“Hiatus” subpopulation resides between early stages of emerging erythroid cells. (A) UMAP visualization of Group_1 to Group_5 in emerging erythroid cells under PHZ and bleeding conditions, respectively. (B) Pie chart (left) and bar graph (right) showing the proportions of each group in PHZ and BLD conditions, respectively. (C) Representative gating strategy of CD52lo high and CD52lo Ery I+II and Ery III fractions within splenic early erythroblasts during bleeding stress. (D) Protein synthesis rate, as determined by OP-Puro incorporation, of the indicated erythroblast populations (CD52hi and CD52lo Ery I+II and Ery III) under bleeding stress. (E) mRNA expression of Gata1, Tal1, Klf1, Nfe2, Abcb10, and Rpl28 isolated from CD52lo and CD52hi Ery I+II and Ery III populations under bleeding stress. Error bars indicate the standard error of the mean (SEM) for 3 independent experiments. (F) Representative gating strategy of CD52lo high and CD52lo Ery I+II and Ery III fractions within splenic early erythroblasts during PHZ stress. (G) Protein synthesis rate, as determined by OP-Puro incorporation, of the indicated erythroblast populations (CD52hi and CD52lo Ery I+II and Ery III) under PHZ stress. (H) mRNA expression of Gata1, Tal1, Klf1, Nfe2, Abcb10, Rpl28 isolated from CD52lo and CD52hi Ery I+II and Ery III populations under PHZ stress. (I) Schematic illustration of the experiment to isolate CD52lo and CD52hi early erythroblasts (Ery I) from mice subjected to bleeding stress and culture in vitro. (J) Adenosine triphosphate content of CD52lo and CD52hi early erythroblasts measured by CellTiter-Glo Luminescent Cell Viability Assay. (K) Proliferation of CD52lo and CD52hi early erythroblasts in the in vitro culture from day 0 to day 5. (L) Erythroid differentiation of CD52lo and CD52hi early erythroblasts in the in vitro culture at day 3 and day 5, measured by flow cytometry analysis. Representative flow cytometric plots were shown on the left. (M) Morphology of CD52lo and CD52hi early erythroblasts after cytospin. Scale bar, 10 μm. (N) Illustration of the proposed model for cell intrinsic differentiation trajectory of emerging erythroid cells during anemic stress. Under steady state, murine spleen contains a reservoir of mature erythrocytes, whereas during anemic stress, mice undergo splenomegaly, and early erythroblasts emerge in the spleen, displaying transition of various biological processes during differentiation, with a “hiatus” subpopulation residing between early stages. Illustration was created via biorender.com. For all quantification, means ± SEMs; ∗ P < .05; ∗∗ P < .01; ∗∗∗ P < .001; ∗∗∗∗ P < .0001 by t test. mRNA, messenger RNA; OP-Puro, O-Propargyl-puromycin.

“Hiatus” subpopulation resides between early stages of emerging erythroid cells. (A) UMAP visualization of Group_1 to Group_5 in emerging erythroid cells under PHZ and bleeding conditions, respectively. (B) Pie chart (left) and bar graph (right) showing the proportions of each group in PHZ and BLD conditions, respectively. (C) Representative gating strategy of CD52lo high and CD52lo Ery I+II and Ery III fractions within splenic early erythroblasts during bleeding stress. (D) Protein synthesis rate, as determined by OP-Puro incorporation, of the indicated erythroblast populations (CD52hi and CD52lo Ery I+II and Ery III) under bleeding stress. (E) mRNA expression of Gata1, Tal1, Klf1, Nfe2, Abcb10, and Rpl28 isolated from CD52lo and CD52hi Ery I+II and Ery III populations under bleeding stress. Error bars indicate the standard error of the mean (SEM) for 3 independent experiments. (F) Representative gating strategy of CD52lo high and CD52lo Ery I+II and Ery III fractions within splenic early erythroblasts during PHZ stress. (G) Protein synthesis rate, as determined by OP-Puro incorporation, of the indicated erythroblast populations (CD52hi and CD52lo Ery I+II and Ery III) under PHZ stress. (H) mRNA expression of Gata1, Tal1, Klf1, Nfe2, Abcb10, Rpl28 isolated from CD52lo and CD52hi Ery I+II and Ery III populations under PHZ stress. (I) Schematic illustration of the experiment to isolate CD52lo and CD52hi early erythroblasts (Ery I) from mice subjected to bleeding stress and culture in vitro. (J) Adenosine triphosphate content of CD52lo and CD52hi early erythroblasts measured by CellTiter-Glo Luminescent Cell Viability Assay. (K) Proliferation of CD52lo and CD52hi early erythroblasts in the in vitro culture from day 0 to day 5. (L) Erythroid differentiation of CD52lo and CD52hi early erythroblasts in the in vitro culture at day 3 and day 5, measured by flow cytometry analysis. Representative flow cytometric plots were shown on the left. (M) Morphology of CD52lo and CD52hi early erythroblasts after cytospin. Scale bar, 10 μm. (N) Illustration of the proposed model for cell intrinsic differentiation trajectory of emerging erythroid cells during anemic stress. Under steady state, murine spleen contains a reservoir of mature erythrocytes, whereas during anemic stress, mice undergo splenomegaly, and early erythroblasts emerge in the spleen, displaying transition of various biological processes during differentiation, with a “hiatus” subpopulation residing between early stages. Illustration was created via biorender.com. For all quantification, means ± SEMs; ∗ P < .05; ∗∗ P < .01; ∗∗∗ P < .001; ∗∗∗∗ P < .0001 by t test. mRNA, messenger RNA; OP-Puro, O-Propargyl-puromycin.

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