NPs is shown in (C). n ≥ 4 organoids from n = 2 batches. Data information: the pixel intensity or the number of positive cells were quantified in 100µm-width boxes, randomly placed across the cortex/organoid neuroepithelia In graphs, data are represented as means ± SEM. 2-way ANOVA test (*P<0.05, **P<0.01, ***P<0.001). Scale bars: 50µm.(D-G) NR2F1 (red) and PAX6 (green) IF in day 40 organoids. Neuroepithelia with high NR2F1 levels show weak PAX6 expression (E,E'), and vice versa (F,F'). Quantification by pixel intensity analysis (G). n ≥ 4 organoids from n = 2 batches. Data information: Nuclei (blue stained with DAPI. the pixel intensity or the number of positive cells were quantified in 100µm-width boxes, randomly placed across the cortex/organoid neuroepithelia In graphs, data are represented as means ± SEM. 2-way ANOVA test (*P<0.05, **P<0.01, ***P<0.001). Scale bars: 50µm.(I-J) NR2F1 (red) and GFP (green) IF in day 40 human brain organoids upon electroporation of the PX458-αNR2F1 plasmid. In (I,I'), GFP+NR2F1- cells (empty arrowheads) and a GFP+NR2F1+ cell (white arrowhead) are shown; quantification in (J). n ≥ 6 organoids from n = 2 batches. Data information: Nuclei (blue stained with DAPI. the pixel intensity or the number of positive cells were quantified in 100µm-width boxes, randomly placed across the cortex/organoid neuroepithelia In graphs, data are represented as means ± SEM. 2-way ANOVA test (*P<0.05, **P<0.01, ***P<0.001). Scale bars: 50µm.(K-M) TUJ1 (red; differentiating neurons) and GFP (green) IF 7 days after electroporation of control (K) and PX458-αNr2f1 plasmids (L). Co-expression of GFP with SOX2 or TUJ1 distinguishes neural progenitors (NPs) from neurons (Ns), as quantified in (M). Arrowheads in (K,L) point to TUJ1+ GFP+ cells. n ≥ 6 organoids from n = 2 batches. Data information: Nuclei (blue stained with DAPI. the number of positive cells was normalized over the total number of GFP+ cells. In graphs, data are represented as means ± SEM. 2-way ANOVA test (*P<0.05, **P<0.01, ***P<0.001). Scale bars: 50µm.(N) Red and green arrowheads in (N) point to a SOX2+ (red) progenitor and a TUJ1+ (blue) neuron, respectively. Scale bars: 50µm.


Figure 1 ( C , D ) Standard replication assays on undamaged ( UD ) , thymine glycol ( Tg ) , and cyclobutane pyrimidine dimer ( CPD ) templates in the presence ( C ) and absence ( D ) of Pol η , Pol ζ , Rev1 , Rad6 - Rad18 , Uba1 , and ubiquitin ( TLS machinery ) . Reactions contained 2 . 5 nM Pol δ .

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

Figure 1 (C, D) Standard replication assays on undamaged (UD), thymine glycol (Tg), and cyclobutane pyrimidine dimer (CPD) templates in the presence (C) and absence (D) of Pol η, Pol ζ, Rev1, Rad6-Rad18, Uba1, and ubiquitin (TLS machinery). Reactions contained 2.5 nM Pol δ.


Figure 2 ( B ) Oligonucleotide competition assay on UD and Tg templates . S ; scrambled oligonucleotide . ( C ) Lane profiles of lanes 4 - 6 in ( B ) normalised to the run off product . FL ; FL - lead , OR ; oligonucleotide restart , S ; Stall , RO ; Run off , OF ; Okazaki fragments . ( D ) Pulse chase reaction on UD and Tg templates performed as shown . Where present , Pol δcat ( 10 nM ) was added directly after addition of the chase . ( E ) Standard replication reaction performed in the absence and presence of RFC / PCNA on the Tg template .

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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O

Figure 2 (B) Oligonucleotide competition assay on UD and Tg templates. S; scrambled oligonucleotide. (C) Lane profiles of lanes 4-6 in (B) normalised to the run off product. FL; FL-lead, OR; oligonucleotide restart, S; Stall, RO; Run off, OF; Okazaki fragments.  (D) Pulse chase reaction on UD and Tg templates performed as shown. Where present, Pol δcat (10 nM) was added directly after addition of the chase. (E) Standard replication reaction performed in the absence and presence of RFC/PCNA on the Tg template.


