Requirement of DNase II for Definitive Erythropoiesis in the Mouse Fetal Liver
10.1126/science.292.5521.1546
Crossref journal-article
American Association for the Advancement of Science (AAAS)
Science (221)
Abstract

Mature erythrocytes in mammals have no nuclei, although they differentiate from nucleated precursor cells. The mechanism by which enucleation occurs is not well understood. Here we show that deoxyribonuclease II (DNase II) is indispensable for definitive erythropoiesis in mouse fetal liver. No live DNase II–null mice were born, owing to severe anemia. When mutant fetal liver cells were transferred into lethally irradiated wild-type mice, mature red blood cells were generated from the mutant cells, suggesting that DNase II functions in a non–cell-autonomous manner. Histochemical analyses indicated that the critical cellular sources of DNase II are macrophages present at the site of definitive erythropoiesis in the fetal liver. Thus, DNase II in macrophages appears to be responsible for destroying the nuclear DNA expelled from erythroid precursor cells.

Bibliography

Kawane, K., Fukuyama, H., Kondoh, G., Takeda, J., Ohsawa, Y., Uchiyama, Y., & Nagata, S. (2001). Requirement of DNase II for Definitive Erythropoiesis in the Mouse Fetal Liver. Science, 292(5521), 1546–1549.

Authors 7
  1. Kohki Kawane (first)
  2. Hidehiro Fukuyama (additional)
  3. Gen Kondoh (additional)
  4. Junji Takeda (additional)
  5. Yoshiyuki Ohsawa (additional)
  6. Yasuo Uchiyama (additional)
  7. Shigekazu Nagata (additional)
References 31 Referenced 321
  1. Nagata S., Exp. Cell Res. 256, 12 (2000). (10.1006/excr.2000.4834) / Exp. Cell Res. by Nagata S. (2000)
  2. D. McIlroy et al. Genes Dev. 14 549 (2000). (10.1101/gad.14.5.549)
  3. Baker K. P., Baron W. F., Henzel W. J., Spencer S. A., Gene 215, 281 (1998). (10.1016/S0378-1119(98)00280-7) / Gene by Baker K. P. (1998)
  4. T. Yasuda et al. J. Biol. Chem. 273 2610 (1998). (10.1074/jbc.273.5.2610)
  5. Wu Y. C., Stanfield G. M., Horvitz H. R., Genes Dev. 14, 536 (2000). (10.1101/gad.14.5.536) / Genes Dev. by Wu Y. C. (2000)
  6. Krieser R. J., Eastman A., Gene 252, 155 (2000). (10.1016/S0378-1119(00)00209-2) / Gene by Krieser R. J. (2000)
  7. A mouse DNase II cDNA (3) was used to screen a genomic library of mouse strain 129/Sv (Stratagene). A DNA fragment carrying the mouse DNase II locus was subcloned and the DNase II–targeting vector was constructed by replacing the 2.3-kb Nru I–Xmn I DNA fragment containing the DNase II gene with the 1.9-kb neo gene driven by the phosphoglycerate kinase–1 gene promoter (Fig. 1A). A DNA fragment encoding diphtheria toxin A fragment under control of the polyoma virus enhancer was inserted downstream of the DNase II gene to generate pKODNII. The pKODNII plasmid (19 μg) was digested with Not I and was introduced into 7.7 × 10 6 R1 ES cells by electroporation. The cells were selected in the presence of 150 μg/ml G-418 for 7 days. G-418–resistant clones were screened for the homologous recombination by PCR and the ES clones carrying the DNase II–mutant allele were introduced into the host embryos by aggregation (28). Embryos that had developed to the blastocyst stage were transplanted into pseudopregnant foster mothers. Chimeric mice were crossed with C57BL/6 mice. Germ-line transmission of the ES cells to their offspring (F 1 ) was judged by the presence of agouti pups and the disrupted alleles were identified by PCR.
  8. Supplementary material is available on Science Online at www.sciencemag.org/cgi/content/full/292/5521/1546/DC1.
  9. Nuez B., Michalovich D., Bygrave A., Ploemacher R., Grosveld F., Nature 375, 316 (1995). (10.1038/375316a0) / Nature by Nuez B. (1995)
  10. Perkins A. C., Sharpe A. H., Orkin S. H., Nature 375, 318 (1995). (10.1038/375318a0) / Nature by Perkins A. C. (1995)
  11. Shiokawa D., Tanuma S., Nucleic Acids Res. 27, 4083 (1999). (10.1093/nar/27.20.4083) / Nucleic Acids Res. by Shiokawa D. (1999)
