|
Fig. 6 scRNAseq analysis of WT and Zbtb11hKO HSCs reveals underrepresentation of Zbtb11hKO HSCs in the cluster characterized by an erythroid signature and dysregulation of niche gene expression. (A) Distribution of cells based on the number, proportion (%), and genotype for each cluster. Clusters 1 and 3 are disproportionately comprised of WT cells, whereas clusters 0 and 4 are disproportionately comprised of KO cells. (B) Uniform Manifold Approximation and Projection plot of unsupervised clustering identified 5 subpopulations of E14.5 FL HSCs (LSK/SLAM). (C) Heatmap of normalized log-expression values for the top 10 cluster-specific marker genes. Column colors represent the cluster identity to which each cell is assigned. (D) Distribution of cell cycle phase within each cluster. (E) Enrichment of log normalized gene expression of top 10 upregulated genes in each cluster was evaluated across individual hematopoietic lineages defined using the Haemopedia Mouse RNAseq data set, which contains 129 samples classified into 13 cell lineages and 57 cell types.46 Expression of marker genes for cluster 1 shows enrichment for erythrocyte lineage (boxed) genes. (F) Differential expression of hematopoietic niche genes in unclustered E14.5 FL Zbtb11hKO HSCs compared with WT HSCs. Y-axis shows the log2FC (fold change) gene expression level, with a false discovery rate < 0.05. Mean fluorescence intensity (MFI) of c-Kit (G) and Flt3 (H) protein in Zbtb11hKO and WT control progenitors at E14.5. WT/controls (green); KO, (red); fluorescence intensity of the protein is shown along the y-axis; data ± standard error of the mean. Two-way analysis of variance with Šídák multiple comparisons: ∗∗∗∗P < .0001.