Figure 3 ( B ) Pulse chase reactions on the Tg template in the absence and presence of 10 nM Pol δ analysed on a denaturing gel . ( D ) Pulse chase reaction on the Tg template in the absence and presence of 10 nM Pol δ analysed on a urea polyacrylamide gel . ( E ) Titration of Pol δ in a pulse chase reaction on the Tg template . Pol δ was added after the 0 min time point was taken . ( F ) Quantification of bypass 5 min after addition of the chase for the data in ( E ) . ( G ) Titration of Pol δ or Pol ε ( 1 , 2 . 5 , 5 , 7 . 5 , 10 nM ) into a primer extension assay on a Tg template in the presence of PCNA and RFC . The primer / template sequence and location of the lesion are shown above .

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

Figure 3 (B) Pulse chase reactions on the Tg template in the absence and presence of 10 nM Pol δ analysed on a denaturing gel.  (D) Pulse chase reaction on the Tg template in the absence and presence of 10 nM Pol δ analysed on a urea polyacrylamide gel.  (E) Titration of Pol δ in a pulse chase reaction on the Tg template. Pol δ was added after the 0 min time point was taken. (F) Quantification of bypass 5 min after addition of the chase for the data in (E).  (G) Titration of Pol δ or Pol ε (1, 2.5, 5, 7.5, 10 nM) into a primer extension assay on a Tg template in the presence of PCNA and RFC. The primer/template sequence and location of the lesion are shown above.


Figure 4 ( B ) Mapping the 3ʹ - end of the stalled nascent leading strand . Pulse chase reaction on Tg and CPD templates . Where present , Pol δ ( 10 nM ) was added immediately after the 0 min time point . Products were cleaved with SwaI and BamHI and resolved alongside a sequencing ladder corresponding to the nascent strand . dd ; dideoxy .

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Figure 4 (B) Mapping the 3ʹ-end of the stalled nascent leading strand. Pulse chase reaction on Tg and CPD templates. Where present, Pol δ (10 nM) was added immediately after the 0 min time point. Products were cleaved with SwaI and BamHI and resolved alongside a sequencing ladder corresponding to the nascent strand. dd; dideoxy.


Figure 5 ( A ) Primer extension reactions on the Tg template with 5 nM Pol δ or Pol ε and either dATP , dCTP , dGTP , or dTTP . ( C ) Analysis of nucleotide incorporation opposite Tg from replication reactions processed as shown in ( B ) . Three individual experiments were performed , two in the presence and one in the absence of Pol δ ( 10 nM ) , giving 99 , 97 , and 95 successful sequencing reads respectively .

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Figure 5 (A) Primer extension reactions on the Tg template with 5 nM Pol δ or Pol ε and either dATP, dCTP, dGTP, or dTTP.  (C) Analysis of nucleotide incorporation opposite Tg from replication reactions processed as shown in (B). Three individual experiments were performed, two in the presence and one in the absence of Pol δ (10 nM), giving 99, 97, and 95 successful sequencing reads respectively.


Figure 6 ( A ) Standard replication assay on undamaged ( UD ) , 8 - oxoguanine ( 8oxoG ) , and thymine glycol ( Tg ) templates in the absence of TLS components . ( B ) Pulse chase reaction on UD and 8oxoG templates . Where present , Pol δcat ( 10 nM ) was added directly after addition of the chase . ( C ) Pulse chase reactions on the 8oxoG template in the absence and presence of 10 nM Pol δ analysed on a denaturing gel .

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Figure 6(A) Standard replication assay on undamaged (UD), 8-oxoguanine (8oxoG), and thymine glycol (Tg) templates in the absence of TLS components. (B) Pulse chase reaction on UD and 8oxoG templates. Where present, Pol δcat (10 nM) was added directly after addition of the chase.  (C) Pulse chase reactions on the 8oxoG template in the absence and presence of 10 nM Pol δ analysed on a denaturing gel.


Figure 2 . A ) Assessment of the detection limit of synthetic SARS - CoV - 2 by the CRISPR - based assay , inferred from fluorescence values after 30 min . Background subtracted fluorescence represents sample minus control fluorescence without target . Detection of synthetic SARS - CoV - 2 RNA using commercially available paper strips ( lateral - flow ) in buffer ( B ) Detection of synthetic SARS - CoV - 2 RNA using commercially available paper strips ( lateral - flow ) in saliva ( C ) .

