Bclaf1 loss results in reduced HSC^LT and increased stress response gene expression signature. (A) Volcano plot shows differentially expressed genes in Vav-Cre:Bclaf1^f/f (n = 3) vs Bclaf1^f/f (n = 4) E17.5 fetal HSCs determined by RNA-seq of bulk-sorted populations. Red and blue dots indicate significantly (P < .05; log2FC ≥1) increased and decreased genes, respectively. (B-G) Single-cell CITE-seq on E17.5 Lin^-Sca1⁺c-Kit⁺ HSPCs from Bclaf1^f/f or Vav-Cre:Bclaf1^f/f fetal livers (cells from 10 embryos pooled for each genotype). (B) UMAP shows Iterative Clustering and Guide-gene Selection version 2 (ICGS2) results of progenitor populations (Bclaf1^f/f, 5551 cells; Vav-Cre:Bclaf1^f/f, 3246 cells). (C) Overlay of surface-marked HSC, MPP2, and MPP3/4 populations defined from barcoded CD150 and CD48 antibodies. (D) Bar graph shows numbers of cells in each ICGS2 cluster by genotype. Genotypes were normalized to cell number in Vav-Cre:Bclaf1^f/f group. Statistical significance was determined by χ² analysis. (E) Volcano plots show differentially expressed genes in Vav-Cre:Bclaf1^f/f vs Bclaf1^f/f cells of indicated hematopoietic populations/clusters determined by pseudobulk gene expression analysis. Red and blue dots indicate significantly (P < .05; log2FC ≥1) increased and decreased genes, respectively. (F) Stress score applied to each cell and overlayed on single-cell RNA-seq UMAP. Higher score (green) indicates increased expression of stress response genes. (G) Quantitation of stress score in Bclaf1^f/f and Vav-Cre:Bclaf1^f/f cells in each cluster. ∗∗P ≤ .01; ∗∗∗∗P ≤ .0001. FC, fold change; FDR, false discovery rate; Mk^P, megakaryocyte progenitors; Mono, monocyte precursor; UMAP, uniform manifold approximation and projection.