( D ) Ndc80 - 9A decreases central MTOCs . number of central MTOCs ( MTOCs that were positioned in middle region of spindle , was determined at each time point ( n = 19 , 21 oocytes , respectively , from at least 3 independent experiments ) .

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(D) Ndc80-9A decreases central MTOCs. The number of central MTOCs (MTOCs that were positioned in the middle region of the spindle , was determined at each time point (n=19, 21 oocytes, respectively, from at least 3 independent experiments).


( A ) Live imaging of Zp3 - Cre Ndc80f / f oocytes injected with Ndc80 - WT , - 9D or - 9A . Z - projection 3D - reconstructed show microtubules ( EGFP - Map4 , green ) chromosomes ( H2B - mCherry , magenta ) . Scale bar , 10 μm .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD I-GENEPROD O O B-CELL_TYPE O O B-GENEPROD O O O O O O O O O O O O O O O O B-SUBCELLULAR O B-GENEPROD O B-GENEPROD O O O B-SUBCELLULAR O B-GENEPROD O B-GENEPROD O O O O O O O O O O

(A) Live imaging of Zp3-Cre Ndc80f/f oocytes injected with Ndc80-WT, -9D or -9A. Z-projection and 3D-reconstructed images show microtubules (EGFP-Map4, green) and chromosomes (H2B-mCherry, magenta). Scale bar, 10 μm.


( B ) Ndc80 - 9D is defective for limiting spindle elongation . aspect ( / width ) of 3D - reconstructed spindles was measured over time ( n = 18 , 18 18 oocytes , respectively , from 3 independent experiments ) .

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(B) Ndc80-9D is defective for limiting spindle elongation. The aspect ratio (length/width) of 3D-reconstructed spindles was measured over time (n=18, 18 and 18 oocytes, respectively, from 3 independent experiments).


( C ) Ndc80 - 9D causes excessive spindle elongation . Spindle lengths measured after 3D reconstruction are shown ( n = 18 , 18 18 oocytes , respectively , from 3 independent experiments ) .

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(C) Ndc80-9D causes excessive spindle elongation. Spindle lengths measured after 3D reconstruction are shown (n=18, 18 and 18 oocytes, respectively, from 3 independent experiments).


( A ) K - are connected to MTOCs extend to spindle poles . Oocytes 6 hours after NEBD were fixed following cold treatment . Microtubules ( α - tubulin , green ) , MTOCs ( pericentrin , magenta ) , kinetochores ( ACA , red ) ( Hoechst333342 ) are shown . Five oocytes from 2 independent experiments were analyzed . Scale bar , 10 μm .

O O O B-SUBCELLULAR I-SUBCELLULAR O O O B-SUBCELLULAR O O B-SUBCELLULAR I-SUBCELLULAR O B-CELL_TYPE O O O O O O O O O O B-SUBCELLULAR O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-SUBCELLULAR O B-GENEPROD O O O O B-SUBCELLULAR O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O B-CELL_TYPE O O O O O O O O O O O O O

(A) K-fibers are connected to MTOCs and extend to spindle poles. Oocytes 6 hours after NEBD were fixed following cold treatment. Microtubules (α-tubulin, green), MTOCs (pericentrin, magenta), kinetochores (ACA, red) and DNA (Hoechst333342) are shown. Five oocytes from 2 independent experiments were analyzed. Scale bar, 10 μm.


( B ) A majority of MTOCs are attached to K - . volumes of MTOCs were measured after 3D reconstruction . sum volume of MTOCs attached to K - relative to total volume of MTOCs was calculated for each oocyte ( n = 5 oocytes from 2 independent experiments ) .

O O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR I-SUBCELLULAR O O O B-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR O O B-SUBCELLULAR I-SUBCELLULAR O O O O O B-SUBCELLULAR O O O O B-CELL_TYPE O O O O B-CELL_TYPE O O O O O O

(B) A majority of MTOCs are attached to K-fibers. The volumes of individual MTOCs were measured after 3D reconstruction. The sum volume of MTOCs attached to K-fibers relative to the total volume of MTOCs was calculated for each oocyte (n=5 oocytes from 2 independent experiments).


( C ) K - extend to spindle poles . of K - between kinetochores a spindle pole ( KT - to - Pole ) were measured . K - fiber relative to KT - to - Pole was calculated ( n = 90 K - of 5 oocytes from 2 independent experiments ) .

O O O B-SUBCELLULAR I-SUBCELLULAR O O B-SUBCELLULAR I-SUBCELLULAR O O B-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O B-CELL_TYPE O O O O O O

(C) K-fibers extend to spindle poles. The length of K-fibers and distance between kinetochores and a spindle pole (KT-to-Pole distance) were measured. K-fiber length relative to the KT-to-Pole distance was calculated (n=90 K-fibers of 5 oocytes from 2 independent experiments).


( B ) Wing variation in absolute centroid ( CS ) relative CS ( adjusted for ) .

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(B) Wing size variation in absolute centroid size (CS) and relative CS (adjusted for body size).


( C ) Reproducibility of - seq experiment . Pairwise Spearman correlation coefficients between showed higher correlations among biological replicates than among non - replicates .

O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O

(C) Reproducibility of RNA-seq experiment. Pairwise Spearman correlation coefficients between RNA levels showed higher correlations among biological replicates than among non-replicates.


( D ) Comparable variability of for genes quantified at both .

(D) Comparable variability of RNA and protein levels for genes quantified at both levels.


( E ) RNAs of genes quantified at both have largest means smallest variabilities among whole RNAs quantified .

(E) RNAs of the genes quantified at both RNA and protein levels have the largest means and smallest variabilities among the whole RNAs quantified.


( F ) Weak correlation between . median Spearman correlation coefficient was 0 . 1 only 18 % of genes compared showed a positive correlation with statistical significance ( p < 0 . 05 ) .

O O O O O O O O O O O O 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) Weak correlation between RNA and protein levels. The median Spearman correlation coefficient was 0.1 and only 18 % of genes compared showed a positive correlation with statistical significance (p < 0.05).


( B ) Association of RNAs with whole wing variation that encompasses both sexes . Volcano plot of p - values against slope of fitted lines . horizontal lines indicate 10 20 % FDR thresholds .

O O O O O O O O B-TISSUE O O O 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) Association of RNAs with the whole wing size variation that encompasses both sexes. Volcano plot of p-values against the slope of the fitted lines. The horizontal lines indicate 10 and 20 % FDR thresholds.


( C ) Association of RNAs with between - wing variation ( absolute / relative CS , adjusted for sex ) .

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(C) Association of RNAs with between-line wing size variation (absolute/ relative CS, adjusted for sex).


( E ) Association of canonical growth regulator transcripts with wing ( absCS relCS ) . P - values from for association type 2 ( ANCOVA ) type 3 ( ANOVA on sex ) are plotted . dashed dotted lines indicate 10 % 20 % FDR thresholds , respectively . Note that canonical growth genes mostly exhibit type - 3 association .

O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) Association of canonical growth regulator transcripts with wing size (absCS and relCS). P-values from the tests for association type 2 (ANCOVA) and type 3 (ANOVA on sex) are plotted. The dashed and dotted lines indicate 10% and 20% FDR thresholds, respectively. Note that canonical growth genes mostly exhibit type-3 association.


( F ) Sexually dimorphic expression pattern of canonical growth genes . Plots of confirm type - 3 association with wing .

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(F) Sexually dimorphic expression pattern of canonical growth genes. Plots of RNA levels confirm type-3 association with wing size.


( G ) Exceptional growth genes that are associated with between - variation ( association type 2 ) .

(G) Exceptional growth genes that are associated with between-line size variation (association type 2).


( C ) of compartment sizes ( P divided by A ) is plotted . 14 genes were knocked down using multiple ( 2 or 3 ) RNAi lines per gene . x - axis indicates RNAi case IDs that represent genes knocked down RNAi type ( KK , GD , or TP as TRiP ) . Detailed information is provided in Dataset EV6 . horizontal indicates in negative control cases . Wilcoxon rank sum evaluated statistical significance against controls ( * : p < 0 . 05 , * * * : p < 0 . 001 , n . s . : not significant , cont . : control cases ) .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) The ratio of compartment sizes (P area divided by A area) is plotted. The 14 genes were knocked down using multiple (2 or 3) RNAi lines per gene. The x-axis indicates RNAi case IDs that represent the genes knocked down and the RNAi line type (KK, GD, or TP as TRiP). Detailed information is provided in Dataset EV6. The horizontal line indicates the size ratio in the negative control cases. Wilcoxon rank sum tests evaluated the statistical significance against controls (*:p <0.05, ***:p<0.001, n.s.: not significant, cont.: control cases).


( D ) Adult wings with RNAi manipulation in P compartment ( shaded ) . Note that boundary lines between A P compartments , which are relatively straight in control cases , bent towards P compartment due to reduced growth in P compartment .

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(D) Adult wings with RNAi manipulation in the P compartment (shaded area). Note that the boundary lines between A and P compartments, which are relatively straight in the control cases, bent towards the P compartment due to the reduced growth and size in the P compartment.


( A ) Cis - association of SNP variation with expression for separately . P - values for eQTLs at FDR < 20 % are plotted against effect of SNPs . top SNPs are labeled with corresponding gene names .

O O O O O O O O O O B-EXP_ASSAY O O O O O 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) Cis-association of SNP variation with RNA expression for females and males separately. P-values for eQTLs at FDR < 20% are plotted against the effect size of the SNPs. The top SNPs are labeled with the corresponding gene names.


( D E ) Cis - association of SNP variation with expression for . P - value for each pQTL is plotted against effect of SNP . pQTLs at FDRs of 20 % 30 % are depicted for , respectively . corresponding are designated with distinct colors . circle indicates that pQTL is also eQTL for same gene .