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Figure 2.A) Assessment of the detection limit of synthetic SARS-CoV-2 by the CRISPR-based assay, inferred from fluorescence values after 30 min. Background subtracted fluorescence represents sample minus control fluorescence without target.Detection of synthetic SARS-CoV-2 RNA using commercially available paper strips (lateral-flow) in buffer (B)Detection of synthetic SARS-CoV-2 RNA using commercially available paper strips (lateral-flow) in saliva (C).


Figure 1Rag1 - / - : Bcl2 and Art - / - : Bcl2 abl pre - B cells were treated with imatinib for 48 h to induce G1 arrest . ( A ) DSBs quantified by qPCR analysis of Igk ( Jκ1 ) genomic DNA . Schematic shows germline ( GL ) Igk locus , unrepaired Jκ1 coding end ( Cut ) and primer location . Results are normalized to Rag1 - / - : Bcl2 abl pre - B cells , which do not generate RAG DSBs and have only germline Igk DNA . Loss of Igk germline product is representative of DSB generation . Rag1 - / - : Bcl2 and Art - / - : Bcl2 abl pre - B cells were treated with imatinib for 48 h to induce G1 arrest . ( B ) Representative images of 53BP1 foci . Scale bar denotes 8 μm . Bar graph shows percentage of cells with indicated number of foci from three biological replicates . In each replicate , 100 cells per condition were quantified . Rag1 - / - : Bcl2 and Art - / - : Bcl2 abl pre - B cells were treated with imatinib for 48 h to induce G1 arrest . ( C ) Western blot analysis of KAP1 phosphorylation ( p - KAP1 ) , NF - κB2 ( p100 and p52 ) and PIM2 . GAPDH is shown as loading control . Rag1 - / - : Bcl2 and Art - / - : Bcl2 abl pre - B cells were treated with imatinib for 48 h to induce G1 arrest . ( D ) Cd40 and Pim2 mRNA expression . Rag1 - / - : Lig4 - / - : Bcl2 abl pre - B cells were transduced with an empty vector ( control ) or vector expressing RAG1 then treated with imatinib for 48 h . ( E ) DSBs quantified by qPCR analysis of Igh ( JH1 ) genomic DNA and analyzed as in A with results normalized to empty vector control , which has only germline Igh DNA . Rag1 - / - : Lig4 - / - : Bcl2 abl pre - B cells were transduced with an empty vector ( control ) or vector expressing RAG1 then treated with imatinib for 48 h . ( F ) Representative images of 53BP1 foci . Scale bar denotes 8 μm . Bar graph shows percentage of cells with indicated number of foci from three biological replicates . In each replicate , 100 cells per condition were quantified . E - H . Rag1 - / - : Lig4 - / - : Bcl2 abl pre - B cells were transduced with an empty vector ( control ) or vector expressing RAG1 then treated with imatinib for 48 h . ( G ) Western blot analysis of FLAG - RAG1 , p - KAP1 , NF - κB2 , and PIM2 . GAPDH is shown as loading control . Rag1 - / - : Lig4 - / - : Bcl2 abl pre - B cells were transduced with an empty vector ( control ) or vector expressing RAG1 then treated with imatinib for 48 h . ( H ) Cd40 and Pim2 mRNA expression .

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Figure 1Rag1-/-:Bcl2 and Art-/-:Bcl2 abl pre-B cells were treated with imatinib for 48 h to induce G1 arrest. (A) DSBs quantified by qPCR analysis of Igk (Jκ1) genomic DNA. Schematic shows germline (GL) Igk locus, unrepaired Jκ1 coding end (Cut) and primer location. Results are normalized to Rag1-/-:Bcl2 abl pre-B cells, which do not generate RAG DSBs and have only germline Igk DNA. Loss of Igk germline product is representative of DSB generation.Rag1-/-:Bcl2 and Art-/-:Bcl2 abl pre-B cells were treated with imatinib for 48 h to induce G1 arrest. (B) Representative images of 53BP1 foci. Scale bar denotes 8 μm. Bar graph shows percentage of cells with indicated number of foci from three biological replicates. In each replicate, 100 cells per condition were quantified.Rag1-/-:Bcl2 and Art-/-:Bcl2 abl pre-B cells were treated with imatinib for 48 h to induce G1 arrest. (C) Western blot analysis of KAP1 phosphorylation (p-KAP1), NF-κB2 (p100 and p52) and PIM2. GAPDH is shown as loading control.Rag1-/-:Bcl2 and Art-/-:Bcl2 abl pre-B cells were treated with imatinib for 48 h to induce G1 arrest. (D) Cd40 and Pim2 mRNA expression.Rag1-/-:Lig4-/-:Bcl2 abl pre-B cells were transduced with an empty vector (control) or vector expressing RAG1 then treated with imatinib for 48 h. (E) DSBs quantified by qPCR analysis of Igh (JH1) genomic DNA and analyzed as in A with results normalized to empty vector control, which has only germline Igh DNA.Rag1-/-:Lig4-/-:Bcl2 abl pre-B cells were transduced with an empty vector (control) or vector expressing RAG1 then treated with imatinib for 48 h. (F) Representative images of 53BP1 foci. Scale bar denotes 8 μm. Bar graph shows percentage of cells with indicated number of foci from three biological replicates. In each replicate, 100 cells per condition were quantified.E-H. Rag1-/-:Lig4-/-:Bcl2 abl pre-B cells were transduced with an empty vector (control) or vector expressing RAG1 then treated with imatinib for 48 h. (G) Western blot analysis of FLAG-RAG1, p-KAP1, NF-κB2, and PIM2. GAPDH is shown as loading control.Rag1-/-:Lig4-/-:Bcl2 abl pre-B cells were transduced with an empty vector (control) or vector expressing RAG1 then treated with imatinib for 48 h. (H) Cd40 and Pim2 mRNA expression.


