Figure 1.
Figure 1. Increased phosphocholine metabolism during terminal mouse fetal erythropoiesis. (A) FACS plot of R1-R5 populations. E14.5 mouse fetal liver cells were sorted into 4 groups (R2: CD71 high, Ter119−; R3: CD71 high, Ter119+; R4: CD71 low, Ter119+; R5: CD71−, Ter119+). (B) Lipids from R2-R5 groups were analyzed and total negative and positive ion abundances were retrieved from LC/MS and plotted. One million cells per group were used for metabolite extraction (n = 3). (C) The lipid composition of mouse R2-R5 cells was analyzed by partial least squares discriminant analysis (PLS). The signal of each class of lipids was normalized by total lipid abundance from the LC/MS results, and the results are shown as colored boxes (from high to low: red to green) and plotted with variable importance in the projection (VIP) score (VIP > 1: significant). PC is bolded and shows its expression from high (red) in R2 cells to low (green) in R5 cells. (D) Polar metabolites from R2-R5 cells were analyzed. Each metabolite signal is normalized to total lipid abundance. Polar metabolites differentiating between the 4 groups are shown as colored boxes and plotted with VIP score (VIP > 1: significant). Phosphocholine and choline are labeled in bold font. Relative metabolite abundance is indicated in the bar, with red representing metabolite accumulation (n = 3 per group). (E) Left, PHOSPHO1 hydrolyzes phosphocholine to choline. Right, Mouse PHOSPHO1 gene expression in each group of cells normalized to 18S ribosomal RNA (rRNA). (n = 3 per group, mean + SEM). (F) Knocking down mPHOSPHO1 in cultures of lineage-negative E14.5 mouse fetal liver erythroblasts using 3 different shRNAs reduces cell proliferation in differentiation medium (n = 3 per group, mean ± SEM). Cells were expanded in maintenance medium for 1 day and differentiated in differentiation medium for 2 days. (G) Knocking down mPHOSPHO1 in lineage-negative E14.5 mouse fetal liver using 3 shRNAs impairs enucleation after 2 days of in vitro differentiation (n = 3 per group, mean + SEM). (H) mPHOSPHO1 gene expression of cells assayed in panels F and G. Messenger RNA (mRNA) was extracted from cells differentiated for 1 day. A.U., arbitrary unit; CerG1, glycosphingolipid; ChE, cholesteryl ester; Co, coenzyme; ctrl, control; DG, diglyceride; LPC, lysophosphatidylcholine; LPE, lysophosphatidylethanolamine; PA, phosphatidic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol; PS, phosphatidylserine; SM, sphingomyelin; So, sphingosine; TG, triglyceride.

Increased phosphocholine metabolism during terminal mouse fetal erythropoiesis. (A) FACS plot of R1-R5 populations. E14.5 mouse fetal liver cells were sorted into 4 groups (R2: CD71 high, Ter119−; R3: CD71 high, Ter119+; R4: CD71 low, Ter119+; R5: CD71−, Ter119+). (B) Lipids from R2-R5 groups were analyzed and total negative and positive ion abundances were retrieved from LC/MS and plotted. One million cells per group were used for metabolite extraction (n = 3). (C) The lipid composition of mouse R2-R5 cells was analyzed by partial least squares discriminant analysis (PLS). The signal of each class of lipids was normalized by total lipid abundance from the LC/MS results, and the results are shown as colored boxes (from high to low: red to green) and plotted with variable importance in the projection (VIP) score (VIP > 1: significant). PC is bolded and shows its expression from high (red) in R2 cells to low (green) in R5 cells. (D) Polar metabolites from R2-R5 cells were analyzed. Each metabolite signal is normalized to total lipid abundance. Polar metabolites differentiating between the 4 groups are shown as colored boxes and plotted with VIP score (VIP > 1: significant). Phosphocholine and choline are labeled in bold font. Relative metabolite abundance is indicated in the bar, with red representing metabolite accumulation (n = 3 per group). (E) Left, PHOSPHO1 hydrolyzes phosphocholine to choline. Right, Mouse PHOSPHO1 gene expression in each group of cells normalized to 18S ribosomal RNA (rRNA). (n = 3 per group, mean + SEM). (F) Knocking down mPHOSPHO1 in cultures of lineage-negative E14.5 mouse fetal liver erythroblasts using 3 different shRNAs reduces cell proliferation in differentiation medium (n = 3 per group, mean ± SEM). Cells were expanded in maintenance medium for 1 day and differentiated in differentiation medium for 2 days. (G) Knocking down mPHOSPHO1 in lineage-negative E14.5 mouse fetal liver using 3 shRNAs impairs enucleation after 2 days of in vitro differentiation (n = 3 per group, mean + SEM). (H) mPHOSPHO1 gene expression of cells assayed in panels F and G. Messenger RNA (mRNA) was extracted from cells differentiated for 1 day. A.U., arbitrary unit; CerG1, glycosphingolipid; ChE, cholesteryl ester; Co, coenzyme; ctrl, control; DG, diglyceride; LPC, lysophosphatidylcholine; LPE, lysophosphatidylethanolamine; PA, phosphatidic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol; PS, phosphatidylserine; SM, sphingomyelin; So, sphingosine; TG, triglyceride.

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