O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D and E) Cis-association of SNP variation with protein expression for females and males. P-value for each pQTL is plotted against the effect size of the SNP. pQTLs at FDRs of 20% and 30% are depicted for females and males, respectively. The corresponding proteins are designated with distinct colors. The circle indicates that the pQTL is also eQTL for the same gene.


( B ) Selection of mediator RNAs linked eQTLs . RNAs co - associated with SNPs wing traits are further tested whether SNPs are also associated with wing traits ( absCS relCS ) for each sex . dotted lines indicate a significance threshold ( p = 0 . 05 ) .

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(B) Selection of mediator RNAs and the linked eQTLs. RNAs co-associated with SNPs and wing size traits are further tested whether the SNPs are also associated with wing size traits (absCS and relCS) for each sex. The dotted lines indicate a significance threshold (p=0.05).


( C D ) RNAi of CG34015 in wing P - compartment significantly reduces in both sexes . are taken from wings . Statistical significance was evaluated by Wilcoxon .

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(C and D) RNAi of CG34015 in the wing P-compartment significantly reduces the size in both sexes. The images are taken from female wings. Statistical significance was evaluated by Wilcoxon test.


( E ) Genome - wide association of CG34015 expression . Manhattan plot shows a strong association of CG34015 with X chromosome in both sexes . P - values are plotted against ordered SNPs along genome . eQTLs associated with CG34015 are designated as red dots .

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(E) Genome-wide association of CG34015 expression. Manhattan plot shows a strong association of CG34015 RNA with X chromosome in both sexes. P-values are plotted against ordered SNPs along the genome. eQTLs associated with CG34015 are designated as red dots.


( F ) Zoomed plot around CG34015 gene region on X chromosome . eQTLs linked to CG34015 are mostly located upstream of CG34015 gene region . red arrow indicates transcriptional direction .

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(F) Zoomed plot around the CG34015 gene region on X chromosome. eQTLs linked to CG34015 are mostly located upstream of the CG34015 gene region. The red arrow indicates the transcriptional direction.


( G ) SNP variation of CG34015 - linked eQTLs . SNP states at each eQTL are depicted for each using different colors ( grey dots : reference SNP , blue dots : alternate SNP with a negative effect on level ) . Lines are ordered from left to right as CG34015 increase . Note that eQTLs are highly linked with each other .

O O O O O O B-GENEPROD O O O O O O O O O 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-EXP_ASSAY O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O

(G) SNP variation of CG34015-linked eQTLs. SNP states at each eQTL are depicted for each line using different colors (grey dots: reference SNP, blue dots: alternate SNP with a negative effect on RNA level). Lines are ordered from left to right as CG34015 RNA levels increase. Note that the eQTLs are highly linked with each other.


( H I ) Segregation of CG34015 wing ( absCS ) by variation of an eQTL ( X _ 16344746 _ SNP ) . P - values are shown . ( J ) Association of eQTL ( X _ 16344746 _ SNP ) with population - wide , wing variation among 143 DGRP lines . P - value R2 are shown for each sex .

O O O O O O B-GENEPROD B-TISSUE O O O O O O O O O O O O O O O O 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-TISSUE O O O O O O O O O O O O O O O O

(H and I) Segregation of CG34015 RNA levels and wing size (absCS) by the variation of an eQTL (X_16344746_SNP). P-values are shown. (J) Association of the eQTL (X_16344746_SNP) with population-wide, wing size variation among 143 DGRP lines. P-value and R2 are shown for each sex.


( A ) Flow cytometry of CD34 + CD1a - thymocytes cultured in FTOCs , in presence of a control or anti - Notch1 blocking antibody . Numbers in dot plots indicate of for corresponding populations . Histograms show TCRγδ expression in CD3 + TCRαβ + ( blue ) CD3 + TCRαβ - ( red ) . ( B , C ) ( B ) Mean frequency ( C ) absolute numbers of human CD4 + CD8β + DP ( black shades ) , CD3 + TCRαβ + ( blue shades ) CD3 + TCRγδ + ( red shades ) thymocytes developed in corresponding cultures from A ( n = 6 independent experiments with different donors , error bars indicate * indicates p < 0 . 05 ns ( not significant ) indicates p > 0 . 05 in a non - parametric paired Wilcoxon ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-CELL_TYPE O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-ORGANISM B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O 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) Flow cytometry analysis of CD34+CD1a- thymocytes cultured in FTOCs, in the presence of a control or anti-Notch1 blocking antibody. Numbers in dot plots indicate percentage of cells for the corresponding populations. Histograms show TCRγδ expression in CD3+TCRαβ+ (blue) and CD3+TCRαβ- (red) cells. (B, C) (B) Mean frequency and (C) absolute cell numbers of human CD4+CD8β+ DP (black shades), CD3+TCRαβ+ (blue shades) and CD3+TCRγδ+ (red shades) thymocytes developed in corresponding cultures from A (n=6 independent experiments with different donors, error bars indicate SEM and * indicates p<0.05 and ns (not significant) indicates p>0.05 in a non-parametric paired Wilcoxon test).


( A ) Flow cytometry of CD34 + CD1a - thymocytes cultured in FTOCs , in presence of a control or anti - Notch1 blocking antibody . Numbers in dot plots indicate of for corresponding populations . Histograms show TCRγδ expression in CD3 + TCRαβ + ( blue ) CD3 + TCRαβ - ( red ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-CELL_TYPE O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O

(A) Flow cytometry analysis of CD34+CD1a- thymocytes cultured in FTOCs, in the presence of a control or anti-Notch1 blocking antibody. Numbers in dot plots indicate percentage of cells for the corresponding populations. Histograms show TCRγδ expression in CD3+TCRαβ+ (blue) and CD3+TCRαβ- (red) cells.


( D ) Flow cytometry of CD34 + CD1a - thymocytes submitted to FTOC with thymic lobes deficient for Dll4 / or Jag2 ( Cre + ) as indicated above dot plots . Numbers in dot plots indicate of for corresponding populations . Histograms show TCRγδ expression in CD3 + TCRαβ + ( blue ) CD3 + TCRαβ - ( red ) . ( E , F ) ( E ) Mean frequency ( F ) absolute numbers of CD3 + TCRαβ + ( blue ) CD3 + TCRγδ + ( red ) thymocytes developed in corresponding cultures from D ( n = 5 - 7 independent experiments , depending on mouse genotype , error bars indicate * indicates p < 0 . 05 ns ( not significant ) indicates p > 0 . 05 in a non - parametric Mann - Whitney U ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-CELL_TYPE O O O O B-TISSUE I-TISSUE O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Flow cytometry analysis of CD34+CD1a- thymocytes submitted to FTOC with fetal thymic lobes deficient for Dll4 and/or Jag2 (Cre+) as indicated above the dot plots. Numbers in dot plots indicate percentage of cells for the corresponding populations. Histograms show TCRγδ expression in CD3+TCRαβ+ (blue) and CD3+TCRαβ- (red) cells. (E, F) (E) Mean frequency and (F) absolute cell numbers of CD3+TCRαβ+ (blue) and CD3+TCRγδ+ (red) thymocytes developed in corresponding cultures from D (n=5-7 independent experiments, depending on the mouse genotype, error bars indicate SEM and * indicates p<0.05 and ns (not significant) indicates p>0.05 in a non-parametric Mann-Whitney U test).


( D ) Flow cytometry of CD34 + CD1a - thymocytes submitted to FTOC with thymic lobes deficient for Dll4 / or Jag2 ( Cre + ) as indicated above dot plots . Numbers in dot plots indicate of for corresponding populations . Histograms show TCRγδ expression in CD3 + TCRαβ + ( blue ) CD3 + TCRαβ - ( red ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-CELL_TYPE O O O O B-TISSUE I-TISSUE O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O

(D) Flow cytometry analysis of CD34+CD1a- thymocytes submitted to FTOC with fetal thymic lobes deficient for Dll4 and/or Jag2 (Cre+) as indicated above the dot plots. Numbers in dot plots indicate percentage of cells for the corresponding populations. Histograms show TCRγδ expression in CD3+TCRαβ+ (blue) and CD3+TCRαβ- (red) cells.


( A ) plots depicting fluctuations in gene expression from vsn normalized probe intensities across human T development for both αβ - γδ - lineages ( n = 2 biological replicates error bars indicate ) .

O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-ORGANISM B-CELL_TYPE O O O O O O O O O O O O O O O O O O O

(A) Line plots depicting fluctuations in gene expression levels from vsn normalized probe intensities across human T cell development for both αβ- and γδ-lineages (n=2 biological replicates and error bars indicate SEM).


( D - E ) Scatterplot displaying up ( red ) or down ( blue ) regulation of probes ( log2 fold change > = 2 ) identified by sequential pairwise comparisons across either αβ ( D ) or γδ ( E ) development .

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(D-E) Scatterplot displaying the up (red) or down (blue) regulation of probes (log2 fold change >= 2) identified by sequential pairwise comparisons across either αβ (D) or γδ (E) development.


( C - D ) Scatterplot displaying up ( red ) or down ( blue ) regulation of miRNA ( log2 fold change > = 2 ) identified by sequential pairwise comparisons starting from CB towards either αβ ( C ) or γδ ( D ) developmental lineages .

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(C-D) Scatterplot displaying the up (red) or down (blue) regulation of miRNA (log2 fold change >= 2) identified by sequential pairwise comparisons starting from CB towards either the αβ (C) or γδ (D) developmental lineages.


( A ) plots displaying averaged NOTCH1 , MYC HES1 expression across T development ( n = 2 biological replicates error bars indicate ) .

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(A) Line plots displaying averaged NOTCH1, MYC and HES1 expression across T cell development (n=2 biological replicates and error bars indicate SEM).


