BM hypoplasia, extramedullary hemopoiesis, and progressive anemia in Ikaros mutant mice. (A) BM and spleen cellularity of Ikaros mutant relative to wild-type mice. BM cellularity is calculated from the cell content of the two hind limbs (30% of total BM). Both BM and spleen cell suspensions were counted after erythrocyte lysis. The cellularity of the wild-type organs is set to 1.0, and mutant values are indicated as a proportion thereof. Numbers of animals included in the analysis were: wild-type, n = 20; Ikaros null, n = 16; and Ikaros DN^−/−, n = 18. (B) Hematocrits in wild-type and Ikaros DN mutant mice are shown as a function of age. Wild-type mice and mutant littermates were bled at the indicated ages and their hematocrits read in a Readacrit centrifuge. Ikaros DN^−/− mutant mice (•) initially had hematocrits similar to those of age-matched control littermates (□), but their hematocrits dropped to <50% of wild type by 6 wk of age. Number of mice bled is as follows: +/+, 2–3 wk old, n = 4; +/+, 3–4 wk old, n = 9; +/+, 4–5 wk old, n = 4; +/+, 5–6 wk old, n = 5; +/+, over 6 wk old, n = 4; −/−, 2–3 wk old, n = 7; −/−, 3–4 wk old, n = 9; −/−, 4–5 wk old, n = 5; −/−, 5–6 wk old, n = 4; and −/−, over 6 wk old, n = 4.

Short-term radioprotection and repopulation ability is unaffected by the Ikaros null mutation but severely compromised by the Ikaros DN mutation. (A) Short-term radioprotective ability of wild-type and Ikaros mutant BM. Ly5a congenic mice were lethally irradiated (9.5 Gy) and injected with 10⁵ wild-type BM (▪), 10⁵ Ikaros null^−/− BM (□), 10⁶ Ikaros DN^−/− BM (▴), or PBS alone (○). The numbers of mice injected per donor group are given in the legend, and their age was 3–4 wk. (B) Donor contribution in the myeloid lineage after transplant with wild-type and Ikaros mutant BM. Ly5a congenic mice were lethally irradiated and injected with 10⁶ wild-type BM (gray bars), 10⁶ Ikaros null BM (left panel, hatched bars), or 6 × 10⁶ Ikaros DN^−/− BM (right panel, hatched bars). At the indicated time points after transplant, recipients were bled and assayed for donor contribution to the myeloid (Mac-1⁺) lineage by FACS™ analysis. A pool of four wild-type or mutant mice was used as a donor population. The experiment was repeated twice, and data shown are the analysis of one representative mouse out of eight recipients. (C) Development of lethal anemia in recipients of Ikaros DN^−/− BM. 5 × 10⁶ Ikaros DN^−/− (•) or wild-type (□) BM cells were transplanted into lethally irradiated recipients, and recipient hematocrits were monitored over 5 wk. Recipients of Ikaros DN^−/− BM (•) eventually died of anemia 5 wk after transplant. Numbers of mice bled were as follows: +/+ recipients, n = 12; and Ikaros DN^−/− recipients, n = 28. Donors were 2–4 wk old.

A progressive reduction in competitive repopulation units from Ikaros null to Ikaros DN^−/− hemopoietic populations. (A) Competitive repopulation assay with wild-type and Ikaros null mutant BM. Lethally irradiated Ly5a mice were coinjected with a mixture of test (wild-type [+/+] or Ikaros null [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.

Hemopoietic lineage repopulation in unconditioned Ikaros mutant mice points to a severe depletion in hemopoietic progenitors. (A) 10⁶ wild-type Ly5a BM cells were injected into 3-wk-old Ly5b Ikaros null mice without prior conditioning. At different time points (3–6 mo) after transplant, recipient mice were killed and analyzed for Ly5a contribution by FACS™ analysis. (B) 10⁶ wild-type Ly5a BM cells were injected intravenously into 12-d-old Ikaros DN^+/− or DN^−/− Ly5b recipients without prior conditioning. At different time points after transplant, recipient mice were killed and analyzed for donor Ly5a contribution by FACS™ analysis. Analysis of representative recipients 5.5 mo after transplant is shown. Top panels correspond to Ikaros DN^+/− recipients, and bottom panels correspond to Ikaros DN^−/− recipients. (C) PCR analysis of the hemopoietic organs of two independent Ikaros DN^−/− recipients of wild-type BM. Total DNA was extracted from the hemopoietic organs of wild-type control mice and Ikaros DN^−/− mutant mice that were transplanted with wild-type BM without prior irradiation. No Ikaros DN mutant DNA (upper band) is detectable in DN^−/− recipients, indicating lack of recipient contribution in any hemopoietic lineage. The DNA titration (lanes 10–13) indicates that the larger mutant band can still be detected in a 1:1,000 dilution into the smaller, and thus more readily amplifiable, wild-type band.

