GMP subpopulations with different lineage potential show distinct NfκB dynamics. (A) Response dynamics frequencies of all GMPs. (B) Exemplary traces. (C) Experimental setup for (D-H). (D) Two-dimensional UMAP representation of the transcriptional space. OSC and TRA cells differ transcriptionally (n = 231 cells, N = 3 biological replicates, 2 independent sequencing runs). (E) Hierarchical clustering on UMAP coordinates is used to quantify distributions within the UMAP in (F-G). (F) Response type fractions in the different clusters. Colors as in (A). (G) Distribution of selected dynamics feature scores across clusters. Confirmation of manual scoring in (D,F). ACF, auto correlation function. (H) Identification of OSC vs TRA surface markers by scRNAseq. (I-N) NfκB dynamics in GMP subpopulations after TNFα stimulation (n = 1298 cells, N = 3 biological replicates). (I) Single-cell heat stripes (vertical) of all measured cells and conditions. Brightness represents nuclear p65 signal intensity. The first 6 hours are shown (12 hours in supplemental Figure 3H). (J) Dynamics frequencies (blind-manual classification). Colors as in (A). (K) The correlation of signaling dynamics and cell cycle stages does not explain HSPC characteristic dynamics. Cell cycle phases are inferred from transcriptomes ∼7 hours after stimulation (see Figure 4). Left: color coding as in (A). (L-N) UMAP graph based on 17 extracted signaling trace features. (L) Right: colors as in (A). (N) Green dots show GMP subpopulation UMAP location. Only TNFα-stimulated cells are shown. (O) Scores for selected features for the 3 subpopulations. See also supplemental Figure 3.