Figure 2A . Rag1 - / - : Lig4 - / - : Bcl2 : iCas9 abl pre - B cells were treated with ( + ) or without ( - ) 2 μM doxycycline ( Dox ) to induce Cas9 as indicated . Western blot shows FLAG - Cas9 and GAPDH ( loading control ) . Rag1 - / - : Lig4 - / - : Bcl2 : iCas9 abl pre - B cells were treated with doxycycline as in A and imatinib to trigger cell cycle arrest for 24 h . Cells were then transfected with indicated gRNA and maintained in doxycycline and imatinib . All analyses were completed 48 h after gRNA transfection . DSBs quantified by qPCR analysis of Eb ( B ) and Gapdh ( C ) genomic DNA . Results are normalized to cells without gRNA ( - ) . Schematic shows germline ( GL ) locus , unrepaired cut end ( Cut ) and location of gRNA and primers . Loss of germline product is representative of DSB generation . ( D ) Representative images of 53BP1 foci . Scale bar denotes 8 μm . Bar graph shows percentage of cells with indicated number of foci from three biological replicates . In each replicate , 100 cells per condition were quantified . ( E ) Western blot analysis of FLAG - tagged Cas9 or RAG1 ( as indicated ) , p - KAP1 , NF - κB2 , and PIM2 . GAPDH is shown as loading control . Responses to RAG DSBs in Rag1 - / - : Lig4 - / - : Bcl2 abl pre - B cells transduced with empty vector or vector expressing RAG1 RAG DSBs from Fig 1G are included for direct comparison . # denotes band for FLAG - Cas9 . * denotes band for FLAG - RAG1 . ( F ) Cd40 and Pim2 mRNA expression . Blue dashed line indicates mRNA expression in response to RAG DSBs from Fig 1H and is included for comparison .

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Figure 2A. Rag1-/-:Lig4-/-:Bcl2:iCas9 abl pre-B cells were treated with (+) or without (-) 2 μM doxycycline (Dox) to induce Cas9 as indicated. Western blot shows FLAG-Cas9 and GAPDH (loading control).Rag1-/-:Lig4-/-:Bcl2:iCas9 abl pre-B cells were treated with doxycycline as in A and imatinib to trigger cell cycle arrest for 24 h. Cells were then transfected with indicated gRNA and maintained in doxycycline and imatinib. All analyses were completed 48 h after gRNA transfection. DSBs quantified by qPCR analysis of Eb (B) and Gapdh (C) genomic DNA. Results are normalized to cells without gRNA (-). Schematic shows germline (GL) locus, unrepaired cut end (Cut) and location of gRNA and primers. Loss of germline product is representative of DSB generation.(D) Representative images of 53BP1 foci. Scale bar denotes 8 μm. Bar graph shows percentage of cells with indicated number of foci from three biological replicates. In each replicate, 100 cells per condition were quantified.(E) Western blot analysis of FLAG-tagged Cas9 or RAG1 (as indicated), p-KAP1, NF-κB2, and PIM2. GAPDH is shown as loading control. Responses to RAG DSBs in Rag1-/-:Lig4-/-:Bcl2 abl pre-B cells transduced with empty vector or vector expressing RAG1 RAG DSBs from Fig 1G are included for direct comparison. # denotes band for FLAG-Cas9. * denotes band for FLAG-RAG1.(F) Cd40 and Pim2 mRNA expression. Blue dashed line indicates mRNA expression in response to RAG DSBs from Fig 1H and is included for comparison.


