EMBO/SourceData huggingface dataset:
Figure 4Bone marrow-derived myeloid cells were treated with BSA-control (Con) or oleate in the presence or absence of 10 nM rapamycin (Rapa). The polarization of myeloid cells was detected via flow cytometry as indicated by CD11b and CD206 expression.Purified Gr1-CD11b+ myeloid cells were co-cultured with CD4+ T cells for proliferation assay. The supernatant was collected for nitric oxide (NO) detection.The oxygen consumption rate (OCR) of 1×105 differentiated myeloid cells were monitored after the addition of oligomycin (OA) (1 µM), the uncoupler carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP, 1 µM), and the electron transport inhibitor rotenone and antimycin A (R/AA, 5 µM) at indicated time points. The basal OCR, basal extracellular acidification rate (ECAR), spare respiratory capacity, ATP production and maximal respiration based on the OCR value were quantified.Mean fluorescence intensity (MFI) of intracellular phosphor-mTOR (pS2448) in polarized myeloid cells as determined by flow cytometryMean fluorescence intensity (MFI) of intracellular phosphor-mTOR (pS2448) in polarized myeloid cells lysed for western blot.
Figure 5Tumor infiltrating immune cells were isolated from the either CT26 or MCA205 tumor bearing model and analyzed for the expression of BODIPY in CD206+ (black) and CD206- (grey) cells.Balb/c mice were inoculated with 5×105 CT26 tumor cells subcutaneously. Since day 6, vehicle or DGAT inhibitors (iDGAT) were administrated via intra-tumoral injection once daily.At day 13, tumor infiltrated immune cells were isolated and stained for CD206 and CD11b.5×105 MCA205 were injected subcutaneously into C57BL/6 mice. PBS, liposome control or iDGAT-liposome was injected into the peritoneum every other day starting at day 7. Tumor size and body weight were measured every 7 days and the mice were sacrificed on day 31 for analysis. Immune cells from spleen, tumor-draining lymph nodes (TDLN) or tumor were isolated and stained for CD11b and with BODIPY for lipid droplets quantification in myeloid cells.Tumor infiltrating CD8+ T cells (red) were stained together with nuclei (DAPI/blue) after sacrificing the tumor bearing mice.
Figure 61×106 CD14+ myeloid cells from healthy donors were polarized by 1 ng/ml GM-CSF in the presence of BSA-control (C), 0.2 mM oleate (O) or 0.2 mM stearate (S) over a 6-day period. The expression of CD206, CD204 and CD38 was analyzed via flow cytometry.The level of lipid droplets was determined via BODIPY staining.The oxygen consumption rate (OCR) of differentiated myeloid cells was monitored as described in Materials and Methods.(D) The purified CFSE-labeled autologous CD4+ T cells were co-cultured with polarized myeloid cells with indicated treatment for T cell proliferation assay.Tumor tissue (tumor) as well as corresponding non-tumorous adjacent tissue (control) were collected from colorectal cancer patients and prepared for histopathology. The expression of CD68 (red), CD206 (green) and ADRP (blue) indicate the lipid droplets in tumor infiltrating myeloid cells. Nuclei (white) were stained using DAPI. The absolute number of positive cells was quantified in 5 high-power fields (hpf) (scale bar = 20 µm).
Figure 2A. Immunofluorescence microscopy of control, parental and patient fibroblasts labeled for LAMP-1. Patient fibroblasts (VPS41S285P/R662* and VPS41c.1423-2A>G/R662*) show more LAMP-1 puncta, which are distributed throughout the cell. (A') Quantification shows a significant increase in the number of LAMP-1 positive compartments for both patients. >15 Cells per condition were quantified (n=3). Scale bars, 10µm.
Figure 3D. HeLaVPS41KO cells transfected with VPS41WT-APEX2-V5, VPS41S285P-APEX2-V5 or VPS41R662*-APEX2-V5 constructs labeled for LAMP-1 and V5 immunofluorescence microscopy. All VPS41 variants co-localize with LAMP-1, indicating that VPS41S285P and VPS41R662* are recruited to late endosomes/lysosomes. Scale bars 10µm; zoom of squared area, 1µm.H. VPS41WT/WT, VPS41WT/R662* and VPS41S285P/R662* primary fibroblasts incubated with Dextran-568 for 0.5, 2, 5, 8 and 24 hours. Colocalization of Dextran-568 and SiR-Lysosome Cathepsin D (SiR-Lyso) reveals a delay in lysosomal delivery in maternal (VPS41WT/R662*) and patient (VPS41S285P/R662*) cells at 2 hours of Dextran uptake. After 5 hours, maternal cells show similar to control colocalization levels. Patient cells show only after 24 hours colocalization levels similar to control, indicating a delay rather than a block in late endosome - lysosome fusion. >10 Cells per cell line were quantified.