( B ) displaying transcript wise scaled expression of NOTCH1 , HES1 , MYC miRNAs from ex vivo isolated subsets correlating ( spearman rho > = 0 . 5 ) with NOTCH1 gene expression Red arrows indicate 6 members of miR - 17 ~ 92 cluster ( n = 2 ) .

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(B) Heatmap displaying transcript wise scaled expression of NOTCH1, HES1, MYC and miRNAs from ex vivo isolated subsets correlating (spearman rho >= 0.5) with NOTCH1 gene expression Red arrows indicate all 6 members of the miR-17~92 cluster (n=2).


( C ) visualizing Notch1 regulated gene expression of in vitro cultured thymocytes across multiple culturing conditions ( OP9 - control , OP9 - JAG1 , OP9 - JAG2 , OP9 - DLL1 OP9 - DLL4 ) time points ( day 0 , 5 10 ) .

O O O O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY O O O O B-CELL_TYPE O O O O O B-CELL_LINE O O O B-CELL_LINE O B-GENEPROD O B-CELL_LINE O B-GENEPROD O B-CELL_LINE O B-GENEPROD B-CELL_LINE O B-GENEPROD O O O O O O O O O O O

(C) Heatmap visualizing Notch1 regulated gene expression of in vitro cultured thymocytes across multiple culturing conditions (OP9-control, OP9-JAG1, OP9-JAG2, OP9-DLL1 and OP9-DLL4) and time points (day 0, 5 and 10).


( D ) NOTCH1 correlating miRNAs ( spearman rho > = 0 . 3 ) of in vitro cultured thymocytes across multiple culturing conditions timepoints . miRNA expression were scaled transcript wise red arrows indicate members of miR - 17 ~ 92 cluster ( n = 2 ) .

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(D) NOTCH1 correlating miRNAs (spearman rho >= 0.3) of in vitro cultured thymocytes across multiple culturing conditions and timepoints. The miRNA expression levels were scaled transcript wise and red arrows indicate members of miR-17~92 cluster (n=2).


( E ) CHIPseq data for Notch1 RBPj binding at miR - 17 ~ 92 locus in CUTTL1 . Red underneath ChIPseq peaks indicate significant enrichment compared to input sample .

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(E) CHIPseq data for Notch1 and RBPj binding at the miR-17~92 locus in CUTTL1 cells. Red line underneath the ChIPseq peaks indicate significant enrichment compared to the input sample.


( B ) Transfection of miR - 17 Pre - miR miRNA Precursor , but not of miR - 92a , specifically targets wild - type ( black bars ) 3 ' UTR of BCL11B in a 3 ' UTR luciferase reporter following cotransfection of HEKT in comparison to controls ( transfection of no miRNA or a control miRNA ) . Mutation of predicted miR - 17 binding sites in 3 ' UTR BCL11B reporter construct prevents miR - 17 dependent targeting ( grey bars ) . Graphs show mean firefly luciferase values ( from 3 ' UTR reporter constructs ) following normalization to cotransfected control Renilla luciferase relative to control sample without miRNA ( n = 5 independent experiments , error bars indicate * indicates p < 0 . 05 ns ( not significant ) indicates p > 0 . 05 in a paired t - ) .

O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O 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) Transfection of miR-17 Pre-miR miRNA Precursor, but not of miR-92a, specifically targets the wild-type (black bars) 3'UTR of BCL11B in a 3'UTR luciferase reporter assay following cotransfection of HEKT cells in comparison to the controls (transfection of no miRNA or a control miRNA). Mutation of the predicted miR-17 binding sites in the 3'UTR BCL11B reporter construct prevents miR-17 dependent targeting (grey bars). Graphs show mean firefly luciferase values (from 3'UTR reporter constructs) following normalization to the cotransfected control Renilla luciferase and relative to control sample without miRNA (n=5 independent experiments, error bars indicate SEM and * indicates p<0.05 and ns (not significant) indicates p>0.05 in a paired t-test).


( C ) Quantitative RT - PCR gene expression of BCL11B in control ( black bar ) or miR - 17 ( grey bar ) transduced CD34 + thymocytes after 24h of culture on coated Fc - DLL1 plates , relative to geometric mean of ACTIN GAPDH relative to control - transduced sample ( n = 3 independent experiments with different donors , error bars indicate * indicates p < 0 . 05 in a paired t - ) .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD O B-CELL_TYPE O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Quantitative RT-PCR gene expression analysis of BCL11B in control (black bar) or miR-17 (grey bar) transduced CD34+ thymocytes after 24h of culture on coated Fc-DLL1 plates, relative to the geometric mean of ACTIN and GAPDH and relative to the control-transduced sample (n=3 independent experiments with different donors, error bars indicate SEM and * indicates p<0.05 in a paired t-test).


( D ) Quantitative real - time RT - PCR gene expression of BCL11B in control ( black bar ) or miR - 17 sponge ( bar ) transduced CD34 + thymocytes , relative to geometric mean of ACTIN GAPDH ( n = 5 independent experiments with different donors , error bars indicate * indicates p < 0 . 05 in a paired t - ) .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD O B-CELL_TYPE O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Quantitative real-time RT-PCR gene expression analysis of BCL11B in control (black bar) or miR-17 sponge (white bar) transduced CD34+ thymocytes, relative to the geometric mean of ACTIN and GAPDH (n=5 independent experiments with different donors, error bars indicate SEM and * indicates p<0.05 in a paired t-test).


( A ) Gene expression pattern of BCL11B ( red dots ) HES1 ( blue squares ) during human T development , derived from microarray data as shown in Figure 2 . ( n = 2 biological replicates error bars indicate ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O B-GENEPROD O O O O O B-ORGANISM B-CELL_TYPE O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O

(A) Gene expression pattern of BCL11B (red dots) and HES1 (blue squares) during human T cell development, derived from microarray data as shown in Figure 2. (n=2 biological replicates and error bars indicate SEM).


Quantitative RT - PCR expression of BCL11B in human after 5 days , respectively , of coculture on OP9 stromal expressing different Notch ligands . Graphs show mean expression , relative to geometric mean of ACTIN GAPDH relative to OP9 - DLL1 cultured ( n = 4 independent experiments with different donors , error bars indicate * indicates p < 0 . 05 ns ( not significant ) indicates p > 0 . 05 in a paired t - ) .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-ORGANISM O O O O O O O O O B-CELL_LINE B-CELL_TYPE O O O O O O O O B-EXP_ASSAY O O O O O O B-GENEPROD I-GENEPROD O O B-CELL_LINE O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Quantitative RT-PCR expression analysis of BCL11B in human after 5 days, respectively, of coculture on OP9 stromal cells expressing different Notch ligands. Graphs show mean expression, relative to the geometric mean of ACTIN and GAPDH and relative to the OP9-DLL1 cultured cells (n=4 independent experiments with different donors, error bars indicate SEM and * indicates p<0.05 and ns (not significant) indicates p>0.05 in a paired t-test).


Quantitative RT - PCR expression of BCL11B in human CD34 + thymocyte progenitors after 2 days , respectively , of coculture on OP9 stromal expressing different Notch ligands . Graphs show mean expression , relative to geometric mean of ACTIN GAPDH relative to OP9 - DLL1 cultured ( n = 4 independent experiments with different donors , error bars indicate * indicates p < 0 . 05 ns ( not significant ) indicates p > 0 . 05 in a paired t - ) .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-ORGANISM B-GENEPROD O B-CELL_TYPE O O O O O O O O O O B-CELL_LINE B-CELL_TYPE O O O O O O O O B-EXP_ASSAY O O O O O O B-GENEPROD I-GENEPROD O O B-CELL_LINE O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Quantitative RT-PCR expression analysis of BCL11B in human CD34+ thymocyte progenitors after 2 days, respectively, of coculture on OP9 stromal cells expressing different Notch ligands. Graphs show mean expression, relative to the geometric mean of ACTIN and GAPDH and relative to the OP9-DLL1 cultured cells (n=4 independent experiments with different donors, error bars indicate SEM and * indicates p<0.05 and ns (not significant) indicates p>0.05 in a paired t-test).


( D ) Quantitative RT - PCR expression of BCL11B in human CD34 + thymocyte progenitors after 2 days of coculture on OP9 - DLL1 stromal in presence of 5 µM DAPT ( GSI ) . Graphs show mean expression , relative to geometric mean of ACTIN GAPDH relative to sample cultured in presence of control DMSO ( n = 4 independent experiments with different donors , error bars indicate * indicates p < 0 . 05 in a paired t - ) .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-ORGANISM B-GENEPROD O B-CELL_TYPE O O O O O O O B-CELL_LINE O B-GENEPROD B-CELL_TYPE O O O O O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Quantitative RT-PCR expression analysis of BCL11B in human CD34+ thymocyte progenitors after 2 days of coculture on OP9-DLL1 stromal cells in the presence of 5 µM DAPT (GSI). Graphs show mean expression, relative to the geometric mean of ACTIN and GAPDH and relative to sample cultured in the presence of control DMSO (n=4 independent experiments with different donors, error bars indicate SEM and * indicates p<0.05 in a paired t-test).


Quantitative RT - PCR expression of miR - 17 in human CB CD34 + Lin - after 5 days , respectively , of coculture on OP9 stromal expressing different Notch ligands Graphs show mean expression , relative to geometric mean of SNORD61 , SNORD95 RNU6 - 2 relative to OP9 - DLL1 cultured ( n = 2 independent experiments with different donors ; error bars indicate - two data points are shown ) .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-ORGANISM B-TISSUE B-GENEPROD O B-GENEPROD O O O O O O O O O O B-CELL_LINE B-CELL_TYPE O O O O O O O B-EXP_ASSAY O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_LINE O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O

Quantitative RT-PCR expression analysis of miR-17 in human CB CD34+Lin- after 5 days, respectively, of coculture on OP9 stromal cells expressing different Notch ligands Graphs show mean expression, relative to the geometric mean of SNORD61, SNORD95 and RNU6-2 and relative to the OP9-DLL1 cultured cells (n=2 independent experiments with different donors; error bars indicate SEM - the two data points are shown).


