Myc haploinsufficiency rescues regeneration, transcriptome, and spliceosome of phenotypically identifiable LT-HSCs. (A-B) Competitive transplantation assay of unfractionated BM cells from 4-month-old WT, Vav^Cre+Rxrab^fl/fl, and Vav^Cre+Rxrab^fl/fl-Myc^+/fl mice (CD45.2⁺) in a 1:1 ratio with C57BL/6 BM cells (CD45.1⁺) into lethally irradiated C57BL/6 mice (CD45.1⁺) (data pooled from 2 independent experiments). (A) Experimental scheme. (B) Percentages of CD45.2⁺ BM LT-HSCs at 16 weeks after transplantation. (C) Frequencies of CD150⁺CD48⁻ LT-HSCs from WT, Vav^Cre+Rxrab^fl/fl, and Vav^Cre+Rxrab^fl/fl-Myc^+/fl mice in the G₀ phase of the cell cycle (pyronin Y⁻/Hoechst 33342⁻; data pooled from 3 independent experiments). (D) Percentage of 5-bromo-2′-deoxyuridine–positive (BrdU⁺) CD150⁺CD48⁻ LT-HSCs from WT, Vav^Cre+Rxrab^fl/fl, and Vav^Cre+Rxrab^fl/fl-Myc^+/fl mice after 1-hour pulse with BrdU. (E-F) Immunofluorescence analysis, showing different division modes according to MYC expression in paired daughter LT-HSCs from WT, Vav^Cre+Rxrab^fl/fl, and Vav^Cre+Rxrab^fl/fl-Myc^+/fl mice. (E) Representative images showing (1) symmetric self-renewal (absence of MYC expression in paired daughter cells), (2) asymmetric division (high vs low MYC expression in paired daughter cells), and (3) symmetric differentiation/commitment (MYC expression in both cells); scale bar, 10 μm. (F) Percentage of LT-HSCs corresponding to division modes 1, 2, and 3 as in E; n = 60 division pairs captured from 5 WT mice; n = 48 division pairs captured from 3 Vav^Cre+Rxrab^fl/fl mice; and n = 37 division pairs captured from 5 Vav^Cre+Rxrab^fl/fl-Myc^+/fl mice. Analyses were performed in 2 independent experiments. (G) Serial CFU plating assay in total BM cells from WT, Vav^Cre+Rxrab^fl/fl, or Vav^Cre+Rxrab^fl/fl-Myc^+/fl mice. (H-L) Bulk transcriptome of WT, Vav^Cre+Rxrab^fl/fl, and Vav^Cre+Rxrab^fl/fl-Myc^+/fl LT-HSCs. Normalized expression values from bulk RNA-seq data are provided in supplemental Tables 12-15. (H) Rescued differentially expressed genes in Vav^Cre+Rxrab^fl/fl-Myc^+/fl vs Vav^Cre+Rxrab^fl/fl LT-HSCs (eBayes t-test P < .05; fold > 1.2), compared with Vav^Cre+Rxrab^fl/fl-Myc^+/fl vs WT LT-HSCs (eBayes t-test P < .05; false discovery rate corrected; fold > 1.5). (I) Heat map of the 435 rescued genes showing silencing of Vav^Cre+Rxrab^fl/fl LT-HSC–induced transcripts by Myc^+/fl expression. On the left are shown example rescued genes (Figure 5D; TFs or RNA-binding regulators). (J) Rescued alternative splicing events in Vav^Cre+Rxrab^fl/fl-Myc^+/fl vs Vav^Cre+Rxrab^fl/fl LT-HSCs, compared with Vav^Cre+Rxrab^fl/fl-Myc^+/fl vs WT LT-HSCs (eBayes t-test P < .05; δ percentage spliced in [PSI] > 0.1 for both comparisons). (K) Heat map of 154 unique splicing events, organized by increased junction PSI value in Vav^Cre+Rxrab^fl/fl vs WT LT-HSCs, with exemplar intron retention events (green tick mark) and genes symbols called out. (L) Number of observed unique annotated splicing events in the 154 rescued splicing events, by exon/intron inclusion or exclusion. Splicing events associated with exon or intron inclusion (increased relative expression) vs exclusion are shown separately. Results are presented as means ± SEM, and dots represent individual animals. Significance was determined by ordinary 1-way analysis of variance, and is represented as follows: ∗P ≤ .05, ∗∗P ≤ .01, and ∗∗∗P ≤ .001. DAPI, 4′,6-diamidino-2-phenylindole; n.s., not significant.