RPL11 haploinsufficient cells prevent expansion of normal erythroid cells on erythroblastic islands. (A) Photomicrograph of 3 erythroblastic islands (EBIs) from wild-type C57Bl/6J mice. Ter119⁺ cells are in cyan; F4/80⁺ macrophages are in red. The indicator bar is 10 μM. (B) Representative ImageStream flow cytometry analysis of enriched bone marrow EBI from a 50:50 Rpl11 chimeric transplant mouse showing both GFP⁺ and Rpl11 haploinsufficient (GFP^–) Ter119⁺ erythroid cells attached within the same EBIs. GFP is overexposed in the panels on the right to aid in resolution of whether the macrophages express GFP or not because macrophage GFP is difficult to detect compared with the very high erythroid GFP expression. The top 3 EBI panels contain GFP⁺ macrophages. About 2/3 of the imaged EBIs included GFP⁺ (therefore control WT) macrophages. Of 18 EBIs examined from Rpl11-chimeras, all contained both GFP⁺ and GFP^– Ter119⁺ erythroid precursors. ImageStream analysis of T2 control and Flvcr-chimeras are in supplemental Figure 4. (C) Representative flow cytometry analysis of mouse bone marrow erythroid precursor cells from a 50:50 Rpl11-chimeric transplant mouse before (whole BM) and after enrichment for erythroblastic islands (BM EBI). Cells are gated to identify BFU-E through reticulocytes as before.¹³ Gating includes lineage-negative precursors (LNPC), erythroid precursor population I includes BFU-E, CFU-E, and proerythroblasts, whereas populations II-V are basophilic, polychromatic, orthochromatic erythroblasts, and reticulocytes, respectively. The isolated EBIs included between 6.6% and 12.8% of the total marrow mononuclear cells recovered from whole marrow in the chimeric mice. Representative flow analysis of T2 control and Flvcr-chimeras are in supplemental Figure 5. (D) Erythroid precursor cell composition of EBIs isolated from 50:50 chimeras. Rpl11-hap:GFP (N = 3), T2 control:GFP (N = 6), Flvcr-del:GFP (N = 4), and Mx control:GFP (N = 2) mice showing the percentage of cre⁺ control or mutant erythroblast cells along with the percentage of GFP⁺ partner cells in each population stage (I-V) of erythroid differentiation in EBI isolated from marrow. (E) Quantitative PCR analysis showing relative expression of heme induced genes Spic, Hmox1, Slc40a1 (ferroportin), and Slc48a1 (HRG1) in F4/80⁺ EBI macrophages isolated from 50:50 chimeric transplant mice (Rpl11 and T2 control chimeras, N = 5 mice; Flvcr and Mx control chimeras, N = 3 mice). The cre control or mutant F4/80⁺ macrophages (GFP^– and their wild-type (GFP⁺) chimeric partner F4/80⁺ macrophages were sorted separately and analyzed as technical replicates to eliminate potential bias from unequal prevalence. (F) Heme content in WT/GFP⁺ polychromatic erythroblasts (population III) isolated from EBI of chimeric mice (N = 3-8 mice). Data are presented as mean ± standard deviation. *P < .05; **P < .01; ***P < .001 from Student t test. BFU-E, burst-forming unit–erythroid; BM, bone marrow.