Quantitative RT - PCR expression of miR - 17 in human CD34 + thymocyte progenitors after 5 2 days , respectively , of coculture on OP9 - DLL1 stromal in presence of 5 µM DAPT ( GSI ) . Graphs show mean expression , relative to sample cultured in presence of control DMSO ( F ) ( n = 2 independent experiments with different donors ; error bars indicate - two data points are shown ) .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-ORGANISM B-GENEPROD O B-CELL_TYPE O O O O O O O O O O O B-CELL_LINE O B-GENEPROD B-CELL_TYPE O O O O O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O

Quantitative RT-PCR expression analysis of miR-17 in human CD34+ thymocyte progenitors after 5 and 2 days, respectively, of coculture on OP9-DLL1 stromal cells in the presence of 5 µM DAPT (GSI). Graphs show mean expression, relative to the sample cultured in the presence of control DMSO (F) (n=2 independent experiments with different donors; error bars indicate SEM - the two data points are shown).


( G ) Quantitative RT - PCR gene expression of BCL11B in control ( black bar ) , GATA3 shRNA or TCF7 shRNA ( grey bars ) transduced CD34 + thymocytes after 48h of coculture on OP9 - DLL1 stromal , relative to geometric mean of ACTIN GAPDH relative to control - transduced sample ( n = 6 independent experiments with different donors for GATA3 shRNA , n = 4 independent experiments with different donors for TCF7 shRNA , error bars indicate * indicates p < 0 . 05 in a paired t - ) .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O B-GENEPROD O B-CELL_TYPE O O O O O B-CELL_LINE O B-GENEPROD B-CELL_TYPE O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O

(G) Quantitative RT-PCR gene expression analysis of BCL11B in control (black bar), GATA3 shRNA or TCF7 shRNA (grey bars) transduced CD34+ thymocytes after 48h of coculture on OP9-DLL1 stromal cells, relative to the geometric mean of ACTIN and GAPDH and relative to the control-transduced sample (n=6 independent experiments with different donors for GATA3 shRNA, n=4 independent experiments with different donors for TCF7 shRNA, error bars indicate SEM and * indicates p<0.05 in a paired t-test).


( A ) Flow cytometry of control miR - 17 transduced CD34 + thymocytes , cultured in ATOs for 3 . Numbers in contour plots indicate of for corresponding populations . Histograms show TCRγδ expression in CD3 + TCRαβ + ( blue ) CD3 + TCRαβ - ( red ) . ( B ) Graphs show mean frequency of CD4 + CD8β + DP , CD3 + TCRαβ + CD3 + TCRγδ + thymocytes of control ( black bar ) or miR - 17 ( grey bar ) transduced CD34 + thymocytes in these cultures ( n = 6 n = 8 independent experiments for control miR - 17 , respectively . Error bars indicate ns ( not significant ) indicates p > 0 . 05 in a non - parametric Mann - Whitney U ) . ( C ) Graphs show mean absolute numbers of total , CD4 + CD8β + DP , CD3 + TCRαβ + CD3 + TCRγδ + thymocytes that developed in these cultures ( n = 6 n = 8 independent experiments for control miR - 17 , respectively . Error bars indicate * indicates p < 0 . 05 in a non - parametric Mann - Whitney U ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-CELL_TYPE O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD O B-CELL_TYPE O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O 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-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-CELL_TYPE O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD 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) Flow cytometry analysis of control and miR-17 transduced CD34+ thymocytes, cultured in ATOs for 3 weeks. Numbers in contour plots indicate percentage of cells for the corresponding populations. Histograms show TCRγδ expression in CD3+TCRαβ+ (blue) and CD3+TCRαβ- (red) cells. (B) Graphs show the mean frequency of CD4+CD8β+ DP, CD3+TCRαβ+ and CD3+TCRγδ+ thymocytes of control (black bar) or miR-17 (grey bar) transduced CD34+ thymocytes in these cultures (n=6 and n=8 independent experiments for control and miR-17, respectively. Error bars indicate SEM and ns (not significant) indicates p>0.05 in a non-parametric Mann-Whitney U test). (C) Graphs show the mean absolute cell numbers of total, CD4+CD8β+ DP, CD3+TCRαβ+ and CD3+TCRγδ+ thymocytes that developed in these cultures (n=6 and n=8 independent experiments for control and miR-17, respectively. Error bars indicate SEM and * indicates p<0.05 in a non-parametric Mann-Whitney U test).


( A ) Flow cytometry of control miR - 17 transduced CD34 + thymocytes , cultured in ATOs for 3 . Numbers in contour plots indicate of for corresponding populations . Histograms show TCRγδ expression in CD3 + TCRαβ + ( blue ) CD3 + TCRαβ - ( red ) . ( B ) Graphs show mean frequency of CD4 + CD8β + DP , CD3 + TCRαβ + CD3 + TCRγδ + thymocytes of control ( black bar ) or miR - 17 ( grey bar ) transduced CD34 + thymocytes in these cultures ( n = 6 n = 8 independent experiments for control miR - 17 , respectively . Error bars indicate ns ( not significant ) indicates p > 0 . 05 in a non - parametric Mann - Whitney U ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-CELL_TYPE O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD O B-CELL_TYPE O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O 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) Flow cytometry analysis of control and miR-17 transduced CD34+ thymocytes, cultured in ATOs for 3 weeks. Numbers in contour plots indicate percentage of cells for the corresponding populations. Histograms show TCRγδ expression in CD3+TCRαβ+ (blue) and CD3+TCRαβ- (red) cells. (B) Graphs show the mean frequency of CD4+CD8β+ DP, CD3+TCRαβ+ and CD3+TCRγδ+ thymocytes of control (black bar) or miR-17 (grey bar) transduced CD34+ thymocytes in these cultures (n=6 and n=8 independent experiments for control and miR-17, respectively. Error bars indicate SEM and ns (not significant) indicates p>0.05 in a non-parametric Mann-Whitney U test).


( A ) Flow cytometry showing mean of Median ( MFI ) of BCL11B in Jurkat , 72h post transduction with control shRNA ( black bar ) , BCL11B shRNA1 or BCL11B shRNA2 ( grey bars ) , each normalized to FMO sample ( BCL11B stained - BCL11B unstained sample ) . ( n = 2 independent experiments ; error bars indicate - two data points are shown ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O B-EXP_ASSAY O O B-GENEPROD O B-CELL_LINE O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O

(A) Flow cytometry analysis showing mean of Median Fluorescence Intensity (MFI) of BCL11B protein levels in Jurkat cells, 72h post transduction with control shRNA (black bar), BCL11B shRNA1 or BCL11B shRNA2 (grey bars), each normalized to the FMO sample (BCL11B stained - BCL11B unstained sample). (n=2 independent experiments; error bars indicate SEM - the two data points are shown).


( B ) Flow cytometry showing mean of MFI of BCL11B in CD4 + CD8β + DP ( black bar ) CD4 - CD8β - DN CD3 + TCRγδ + ( grey bar ) ex vivo thymocytes , normalized to FMO sample ( BCL11B stained - BCL11B unstained sample ) ( n = 4 independent experiments with different donors , error bars indicate * indicates p < 0 . 05 in a paired t - ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O B-CELL_TYPE O O O O O O B-GENEPROD B-EXP_ASSAY O B-GENEPROD O O O O O O 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) Flow cytometry analysis showing mean of MFI of BCL11B protein levels in CD4+CD8β+ DP (black bar) and CD4-CD8β- DN CD3+TCRγδ+ (grey bar) ex vivo thymocytes, normalized to FMO sample (BCL11B stained - BCL11B unstained sample) (n=4 independent experiments with different donors, error bars indicate SEM and * indicates p<0.05 in a paired t-test).


( C ) Mean total numbers of control shRNA ( black bar ) , BCL11B shRNA1 or BCL11B shRNA2 ( grey bars ) transduced CD34 + thymocytes , cultured in ATOs for 3 ( n = 5 independent experiments with different donors , error bars indicate * indicates p < 0 . 05 in a non - parametric paired Wilcoxon ) .

O O O O O B-EXP_ASSAY O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O B-GENEPROD O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Mean total cell numbers of control shRNA (black bar), BCL11B shRNA1 or BCL11B shRNA2 (grey bars) transduced CD34+ thymocytes, cultured in ATOs for 3 weeks (n=5 independent experiments with different donors, error bars indicate SEM and * indicates p<0.05 in a non-parametric paired Wilcoxon test).


