Fig. 3: Single cell multiomic analyses of mutant eNSCs in vitro uncovers deregulated metabolism and neuronal fate commitment.

A Inferred ordering of eNSC genotypes by the number of genes expressed in single cells, RNA content, and association with transcriptional covariance across top genes during predicted trajectory (CytoTRACE²¹). Box plots indicate the median (center line), interquartile range (hinges), and 1.5x interquartile range (whiskers). B Gene ontology analysis of genes correlated with terminal states predicted by RNA content (n = 405 genes). Circle size represents number of genes within each ontology, and color indicates log-transformed FDR. C Seahorse XF analysis in WT and PRO eNSCs of glycolysis-dependent extracellular acidification rate (left) and spare respiratory capacity (right), a measure of metabolic fitness and adaptation to energetic demands. Data represent mean ± SEM in independent replicates (n = 3, ANOVA, **P < 0.01). D UMAP reduction of eNSC transcriptomic data by RNA velocity in single cells colored by genotype (left) and overlaid with the RNA velocity vector field (right). E Cluster analysis of velocity-based UMAP (left) and expression topography analysis of stable gene networks exhibiting elevated selective potential by dynamical modeling (right). Expression nodes are numbered and represent fixed points within the landscape. They are colored by cell-wise statistical confidence of identification. Half circles are saddle points within the transcriptomic space while full circles are stable points. Font color indicates absorbing fixed points (black), emitting fixed points (red), and unstable fixed points (blue). Larger nodes represent dominant fixed stable points during fate trajectory simulation using WT eNSCs as the initiating population (Dynamo²²). F Venn diagram of putative tumorigenic clusters falling within the stable absorbing node identified by RNA velocity expression topography analysis. Each circle represents the differentially expressed genes (DEGs) in clusters 3, 12, and 21, and their overlap of shared or unique genes. Shown are representative GBM- or development-related genes identified. G Gene ontology analysis of shared DEGs between clusters 3, 12, and 21 (n = 201 genes). Circle size represents number of genes within each ontology, and color indicates log-transformed FDR. H Joint analysis of gene expression and chromatin accessibility in single eNSCs for transcription factor motif enrichment and activation in PRO eNSCs compared to WT NSCs. Source data are provided as a Source Data file, including exact statistical analyses and p values.