Figure 4A. Western blot of LC3 expression levels in control and patient fibroblasts. In steady state (0 hours) conditions, patient fibroblasts have a higher ratio of lipidated LC3 (LC3II):LC3I than control cells. Induction of autophagy by nutrient starvation (2 hours incubation with minimal EBSS medium) results in a raise in LC3II:LC3I ratio in both control and patient cells, but in VPS41S285P/R662* fibroblasts this increase is only modest (quantified in A') (n=2).E. Immunofluorescence of HeLaWT and HeLaVPS41KO cells transfected with LC3GFP/RFP tandem construct. The increased percentage of GFP/RFP-positive compartments in HeLaVPS41KO cells indicate a block in autophagic flux (quantified in E'). >12 Cells per cell line were quantified. Scale bars 10µm; zoom, 1µm.
Figure 5A. Immunofluorescence of control, parental and patient fibroblasts labeled for LAMP-1 and mTOR. In steady state conditions (0 hours) VPS41S285P/R662* fibroblasts show less colocalization between mTOR and LAMP-1. VPS41WT/WT, VPS41WT/S285P and VPS41WT/R662* show an appropriate mTOR response upon nutrient deprivation (2 hours) or restimulation (2 hours starvation followed by 15 minutes restimulation) (Appendix Fig S5A). >10 Cells per cell line were quantified (A'). Quantifications are performed relative to colocalization under steady state conditions per cell line (n=3). Scale bars, 10µm; zoom, 1µm.D. Rescue experiments of HeLaVPS41KO cells. Expression of VPS41WT-APEX2-V5, VPS41S285P-APEX2-V5 or VPS41R662*-APEX2-V5. Reintroduction of VPS41WT rescues TFE3 localization, whereas expression of mutant VPS41 has no effect (quantified in D'). >83 Cells per condition were quantified (n=3). Scale bars, 10µm.
Figure 6B. Western blot on cellular or secreted secretogranin II (SgII) of PC12 cells VPS41KO, or VPS41KO transduced with HA-VPS41WT or HA-VPS41S285P lentivirus. Cells were washed and incubated for 30 minutes in Tyrode's solution containing 2.5 mM K+ (basal) or 90 mM K+ (stimulated). No difference in secreted sgII levels was observed between VPS41WT and VPS41S285P.
Figure 7C. Graph representing the percentage of animals with 6 normal dopaminergic neurons in the anterior region of Pdat-1::GFP; Pdat-1:: α-syn (strain UA44) animals with heterozygous expression of hVPS41 variants. Heterozygous expression of hVPS41WT with either variant (strains UA389 or UA390) significantly rescues neurons from α-synuclein-induced degeneration at day 7 whereas compound heterozygous expression (strain UA391) fails to rescue neurodegeneration. At day 10, none of the heterozygous backgrounds significantly rescues α-synuclein-induced neurodegeneration. n=30 Adult worms for each of 3 independent experiments for the α-synuclein (α-syn) strain (total of 90 worms) and n=90 for each independent transgenic strain (30 worms/trial x 3 independent transgenic lines = 270 worms) for each of 3 independent experiments. D. Representative images of DA neurons from C. elegans expressing Pdat-1::GFP; Pdat-1:: α-syn, with or without hVPS41 variants, as described in (C). Intact DA neurons are indicated with arrowheads and missing neurons are indicated with arrows. Scale bar 20µm.