Figure 3A - B . DSBs quantified by qPCR analysis of Jκ1 ( A ) and JH1 and JHD1 - 1 ( B ) genomic DNA . Schematics show germline ( GL ) locus and location of respective gRNAs and primers . PCR is normalized to cells without gRNA ( - ) . Loss of germline product is representative of DSB generation . C . Representative images of 53BP1 foci . Scale bar denotes 8 μm . Bar graph shows percentage of cells with indicated number of foci from three biological replicates . In each replicate , 100 cells per condition were quantified . D . Western blot analysis of FLAG - Cas9 , p - KAP1 , NF - κB2 , and PIM2 . GAPDH is shown as loading control . E . CD40 and Pim2 mRNA expression . Blue dashed line indicates mRNA expression in response to RAG DSBs from Fig 1H and is included for comparison .

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Figure 3A-B. DSBs quantified by qPCR analysis of Jκ1 (A) and JH1 and JHD1-1 (B) genomic DNA. Schematics show germline (GL) locus and location of respective gRNAs and primers. PCR is normalized to cells without gRNA (-). Loss of germline product is representative of DSB generation.C. Representative images of 53BP1 foci. Scale bar denotes 8 μm. Bar graph shows percentage of cells with indicated number of foci from three biological replicates. In each replicate, 100 cells per condition were quantified.D. Western blot analysis of FLAG-Cas9, p-KAP1, NF-κB2, and PIM2. GAPDH is shown as loading control.E. CD40 and Pim2 mRNA expression. Blue dashed line indicates mRNA expression in response to RAG DSBs from Fig 1H and is included for comparison.


Figure 4B . DSBs quantified by qPCR analysis of Igh ( JH1 ) genomic DNA and analyzed as in Fig 1E with results normalized to empty vector control , which has only germline Igh DNA . There is no statistically significant difference in DSBs generated by cRAG1 or RAG1P326G compared to full - length RAG1 . C . Representative images of 53BP1 foci . Scale bar denotes 8 μm . Bar graph shows percentage of cells with indicated number of foci from three biological replicates . In each replicate , 100 cells per condition were quantified . D . Western blot analysis of FLAG - RAG1 , p - KAP1 , NF - κB2 , and PIM2 . GAPDH is shown as loading control . E . Cd40 and Pim2 mRNA expression .

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Figure 4B. DSBs quantified by qPCR analysis of Igh (JH1) genomic DNA and analyzed as in Fig 1E with results normalized to empty vector control, which has only germline Igh DNA. There is no statistically significant difference in DSBs generated by cRAG1 or RAG1P326G compared to full-length RAG1.C. Representative images of 53BP1 foci. Scale bar denotes 8 μm. Bar graph shows percentage of cells with indicated number of foci from three biological replicates. In each replicate, 100 cells per condition were quantified.D. Western blot analysis of FLAG-RAG1, p-KAP1, NF-κB2, and PIM2. GAPDH is shown as loading control.E. Cd40 and Pim2 mRNA expression.


Figure 1Photograph of male patient with FMR1G - ins . allele . Typical physical and behavioral features of FXS were noted in the patient , who shows moderate to severe intellectual disability . FMR1 mRNA levels were analyzed via RT - qPCR in EBV - transformed lymphocyte cells derived from the patient ' s blood samples . Patient cells showed a ˜ 60 % decrease in FMR1 mRNA compared to control cells ( * P = 0 . 010 , 0 . 383 ± 0 . 110 SD , n = 3 ) . Treatment with translational blocker puromycin restored FMR1 mRNA levels in patient cells ( * P = 0 . 048 , 0 . 383 ± 0 . 110 SD versus 1 . 25 ± 0 . 166 SD , n = 3 ) , suggesting that the reduction of FMR1 mRNA in these cells is primarily due to nonsense - mediated decay . RT - qPCR reactions were run in triplicate in three independent experiments . Fold changes in FMR1 expression , normalized to HRPT expression , were calculated using the ΔΔCT method , and analyzed statistically with a two - tailed t - test ( GraphPad ) . Error bars represent mean values with SD . Western blot analysis shows a significant decrease ( > 90 % ) in FMRP protein levels in patient - derived cells ( * * * P = 0 . 008 , 0 . 13 ± 0 . 031 SD , n = 2 ) , and the patient FMRP migrates slightly lower than the wild - type protein . Band intensities for the different cell lines were quantified , normalized for actin and analyzed statistically with a twot - tailed t - test ( GraphPad ) . Error bars represent mean values with SD .

