Single-cell analysis of mouse BFU-E cells identifies TβRIII as a marker that differentiates early and late BFU-E progenitor cells. (A) Unbiased hierarchical clustering of the 96 genes identified by PCA as best separating the transcriptome of the 48 mouse BFU-E cells. Red asterisk, Gata1; green asterisk, Tgfbr3. (B) Gene expression of Gata1 and Tgfbr3 in each single cell is plotted in the PCA analysis. PC, principal component. (C) Colony-forming assays on unfractionated, TβRIII^10%hi and TβRIII^10%lo BFU-E populations. Large BFU-E colonies have >12 clusters, and small BFU-Es have 5 to 12 clusters as described (*P < .05.). (D) Total BFU-E (c-Kit⁺CD71^{10% lo}) cells were separated by flow cytometry, and the TβRIII^10%hi and TβRIII^10%lo populations were subsequently isolated by FACS. Purified TβRIII^10%hi or TβRIII^10%lo cells were seeded in SFELE medium in the presence or absence of 100 nM DEX. The production of erythroblasts from total BFU-E, purified TβRIII^10%hi, and TβRIII^10%lo BFU-E cells was quantified over time. Error bars represent mean ± standard deviation from 3 biological replicates (**P < .01; ***P < .001). (E) Colony-forming assays were conducted to determine BFU-E colony numbers from 100 TβRIII^10%hi or TβRIII^10%lo BFU-E cells cultured under the indicated conditions. Colony-forming assays were performed at 24-hour intervals (**P < .01).