Figure 2Flow cytometry analysis and absolute numbers of B220loCD138+ (plasmablasts) (A, B) from HET-cd19 (n=6), MaxKO-cd19 (n=8), MycKO-cd19 (n=4), and DKO-cd19 (n=5) mice. Spleen B220+GFP+ B lymphocytes were sorted and activated in vitro with LPS plus IL-4 for 72 h. Cells were stained with monoclonal antibodies against the indicated markers and analyzed by flow cytometry. *p<0.05, **p<0.01. Columns represent mean values and error bars standard deviations. Experiment representative of at least three independent experiments.Flow cytometry analysis and absolute numbers of B220+IgG1+ switched B cells (C, D) from HET-cd19 (n=6), MaxKO-cd19 (n=8), MycKO-cd19 (n=4), and DKO-cd19 (n=5) mice. Spleen B220+GFP+ B lymphocytes were sorted and activated in vitro with LPS plus IL-4 for 72 h. Cells were stained with monoclonal antibodies against the indicated markers and analyzed by flow cytometry. *p<0.05, **p<0.01. Columns represent mean values and error bars standard deviations. Experiment representative of at least three independent experiments.E Spleen B220+GFP+ B lymphocytes were stained with 7AAD and annexin-V to monitor apoptosis. 7-AAD- annexin-v- (alive), 7-AAD- annexin-v+ (early apoptotic), 7-AAD+ annexin-v+ (late apoptotic), 7-AAD+ annexin-v- (necrosis). F Relative numbers of live, early apoptotic, late apoptotic and necrotic cells from E. G CD69 surface expression in purified B220+GFP+ mature spleen cells. Sorted B cells were activated in vitro with LPS plus IL-4 for 72 h, stained and analyzed by flow cytometry. The solid grey peak represents the expression levels of CD69 marker in non-activated mature B lymphocytes at day 0. H Relative numbers of CD69+ B lymphocytes in the cultures from G (n=4). Experiment representative of at least three independent experiments. I Analysis of germinal center (GC) formation in the spleen of MaxKO-cd19, Myc-KO-cd19, DKO-cd19 and heterozygous control mice immunized with TNP-KLH. Representative images of frozen spleen sections stained with IgM (grey/blue), PNA (GC marker; red), and GFP (Max-, c-Myc- or c-Myc/Max-deficient B cells; green). Scale bar, 80µm.J Upper, Number of GCs per mouse in spleen sections of immunized MaxKO-cd19 (n=3 mice, n=186 follicles analyzed), MycKO-cd19 (n=3 mice, n=178 follicles analyzed), DKO-cd19 (n=3 mice, n=191 follicles analyzed) and heterozygous control mice (n=3 mice, n=203 follicles analyzed). Lower, Frequencies of GC (PNA+) GFP+ (deleted cells) or GFP- (non-deleted cells) in immunized MaxKO-cd19 (n=13 GCs), MycKO-cd19 (n=21 GCs), DKO-cd19 (n=27 GCs) and heterozygous control (n=62 GCs) mice. Analysis was performed at 13 days post-immunization. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
Figure 3A Number of cell divisions in activated mature B lymphocytes. Sorted B220+GFP+ spleen cells from HET-cd19 (n=5), MaxKO-cd19 (n=5), MycKO-cd19 (n=3), and DKO-cd19 (n=3) mice were stained with a cell tracer, activated in vitro with LPS plus IL-4 for 72 h and analyzed by flow cytometry. Experiment representative of at least three independent experiments B Cell counts from the cultures C Photographs of B cell cultures Scale bar, 500 µm. 4× magnification. D Forward scatter (FSC) histograms of activated B lymphocytes An arbitrary dashed line is shown for comparison.Cell cycle analysis of sorted B220+GFP+ B lymphocytes activated in vitro. Cells were activated and EdU was added 4 h before harvesting. Cells were stained with propidium iodide to measure DNA content. Experiment representative of at least three independent experiments. An arbitrary dashed line shows the difference of EdU MFI. S phase mean fluorescence intensity (MFI): 7871 HET, 6512 MaxKO, 5860 MycKO, 6420 DKORepresentation of labeling with nucleotide analogues CldU (red) and IdU (green) and photographs of DNA fibers from sorted B220+GFP+ B lymphocytes activated for 48 h. Scale bar, 10 µm.H Fork rate of DNA fibres from primary B lymphocytes sorted and activated Data is pooled from three replicate experiments. Horizontal bars represent median values. 150-250 forks were measured per condition. ***, p<0.001; **, p<0.01I Western blotting of DNA replication proteins in activated B lymphocytes Sorted B220+GFP+ spleen cells were activated in vitro with LPS plus IL-4 for 72 h. MEK2, SMC1 are shown as loading controls. The blots shown are representative of three independent experiments.