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Figure 1Photograph of male patient with FMR1G-ins. allele. Typical physical and behavioral features of FXS were noted in the patient, who shows moderate to severe intellectual disability.FMR1 mRNA levels were analyzed via RT-qPCR in EBV-transformed lymphocyte cells derived from the patient's blood samples. Patient cells showed a ˜60% decrease in FMR1 mRNA compared to control cells (*P = 0.010, 0.383 ± 0.110 SD, n = 3). Treatment with translational blocker puromycin restored FMR1 mRNA levels in patient cells (*P = 0.048, 0.383 ± 0.110 SD versus 1.25 ± 0.166 SD, n = 3), suggesting that the reduction of FMR1 mRNA in these cells is primarily due to nonsense-mediated decay. RT-qPCR reactions were run in triplicate in three independent experiments. Fold changes in FMR1 expression, normalized to HRPT expression, were calculated using the ΔΔCT method, and analyzed statistically with a two-tailed t-test (GraphPad). Error bars represent mean values with SD.Western blot analysis shows a significant decrease (> 90%) in FMRP protein levels in patient-derived cells (***P = 0.008, 0.13 ± 0.031 SD, n = 2), and the patient FMRP migrates slightly lower than the wild-type protein. Band intensities for the different cell lines were quantified, normalized for actin and analyzed statistically with a twot-tailed t-test (GraphPad). Error bars represent mean values with SD.


Figure 2A - C HEK293 ( A ) and SH - Sy5y ( B ) cells , as well as primary neurons from FMR1 KO mice ( C ) , were transfected with vectors encoding the GFP - tagged wild - type FMRP ( GFP - FMR1WT ) or the GFP - tagged patient FMRP ( GFP - FMR1G - insertion ) under the control of a β - actin promoter . Wild - type FMRP localizes predominantly in the cytoplasm , whereas the patient FMRP forms nuclear and nucleolar aggregates that colocalize with nucleophosmin . Scale bars in ( A , B ) represent 10 μm . Scale bar in ( C ) represents 5 μm .

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Figure 2A-C HEK293 (A) and SH-Sy5y (B) cells, as well as primary neurons from FMR1 KO mice (C), were transfected with vectors encoding the GFP-tagged wild-type FMRP (GFP-FMR1WT) or the GFP-tagged patient FMRP (GFP-FMR1G-insertion) under the control of a β-actin promoter. Wild-type FMRP localizes predominantly in the cytoplasm, whereas the patient FMRP forms nuclear and nucleolar aggregates that colocalize with nucleophosmin. Scale bars in (A, B) represent 10 μm. Scale bar in (C) represents 5 μm.


Figure 3Transfected HEK293 cells expressing a GFP - tagged FMRP in which the C - terminus is truncated following the G - ins . mutation site ( GFP - FMR1ΔCt ) . GFP - FMR1ΔCt protein is exclusively cytoplasmic , suggesting that the truncation of the C - terminus alone is not sufficient to explain the localization change observed for the patientFMRP . Transfected HEK293 cells expressing a modified version of GFP - tagged patientFMRP ( GFP - FMR1G - ins . [ NLSmutated ] ) . The novel amino acid sequence in the C - terminus of patientFMRP [ RKRTRRKRTWRRLQRKRRSLPNR ] contains stretches of arginines and lysines predicted to function as a nuclear localization signal ( based on Expasy protein motif scanner , ScanProsite ) . Mutating adjacent R and K residues into alanines [ RARTRAARTWARLQRAARSLPNR ] abolishes the nucleolar localization of the patientFMRP , strongly suggesting that the novel amino acid sequence present in the patientFMRP C - terminus is a functional nuclear localization signal ( NLS ) .

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Figure 3Transfected HEK293 cells expressing a GFP-tagged FMRP in which the C-terminus is truncated following the G-ins. mutation site (GFP-FMR1ΔCt). GFP-FMR1ΔCt protein is exclusively cytoplasmic, suggesting that the truncation of the C-terminus alone is not sufficient to explain the localization change observed for the patientFMRP.Transfected HEK293 cells expressing a modified version of GFP-tagged patientFMRP (GFP-FMR1G-ins. [NLSmutated]). The novel amino acid sequence in the C-terminus of patientFMRP [RKRTRRKRTWRRLQRKRRSLPNR] contains stretches of arginines and lysines predicted to function as a nuclear localization signal (based on Expasy protein motif scanner, ScanProsite). Mutating adjacent R and K residues into alanines [RARTRAARTWARLQRAARSLPNR] abolishes the nucleolar localization of the patientFMRP, strongly suggesting that the novel amino acid sequence present in the patientFMRP C-terminus is a functional nuclear localization signal (NLS).