Reduction in hemopoietic progenitors (CFU-S₁₄) caused by the Ikaros mutations. (A) Absolute number of CFU-S₁₄ in the two hind limbs (left) and spleens (right) of wild-type and Ikaros mutant mice. Numbers do not include seeding efficiency conversions. Each square represents the average of four recipients of either 5 × 10⁴ BM or 2.5 × 10⁵ spleen cells from one donor mouse. The number of donor mice is as follows: +/+ BM, n = 12; +/+ spleen, n = 12; DN BM, n = 14; DN spleen, n = 14; null −/− BM, n = 10; and null −/− spleen, n = 10. Donors were 2–4 wk old. Spleen colonies generated upon injection of wild-type and Ikaros mutant BM and spleen are shown in the bottom panels. (B) CFU-S14 content of day 10 YS (Yolk Sac-D10), day 10 AGM (AGM-D10), and day 14 fetal liver (Fetal Liver-D14) without seeding factor correction is shown for wild-type 67 and Ikaros DN^−/− embryos 4.

Dynamics in myeloid differentiation in Ikaros mutant mice. (A) Proportion of absolute CFCs recovered from BM and spleens (Sp) of Ikaros null and DN^−/− mutants relative to wild type (dashed line) is shown. The data represent the average ±SEM from a total of eight animals per genotype. Precursor classes are defined in Materials and Methods. (B) Frequency of CFCs (i.e., number per 10⁵ cells) in Ikaros null and Ikaros DN^−/− BM and spleens is shown. Data are standardized to wild-type frequencies, represented by the dashed line. (C) The combined CFC content of BM and spleen relative to wild type is shown. CFC content for the entire bodily BM compartment is included in the calculation by assuming that one femur represents 10% of total BM.

Analysis of lin⁻ Ikaros mutant hemopoietic cells. (A) Total BM cells from 2–4 wk-old wild-type (+/+), Ikaros null (Ik −/−), and Ikaros (Ik) DN^−/− mice were lineage depleted and stained for c-kit and Sca-1 (as described in Materials and Methods). The c-kit^hi, c-kit^lo, and c-kit⁺Sca-1⁺ populations are boxed and percentages are indicated. (B) Histograms of cell surface c-kit expression in Ikaros mutant lin⁻ BM populations (dashed lines) and wild type (solid lines) are shown. The left and right panels contrast Ikaros null and Ikaros DN^−/− cells, respectively, with wild-type cells. (C) RT-PCR analysis of sorted hemopoietic cells from wild-type and Ikaros mutant mice. BM cells were lineage depleted and sorted according to expression of c-kit and Sca-1, as indicated in A. cDNA was obtained from each sorted population and PCR amplified for the indicated genes. cDNA levels were normalized to HPRT levels.

Effects of Ikaros deficiency on the hemopoietic progenitor/precursor hierarchy. Commitment of a mesodermal precursor to the HSC fate, self-renewal (curved arrow), and subsequent progression through intermediate steps that involve long- and short-term repopulating progenitors and precursors to terminally differentiated progeny is illustrated. The mesodermal precursor commitment step as a possible target for the Ikaros deficiencies and a cause for the reduction in HSCs is indicated as the perpendicular intersection of two broken lines, which also indicates a possible defect in HSC renewal. Complete blocks in the differentiation of Ikaros-deficient progenitors toward the B cell and NK lineages are shown as the perpendicular intersection of two solid lines. The increase in the differentiation output of these mutant precursor populations toward the erythromyeloid lineage is depicted with green arrows.