( D ) Flow cytometry of CD4 + CD8β + DP , CD3 + TCRαβ + ( blue ) CD3 + TCRαβ - ( red ) thymocytes that developed in corresponding cultures from C . Numbers in contour plots indicate of for corresponding populations . Histograms show TCRγδ expression in CD3 + TCRαβ + ( blue ) CD3 + TCRαβ - ( red ) . ( E ) Graphs show mean frequency of CD3 + TCRαβ + CD3 + TCRγδ + thymocytes that developed in these cultures ( n = 5 independent experiments with different donors , error bars indicate * indicates p < 0 . 05 in a paired t - ) . ( F - G ) Mean absolute numbers of ( F ) CD3 + TCRαβ + ( G ) CD3 + TCRγδ + thymocytes that developed in these cultures ( n = 5 independent experiments with different donors , error bars indicate * indicates p < 0 . 05 in a non - parametric paired Wilcoxon ) . ( H ) CD3 + TCRγδ + / CD3 + TCRαβ + from cultures depicted in D ( n = 5 independent experiments with different donors , error bars indicate * indicates p < 0 . 05 in a paired t - ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-CELL_TYPE O O O O O O O O O O O O O O O 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-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O B-CELL_TYPE O O O O O O O O O O O O O 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-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Flow cytometry analysis of CD4+CD8β+ DP, CD3+TCRαβ+ (blue) and CD3+TCRαβ- (red) thymocytes that developed in corresponding cultures from C. Numbers in contour plots indicate the percentage of cells for the corresponding populations. Histograms show TCRγδ expression in CD3+TCRαβ+ (blue) and CD3+TCRαβ- (red) cells. (E) Graphs show the mean frequency of CD3+TCRαβ+ and CD3+TCRγδ+ thymocytes that developed in these cultures (n=5 independent experiments with different donors, error bars indicate SEM and * indicates p<0.05 in a paired t-test). (F-G) Mean absolute cell numbers of (F) CD3+TCRαβ+ and (G) CD3+TCRγδ+ thymocytes that developed in these cultures (n=5 independent experiments with different donors, error bars indicate SEM and * indicates p<0.05 in a non-parametric paired Wilcoxon test). (H) CD3+TCRγδ+/CD3+TCRαβ+ ratio from cultures depicted in D (n=5 independent experiments with different donors, error bars indicate SEM and * indicates p<0.05 in a paired t-test).


( D ) Flow cytometry of CD4 + CD8β + DP , CD3 + TCRαβ + ( blue ) CD3 + TCRαβ - ( red ) thymocytes that developed in corresponding cultures from C . Numbers in contour plots indicate of for corresponding populations . Histograms show TCRγδ expression in CD3 + TCRαβ + ( blue ) CD3 + TCRαβ - ( red ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O

(D) Flow cytometry analysis of CD4+CD8β+ DP, CD3+TCRαβ+ (blue) and CD3+TCRαβ- (red) thymocytes that developed in corresponding cultures from C. Numbers in contour plots indicate the percentage of cells for the corresponding populations. Histograms show TCRγδ expression in CD3+TCRαβ+ (blue) and CD3+TCRαβ- (red) cells.


( D ) Fragment distributions of human - ( pink ) mouse - ( blue ) specific reads from a DBS sample . Two vertical lines indicate 146 166 bp , observed peaks for ctDNA cfDNA , respectively .

O O O O O O B-ORGANISM O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O

(D) Fragment length distributions of human- (pink) and mouse- (blue) specific reads from a DBS sample. Two vertical lines indicate 146 and 166 bp, the observed peaks for ctDNA and cfDNA, respectively.


( E ) Example of an absolute copy number ( ACN ) profile successfully generated from human - specific reads from a DBS collected from a PDX mouse of 828 . ( F ) Matching ACN profile generated from sWGS of PDX tumour tissue .

O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-ORGANISM O O O O O O O O O O B-ORGANISM O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O O O O O

(E) Example of an absolute copy number (ACN) profile successfully generated from human-specific reads from a DBS collected from a PDX mouse of patient line 828. (F) Matching ACN profile generated from sWGS of PDX tumour tissue.


( B ) xTF change from baseline during first 29 days since treatment start for each PDX . xTFs were normalised to baseline ( day 1 ) xTF values for each mouse ( dashed lines ) . Carboplatin - treated mice are shown in purple , control mice are shown in teal . Bold lines show linear - model fitted across mice within same treatment group . Horizontal dashed lines at y = 1 indicate normalised baseline .

O O O B-EXP_ASSAY O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O B-EXP_ASSAY O O O B-ORGANISM O O O O O B-SMALL_MOLECULE O O B-ORGANISM O O O O O O B-ORGANISM O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O

(B) xTF change from baseline during the first 29 days since treatment start for each PDX patient line. xTFs were normalised to baseline (day 1) xTF values for each mouse (dashed lines). Carboplatin-treated mice are shown in purple, control mice are shown in teal. Bold lines show the linear-model fitted line across all mice within the same treatment and patient group. Horizontal dashed lines at y=1 indicate normalised baseline.


( C ) Fraction of blood spot samples in which putative driver amplifications were detected over time . fraction of samples with detected gene amplifications decreases in carboplatin treated group , while increasing in control group over time .

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(C) Fraction of blood spot samples in which putative driver amplifications were detected over time. The fraction of samples with detected gene amplifications decreases in carboplatin treated group, while increasing in the control group over time.


( D ) Correlation between xTF values tumour volumes of nearest matched time - point for both untreated ( Pearson ' s R = 0 . 45 , p = 0 . 00018 ) , carboplatin - treated ( Pearson ' s R = 0 . 056 , p = 0 . 78 ) PDX mice .

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(D) Correlation between xTF values and tumour volumes of the nearest matched time-point for both untreated (Pearson's R = 0.45, p = 0.00018), and carboplatin-treated (Pearson's R = 0.056, p = 0.78) PDX mice.


( A - D ) Weekly measured tumour volumes ( mm3 ) for PDX mice over time . Treatment start is indicated by grey solid vertical . Solid coloured lines show modelled tumour growth curves using heteroscedastic point - wise random intercept linear mixed models ( see Methods ) . Growth curve inflection points were determined ( see Methods ) to estimate start of treatment effect tumour regrowth ( dashed vertical purple lines labelled t1 t2 , respectively ) .

O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O

(A-D) Weekly measured tumour volumes (mm3) for PDX mice over time. Treatment start is indicated by grey solid vertical line. Solid coloured lines show modelled tumour growth curves using heteroscedastic point-wise random intercept linear mixed models (see Methods). Growth curve inflection points were determined (see Methods) to estimate the start of treatment effect and tumour regrowth (dashed vertical purple lines labelled t1 and t2, respectively).


( E ) mean xTF slope was estimated for each treatment group across four lines ( Fig . 2B ) compared to mean time to endpoint ( Pearson ' s R = - 0 . 73 , p = 0 . 039 ) tumour regrowth ( Pearson ' s R = - 0 . 97 , p = 0 . 025 ) . four different lines are indicated by different colours .

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(E) The mean xTF slope was estimated for each treatment group across the four patient lines (Fig. 2B) and compared to the mean time to endpoint (Pearson's R = -0.73, p = 0.039) and tumour regrowth (Pearson's R = -0.97, p = 0.025). The four different patient lines are indicated by different colours.


G - I ) Experimental validation of collective survival . populations grew to high densities in absence of ( Blue curves ) . In presence of , only populations with initial densities greater than NCT grew . NCT was increased with of . Engineered with QS - BlaM circuit exhibited a significantly higher NCT than those with BlaM or QS - CAT circuits . Carbenicillin was used for BlaM QS - BlaM circuits ; chloramphenicol for QS - CAT circuit . Each error bar represents standard deviation from triplicate measurements .

O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O B-GENEPROD O O B-GENEPROD O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O

G-I) Experimental validation of collective survival. All populations grew to high cell densities in the absence of antibiotics (Blue curves). In the presence of antibiotics, only populations with initial densities greater than NCT grew. NCT was increased with the concentration of antibiotics. Engineered bacteria with the QS-BlaM circuit exhibited a significantly higher NCT than those with the BlaM or the QS-CAT circuits. Carbenicillin was used for the BlaM and QS-BlaM circuits; chloramphenicol for the QS-CAT circuit. Each error bar represents the standard deviation from triplicate measurements.


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


( G - I ) Experimental validation of collective survival . populations grew to high densities in absence of ( Blue curves ) . In presence of , only populations with initial densities greater than NCT grew . NCT was increased with of . Engineered with QS - BlaM circuit exhibited a significantly higher NCT than those with BlaM or QS - CAT circuits . Carbenicillin was used for BlaM QS - BlaM circuits ; chloramphenicol for QS - CAT circuit . Each error bar represents standard deviation from triplicate measurements .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O

(G-I) Experimental validation of collective survival. All populations grew to high cell densities in the absence of antibiotics (Blue curves). In the presence of antibiotics, only populations with initial densities greater than NCT grew. NCT was increased with the concentration of antibiotics. Engineered bacteria with the QS-BlaM circuit exhibited a significantly higher NCT than those with the BlaM or the QS-CAT circuits. Carbenicillin was used for the BlaM and QS-BlaM circuits; chloramphenicol for the QS-CAT circuit. Each error bar represents the standard deviation from triplicate measurements.