Figure 4A Bar plot showing the number of DEGs (adj. p-value <0.01, FC +1.5 in red, 1.5 < FC >-1.5 in white, FC< -1.5 in blue). Sorted B220+GFP+ spleen B cells from MaxKO-cd19, MycKO-cd19, DKO-cd19 and control HET-cd19 mice were activated with LPS plus IL-4 for 72 h and analyzed. Two samples for each condition were used.C Hierarchical clustering heat map of DEGs in any comparison (two samples per condition). Dark blue in left panel indicates significance for that contrast column. Colour code: red represents upregulation, white, unchanged, and blue downregulation of expression. Gender: colour code for genetic condition. Pink are female and blue colour male mice.D Venn diagrams of up- (left) and down-regulated genes (right) for all three conditions (+/-1.5-FC and p<0.01). Heatmaps of genes only deregulated in MaxKO-cd19, MycKO-cd19, or DKO-cd19 mice are shown.
Figure 5B Heatmaps of genes involved in the relevant pathways Colour code: red represents upregulation, white, unchanged, and blue downregulation of expression.C qPCR analysis of up- and down-regulated genes Pooled RNA was prepared from sorted B220+GFP+ spleen B cells of MaxKO-cd19 (n=4), MycKO-cd19 (n=4), or DKO-cd19 (n=4) and control HET-cd19 (n=4) mice activated Genes were randomly selected and qPCR was performed using specific primers for cd72, med13l, card11, ssh2, cdc7, mrpl12, rpl39, and cysc. max and c-myc were included as controls and actin was used for normalization.
Figure 4Bacterial growth assessed by color change due to medium acidification for both the undefined ATCC 1161 and semi-defined CSY media with or without sucrose, 4% and 1% respectively. (+), inoculated wells; (-), non-inoculated control. The picture was taken after a 24-hour incubation period.Impact of horse serum (HS) and yeast extract (YE) dilution on M. florum doubling time. The green and red dotted lines indicate HS and YE concentrations found in ATCC 1161 medium (20% HS, 1.35% YE) and CSY medium (0.313% HS, 0.02% YE), respectively. Doubling times were measured in a CMRL-1066 base medium using colorimetric assays. Dots and error bars indicate the mean and standard deviation calculated from three biological replicates.The maximal biomass concentration observed for M. florum cultures growing in CSY medium with varying initial concentrations of sucrose. Biomass was measured using colony forming units (CFU/ml; left axis) and converted to grams of dry weight (gDW/L; right axis). A rectangular hyperbola fit is shown (yellow), and the dotted line represents the maximal biomass value predicted by the fit (1e8 x 5.95 CFU/ml; 0.013 gDW/L).Sucrose specific uptake rate (e) and combined lactate/acetate specific secretion rates (f) at varying initial sucrose concentrations in CSY medium. Boxplots represent the median and interquartile range of uptake or secretion rate values calculated at different time intervals during the exponential growth phase of M. florum cultures. Whiskers indicate minimal and maximal values. Dotted lines indicate the selected uptake (-5.26 mmol∙gDW-1∙hr-1) and secretion rate (8.69 mmol∙gDW-1∙hr-1) values used for modeling. Sucrose quantifications were performed in technical duplicate, whereas lactate and acetate quantifications were performed in single replicates. See Appendix Figure S5 for further details.
Figure 1Week 10 COs stained for the ventricular radial glia (vRGs) marker CRYAB, together with WWOX and the pan-radial glia marker SOX2. The images on the right are enlargements of the boxed area. The dashed line defines the ventricular space. Scale= 50µm (left), 25µm (right) (WT: n=8 from 3 batches, KO: n=8 from 3 batches).Immunofluorescent (IF) staining for the glutamatergic neurons marker VGLUT1 and GABAergic neurons marker GAD67 (GAD1) in week 10 COs (WT: n=8 from 3 batches, KO: n=8 from 3 batches, W-AAV: n=4 from 1 batch). Scale = 100µm.   Quantification of the images represented in figure 1E. VGLUT1 and GAD67 were quantified as the surface area covered by the staining and were normalized to the number of nuclei in each image. The boxplot represents the 1st and 3rd quartile, with its whiskers showing the minimum and maximum points and a central band representing the median. As the samples were not normally distributed, statistical significance was determined using Kruskal-Wallis test with Dunn's multiple comparisons test. Outliers were removed using the ROUT test (Q=1%). Data information: n.s (non-significant), *p ≤ 0.05, ****p ≤ 0.0001.