Figure 4Transfected HEK293 cells expressing GFP - tagged wild - type FMRP fused to the patient NLS motif ( GFP - FMR1wt + NLS ) . Unlike the patient FMRP , GFP - FMR1wt + NLS protein is predominantly cytoplasmic and does not aggregate in the nucleus . However , treatment of HEK293 cells expressing GFP - FMR1wt + NLS with leptomycin B - an inhibitor of nuclear export - resulted in the appearance of nucleolar inclusions . This suggests that the presence of a full - length C - terminus facilitates the nuclear export of FMRP in this context . Transfected HEK293 cells expressing a modified version of GFP - tagged patient FMRP , in which the truncation of the C - terminus is reverted by restoring the open reading frame ( GFP - FMR1G - ins . [ Ct restored ] ) . The patient protein is not detected in the nucleus when the C - terminus is intact ; however , nucleolar retention of the protein is observed upon leptomycin B treatment of the transfected cells . In line with results from ( A ) , this suggests that an intact C - terminus enables nuclear export of the FMR1 protein . Data information : Scale bar represents 10 μm .

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Figure 4Transfected HEK293 cells expressing GFP-tagged wild-type FMRP fused to the patient NLS motif (GFP-FMR1wt+NLS). Unlike the patient FMRP, GFP-FMR1wt+NLS protein is predominantly cytoplasmic and does not aggregate in the nucleus. However, treatment of HEK293 cells expressing GFP-FMR1wt+NLS with leptomycin B-an inhibitor of nuclear export-resulted in the appearance of nucleolar inclusions. This suggests that the presence of a full-length C-terminus facilitates the nuclear export of FMRP in this context.Transfected HEK293 cells expressing a modified version of GFP-tagged patient FMRP, in which the truncation of the C-terminus is reverted by restoring the open reading frame (GFP-FMR1G-ins.[Ct restored]). The patient protein is not detected in the nucleus when the C-terminus is intact; however, nucleolar retention of the protein is observed upon leptomycin B treatment of the transfected cells. In line with results from (A), this suggests that an intact C-terminus enables nuclear export of the FMR1 protein.Data information: Scale bar represents 10 μm.


Figure 5The morphology of small lateral ventral neurons ( sLNvs ) is sensitive to dfmr1 activity . Overexpression of wild - type dfmr1 in the sLNv neurons results in a consistent " axonal collapse " phenotype , where the branching of axonal termini of sLNvs is reduced . Scale bars represent 50 and 10 μm ( magnified images ) .

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Figure 5The morphology of small lateral ventral neurons (sLNvs) is sensitive to dfmr1 activity. Overexpression of wild-type dfmr1 in the sLNv neurons results in a consistent "axonal collapse" phenotype, where the branching of axonal termini of sLNvs is reduced. Scale bars represent 50 and 10 μm (magnified images).


Figure 6Overexpression of wild - type dfmr1 ( UAS - dfmr1wt ) using a LNv neuron - specific driver line ( Pdf - Gal4 ) causes reduction of branching area - " axonal collapse " - in the sLNv axonal termini . dfmr1ΔCt and dfmr1wt + NLS overexpression phenocopies overexpression of dfmr1wt , suggesting that the truncation of the C - terminus or the presence of the patient NLS alone does not impair or alter the functional efficacy of dfmr1 in this context . However , overexpression of dfmr1ΔCt + NLS fails to cause collapse of sLNv termini , indicating a significant loss or change of Dfmrp protein function . Axonal morphology is visualized by expressing CD8 - GFP under the control of the same neuronal driver . Scale bar represents 10 μm . Quantification of axonal termini branching in LNv neurons overexpressing various dfmr1 alleles . Overexpression of dfmr1wt , dfmr1ΔCt , or dfmr1wt + NLS significantly reduced axonal branching area [ * * * * P < 0 . 0001 ; control 101 . 3 ± 29 . 76 SD , n = 30 ; UAS - dfmrwt 45 . 41 ± 21 . 69 SD , n = 29 ; UAS - dfmrΔCt 52 . 31 ± 14 . 67 SD , n = 32 ; UAS - dfmrwt + NLS 72 . 45 ± 21 . 83 SD , n = 29 ; UAS - dfmrΔCt + NLS 117 . 8 ± 36 . 96 SD , n = 28 ] . dfmr1ΔCt + NLS overexpression does not result in a significant change of axonal branching area compared to the background control [ Pdf - Gal4 , UAS - GFP ] ( P = 0 . 672 ) . For quantification purposes , each branching area ( n ) was manually outlined , starting from the first point of defasculation of the axonal bundle . These measurements were normalized for variability in brain size across samples using LNv commissure length measurements for each brain . Two - tailed t - tests using Welch ' s correction were then used to compare controls with mutant phenotypes ( GraphPad ) . Error bars represent mean values with SD . Dots visible for box plots of UAS - dfmrwt and UAS - dfmrΔCt represent samples that outlier the whiskers . Overexpression of dfmr1ΔCt + NLS results in aberrant axonal termini morphology . Axonal guidance defects were frequently observed ( arrowheads ) and scored manually for bifurcations of the axon bundle ( 9 / 48 ) , tangling of axons in the termini ( 13 / 48 ) , and apparent looping of axons ( 7 / 48 ) .

