Expansion of megakaryocyte–myeloid-biased stem cells in RXRα;RXRβ-deficient mice. (A-E) Single-cell RNA sequencing (10× Genomics) of sorted LSKs from WT and Vav^Cre+Rxrab^fl/fl BM cells (shown are clusters derived from gene set enrichment of a compendium of prior HSC/P subsets from single-cell functional studies).²⁷ (A-B) UMAP plots of HSC/P subsets derived from Vav^Cre+Rxrab^fl/fl and WT BM LSKs (A), and of subclustering of the broader HSC-MPP cluster (B). (C) Top HSC-MPP cluster marker genes. (D) HSC-MPP population frequencies, showing significant differences between Vav^Cre+Rxrab^fl/fl and WT mice (Fischer exact P-value thresholds are indicated). (E) Heat map showing differentially expressed genes identified by cellHarmony analysis of each HSC-MPP subcluster in Vav^Cre+Rxrab^fl/fl vs WT mice. On the left of the associated clusters are shown enriched prior-defined HSC/P functional subsets (blue), pathways (purple), or curated transcription factor targets (green) (GO-Elite software); on the right of the associated clusters are shown matching bulk RNA-seq regulated genes. (F-K) Flow cytometry of BM from 5-month-old WT and Vav^Cre+Rxrab^fl/fl mice. (F-G) Absolute numbers of HSC subpopulations per femur, according to the “34F” or “SLAM” stain codes. (H) Absolute numbers of common lymphoid progenitors (CLPs) per femur. (I) Frequency of CD41⁺ and CD41⁻ cells in the CD150⁺CD48⁻ LT-HSC subset. (J) Absolute numbers of MK progenitor per femur. (K) Annotated t-SNE plots for the identified lineage⁻ BM-cell populations from WT and Vav^Cre+Rxrab^fl/fl mice using the “SLAM” stain code as in G. Insets in the left panels show magnifications of the plotted HSC/P subpopulations; plots in the right panels show overlaid biexponential transformed marker expression levels (n = 3-4 per genotype). (L-M) Colony-forming unit (CFU) assay in total BM cells from 5-month-old WT and Vav^Cre+Rxrab^fl/fl mice. (L) Representative images of hematopoietic colonies identified in Vav^Cre+Rxrab^fl/fl mice after 7 days of incubation: burst-forming unit-erythroid (BFU), unipotent CFU-M (monocyte), CFU-MK (megakaryocyte), CFU-G (granulocyte), and CFU–pre-B (B lymphocyte) progenitors; bipotent CFU-GM (granulocyte, monocyte) progenitors; and multipotent CFU-GEMM (granulocyte, erythrocyte, monocyte, and megakaryocyte) progenitors. (M) Number of colonies per 20 × 10³ plated BM cells (n = 6; 3 mice per genotype, with 2 technical replicates per mouse; data are representative of 2 independent experiments). (N) Lymphocyte output after 8 days of OP9NL1 cell coculture with BM cells from 5-month-old WT and Vav^Cre+Rxrab^fl/fl mice; graph shows the percentage of T cells (DN1 + DN2 + DN3 + DN4 + CD3⁺ cells; for gating strategy, see supplemental Figure 6F) and Cd11b⁺ cells within CD45⁺ lymphocytes. Data are shown as means ± SEM, and dots represent individual animals. Significance was determined by unpaired Student t test, and is represented as follows: ∗P ≤ .05, ∗∗P ≤ .01, ∗∗∗P ≤ .001, and ∗∗∗∗P ≤ .0001. n.s., not significant.