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Defects in hemopoietic stem cell activity in Ikaros mutant mice.

Nichogiannopoulou A, Trevisan M, Neben S, Friedrich C, Georgopoulos K - J. Exp. Med. (1999)

Bottom Line: The defect in HSC activity is also illustrated by the ability of wild-type marrow to repopulate unconditioned Ikaros mutants.A progressive reduction in multipotent CFU-S(14) (colony-forming unit-spleen) progenitors and the earliest erythroid-restricted precursors (BFU-E [burst-forming unit-erythroid]) is also detected in the Ikaros mutant strains consistent with the reduction in HSCs.Nonetheless, the more mature clonogenic erythroid and myeloid precursors are less affected, indicating either the action of a compensatory mechanism to provide more progeny or a negative role of Ikaros at later stages of erythromyeloid differentiation.

View Article: PubMed Central - PubMed

Affiliation: Cutaneous Biology Research Center, Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts 02129, USA.

ABSTRACT
Here we provide evidence that the Ikaros family of DNA binding factors is critical for the activity of hemopoietic stem cells (HSCs) in the mouse. Mice homozygous for an Ikaros mutation display a >30-fold reduction in long-term repopulation units, whereas mice homozygous for an Ikaros dominant negative mutation have no measurable activity. The defect in HSC activity is also illustrated by the ability of wild-type marrow to repopulate unconditioned Ikaros mutants. A progressive reduction in multipotent CFU-S(14) (colony-forming unit-spleen) progenitors and the earliest erythroid-restricted precursors (BFU-E [burst-forming unit-erythroid]) is also detected in the Ikaros mutant strains consistent with the reduction in HSCs. Nonetheless, the more mature clonogenic erythroid and myeloid precursors are less affected, indicating either the action of a compensatory mechanism to provide more progeny or a negative role of Ikaros at later stages of erythromyeloid differentiation. In Ikaros mutant mice, a decrease in expression of the tyrosine kinase receptors flk-2 and c-kit is observed in the lineage-depleted c-kit(+)Sca-1(+) population that is normally enriched for HSCs and may in part contribute to the early hemopoietic phenotypes manifested in the absence of Ikaros.

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A progressive reduction in competitive repopulation units from Ikaros  to Ikaros DN−/− hemopoietic populations. (A) Competitive repopulation assay with wild-type and Ikaros  mutant BM. Lethally irradiated Ly5a mice were coinjected with a mixture of test (wild-type [+/+] or Ikaros  [Ik−/−]) Ly5b BM and a constant dose of 105 cells of competitor Ly5a BM at the indicated ratios. At various times (3–12 mo) after transplant, recipient mice were killed and analyzed for the hemopoietic contribution to marrow Mac-1+ cells from test and competitor populations by FACS™ analysis. One representative out of four recipient mice killed is shown. Donors were 4 wk old. (B) Lack of competitive LTR activity in Ikaros DN−/− BM. 5 × 106 Ikaros DN−/− Ly5b BM cells were transplanted along with 5 × 104 competitor Ly5a BM cells into lethally irradiated Ly5a recipients. At the indicated time points after transplant, recipient mice were bled and contribution to the Mac-1 lineage from each injected population was determined by FACS™ analysis. The average of six recipients per group is shown for each time point. Donors were 2–3 wk old.
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Figure 3: A progressive reduction in competitive repopulation units from Ikaros to Ikaros DN−/− hemopoietic populations. (A) Competitive repopulation assay with wild-type and Ikaros mutant BM. Lethally irradiated Ly5a mice were coinjected with a mixture of test (wild-type [+/+] or Ikaros [Ik−/−]) Ly5b BM and a constant dose of 105 cells of competitor Ly5a BM at the indicated ratios. At various times (3–12 mo) after transplant, recipient mice were killed and analyzed for the hemopoietic contribution to marrow Mac-1+ cells from test and competitor populations by FACS™ analysis. One representative out of four recipient mice killed is shown. Donors were 4 wk old. (B) Lack of competitive LTR activity in Ikaros DN−/− BM. 5 × 106 Ikaros DN−/− Ly5b BM cells were transplanted along with 5 × 104 competitor Ly5a BM cells into lethally irradiated Ly5a recipients. At the indicated time points after transplant, recipient mice were bled and contribution to the Mac-1 lineage from each injected population was determined by FACS™ analysis. The average of six recipients per group is shown for each time point. Donors were 2–3 wk old.

Mentions: The LTR potential of Ikaros and DN−/− BM was analyzed in a competitive repopulation assay 2324. Ikaros mutant Ly5b BM cells were injected into lethally irradiated Ly5a recipients along with a constant competitor dose of 105 Ly5a marrow. When Ikaros BM (105) was transplanted with an equal amount of wild-type competitor marrow, it failed to contribute to myeloid (Mac-1+) cells in the BM or the periphery of recipients (Fig. 3 A, top right panel). In sharp contrast, wild-type BM contributed to 44% of the BM myeloid (Mac-1+) populations (Fig. 3 A, top left panel). Ikaros BM injected in 10-fold excess (106) over wild-type competitor gave 17% contribution to the myeloid (Mac-1+) lineage in the BM (Fig. 3 A, center panels). No significant contribution to the T cell lineage was observed in the thymus or the spleen (data not shown). In comparison, wild-type BM coinjected at a similar dose contributed to >99% of the myeloid (Fig. 3 A, center panels) and lymphoid (data not shown) populations in the BM and periphery. When 7.5 × 106 Ikaros BM cells were injected alongside 105 competitor BM cells, donor-derived contribution to the myeloid lineage increased to 75.3% (Fig. 3 A, bottom panels). This analysis indicates that there is a 30–40-fold decrease in LTR activity in the Ikaros BM.


Defects in hemopoietic stem cell activity in Ikaros mutant mice.

