PML-RARA expression drives the formation of a unique population of preleukemic myeloid progenitors that express Gata2. (A) t-Distributed stochastic neighbor embedding (t-SNE) plots of scRNA-seq data from whole bone marrow cells from young nonleukemic WT mice (left panel) or Ctsg-PML-RARA mice (right panel) mice. Known hematopoietic cell types are labeled based on Haemopedia gene expression profiling40,58). Major cell populations (early stem and progenitors, GMPs, neutrophils, monocytes, macrophages, T cell, DCs, mature B, and progenitor B) are inferred, outlined, and labeled based on Haemopedia lineage assignment software and based on graph-based clustering analysis. A unique population of myeloid precursor cells that are only present in the bone marrow from Ctsg-PML-RARA mice is outlined in blue (“PML-RARA specific”). (B) t-SNE plots of the relative expression of Ctsg in whole bone marrow cells from WT mice (left panel) or Ctsg-PML-RARA mice (right panel) by scRNA-seq. (C) t-SNE plots of the relative expression of Gata2 in whole bone marrow cells from WT mice (left panel) or Ctsg-PML-RARA mice (right panel) by scRNA-seq. (D) Heat map of the 1090 DEGs between Ctsg+ myeloid precursors from Ctsg-PML-RARA mice compared with those from WT littermates by scRNA-seq. Replicate littermate pairs are labeled as Rep 1 and Rep 2 (fold change ≥2 and FDR <0.05 by ANOVA). (E) Volcano plot of expression changes between Ctsg+ myeloid progenitors from Ctsg-PML-RARA vs WT mouse bone marrow. Expression changes in Bcl2, Zbtb16 (Plzf1), Hgf, Pdgfrb, Hdc, Elane, Prtn3, and Cd177 are labeled. (F) Relative Gata2 expression by scRNA-seq in the various lineage populations from panel A in WT or Ctsg-PML-RARA (PR) bone marrow. Data in panels A-F are from 2 pairs of mice that were littermate matched and 8 to 12 weeks of age. Samples of the same genotype were concatenated for the analyses in panels A-C and E-F. **P = 1.37E-4, ***P = 1.73E-142, ****P < 1.00E-298; ANOVA. CFU-E, colony forming unit-erythroid; CLP, common lymphoid progenitor; DC, dendritic cell; GMPs, granulocyte-monocyte progenitors; MDP, monocyte dendritic cell progenitor; MEP, megakaryocyte erythroid progenitor; MPP, multipotent progenitor; NP, neutrophil progenitor; RBC, red blood cell; ST-HSC, short-term hematopoietic stem cell.