Fig 2: 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.

Fig 3: Productive infection of primary neuronal cultures with HEVA71 causes neuronal cell death.a Immunostaining of UI-, S41- or C2-infected DIV7 neuronal cultures with antibodies against dsRNA and Tau. Scale bars represent 20 µm. Arrowheads indicated dsRNA-positive neurons. b Quantification of dsRNA-positive cells in infected DIV7 neuronal cultures at 6 hpi. Two hundred cells taken from random fields were manually counted for each condition. Percentages of dsRNA-positive cells were highest in S41- or C2-infected neurons but almost negligible in UI-infected controls. c Immunoblotting of cell lysates from S41-infected neuronal cultures for VP0 and VP2. ß-actin was used as a loading control. VP0 and VP2 bands appeared at 6 hpi and continued to increase over the next 42 h. d Quantification of progeny viral particles release into supernatants of S41- or C2-infected neuronal cultures, where maximal viral titers were detected at 24 hpi in DIV7 neuronal cultures. Peak viral titers were similar in both infected neuronal cultures and RD. e Apoptotic percentages of caspase 3- or TUNEL-positive cells were highest in S41- or C2-infected neuronal cultures that were similar to the positive controls (staurosporine or TAC nuclease). Two hundred cells taken from random fields were manually counted for each condition. Scale bars represent 20 µm. MOI of 1 was used for all experiments. Data are expressed as the average with error bars representing ±SD from 3 independent experiments (n = 3). Statistical analyses were performed with two-way ANOVA corrected using Bonferroni post-test. (****P < 0.0001). Source data are provided as a Source Data file.

Fig 4: 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.

Fig 5: 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.