Figure 2.
Reg1 and Reg3 control lineage biases within HSPCs. (A) Schematic representation of experimental strategy for single-cell sorting. (B) Gating strategy for WT and Reg1–/–Reg3–/– (DKO) LSK cells sorted from the identical mouse receiving WT (CD45.1) and DKO (CD45.2) FL cells. (C) A 2-dimensional projection of 2241 single cells sorted from WT (CD45.1) LSK and 2242 single cells from DKO (CD45.2) LSK onto the reference model. Cells are colored according to assignment to reference model meta cells (see “Methods”). (D) Difference in meta-cell abundance between single cells sorted from DKO and WT mice. Each circle represents a reference meta cell. Circle color represents log2 (fold change) in meta-cell abundance between the 2 genetic backgrounds. (E) Cell type distribution of single cells sorted from WT and DKO mice. ∗P < .05; ∗∗P < .001; ∗∗∗P < .00001. (F) Differential gene expression between pooled cells from DKO cells compared with WT cells. Gold dots indicate differentially expressed genes (FDR corrected P < .001 under χ2 test) with fold change >2. Diagonal lines show log2 (fold change) = −1 or 1. Values represent log2 size–normalized expression. (G) Total expression of specific genes in different genetic backgrounds. Values represent size-normalized total transcripts per 100 cells. Colors represent relative contribution from the different hematopoietic core subsets. (H) Luciferase activity was assessed by using pGL3 plasmids containing the 3’ UTR of Nfkbiz and Ehd3, together with mock or plasmids for WT or nuclease-dead mutant for Reg1 and Reg3 (n = 3 each). Data are representative of at least 3 independent experiments. (I) mRNA stability of Nfkbiz and Ehd3 in Lin– cells from control and DKOCreERT2 mice. Cells were stimulated with IL-1β for 2 hours, followed by addition of actinomycin D (ActD). The remaining transcripts relative to the 0-hour time point were measured by quantitative RT-PCR. Data are representative of 2 independent experiments (H-I). Data are presented as mean ± SD (H) or ± standard error of the mean (I). Statistical significance was calculated by 2-tailed Fisher exact test (E), 1-way analysis of variance (ANOVA) with Holm-Sidak multiple comparisons test (H), or unpaired 2-tailed Student t test (I). ND, nuclease dead.

Reg1 and Reg3 control lineage biases within HSPCs. (A) Schematic representation of experimental strategy for single-cell sorting. (B) Gating strategy for WT and Reg1–/–Reg3–/– (DKO) LSK cells sorted from the identical mouse receiving WT (CD45.1) and DKO (CD45.2) FL cells. (C) A 2-dimensional projection of 2241 single cells sorted from WT (CD45.1) LSK and 2242 single cells from DKO (CD45.2) LSK onto the reference model. Cells are colored according to assignment to reference model meta cells (see “Methods”). (D) Difference in meta-cell abundance between single cells sorted from DKO and WT mice. Each circle represents a reference meta cell. Circle color represents log2 (fold change) in meta-cell abundance between the 2 genetic backgrounds. (E) Cell type distribution of single cells sorted from WT and DKO mice. ∗P < .05; ∗∗P < .001; ∗∗∗P < .00001. (F) Differential gene expression between pooled cells from DKO cells compared with WT cells. Gold dots indicate differentially expressed genes (FDR corrected P < .001 under χ2 test) with fold change >2. Diagonal lines show log2 (fold change) = −1 or 1. Values represent log2 size–normalized expression. (G) Total expression of specific genes in different genetic backgrounds. Values represent size-normalized total transcripts per 100 cells. Colors represent relative contribution from the different hematopoietic core subsets. (H) Luciferase activity was assessed by using pGL3 plasmids containing the 3’ UTR of Nfkbiz and Ehd3, together with mock or plasmids for WT or nuclease-dead mutant for Reg1 and Reg3 (n = 3 each). Data are representative of at least 3 independent experiments. (I) mRNA stability of Nfkbiz and Ehd3 in Lin– cells from control and DKOCreERT2 mice. Cells were stimulated with IL-1β for 2 hours, followed by addition of actinomycin D (ActD). The remaining transcripts relative to the 0-hour time point were measured by quantitative RT-PCR. Data are representative of 2 independent experiments (H-I). Data are presented as mean ± SD (H) or ± standard error of the mean (I). Statistical significance was calculated by 2-tailed Fisher exact test (E), 1-way analysis of variance (ANOVA) with Holm-Sidak multiple comparisons test (H), or unpaired 2-tailed Student t test (I). ND, nuclease dead.

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