Fig. 3: Direct and conventional thrombopoiesis pathways proceed via distinct progenitors.

a RFP labeling of cell populations from Fgd5^{ZsGreen:CreERT2}/R26^LSL-tdRFP mice (same as in Fig. 1c–e) shown as fraction of label in the MPPs of the same animal at final analysis. b Model scheme with direct route to MkPs (LS⁻K CD41⁺CD150⁺) from CD201^−/lo Sca-1^lo HSCs. c Fgd5^{ZsGreen:CreERT2}/R26^LSL-tdRFP mice were TAM-induced and indicated cell populations were analyzed at 10, 59, and 120 days (n = 5 animals/ time point, means and SEM are shown). d PHATE embedding of single-cell transcriptomes of index-sorted hematopoieitic stem and progenitor cells (n = 820 cells total) isolated from 6 R26^rtTA/Col1A1^H2B-GFP animals, colored by cell type and expression of lineage markers (blue: low, yellow: high). e HSCs, MPPs, and HPCs-1 show a decrease of Sca-1 surface expression in proximity of MkPs and myeloid-committed progenitors. f PHATE plots showing Vwf and Itga2b expression. g Lower surface expression of CD48 in MkPs residing closer to HSCs in the transcriptional landscape. h Proximity in transcriptional space of MkP subsets to HSCs (left), and of HSC subsets to CD48^−/lo MkPs (right), placing CD48^−/lo MkPs close to Sca-1^lo HSCs. (two-sided Wilcoxon test; left plot: n = 135 cells, right plot: n = 279 cells; p-value ≤ 10⁻⁴ for both comparisons; boxplot description: central line indicates median, upper (lower) hinges indicate third (first) quartiles, top (bottom) whisker extends to largest value within a 1.5× inter-quantile range from top (bottom) hinge). i PAGA embedding of surface marker-defined populations, displaying transcriptional proximity of CD201^−/lo Sca-1^lo HSC to CD48^−/lo MkP (green link), and a distinct differentiation path to CD48^hi MkP (blue) via myeloid (MyP, LS⁻K CD41⁻ CD150⁻) and erythroid progenitors (EryP, LS⁻K CD41⁻CD150⁺). Red PAGA links were previously inferred by statistical model selection (see Fig. 2c). Cell populations were identified via indexed surface markers, except for lymphoid-biased HPCs-1, which were identified by their transcriptional signature. j Confidence score for PAGA links. k Comparison between Mk score and pseudotime shows parallel Mk maturation trajectories in CD48^−/lo and CD48^hi MkP subsets. Gray areas represent 95% confidence bounds for linear regression, R indicates the Pearson correlation coefficient, p-value is relative to a two-sided alternative hypothesis.