Subclustering of HSPC1s demonstrates the loss of gata2b-expressing quiescent subcluster and appearance of proliferative subcluster. (A) Cluster selection for subclustering. (B) Reclustering of the HSPC1 population split between WT and gata2b^−/− cells. (C) Genotype distribution of each of the clusters, with WT cells in white and gata2b^−/− cells in black. (D) Quantitation of proportions of distribution between WT and gata2b^−/− cells in the different clusters. Significant differences are indicated in red. (E-F) WT and gata2b^−/− feature analysis with gradual gene expression in shades of blue within HSPC1s of gata2b (E) and mki67 (F). (G) Volcano plot comparing HSPC1 gata2b^−/− vs WT. At the left of the y-axis is gene expression in gata2b^−/− HSPC1s with an average logarithmic fold change less than −0.25, and to the right is gene expression with a logarithmic fold change >0.25 compared with WT HSPC1s. Each dot represents a gene. (H) Lineage differentiation trajectory depicted on uniform manifold approximation projection, with WT cells in blue and gata2b^−/− cells in pink. (I) Pseudotime analysis assuming the quiescent population as starting point. (J-O) Gene expression analysis on pseudotime analysis with meis1b (J), gata2b (K), GFP (L), gata2a (M), pcna (N), and ki67 (O). (P) WT and gata2b^−/− feature analysis with gradual gene expression of GFP in shades of blue within HSPC1 cells. Dotted circles indicate the quiescent and HSC subcluster. (Q) Cell-cycle analysis by flow cytometry of Ki67 and DAPI staining of CD41:GFP^low cells in adult WT and gata2b^−/− KM cells. (R) Bar graph representing the quantitation of cell cycle of CD41:GFP^low cells in adult WT and gata2b^−/− KM cells. Bars represent mean ± standard error of the mean; each dot indicates analysis from 1 zebrafish. *P < .05, **P < .01. FDR, false discovery rate; n.s., not significant.