Figure 2Sample recordings from 7-week-old hESC-derived Cerebral Organoids (COs). Sample traces show visible differences in local field potential, with WWOX-KO COs (Red) showing increased activity compared to WT (Blue) in baseline condition (Left) and in the presence of 100µM 4AP (Right).Sample recordings from 7-week-old hESC-derived Cerebral Organoids (COs). WWOX's coding sequence was re-introduced into week 6 WWOX-KO COs using lentiviral transduction (lenti-WWOX) (F) Immunofluorescent staining showing WWOX expression in different populations in WWOX-KO organoids following infection with lentivirus. NT= non-treated. Scale = 50µm .
Figure 3Week 15 and week 24 COs stained for the astrocytic and radial glia marker GFAP, and the astrocyte-specific marker S100β (WT W15: n=9 from 3 batches, KO W15: n=16 from 3 batches, W-AAV W15: n=4 organoids from 1 batch. WT W24: n=10 from 4 batches, KO W24: n=9 from 3 batches, W-AAV W24: n=4 from 1 batch). Scale = 100µmIF staining of week 6 COs for astrocytic markers in the surrounding of VZs (WT: n=6 from 2 batches, KO: n=6 from 2 batches). Scale = 100µm (left) , 50µm (right).   Staining for the DNA damage markers γH2AX and 53BP1 in the nuclei of cells in the VZ of week 6 COs at physiological conditions, together with the pan-radial glia marker SOX2 (WT: n=8 from 3 individual batches, KO: n=12 from 3 individual batches, W-AAV: n=4 from 1 batch). Scale = 50µm (left), 25µm (right).   Quantification of γH2AX (top) and 53BP1 foci (bottom) in the nuclei of cells composing the innermost layer of the VZ, normalized to the total number of nuclei in this layer. The boxplot represents the 1st and 3rd quartile, with its whiskers showing the minimum and maximum points and a central band representing the median. Statistical significance was determined using one-way ANOVA with Tukey's multiple comparisons test (WT: n=8 organoids from 3 batches, KO: n=12 organoids from 3 batches, W-AAV: n=4 from 1 batch).  Data information: n.s (non-significant), *p ≤ 0.05, **p ≤ 0.01, ****p ≤ 0.0001.
Figure 4RNA sequencing (RNA-seq) of week 15 COs and transcriptome analysis (WT: n=2, KO: n=4). qPCR analysis for selected Wnt target genes validating the results of the RNA- seq. Y-axis indicates relative expression fold change. Data are represented as mean ± SEM. Statistical significance was determined using one-way ANOVA with Tukey's multiple comparisons test (WT: n=4 from 1 batch, KO: n=4 from 1 batch, W-AAV: n=4 from 1 batch). Data information: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.Week 16 COs were sub-fractionated into a cytoplasmic (C) and nuclear (N) fractions. The experiment was run twice with a total of 2 WT organoids and 4 KOs organoids (2 for each KO line). KAP-1 marks the nucleus and HSP90 marks the cytoplasm. The numbers at the bottom are a quantification of the nuclear fraction of β-catenin band intensities normalized to the cytosolic fraction [β-C (N/C)].IF staining in week 15 COs validating the decreased levels of the deep-layer cortical markers CTIP2 (BCL11B) and TBR1, and superficial-layer marker SATB2 (WT: n=3, KO: n=4, W-AAV: n=4). Scale = 50µm.   Quantification of the cortical markers seen in G, normalized to the total number of nuclei. Y-axis indicates the fold change compared to the average of the WT COs. The boxplot represents the 1st and 3rd quartile, with its whiskers showing the minimum and maximum points and a central band representing the median. Statistical significance was determined using one-way ANOVA with Tukey's multiple comparisons test (WT: n=9 from 3 batches, KO: n=16 from 3 batches, W-AAV: n=4 organoids from 1 batch).   Data information: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.