Nichogiannopoulou A, Trevisan M, Neben S, Friedrich C, Georgopoulos K - J. Exp. Med. (1999)

A progressive reduction in competitive repopulation units from Ikaros  to Ikaros DN−/− hemopoietic populations. (A) Competitive repopulation assay with wild-type and Ikaros  mutant BM. Lethally irradiated Ly5a mice were coinjected with a mixture of test (wild-type [+/+] or Ikaros  [Ik−/−]) Ly5b BM and a constant dose of 105 cells of competitor Ly5a BM at the indicated ratios. At various times (3–12 mo) after transplant, recipient mice were killed and analyzed for the hemopoietic contribution to marrow Mac-1+ cells from test and competitor populations by FACS™ analysis. One representative out of four recipient mice killed is shown. Donors were 4 wk old. (B) Lack of competitive LTR activity in Ikaros DN−/− BM. 5 × 106 Ikaros DN−/− Ly5b BM cells were transplanted along with 5 × 104 competitor Ly5a BM cells into lethally irradiated Ly5a recipients. At the indicated time points after transplant, recipient mice were bled and contribution to the Mac-1 lineage from each injected population was determined by FACS™ analysis. The average of six recipients per group is shown for each time point. Donors were 2–3 wk old.
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Related In: Results  -  Collection

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Figure 3: A progressive reduction in competitive repopulation units from Ikaros to Ikaros DN−/− hemopoietic populations. (A) Competitive repopulation assay with wild-type and Ikaros mutant BM. Lethally irradiated Ly5a mice were coinjected with a mixture of test (wild-type [+/+] or Ikaros [Ik−/−]) Ly5b BM and a constant dose of 105 cells of competitor Ly5a BM at the indicated ratios. At various times (3–12 mo) after transplant, recipient mice were killed and analyzed for the hemopoietic contribution to marrow Mac-1+ cells from test and competitor populations by FACS™ analysis. One representative out of four recipient mice killed is shown. Donors were 4 wk old. (B) Lack of competitive LTR activity in Ikaros DN−/− BM. 5 × 106 Ikaros DN−/− Ly5b BM cells were transplanted along with 5 × 104 competitor Ly5a BM cells into lethally irradiated Ly5a recipients. At the indicated time points after transplant, recipient mice were bled and contribution to the Mac-1 lineage from each injected population was determined by FACS™ analysis. The average of six recipients per group is shown for each time point. Donors were 2–3 wk old.
Mentions: The LTR potential of Ikaros and DN−/− BM was analyzed in a competitive repopulation assay 2324. Ikaros mutant Ly5b BM cells were injected into lethally irradiated Ly5a recipients along with a constant competitor dose of 105 Ly5a marrow. When Ikaros BM (105) was transplanted with an equal amount of wild-type competitor marrow, it failed to contribute to myeloid (Mac-1+) cells in the BM or the periphery of recipients (Fig. 3 A, top right panel). In sharp contrast, wild-type BM contributed to 44% of the BM myeloid (Mac-1+) populations (Fig. 3 A, top left panel). Ikaros BM injected in 10-fold excess (106) over wild-type competitor gave 17% contribution to the myeloid (Mac-1+) lineage in the BM (Fig. 3 A, center panels). No significant contribution to the T cell lineage was observed in the thymus or the spleen (data not shown). In comparison, wild-type BM coinjected at a similar dose contributed to >99% of the myeloid (Fig. 3 A, center panels) and lymphoid (data not shown) populations in the BM and periphery. When 7.5 × 106 Ikaros BM cells were injected alongside 105 competitor BM cells, donor-derived contribution to the myeloid lineage increased to 75.3% (Fig. 3 A, bottom panels). This analysis indicates that there is a 30–40-fold decrease in LTR activity in the Ikaros BM.

Bottom Line: The defect in HSC activity is also illustrated by the ability of wild-type marrow to repopulate unconditioned Ikaros mutants.A progressive reduction in multipotent CFU-S(14) (colony-forming unit-spleen) progenitors and the earliest erythroid-restricted precursors (BFU-E [burst-forming unit-erythroid]) is also detected in the Ikaros mutant strains consistent with the reduction in HSCs.Nonetheless, the more mature clonogenic erythroid and myeloid precursors are less affected, indicating either the action of a compensatory mechanism to provide more progeny or a negative role of Ikaros at later stages of erythromyeloid differentiation.

View Article: PubMed Central - PubMed

Affiliation: Cutaneous Biology Research Center, Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts 02129, USA.

ABSTRACT
Here we provide evidence that the Ikaros family of DNA binding factors is critical for the activity of hemopoietic stem cells (HSCs) in the mouse. Mice homozygous for an Ikaros mutation display a >30-fold reduction in long-term repopulation units, whereas mice homozygous for an Ikaros dominant negative mutation have no measurable activity. The defect in HSC activity is also illustrated by the ability of wild-type marrow to repopulate unconditioned Ikaros mutants. A progressive reduction in multipotent CFU-S(14) (colony-forming unit-spleen) progenitors and the earliest erythroid-restricted precursors (BFU-E [burst-forming unit-erythroid]) is also detected in the Ikaros mutant strains consistent with the reduction in HSCs. Nonetheless, the more mature clonogenic erythroid and myeloid precursors are less affected, indicating either the action of a compensatory mechanism to provide more progeny or a negative role of Ikaros at later stages of erythromyeloid differentiation. In Ikaros mutant mice, a decrease in expression of the tyrosine kinase receptors flk-2 and c-kit is observed in the lineage-depleted c-kit(+)Sca-1(+) population that is normally enriched for HSCs and may in part contribute to the early hemopoietic phenotypes manifested in the absence of Ikaros.

Show MeSH
Related in: MedlinePlus