EMBO/SourceData huggingface dataset:
Figure 2(A) Live imaging of MTOC and kinetochore dynamics. MTOCs (mNG-Cep192, green) and kinetochores (tdTomato-CENP-C, magenta) in B6D2F1 oocytes are shown. Time in h:mm after NEBD. Circles indicate central MTOCs, which are positioned in the middle region of the spindle later than 4 hours after NEBD. Arrows indicate displacement of the MTOCs. Scale bar, 10 μm.(B) Tracking analysis of central MTOCs. In the left graph, the positions of central MTOCs along the spindle axis are shown over time. MTOC positions were used to calculate distance to the spindle equator. In the right graph, lines showing temporal changes in MTOC-equator distance are aligned based on the time when the MTOC reached a position closest to the spindle equator. Horizontal lines at 7 and at −7 μm denote the thresholds used for the definition of the middle and polar regions of the spindle.(C) Central MTOCs originate from spindle poles and transiently stay in the middle region of the spindle. trajectories of central MTOCs (n=25 MTOCs of 8 oocytes from 3 independent experiments) were analyzed and their temporal changes in MTOC-equator distance are shown (Left graph). Central MTOCs were categorized into two groups: (1) ones that came from polar regions of the spindle (n=21, middle graph) and (2) others that stayed in the middle region throughout the period before reaching a position closest to the equator (n=4, right graph).(E) Destination of central MTOCs. The tracks of central MTOCs that originated from polar regions were used (n=21 MTOCs). The pole of origin was defined as Pole 1, while the other pole was defined as Pole 2. MTOC positions along the spindle axis (positive values for those closer to Pole 1) over time are shown. Horizontal lines at 7 and at −7 μm denote the thresholds used for the definition of the middle and polar regions of the spindle. Note that central MTOCs moved back to the original pole (Pole 1) or switched their positions to the opposite pole (Pole 2). (F) Destination of central MTOCs that originated from a polar region was categorized as in (E).
Figure 3(A) Live imaging of Zp3-Cre Ndc80f/f oocytes injected with Ndc80-WT, Ndc80-9D or Ndc80-9A. Z-projection and 3D-reconstructed images show MTOCs (mNeonGreen-Cep192, green) and chromosomes (H2B-mCherry, magenta). Time in h:mm after NEBD. Scale bar, 10 μm.(B) Ndc80-9D is defective for MTOC sorting. The density map of MTOCs along the spindle axis in Zp3-Cre Ndc80f/f oocytes expressing Ndc80-WT, Ndc80-9D or Ndc80-9A. The color represents the percentage of MTOC volume coded from dark (0%) to white (50% or more of the total volume).(C) MTOCs do not accumulate at the spindle poles in Ndc80-9D-expressing oocytes. Ratio of MTOCs (green) or chromosomes (magenta) in the polar region versus those in the middle region of the spindle at 6 hours after NEBD (n=19, 19 and 22 oocytes, respectively, from at least 3 independent experiments).(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).
Figure 4(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. 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 and 18 oocytes, respectively, from 3 independent experiments).
Figure 5(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-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-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).
Figure 1(B) Wing size variation in absolute centroid size (CS) and relative CS (adjusted for body size).(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 RNA and protein levels for genes quantified at both levels.(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 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).
Figure 2(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-line wing size variation (absolute/ relative CS, adjusted for sex).(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 RNA levels confirm type-3 association with wing size.(G) Exceptional growth genes that are associated with between-line size variation (association type 2).
Figure 3(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 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.
Figure 4(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 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.
Figure 5(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 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 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 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 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 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.
Figure 1 (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).  (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).
Figure 2(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 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.
Figure 3 (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.
Figure 4(A) Line plots displaying averaged NOTCH1, MYC and HES1 expression across T cell development (n=2 biological replicates and error bars indicate SEM). (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) 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 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 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.
Figure 5(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 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 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).
Figure 6 (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 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 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 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 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 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 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).
Figure 7 (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).
Figure 8(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 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 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 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).
Figure 1(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 patient line 828. (F) Matching ACN profile generated from sWGS of PDX tumour tissue.
Figure 2(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. 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 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.
Figure 3(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) 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.
Figure 2G-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.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.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.(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.