Fig. 2

Oligodendroglia cluster segregation and validation. a Feature plots of subsetted oligodendroglia that qualitatively show the expression of lineage marker and cluster marker genes (scale bars show LogNormalized counts). PLP1 marks oligodendrocytes (Oligo) and PDGFRA oligodendrocyte precursor cells (OPCs). b Dot plot of a selection of marker genes for committed oligodendrocyte precursor cells (COP_A-C), oligodendrocyte precursors (OPC_A & B) and oligodendrocytes (Oligo_A-F) showing cluster segregation. The dendrogram visualizes the relationship of clusters based on 2000 most variable genes. c Validation of oligodendrocyte cluster markers by immuno-fluorescence stainings at the protein level. OLIG2-positive oligodendrocytes show either one cluster marker (fat arrow) or a second cluster marker (arrowhead), only with the occasional double-positive cell (thin arrow). SPARC-positive Oligo_F cells also express HCN2. d BaseScope duplex stainings for OPC cluster validation. Dotted line delineates the cell boundary with arrow pointing at cluster marker signal. PDGFRA marks all OPCs with PAX3 labelling OPC_A and NELL1, OPC_B. e, f Proportion of oligodendroglia expressing cluster markers by pairs in our snRNAseq dataset e colour-coded by single markers or overlap and f in the validation datasets by immunofluorescence (oligodendrocytes (OL), OLIG2 +) or BaseScope Duplex (OPCs – PDGFRA +), showing good concordance. For example, OL_F (cluster Oligo_F) is SPARC- positive but RBFOX1-negative (second bar in f) and as positive for HCN2 and SPARC (5th bar in (f)). IF immuno-fluorescence, ISH In situ hybridization. g Violin plot showing that most HCN2 expression is in the Oligo_F cluster