Configuration of two - compartment model . microenvironment of microbial swarmbot is separated from that of chamber by its permeable hydrogel shell ( orange ) , resulting in two separate compartments , defined as swarmbot environment ( chamber ) . Four major components involved in population dynamics are explored in model , including density ( n ) , nutrient ( s ) , carbenicillin ( a ) BlaM ( b ) . Each component has a different transport rate , depending on molecule . Simulated microbial swarmbot dynamics under a static condition . a = 0 . 4 , a microbial swarmbot with a low n0 cannot initiate growth ( red lines ) but one with a high n0 leads to growth both in swarmbot , after a delay , in chamber ( green lines ) . While ( C ) swarmbots with both low high initial densities lead to eventual full growth in chamber in absence of antibiotic ( a = 0 ) . Solid lines : density inside swarmbots ; dashed lines : density in chambers . ( D ) A microfluidic device for controlling monitoring population dynamics of microbial swarmbot . device consists of an input channel leading to a culturing chamber a trapping slot . chamber is used for culturing engineered populations both inside outside swarmbot , while trapping slot is for trapping swarmbot due to its smaller but allowing medium to flow through . Input flow patterns ( static or pulsing ) are controlled by a programmable syringe pump . ( E ) Experimental validation of model prediction . At 100µg / ml carbenicillin , a microbial swarmbot with a high initial density led to robust growth in capsule , after a delay , in chamber ( green lines ) . While a swarmbot with a low initial density did not exhibit growth either in swarmbot or in chamber at same carbenicillin ( red lines ) . ( F ) In contrast , both swarmbot culturing chamber reached high densities in absence of carbenicillin , regardless of initial densities .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Configuration of the two-compartment model. The microenvironment of microbial swarmbot is separated from that of the chamber by its permeable hydrogel shell (orange), resulting in two separate compartments, defined as swarmbot and environment (chamber). Four major components involved in the population dynamics are explored in the model, including cell density (n), nutrient (s), carbenicillin (a) and BlaM concentration (b). Each component has a different transport rate, depending on the molecule size.Simulated microbial swarmbot dynamics under a static condition. a = 0.4, a microbial swarmbot with a low n0 cannot initiate growth (red lines) but one with a high n0 leads to growth both in the swarmbot and, after a delay, in the chamber (green lines). While (C) swarmbots with both low and high initial densities lead to eventual full growth in the chamber in the absence of the antibiotic (a = 0). Solid lines: cell density inside swarmbots; dashed lines: cell density in the chambers.(D) A microfluidic device for controlling and monitoring population dynamics of microbial swarmbot. The device consists of an input channel leading to a culturing chamber and a trapping slot. The chamber is used for culturing engineered bacterial populations both inside and outside the swarmbot, while the trapping slot is for trapping the swarmbot due to its smaller size but allowing the medium to flow through. Input flow patterns (static or pulsing) are controlled by a programmable syringe pump.(E) Experimental validation of model prediction. At 100µg/ml carbenicillin, a microbial swarmbot with a high initial density led to robust growth in the capsule and, after a delay, in the chamber (green lines). While a swarmbot with a low initial cell density did not exhibit growth either in the swarmbot or in the chamber at the same carbenicillin concentration (red lines). (F) In contrast, both the swarmbot and culturing chamber reached high cell densities in absence of carbenicillin, regardless of the initial cell densities.


( G ) Time - lapse of microbial swarmbot dynamics . population initiated growth inside swarmbot subsequently in chamber at a high initial density . But no growth occurred for a swarmbot with a low initial density at same antibiotic ( rightmost ) . of carbenicillin was 100 µg / ml scale bar represents 200 µm . ( H ) control lacking BlaM circuit . population could not survive at same of carbenicillin at high initial density .

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(G) Time-lapse images of microbial swarmbot dynamics. The population initiated growth inside the swarmbot and subsequently in the chamber at a high initial density. But no growth occurred for a swarmbot with a low initial density at the same antibiotic concentration (rightmost). The concentration of carbenicillin was 100 µg/ml and the scale bar represents 200 µm.(H) The control strain lacking BlaM circuit. The population could not survive at the same concentration of carbenicillin at the high initial density.


Time courses of swarmbots treated with varying concentrations of carbenicillin glucose . Solid lines indicate densities inside swarmbots ; dotted lines indicate those in chamber . Safeguard was established for lower nutrient ( 0 . 004 % or 0 . 04 % glucose ) along with carbenicillin treatment . At a high nutrient level ( 0 . 4 % glucose ) , safeguard was lost due to either growth in chamber ( panels 3 6 ) or gradual killing of population inside swarmbot ( solid in panel 9 ) . These results are qualitatively consistent with model predictions ( Fig 4 ) .

O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Time courses of swarmbots treated with varying concentrations of carbenicillin and glucose. Solid lines indicate cell densities inside swarmbots; dotted lines indicate those in the chamber. Safeguard was established for lower nutrient levels (0.004% or 0.04% glucose) along with carbenicillin treatment. At a high nutrient level (0.4% glucose), the safeguard was lost due to either growth in the chamber (panels 3 and 6) or gradual killing of the population inside swarmbot (solid line in panel 9). These results are qualitatively consistent with the model predictions (Fig 4).


Snapshots of safeguard performance at end time point of ( A ) . red arrow in panel 6 indicates significant growth in chamber ; dimmer of swarmbot in panel 9 indicates killing of population .

O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O

Snapshots of safeguard performance at the end time point of (A). The red arrow in panel 6 indicates significant growth in the chamber; the dimmer fluorescence of the swarmbot in panel 9 indicates killing of the population.


Figure 6 . Experimental control of microbial swarmbot safeguard by other circuits . Demonstration of modular safeguard performance of swarmbots with different collective survival systems . For swarmbots containing carrying QS - CAT circuit , pulsing flow of medium containing 100µg / ml chloramphenicol established safeguard control but not for static condition . For swarmbots with QS - BlaM circuit , static condition of 100 µg / ml carbenicillin was sufficient for safeguard control . For conditions , safeguard did not occur in absence of antibiotic ( top panels from each condition ) . were from swarmbots cultured at 37 ˚ C for 16 hours . scale bar is 250 µm .

O O O O O O O O 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-EXP_ASSAY O 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-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Experimental control of microbial swarmbot safeguard by other circuits.Demonstration of modular safeguard performance of swarmbots with different collective survival systems. For the swarmbots containing cells carrying the QS-CAT circuit, pulsing flow of medium containing 100µg/ml chloramphenicol established safeguard control but not for the static condition. For the swarmbots with the QS-BlaM circuit, the static condition of 100 µg/ml carbenicillin was sufficient for safeguard control. For all the conditions, safeguard did not occur in the absence of antibiotic (top panels from each condition). All the images were from the swarmbots cultured at 37˚C for 16 hours. The scale bar is 250 µm.


( a ) Immunostaining of TCF20 , NESTIN , merged with DAPI in E13 E16 embryonic brain sections . TCF20 is widely expressed in brain . Data information : Scale bar in ( A ) e13 , 50 μm , e16 100

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(a) Immunostaining of TCF20, NESTIN, and merged with DAPI in E13 and E16 embryonic brain sections. TCF20 is widely expressed in the brain. Data information: Scale bar in (A) e13, 50 μm, e16 100 μm


( b ) Costaining with NSC markers NESTIN , SOX2 , PAX6 reveals that TCF20 is expressed in NSCs .

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(b) Costaining with the NSC markers NESTIN, SOX2, and PAX6 reveals that TCF20 is expressed in NSCs.


( c ) Absolute quantitative PCR of TCF20 in different developmental stages from E12 to P0 . n = 4 independently repeated .

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(c) Absolute quantitative PCR of TCF20 in different developmental stages from E12 to P0. n=4 independently repeated tests.


( d ) Western blotting results show changes in TCF20 expression level during brain development . ( e ) Statistical of normalized band in western blot results . n = 4 samples for each group .

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(d) Western blotting results show the changes in TCF20 protein expression level during brain development. (e) Statistical analysis of normalized band intensity in the western blot results. n= 4 samples for each group.


( f ) In utero electroporation of TCF20 shRNAs in E13 cerebral cortex led to abnormal distribution . These electroporated were collected at E16 . ( g ) Statistical of distributions of GFP - positive in different brain regions . of GFP - positive in CP ( cortical plate ) , IZ ( intermediate zone ) , VZ / SVZ ( ventricle zone subventricle zone ) was analyzed . n = 7 brains . Data information : Error bars represent means ± S . E . M . Two unpaired T - were used to analyze data , n . s . ( no significant difference ) , p < 0 . 01 ( * * ) , p < 0 . 001 ( * * * ) . Scale bar in ( F ) 50 μm .

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(f) In utero electroporation of TCF20 shRNAs in the E13 cerebral cortex led to abnormal cell distribution. These electroporated embryos were collected at E16. (g) Statistical analysis of the distributions of GFP-positive cells in different brain regions. The percentage of GFP-positive cells in the CP (cortical plate), IZ (intermediate zone), and VZ/SVZ (ventricle zone and subventricle zone) was analyzed. n=7 brains. Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.01(**), p<0.001(***). Scale bar in (F) 50 μm.


( b ) Immunostaining of BrdU . arrows represent BrdU GFP double - positive . Scale bar : 50 μm .

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(b) Immunostaining of BrdU. White arrows represent BrdU GFP double-positive cells. Scale bar: 50 μm.


( c ) Statistical of 2 - hour BrdU labeling experiments . n = 6 brains Data information : Error bars represent means ± S . E . M . Two unpaired T - were used to analyze data , n . s . ( no significant difference ) , p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) , p < 0 . 001 ( * * * ) .

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(c) Statistical analysis of 2-hour BrdU labeling experiments. n=6 brains Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**), p<0.001(***).


( d ) Western blotting indicated an increase in proliferation marker Sox2 in TCF20 depleted NSCs .

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(d) Western blotting analysis indicated an increase in the proliferation marker Sox2 in TCF20 depleted NSCs.


( e ) Immunostaining with pax6 in E16 electroporated brains . arrows represent PAX6 , GFP double - positive . 1 , 2 are 2x enlarged as dashed boxes showed . Scale bar : 50 μm . ( f ) Statistical of of PAX6 + GFP + among total GFP + . n = 3 brains for each group . Data information : Error bars represent means ± S . E . M . Two unpaired T - were used to analyze data , n . s . ( no significant difference ) , p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) , p < 0 . 001 ( * * * ) .

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(e) Immunostaining with pax6 in E16 electroporated embryo brains. White arrows represent PAX6, GFP double-positive cells. 1,2 are 2x enlarged as the dashed boxes showed. Scale bar: 50 μm. (f) Statistical analysis of the percentage of PAX6+GFP+ cells among the total GFP+ cells. n=3 brains for each group. Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**), p<0.001(***).


( g ) Immunostaining with sox2 pax6 in E16 electroporated brains . arrows represent GFP , sox2 double positive ; Red arrows represent GFP , TBR2 double positive . . Scale bar : 50 μm . ( h ) Statistical of of SOX2 + GFP + among total GFP + . n = 8 brains for each group . ( i ) Statistical of of TBR2 + GFP + among total GFP + . n = 5 brains for each group . Data information : Error bars represent means ± S . E . M . Two unpaired T - were used to analyze data , n . s . ( no significant difference ) , p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) , p < 0 . 001 ( * * * ) .