O B-PANEL_START 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 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 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 B-PANEL_START 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

Figure 6Overexpression of wild-type dfmr1 (UAS-dfmr1wt) using a LNv neuron-specific driver line (Pdf-Gal4) causes reduction of branching area-"axonal collapse"-in the sLNv axonal termini. dfmr1ΔCt and dfmr1wt+NLS overexpression phenocopies overexpression of dfmr1wt, suggesting that the truncation of the C-terminus or the presence of the patient NLS alone does not impair or alter the functional efficacy of dfmr1 in this context. However, overexpression of dfmr1ΔCt+NLS fails to cause collapse of sLNv termini, indicating a significant loss or change of Dfmrp protein function. Axonal morphology is visualized by expressing CD8-GFP under the control of the same neuronal driver. Scale bar represents 10 μm.Quantification of axonal termini branching in LNv neurons overexpressing various dfmr1 alleles. Overexpression of dfmr1wt, dfmr1ΔCt, or dfmr1wt+NLS significantly reduced axonal branching area [****P < 0.0001; control 101.3 ± 29.76 SD, n = 30; UAS-dfmrwt 45.41 ± 21.69 SD, n = 29; UAS-dfmrΔCt 52.31 ± 14.67 SD, n = 32; UAS-dfmrwt+NLS 72.45 ± 21.83 SD, n = 29; UAS-dfmrΔCt+NLS 117.8 ± 36.96 SD, n = 28]. dfmr1ΔCt+NLS overexpression does not result in a significant change of axonal branching area compared to the background control [Pdf-Gal4, UAS-GFP] (P = 0.672). For quantification purposes, each branching area (n) was manually outlined, starting from the first point of defasculation of the axonal bundle. These measurements were normalized for variability in brain size across samples using LNv commissure length measurements for each brain. Two-tailed t-tests using Welch's correction were then used to compare controls with mutant phenotypes (GraphPad). Error bars represent mean values with SD. Dots visible for box plots of UAS-dfmrwt and UAS-dfmrΔCt represent samples that outlier the whiskers.Overexpression of dfmr1ΔCt+NLS results in aberrant axonal termini morphology. Axonal guidance defects were frequently observed (arrowheads) and scored manually for bifurcations of the axon bundle (9/48), tangling of axons in the termini (13/48), and apparent looping of axons (7/48).


Figure 7A - D Subcellular localization of transgenically expressed dfmr1wt , dfmr1ΔCt , dfmr1wt + NLS , and dfmr1ΔCt + NLS proteins in LNv neurons . DfmrpΔCt + NLS shows nuclear localization ( A ) , colocalizing with the genetically encoded nuclear marker RedStinger , while Dfmrpwt , DfmrpΔCt , and Dfmrpwt + NLS are predominantly cytoplasmic ( B - D ) . These results suggest that an intact C - terminus can mediate the nuclear export of the Dfmrp protein . Scale bar represents 5 μm .

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Figure 7A-D Subcellular localization of transgenically expressed dfmr1wt, dfmr1ΔCt, dfmr1wt+NLS, and dfmr1ΔCt+NLS proteins in LNv neurons. DfmrpΔCt+NLS shows nuclear localization (A), colocalizing with the genetically encoded nuclear marker RedStinger, while Dfmrpwt, DfmrpΔCt, and Dfmrpwt+NLS are predominantly cytoplasmic (B-D). These results suggest that an intact C-terminus can mediate the nuclear export of the Dfmrp protein. Scale bar represents 5 μm.