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	Figure EV1. Characterization of EML1 patient‐ and EML1‐heKO‐derived iPS cells A. B. C. D. E. F.
	Figure 1. Deficiency of EML1 causes ectopic neural rosettes and neuronal heterotopia in cerebral organoids Day 33 ± 2 control‐ and EML1 patient‐derived cerebral organoids stained for ß‐III‐tubulin and DAPI. VZ and CP areas are marked by white brackets, ectopic neural rosettes by red bracket, and heterotopic neurons by red arrows.Day 33 ± 2 control‐ and EML1 patient‐derived cerebral organoid stained for the adherens junction marker N‐cadherin (NCAD) expressed at the VZ surface and within the ectopic neural rosettes.Day 20 ± 2 cerebral organoids derived from controls, patients, and EML1‐heKOs stained for the neuronal marker MAPT and the adherens junction marker NCAD, counterstained with DAPI. Ectopic neural rosettes and neuronal heterotopia are highlighted with dotted yellow lines.Quantification of VZ areas with ectopic neural rosettes (three batches, three organoids analyzed per batch, significance based on Kruskal–Wallis test, P = 0.0001, Dunn’s post hoc test for multiple comparisons performed to define statistical differences between genotypes, and single data points presented are colored by batch).Quantification of heterotopic, disorganized, or organized cortical areas (three batches, three organoids per batch, significance based on Kruskal–Wallis test, P = 0.0001 for “organized” and “heterotopic”; no significant difference for “disorganized,” Dunn’s post hoc test performed for multiple comparisons to define statistical differences between genotypes). Data information: * marks statistical significance in relation to Control 1, # in relation to Control 2. P‐values: ***/###< 0.001. VZ, ventricular zone; CP, cortical plate. Data in graphs are represented as means ± SD. Scale bars: (A) 100 µm; (B) 100 µm, enlarged 25 µm; and (C) 50 µm. Source data are available online for this figure.
	Figure 2. Ectopic progenitor cells originate from apical RG with perturbed cell intrinsic behavior Hybrid organoids generated from controls mixed with control‐EGFP+ or EML1‐heKO‐EGFP+ cells (day 20 ± 2). Yellow dotted lines indicate VZ areas.Quantification of control‐EGFP+ or EML1‐heKO‐EGFP+ cells within or at the basal side of VZ areas (three batches, three organoids each, significance based on Mann–Whitney test, ***P = 0.0001 for “EGFP+ cells at the basal side of VZ areas”).Representative images and scheme of cleavage plan orientation. Upper panel: cells immunostained for p‐Vim (red) and TPX2 (green), counterstained with DAPI, white dotted line indicates cleavage plane orientation, and yellow dotted line VZ surface.Quantification of horizontal, vertical, and oblique plane of cell divisions in control and EML1‐deficient organoids (day 20 ± 2; three batches and three organoids each).Quantification of primary cilia length in control and EML1‐deficient cortical progenitors (* marks statistical significance in relation to Control 1, # in relation to Control 2, three biological replicates, four areas per sample, significance based on Kruskal–Wallis test and Dunn’s post hoc test, ***/### P = 0.0001).Electron microscopy of primary cilia derived from control or EML1‐deficient cortical progenitors in DMSO control or following EpothiloneD (EpoD) exposure (blue arrow: basal body, yellow arrow: appendages, and red arrow: ciliary pockets).Quantification of primary cilia length in control and EML1‐deficient cortical progenitors with/without EpoD exposure. Three biological replicates, four areas per sample, significance based on two‐way ANOVA, and Sidak’s multiple comparisons post hoc test, P = 0.0213. Data information: Data in graphs are represented as means ± SD. Scheme in C was designed using biorender.com. Scale bars: (A) 50 µm; (C) 5 µm; and (F) 0.5 µm. Source data are available online for this figure.
	Figure EV2. RG cell division mode and EpoD‐mediated tubulin stabilization Immunocytochemical analyses of day 20 ± 2 control‐ and EML1‐deficient cerebral organoids stained for p‐Vim (mitotic cells) and TPX2 (labeling the mitotic spindle). Dotted lines indicate the VZ surface and RG‐cell division mode, squares indicate areas enlarged below.PAX6‐positive cortical progenitors derived from control, EML1‐heKO, and EML1‐heKO treated with EpoD exhibit ARL13B‐positive primary cilia.Immunoblot for acetylated tubulin in control and EML1‐deficient cortical progenitors under control‐ (−) or EpoD‐treated (+) condition.Quantification of relative protein signal intensities of the immunoblot shown in C based on n = 3 biological replicates of independent experiments. Signals were normalized to actin. Data information: Bar graphs show mean with SEM, significance based on two‐way ANOVA with Bonferroni’s multiple‐comparison test, **P = 0.0047. For individual P‐values, see the source data for this figure. Scale bars: (A) 50 μm, enlarged 10 µm; (B) 5 μm.