O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O O B-EXP_ASSAY B-TISSUE O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O B-TISSUE O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(g) Immunostaining with sox2 and pax6 in E16 electroporated embryo brains. White arrows represent GFP, sox2 double positive cells; Red arrows represent GFP, TBR2 double positive cells. area. Scale bar: 50 μm. (h) Statistical analysis of the percentage of SOX2+GFP+ cells among the total GFP+ cells. n=8 brains for each group. (i) Statistical analysis of the percentage of TBR2+GFP+ cells among the total GFP+ cells. n=5 brains for each group. Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**), p<0.001(***).


( a ) SATB2 revealed a reduction in SATB2 GFP colocalization when TCF20 was depleted . arrows represent SATB2 , GFP double - positive . ( b ) Statistical of SATB2 GFP double - positive in GFP positive . n = 3 brains . Data information : Error bars represent means ± S . E . M . Two unpaired T - were used to analyze data , n . s . ( no significant difference ) , p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) , p < 0 . 001 ( * * * ) . Scale bar : 25

O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD B-EXP_ASSAY O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) SATB2 staining revealed a reduction in SATB2 and GFP colocalization when TCF20 was depleted. White arrows represent SATB2, GFP double-positive cells. (b) Statistical analysis of the SATB2 GFP double-positive cell ratio in GFP positive cells. n=3 brains. Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**), p<0.001(***). Scale bar: 25 μm


( c ) with CTIP2 TBR1 antibodies shows TCF20 knockdown decreases colocalization of CTIP2 TBR1 with GFP in E13 . 5 - E16 . 5 brains . arrows represent CTIP2 , GFP double - positive . arrow heads represent TBR1 , GFP double - positive . ( d , e ) Statistical of CTIP2 , GFP TBR1 , GFP double - positive in GFP positive . n = 3 brains . Data information : Error bars represent means ± S . E . M . Two unpaired T - were used to analyze data , n . s . ( no significant difference ) , p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) , p < 0 . 001 ( * * * ) . Scale bar : ( d ) 50

O O O O B-GENEPROD I-GENEPROD O O B-GENEPROD O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O B-TISSUE O O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(c) Staining with CTIP2 and TBR1 antibodies shows TCF20 knockdown decreases the colocalization of CTIP2 and TBR1 with GFP in E13.5-E16.5 brains. White arrows represent CTIP2, GFP double-positive cells. White arrow heads represent TBR1, GFP double-positive cells. (d, e) Statistical analysis of CTIP2, GFP and TBR1, GFP double-positive cell ratio in GFP positive cells. n=3 brains. Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**), p<0.001(***). Scale bar: (d) 50 μm


( f ) A diagram of BrdU labeling time point in electroporation experiments . ( g ) Immunostaining of BrdU . arrows represent BrdU GFP double - positive . ( h ) of BrdU GFP double - positive in GFP - positive n = 3 brains for each group Data information : Error bars represent means ± S . E . M . Two unpaired T - were used to analyze data , n . s . ( no significant difference ) , p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) , p < 0 . 001 ( * * * ) . Scale bar : f ) 25

O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE B-GENEPROD O O O O O O O O B-SMALL_MOLECULE B-GENEPROD O O O O B-GENEPROD O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) A diagram of the BrdU labeling time point in electroporation experiments. (g) Immunostaining of BrdU. White arrows represent BrdU GFP double-positive cells. (h) Percentage of BrdU and GFP double-positive cells in GFP-positive cells n=3 brains for each group Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**), p<0.001(***). Scale bar: f) 25 μm


( b ) Western blotting of TCF20 in TCF20 WT , HET , KO mice reveals that TCF20 was deleted . ( c ) Statistical of normalized band of TCF20 . n = 3 samples for each genotype . Data information : Error bars represent means ± S . E . M . Two unpaired T - were used to analyze data , n . s . ( no significant difference ) , p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) , p < 0 . 001 ( * * * ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O B-ORGANISM O O B-GENEPROD O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Western blotting analysis of TCF20 in TCF20 WT, HET, KO mice reveals that TCF20 was deleted. (c) Statistical analysis of the normalized band intensity of TCF20. n=3 samples for each genotype. Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**), p<0.001(***).


( d ) RT - qPCR of TCF20 in TCF20 WT , HET , KO mice reveals that TCF20 was deleted . n = 4 samples . Data information : Error bars represent means ± S . E . M . Two unpaired T - were used to analyze data , n . s . ( no significant difference ) , p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) , p < 0 . 001 ( * * * ) .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O B-ORGANISM O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(d) RT-qPCR analysis of TCF20 in TCF20 WT, HET, and KO mice reveals that TCF20 was deleted. n=4 samples. Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**), p<0.001(***).


( e ) Immunostaining of E16 WT KO mouse brain slices revealed that TCF20 was successfully knocked out . Scale bar : 50 μm . ( f ) Statistical of normalized of TCF20 . n = 6 samples for each genotype . Data information : Error bars represent means ± S . E . M . Two unpaired T - were used to analyze data , n . s . ( no significant difference ) , p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) , p < 0 . 001 ( * * * ) .

O O O B-EXP_ASSAY O O O O B-ORGANISM B-TISSUE O O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(e) Immunostaining of E16 WT and KO mouse brain slices revealed that TCF20 was successfully knocked out. Scale bar: 50 μm. (f) Statistical analysis of the normalized fluorescence intensity of TCF20. n=6 samples for each genotype. Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**), p<0.001(***).


( g ) Western blotting of neural proliferation differentiation markers in WT HET KO mice showed that TCF20 deficits promote proliferation inhibit differentiation .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE O O O O O O O B-ORGANISM O O B-GENEPROD O O O O O O

(g) Western blotting analysis of neural proliferation and differentiation markers in WT HET and KO mice showed that TCF20 deficits promote cell proliferation and inhibit cell differentiation.


( h ) Immunostaining of different cortical layer markers TBR1 , CTIP2 , SATB2 , dash represents positive . Scale bar : 50 μm . ( i - k ) Statistical of different layers in TCF20 WT , HET KO mice . n = 7 brains of each group . Data information : Error bars represent means ± S . E . M . Two unpaired T - were used to analyze data , n . s . ( no significant difference ) , p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) , p < 0 . 001 ( * * * ) .

O O O B-EXP_ASSAY O O B-TISSUE O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-ORGANISM O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(h) Immunostaining of different cortical layer markers TBR1, CTIP2, and SATB2, White dash line represents the positive area. Scale bar: 50 μm. (i-k) Statistical analysis of different cells layers in TCF20 WT, HET and KO mice. n=7 brains of each group. Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**), p<0.001(***).


( l ) TCF20 deletion impairs neurogenesis results in abnormal distribution . Scale bar : 50 μm . ( m ) Statistical of GFP + distribution in different zones . n = 3 brains for each genotype . Data information : Error bars represent means ± S . E . M . Two unpaired T - were used to analyze data , n . s . ( no significant difference ) , p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) , p < 0 . 001 ( * * * ) .

O O O B-GENEPROD O O O O O O B-EXP_ASSAY O O O O O O O O O O O O B-GENEPROD O B-EXP_ASSAY O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(l) TCF20 deletion impairs neurogenesis and results in abnormal cell distribution. Scale bar: 50 μm. (m) Statistical analysis of GFP+ distribution in different zones. n=3 brains for each genotype. Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**), p<0.001(***).


( a ) Diagram of pup ultrasonic vocalization at frequency level after Fourier transforms ( b - d ) ( b ) Total call durations , ( c ) Call durations ( d ) Call numbers of WT , HET , KO mouse pups . nWT = 13 mice , nHET = 16 mice , nKO = 18 mice . Data information : Error bars represent means ± S . E . M . Two unpaired T - were used to analyze data , n . s . ( no significant difference ) , p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) , p < 0 . 001 ( * * * ) .

O O O O O B-ORGANISM B-EXP_ASSAY I-EXP_ASSAY O O 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-EXP_ASSAY I-EXP_ASSAY O O O O O O B-ORGANISM I-ORGANISM O O O O B-ORGANISM O O O O B-ORGANISM O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Diagram of pup ultrasonic vocalization at the frequency level after Fourier transforms (b-d) (b) Total call durations, (c) Call durations and (d) Call numbers of WT, HET, and KO mouse pups. nWT=13 mice, nHET=16 mice, and nKO=18 mice. Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**), p<0.001(***).


( e ) A sketch of mouse trace in open field . ( f - h ) ( f ) Statistical of time spent in center , ( g ) Statistical of total mouse movement in 5 min , ( h ) of mice in center movement to total . n for WT = 15 adult mice , n for HET = 12 adult mice . Data information : Error bars represent means ± S . E . M . Two unpaired T - were used to analyze data , n . s . ( no significant difference ) , p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) , p < 0 . 001 ( * * * ) .

O O O O O O B-ORGANISM O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O B-ORGANISM O O O O O O O O O O O O B-ORGANISM O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(e) A sketch of the mouse trace in the open field test. (f-h) (f)Statistical analysis of time spent in the center area, (g) Statistical analysis of total mouse movement distance in 5 min, (h) The ratio of mice in the center area movement distance to the total distance. n for WT=15 adult mice, n for HET=12 adult mice. Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**), p<0.001(***).