  12. V. Appierto et al. Gene 188 119 (1997). (10.1016/S0378-1119(96)00794-9)
  13. To measure the DNase II activity we incubated 2 μg of plasmid DNA (pBluescript) at 37°C for 1 hour with the cell extracts in 300 μl of 10 mM tris-HCl buffer (pH 5.7) containing 10 mM EDTA. After phenol extraction DNA was subjected to electrophoresis on a 1% agarose gel. The assay for DNase I activity was similar to that described above for DNase II except that the reaction mixture contained 10 mM tris-HCl buffer (pH 7.4) 10 mM CaCl 2 and 10 mM MgCl 2 .
  14. K. Kawane H. Fukuyama S. Nagata data not shown.
  15. For transplantation of the fetal liver cells single-cell suspensions prepared from E12.5 fetal liver of the wild-type or DNase II −/− embryos (seven to eight embryos each) were pooled. Three-month-old female B6.SJL mice (Taconic) were lethally irradiated (900 cGy) by a 137 Cs source and were intravenously injected with 1 × 10 6 fetal liver cells. The mice were maintained on aqueous antibiotics (1000 U/ml polymixin B sulfate and 1.1 mg/ml neomycin sulfate).
  16. Russell E. S., McFarland E. C., Ann. N.Y. Acad. Sci. 241, 25 (1974). (10.1111/j.1749-6632.1974.tb21864.x) / Ann. N.Y. Acad. Sci. by Russell E. S. (1974)
  17. Austyn J. M., Gordon S., Eur. J. Immunol. 11, 805 (1981). (10.1002/eji.1830111013) / Eur. J. Immunol. by Austyn J. M. (1981)
  18. Crocker P. R., Gordon S., J. Exp. Med. 162, 993 (1985). (10.1084/jem.162.3.993) / J. Exp. Med. by Crocker P. R. (1985)
  19. V. A. Fadok et al. J. Clin. Invest. 101 890 (1998). (10.1172/JCI1112)
  20. Dybedal I., Jacobsen S. E., Blood 86, 949 (1995). (10.1182/blood.V86.3.949.949) / Blood by Dybedal I. (1995)
  21. W. Wiktor-Jedrzejczak et al. Proc. Natl. Acad. Sci. U.S.A. 87 4828 (1990). (10.1073/pnas.87.12.4828)
  22. H. Yoshida et al. Nature 345 442 (1990). (10.1038/345442a0)
  23. 10.1126/science.8079170
  24. 10.1126/science.279.5348.230
  25. A. M. Lichanska et al. Blood 94 127 (1999). (10.1182/blood.V94.1.127.413k07_127_138)
  26. M. G. Cecchini et al. Development 120 1357 (1994). (10.1242/dev.120.6.1357)
  27. Platt N., da Silva R. P., Gordon S., Trends Cell Biol. 8, 365 (1998). (10.1016/S0962-8924(98)01329-4) / Trends Cell Biol. by Platt N. (1998)
  28. Kondoh G., et al., J. Biochem. Biophys. Methods 39, 137 (1999). (10.1016/S0165-022X(99)00008-1) / J. Biochem. Biophys. Methods by Kondoh G. (1999)
  29. A. Torriglia et al. J. Biol. Chem. 270 28579 (1995). (10.1074/jbc.270.48.28579)
  30. Alter B. P., Campbell A. S., Hemoglobin 6, 517 (1982). (10.3109/03630268209083764) / Hemoglobin by Alter B. P. (1982)
  31. We thank Y. Seto for maintaining the mice K. Miwa for PCR typing of the mouse embryos S. Aoyama for secretarial assistance and Y. Kanakura and T. Nakahata for discussion. Supported in part by Grants-in-Aid from the Ministry of Education Science Sports and Technology of Japan. K.K. was supported by a research fellowship of the Japan Society for the Promotion of Science.
Dates
Type When
Created 23 years, 1 month ago (July 27, 2002, 5:52 a.m.)
Deposited 1 year, 7 months ago (Jan. 13, 2024, 4:39 a.m.)
Indexed 4 weeks, 1 day ago (Aug. 6, 2025, 9:47 a.m.)
Issued 24 years, 3 months ago (May 25, 2001)
Published 24 years, 3 months ago (May 25, 2001)
Published Print 24 years, 3 months ago (May 25, 2001)
Funders 0

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@article{Kawane_2001, title={Requirement of DNase II for Definitive Erythropoiesis in the Mouse Fetal Liver}, volume={292}, ISSN={1095-9203}, url={http://dx.doi.org/10.1126/science.292.5521.1546}, DOI={10.1126/science.292.5521.1546}, number={5521}, journal={Science}, publisher={American Association for the Advancement of Science (AAAS)}, author={Kawane, Kohki and Fukuyama, Hidehiro and Kondoh, Gen and Takeda, Junji and Ohsawa, Yoshiyuki and Uchiyama, Yasuo and Nagata, Shigekazu}, year={2001}, month=may, pages={1546–1549} }