Fig. 1: Single-cell RNA-Sequencing identifies RNA dysregulation specific to motor neurons with FUS mutations in a dose-dependent manner.
a Schematic showing generation of the different iPSC-lines using CRISPR-Cas9 gene editing, subsequent differentiation into motor neurons and interneurons, sorting and single-cell RNA sequencing. b–e UMAP projections of all cells of the included cell lines that passed quality control. The two UMAPs on the left are colored by (b) assigned cell type or (c) cell line, the two on the right contain neuronal cells only, again colored by assigned (d) cell type or (e) cell line. f Gene expression heatmaps of all neuronal cells, showing expression data of several (motor-) neuronal marker genes, progenitor markers and housekeeping genes. g Gene expression heatmaps of specific motor- and interneuron subtype marker genes. h Violin plots of the FUS mRNA expression in the control and ALS mutant lines across cell types. The normalized expression is given as log10(RPKM + 1). Differential expression was performed within cell type, across lines. Error bars show mean ± standard error of the mean (SEM). * p < 0.05, ** p < 0.01, *** p < 0.001, derived from two-sided DEA testing using DESeq2 and corrected for an FDR of 0.05. The p-values are 6.48E-5 for control vs. FUS P525L in motor neurons, and 1.86E-4 for FUS P525L homozygous motor neuron vs. FUS P525L homozygous V2a. i Upset plots of the intersections between DEGs in motor neurons, V2a interneurons and other interneurons between the FUS mutant lines versus the control cell line. Each column represents a section of a Venn diagram of the cell types as indicated in the table below. The corresponding Venn diagram is shown in the insets. The red (top) and blue (bottom) columns represent upregulated and downregulated genes, respectively. Genes with an adjusted p-value of <0.05 were considered differentially expressed.