( i ) Statistical of Y maze . S , O , N represent start Arm , old Arm new Arm . of time that mice stayed in each arm was recorded analyzed ; n for WT = 11 adult mice , n for HET = 11 adult mice . Whiskers represent minimum to maximum value ; central lines represent median values ; boxes represent values between upper quartile lower quartile . ( j ) Results of elevated - plus maze . Time that mice spent in open arms ; n for WT = 8 adult mice , n for HET = 11 adult mice . ( k ) Results of elevated - plus maze . Time that mice spent in closed arms ; n for WT = 8 adult mice , n for HET = 11 adult mice . Data information : Error bars represent means ± S . E . M . Two unpaired T - were used to analyze data , n . s . ( no significant difference ) , p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) , p < 0 . 001 ( * * * ) .

O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O B-ORGANISM O O O O O O O B-ORGANISM O O O O O O O O O O O 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-ORGANISM O O O O O O O O O O O B-ORGANISM O O O O O O O B-ORGANISM O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O B-ORGANISM O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(i) Statistical analysis of the Y maze test. S, O, and N represent the start Arm, old Arm and new Arm. The percent of time that mice stayed in each arm was recorded and analyzed; n for WT=11 adult mice, n for HET=11 adult mice. Whiskers represent the minimum to maximum value; central lines represent median values; boxes represent the values between upper quartile and lower quartile. (j) Results of the elevated-plus maze. Time that mice spent in open arms; n for WT=8 adult mice, and n for HET=11 adult mice. (k) Results of elevated-plus maze. Time that mice spent in closed arms ; n for WT=8 adult mice, and n for HET=11 adult mice.Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**), p<0.001(***).


( l ) Numbers of marbles buried in marble burying ; n for WT = 10 adult mice , n for HET = 10 adult mice . Data information : Error bars represent means ± S . E . M . Two unpaired T - were used to analyze data , n . s . ( no significant difference ) , p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) , p < 0 . 001 ( * * * ) .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-ORGANISM O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(l) Numbers of marbles buried in the marble burying test; n for WT=10 adult mice, and n for HET=10 adult mice. Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**), p<0.001(***).


( m ) Diagram of three - chamber social interaction mouse movement trace . HET mice did not show preference or stay longer in stranger1 or stranger2 chambers than WT mice did in two phases . ( n ) Statistical of of time spent in each chamber with one empty chamber another chamber containing stranger1 ; n for WT = 16 adult mice , n for HET = 13 adult mice . ( o ) Comparison between time that WT HET mice spent in stranger1 chamber . n for WT = 16 adult mice , n for HET = 13 adult mice . ( p ) Time spent in each chamber when one contains familiar mouse stranger1 , as well as new mouse stranger2 . n for WT = 16 adult mice , n for HET = 13 adult mice . ( q ) Comparison between time of WT HET mice staying in stranger2 chamber . n for WT = 16 adult mice , n for HET = 13 adult mice . Data information : Error bars represent means ± S . E . M . Two unpaired T - were used to analyze data , n . s . ( no significant difference ) , p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) , p < 0 . 001 ( * * * ) .

O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY B-ORGANISM O O O O B-ORGANISM O O O O O O O O O O O O O O B-ORGANISM O O O O O O O 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-ORGANISM O O O O O O O B-ORGANISM O O O O O O O O O O B-ORGANISM O O O O O O O O O O O B-ORGANISM O O O O O O O B-ORGANISM O O O O O O O O O O O O O B-ORGANISM O O O O O O B-ORGANISM O O O O O O O O B-ORGANISM O O O O O O O B-ORGANISM O O O O O O O O O O B-ORGANISM O O O O O O O O O O O B-ORGANISM O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(m) Diagram of the three-chamber social interaction test and mouse movement trace. HET mice did not show preference or stay longer in the stranger1 or stranger2 chambers than the WT mice did in the two test phases. (n) Statistical analysis of the percentage of time spent in each chamber with one empty chamber and another chamber containing stranger1; n for WT=16 adult mice, n for HET=13 adult mice. (o) Comparison between the time that WT and HET mice spent in stranger1 chamber. n for WT=16 adult mice, n for HET=13 adult mice. (p)Time spent in each chamber when one contains familiar mouse stranger1, as well as new mouse stranger2. n for WT=16 adult mice, n for HET=13 adult mice. (q) Comparison between the time of WT and HET mice staying in the stranger2 chamber. n for WT=16 adult mice, n for HET=13 adult mice. Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**), p<0.001(***).


( c ) Heat map of significantly different genes .

(c) Heat map of significantly different genes.


( d ) TDG is significantly decreased after TCF20 deletion .

(d) TDG is significantly decreased after TCF20 deletion.


( e ) Volcano map showing that TDG is significantly decreased .

(e) Volcano map showing that TDG is significantly decreased.


( f , g ) Electroporation of GFP at E13 . 5 to E16 . 5 in TCF20 KO mice impairs distribution . n = 3 brains . Scalebar 25 μm Data information : Error bars represent means ± S . E . M . Two unpaired T - were used to analyze data , n . s . ( no significant difference ) , p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) .

O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O B-GENEPROD O B-ORGANISM O B-EXP_ASSAY O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, g) Electroporation of GFP at E13.5 to E16.5 in TCF20 KO mice impairs cell distribution. n=3 brains. Scalebar 25 μm Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**).


( h ) Chip - qPCR of TCF20 binding to TDG promoter . n = 4 biological repeats . Data information : Error bars represent means ± S . E . M . Two unpaired T - were used to analyze data , n . s . ( no significant difference ) , p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) .

O O O B-EXP_ASSAY O B-EXP_ASSAY O B-GENEPROD B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(h) Chip-qPCR of TCF20 binding to the TDG promoter. n= 4 biological repeats. Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**).


( b ) TDG ChIP - qPCR revealed that TDG mainly binds to TCF - 4 CpG islands . Seven regions ( from - 5 kbp to - 200 bp ) of promoter were analyzed . - 500 bp - 200 bp regions were located in CpG island of TCF - 4 promoter . ( n = 4 biological repeats ) . Data information : Error bars represent means ± S . E . M . Two unpaired T - were used to analyze data , n . s . ( no significant difference ) , p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) . p < 0 . 001 ( * * * ) .

O O O B-GENEPROD B-EXP_ASSAY O B-EXP_ASSAY O O B-GENEPROD O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O 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-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) TDG ChIP-qPCR revealed that TDG mainly binds to TCF-4 CpG islands. Seven regions (from -5 kbp to -200 bp) of the promoter were analyzed. -500 bp and -200 bp regions were located in the CpG island of the TCF-4 promoter. (n=4 biological repeats). Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**). and p<0.001(***).


( c - f ) MeDIP - qPCR showed that 5fC , 5caC , 5mC are increased in TCF - 4 promoter , while 5hmC is decreased when TCF20 is insufficient . MeDIP measure region - 500 bp from TCF - 4 promoter . ( n = 4 biological repeats ) Data information : Error bars represent means ± S . E . M . Two unpaired T - were used to analyze data , n . s . ( no significant difference ) , p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) . p < 0 . 001 ( * * * ) .

O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-SMALL_MOLECULE O O O B-GENEPROD O O O B-EXP_ASSAY O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(c-f) MeDIP-qPCR showed that 5fC, 5caC, and 5mC are increased in the TCF-4 promoter, while 5hmC is decreased when TCF20 is insufficient. The MeDIP assay measure the region -500 bp from the TCF-4 promoter. (n=4 biological repeats) Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**). and p<0.001(***).


( g ) NSCs were isolated from C57B6 / J E13 . 5 embryonic brains then were infected with a lentivirus containing a TCF20 shRNA or with a control plasmids . Immunostaining of 5fC shows that TCF20 depletion increased 5fC . dashed lines show infected with lentivirus . Scalebar 25 μm ( h ) Statistical of 5fC level . n = 20 , P < 0 . 0001 Data information : Error bars represent means ± S . E . M . Two unpaired T - were used to analyze data , n . s . ( no significant difference ) , p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) . p < 0 . 001 ( * * * ) .

O O O B-CELL_TYPE O O O O O O O O O B-TISSUE I-TISSUE O O O O O B-ORGANISM O O B-GENEPROD O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O B-GENEPROD O O B-SMALL_MOLECULE O O O O O O B-ORGANISM O O O O O O O O O B-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(g) NSCs were isolated from C57B6/J E13.5 embryonic brains and then were infected with a lentivirus containing a TCF20 shRNA or with a control plasmids. Immunostaining of 5fC shows that TCF20 depletion increased 5fC levels. White dashed lines show the cells infected with lentivirus. Scalebar 25 μm (h)Statistical analysis of 5fC fluorescence level. n=20 cells, and P<0.0001 Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**). and p<0.001(***).


( i ) Immunostaining of 5caC shows that TCF20 depletion increased 5caC level . dashed lines show infected with lentivirus . Scalebar 25 μm . ( j ) Statistical of 5caC level . n = 20 , P < 0 . 0001 . Data information : Error bars represent means ± S . E . M . Two unpaired T - were used to analyze data , n . s . ( no significant difference ) , p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) . p < 0 . 001 ( * * * ) .

O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O B-GENEPROD O O B-SMALL_MOLECULE B-EXP_ASSAY O O O O O O B-ORGANISM O O O O O O O O O O B-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(i) Immunostaining of 5caC shows that TCF20 depletion increased 5caC level. White dashed lines show the cells infected with lentivirus. Scalebar 25 μm. (j) Statistical analysis of the 5caC fluorescence level. n=20 cells, and P<0.0001. Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**). and p<0.001(***).


( k , l ) Overexpression of TDG TCF - 4 rescued TCF20 knockdown phenotype in brains ; n = 3 brains . Scalebar 50 μm Data information : Error bars represent means ± S . E . M . Two unpaired T - were used to analyze data , n . s . ( no significant difference ) , p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) . p < 0 . 001 ( * * * ) .

O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-EXP_ASSAY O B-TISSUE O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(k, l) Overexpression of TDG and TCF-4 rescued the TCF20 knockdown phenotype in brains; n=3 brains. Scalebar 50 μm Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**). and p<0.001(***).