Figure 5Peripheral blood mononuclear cells (PBMCs) were isolated from a WOREE patient and from his healthy parents and were reprogrammed into iPSCs, and subsequently were differentiated into COs.   Week 10 WSM COs stained for the progenitor marker SOX2, neuronal marker β3-Tubulin and WWOX (WSM F1: n=2 from 1 batch, WSM M2: n=2 from 1 batch, WSM S2: n=2 from 1 batch, WSM S5: n=2 from 1 batch, WSM S5 W-AAV3: n=2 from 1 batch, WSM S5 W-AAV6: n=2 from 1 batch). Scale = 50µm.   Peripheral blood mononuclear cells (PBMCs) were isolated from a WOREE patient and from his healthy parents and were reprogrammed into iPSCs, and subsequently were differentiated into COs. Average firing rate over 24 neurons from WSM P COs (4 organoids), 41 neurons from WSM S COs (3 organoids) and 40 neurons from WSM S W-AAV organoids (3 organoids). Statistical significance was determined using one-way ANOVA with Tukey's multiple comparisons test. Bars represents the mean ± SEM. Data information: ns (non-significant), *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.Peripheral blood mononuclear cells (PBMCs) were isolated from a WOREE patient and from his healthy parents and were reprogrammed into iPSCs, and subsequently were differentiated into COs. GAD67 and VGLUT1 immunostaining in week 10 WSM COs (WSM F1: n=2 from 1 batch, WSM M2: n=2 from 1 batch, WSM S2: n=2 from 1 batch, WSM S5: n=2 from 1 batch, WSM S5 W-AAV3: n=2 from 1 batch, WSM S5 W-AAV6: n=2 from 1 batch). Scale = 50µm.   Quantification of the data shown in fig 5D. Statistical significance was determined using one-way ANOVA with Tukey's multiple comparisons test (WSM F1: n=2 from 1 batch, WSM M2: n=2 from 1 batch, WSM S2: n=2 from 1 batch, WSM S5: n=2 from 1 batch, WSM S5 W-AAV3: n=2 from 1 batch, WSM S5 W-AAV6: n=2 from 1 batch).  Data information: ns (non-significant), *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.
Figure 6Week 15 WSM COs stained for astrocytic markers GFAP and S100β. (WSM F1: n=2 from 1 batch, WSM M2: n=3 from 1 batch, WSM S2: n=2 from 1 batch, WSM S5: n=2 from 1 batch, WSM S5 W-AAV3: n=2 from 1 batch, WSM S5 W-AAV6: n=2 from 1 batch). Scale = 50µm.   Quantification of DNA damage foci, marked by γH2AX (left) and 53BP1 (right), in the nuclei of the cells in the VZs of week 6 WSM COs. The boxplot represents the 1st and 3rd quartile, with its whiskers showing the minimum and maximum points and a central band representing the median. Statistical significance was determined using one-way ANOVA with Tukey's multiple comparisons test (WSM F1: n=4 from 1 batch, WSM M2: n=4 from 1 batch, WSM S2: n=4 from 1 batch, WSM S5: n=4 from 1 batch, WSM S5 W-AAV3: n=4 from 1 batch, WSM S5 W-AAV6: n=4 from 1 batch).   Data information: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. qPCR analysis for selected Wnt target genes in week 10 WSM COs. Y-axis indicates relative expression fold change. Data are represented as mean ± SEM. Statistical significance was determined using one-way ANOVA with Tukey's multiple comparisons test (WSM P: n=6 from 1 batch, WSM S: n=4 from 1 batch, WSM S W-AAV: n=4 from 1 batch). Data information: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. Week 15 WSM COs stained for the cortical layers' markers CTIP2 and SATB2. (WSM F1: n=2 from 1 batch, WSM M2: n=3 from 1 batch, WSM S2: n=2 from 1 batch, WSM S5: n=2 from 1 batch, WSM S5 W-AAV3: n=2 from 1 batch, WSM S5 W-AAV6: n=2 from 1 batch). Scale = 50µm.   Quantification of the staining presented in E. The boxplot represents the 1st and 3rd quartile, with its whiskers showing the minimum and maximum points and a central band representing the median. Statistical significance was determined using one-way ANOVA with Tukey's multiple comparisons test (WSM F1: n=2 from 1 batch, WSM M2: n=3 from 1 batch, WSM S2: n=2 from 1 batch, WSM S5: n=2 from 1 batch, WSM S5 W-AAV3: n=2 from 1 batch, WSM S5 W-AAV6: n=2 from 1 batch).  Data information: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. E