	Figure EV3. Overview of scRNAseq experiment, EML1 expression, and further characterization of RG to bRG cells Schematic of scRNA‐seq experiment procedure. VZ areas from nine control and nine EML1‐heKO‐derived organoids from three independent batches were dissected and pooled. Image was designed using biorender.com.Dotplot of known marker gene expression in control‐derived cells. Scaled average normalized expression for the respective gene in each cell type is indicated by a continuous color scale. Number of cells in the respective cluster expressing the genes is represented by the size of the dots.Normalized expression of EML1 in day 33 ± 2 control‐derived cerebral organoid cells per cell type investigated by scRNAseq (three pooled batches and three organoids each).Heatmap of z‐scaled gene expression of extracellular matrix (ECM) and basal radial glial (bRG) genes in the RG to bRG cell cluster derived from either control or EML1‐heKO organoids.Overview of a day 33 ± 2 cortical structure derived from control or EML1‐heKO stained for the pan‐neuronal marker MAPT, counterstained with DAPI. The VZ is encircled; the heterotopic region is highlighted by a dotted line and labeled with #.COL1A2, MEIS2, and MAPT magnification for control and EML1‐heKO, counterstained with DAPI. The VZ is encircled; The heterotopic region is highlighted by a dotted line and labeled with #. Data information: Scale bars: (E and F) 50 μm. Source data are available online for this figure.
	Figure 3. ScRNA‐seq reveals cellular identity of ectopic cells in EML1‐heKO cerebral organoids scRNA‐seq of 2,335 control and 3,358 EML1‐heKO cells (three pooled independent batches and three organoids each). Cells shown in UMAP plot and colored by annotated cell type.Comparison of cell type composition between control and EML1‐heKO. Numbers indicate percentage of total cells belonging to the respective cell type.Normalized expression values of COL1A2, COL3A1, LUM, and MEIS2 per cell type (three pooled batches and three organoids each).Gene ontology (GO) term analysis of RG to bRG cells in control and EML1‐heKO shows percentage of counts belonging to the set of genes associated with the respective GO term (three pooled batches and three organoids each).Control‐ and EML1‐heKO‐derived cerebral organoid stained for COL1A2 and MEIS2. VZ areas are encircled, and squares indicate areas enlarged in F.Control‐ and EML1‐heKO‐derived cerebral organoid stained for COL1A2, MEIS2, and MAPT.Representative images showing EGFP control and EGFP‐EML1‐heKO hybrid cerebral organoid stained for EGFP, FAM107A, and NCAD. Yellow dotted lines indicate morphology of EGFP+ cells (4 independent batches each, 3 organoids per batch, at least 30 ectopic rosettes in total). Data information: RG1: radial glia1, RG to bRG: radial glia to basal radial glia, RG2: radial glia to young neurons, IP, intermediate progenitors; YN, young neurons; MLC, mesenchymal‐like cells. (C and D) Asterisks indicate Bonferroni corrected P‐values. Wilcoxon rank sum test: ***< 0.001, *< 0.05, ns, not significant. Scale bars: (E) 50 µm; (F) 10 µm; and (G) 50 µm, enlarged 10 µm. Source data are available online for this figure.
	Figure 4. YAP signaling drives ectopic progenitor cell expansion in EML1‐deficient organoids A. B. C. D, E. F. G. H. I. J.
	Figure EV4. Ectopic progenitor cell proliferation in EML1‐deficient organoids and Fluvastatin‐mediated YAP inhibition Immunocytochemical analyses of control and EML1‐deficient organoids (day 20 ± 2) for phosphorylated vimentin (p‐VIM) counterstained with DAPI. Note: Dividing p‐Vim‐positive cells located at the apical surface of VZ areas in control organoids. EML1 patients‐ and Eml1‐heKO‐derived organoids reveal ectopically localized p‐VIM‐positive cells outside VZ areas. Apical surface of VZ areas highlighted by dotted line and p‐VIM‐positive cells outside the VZ areas indicated with arrowheads.Quantification of mitotic cells located at the apical side of VZ areas in control and EML1‐deficient conditions (three batches, three organoids each, significance based on Kruskal–Wallis test and Dunn’s post hoc test, P = 0.0001; for detailed statistical information, see the source data for this figure).Immunocytochemical analyses of control and EML1‐deficient organoids (day 20 ± 2) for BrdU and Ki67 counterstained with DAPI.Normalized expression of YAP1, CCND3, CDK6, and CYR61 in all cell types. Split violins show expression in control and EML1‐heKO (three pooled batches and three organoids each). Asterisks indicate Bonferroni corrected P‐values (Wilcoxon rank sum test: ***< 0.001, **< 0.01, *< 0.05, ns, not significant; for detailed statistical information, see the source data for this figure).Immunocytochemical analyses of p‐VIM and YAP1 in EML1‐heKO‐derived organoids in DMSO‐ or Fluvastatin‐treated condition. Squares indicate areas enlarged in adjacent parts of the panel.Immunocytochemical analyses of NCAD in EML1‐heKO‐derived organoids in DMSO‐ or Fluvastatin‐treated condition counterstained with DAPI. Data information: Scale bars: (A, C, E, and F) 50 μm, (E) enlarged 10 μm. Source data are available online for this figure.

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