Abstract
Definitive erythropoiesis usually occurs in the bone marrow or fetal liver, where erythroblasts are associated with a central macrophage in anatomical units called ‘blood islands’1,2. Late in erythropoiesis, nuclei are expelled from the erythroid precursor cells and engulfed by the macrophages in the blood island2,3. Here we show that the nuclei are engulfed by macrophages only after they are disconnected from reticulocytes, and that phosphatidylserine, which is often used as an ‘eat me’ signal for apoptotic cells, is also used for the engulfment of nuclei expelled from erythroblasts. We investigated the mechanism behind the enucleation and engulfment processes by isolating late-stage erythroblasts from the spleens of phlebotomized mice. When these erythroblasts were cultured, the nuclei protruded spontaneously from the erythroblasts. A weak physical force could disconnect the nuclei from the reticulocytes. The released nuclei contained an undetectable level of ATP, and quickly exposed phosphatidylserine on their surface. Fetal liver macrophages efficiently engulfed the nuclei; masking the phosphatidylserine on the nuclei with the dominant-negative form of milk-fat-globule EGF8 (MFG-E8) prevented this engulfment.
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Acknowledgements
We thank H. Fukuyama for help at the initial stage of this work; H. Suzuki, I. Ito and S. Hanasaki for taking the time-lapse video; T. Yanagida for discussion; and M. Fujii and M. Harayama for secretarial assistance. This work was supported in part by Grants-in-Aid from the Ministry of Education, Science, Sports and Culture in Japan.Author Contributions H.Y. performed most of the experiments. M.K. carried out the electron microscopy analysis, and Y.M. took the time-lapse video. K.K., Y.U. and S.N. contributed to the data analysis. S.N. was responsible for planning the project.
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Supplementary information
Supplementary Video S1
Time-lapse video of the enuclation of an erythroblast. Erythroblasts (1 X 106 cells/ml) were incubated at 37°C in the presence of erythropoietin and transferin. The morphological change of one erythroblast was followed for 3 h, and is reproduced in a 10-s video. (MOV 947 kb)
Supplementary Video S2
Time-lapse video of the engulfment of nuclei by macrophages. Fetal liver macrophages (2 x 104 cells) from DNase II-/- embryos were co-cultured at 37°C with 5 x 105 nuclei isolated from erythroblasts in the absence of 0.5 µg/ml of the D89E mutant of MFG-E8. The engulfment of nuclei by one macrophage was followed for 3 h, and reproduced in a 10-s video. At the end of the incubation, the cells were stained with SYTOX to visualize the engulfed nuclei. (MOV 664 kb)
Supplementary Video S3
Time-lapse videos of the engulfment of nuclei by macrophages. Fetal liver macrophages (2 x 104 cells) from DNase II-/- embryos were co-cultured at 37°C with 5 x 105 nuclei isolated from erythroblasts in the presence of 0.5 µg/ml of the D89E mutant of MFG-E8. The engulfment of nuclei by one macrophage was followed for 3 h, and reproduced in a 10-s video. At the end of the incubation, the cells were stained with SYTOX to visualize the engulfed nuclei. (MOV 637 kb)
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Yoshida, H., Kawane, K., Koike, M. et al. Phosphatidylserine-dependent engulfment by macrophages of nuclei from erythroid precursor cells. Nature 437, 754–758 (2005). https://doi.org/10.1038/nature03964
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DOI: https://doi.org/10.1038/nature03964
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