Analysis of dose response and strain-specific effects of 5FU. Each group of mice consists of two subgroups of 8 to 10 animals, one subgroup was tested at D0, 4, 8, 12, 16, and the second was tested at D0, 2, 6, 10, 14. The two subgroups were combined to create one group represented by a single recovery curve, which includes the baseline and data sampled every 2 days post-treatment. (A) 5FU dose response. C57BL/6J females were injected intraperitoneally with 5FU (100, 120, or 150 μg/g) and the recovery curves for RBC, PLT, and WBC were determined (left, center, and right, respectively). The degree of anemia was assessed for each dose by comparing the RBC at the nadir for each recovery curve (RBC nadir t-test: 120 vs 150, p = 0.412 NS; 120 or 150 vs 100, p < 0.0005). The 5FU dose of 100 and 120 μg/g induced similar levels of thrombocytopenia (PLT nadir t-test: 100 D6 vs 120 D6: p = 0.16) and PLT overshoot (PLT peak of overshoot: 100 D10 vs 120 D12; p = 0.75). The dose of 150 mg/g 5FU induced a unique PLT recovery curve compared to 100 μg/g and 120 μg/g (two-way repeat measures ANOVA: 120 vs 150; p <0.0001; 100 vs 150; p =0.0001). The overall WBC recovery kinetics were different in mice treated with 150 μg/g 5FU (two-way repeat measures ANOVA: 100 vs 120; p =0.656; 100 vs 150 and 120 vs 150; p <0.005). The WBC overshoot was higher for mice treated with 150 μg/g for each (peak WBC values t-test: 100 D10 vs 120 D12; p =0.559; 150 vs 100 or 120; p <0.005). (B) Effect of mouse strain on 5FU-induced cytopenia. C57BL/6J, BALB/cJ, 129S1/SVImJ, and C3H/HeJ female mice were used for this comparison. The RBC recovery was compared between the four strains at baseline (one-way ANOVA: p < 0.0001), and C3H/HeJ was lower than other strains tested (t-test: C3H/HeJ vs C57BL/6J, BALB/cJ, or 129S1/SVImJ; p < 0.05); and the degree of anemia (RBC nadir: ANOVA, p < 0.0001). The PLT recovery kinetics between the four mouse strains were analyzed for the following components: baseline (ANOVA, p < 0.002); induction of thrombocytopenia or PLT overshoot (one-way repeated measures ANOVA and Dunnett’s post-test, p < 0.05: C57BL/6J D8-14; BALB/cJ D10-14; 129S1/SVImJ D4-6, D10-14; C3H/HeJ D4, D8-14); and comparing overall PLT recovery kinetics (two-way repeated measures ANOVA, p < 0.05: C3H/HeJ vs 129S1/SVImJ or C57BL/6J and C57BL/6J vs 129S1/SVImJ). The ability of 5FU to induce leukopenia or WBC overshoot in the four strains was analyzed (one-way repeat measures ANOVA with Dunnett’s post-test, p < 0.05: C57BL/6J D6-10; BALB/cJ D4-8 D14; 129S1/SVImJ D4-8, C3H/HeJ D2-4 D8). The differences in WBC recovery between the mouse strains were observed at baseline (one-way ANOVA, p < 0.03) and the height of WBC overshoot (one-way ANOVA, p < 0.05).

Time course examining the alterations in the KTLS population and profile post-cytopenia induction. Each experimental cohort is represented by two groups of three C57BL/6J female mice treated at 5 weeks of age, one group was treated with phosphate-buffered saline (PBS) only and one with 5FU. Two to three cohorts were examined at 18 hours, 7 days, or 14 days after treatment. At each time point, BM cells were analyzed by flow cytometry for KTLS expression (c-kit⁺, Thy1^lo, Lin⁻, Sca-1⁺). All animals were analyzed individually. Each flow plot shows a Lin⁻Thy1^lo population on a c-kit and Sca-1 plot and the red box represents the KTLS gate, while the proportion of cells inside each box is shown as a percentage of total cell number and represents the average for a group in an individual experiment. The c-kit^hi KTLS–positive population is shown in the green box, percentage of this population shown in right corner. 5FU-treated mice, 18 hours post-treatment, showed a loss of c-kit^hi-expressing cells. A statistically significant difference was observed between PBS only and 5FU-treated groups in the c-kit^hi (t-test p < 0.05 in each cohort). The KTLS population increased in the 5FU-treated group on D7 (t-test 0.1 < p < 0.004 in two cohorts).

The effect of 5FU on BM and splenic-derived hematopoietic progenitors. Each experimental cohort was represented by two groups of three C57BL/6J female mice treated at 5 weeks of age, and each group was treated with either phosphate-buffered saline or 5FU. Three cohorts were examined at each time point of 18 hours, 7 days, and 14 days post-treatment. Data plotted represents the average ± standard error of mean for all three cohorts, representing a total of nine animals for each treatment at each time point. Statistically significant differences (p < 0.05) from baseline (D0) are indicated by a line over each group and an asterisk. (A) BM cellularity was assessed by the total number of cells isolated per femur flushed with Iscove’s modified Dulbecco’s medium + fetal bovine serum. (B) CFU-C frequency is shown as the number of CFU-C colonies per 2 × 10⁴ BM cells. Each bar indicates total CFU-C number along with the frequency of CFU-granulocyte (CFU-G), CFU-macrophage (CFU-M), CFU-granulocyte, erythrocyte, monocyte, megakaryocyte (CFU-GEMM), CFU-granulocyte macrophage (CFU-GM), and BFU-E colonies. (C) Number of CFU-C per femur was calculated from (CFU-C freq/cell plated) × total BM cells per femur. (D) The frequency of CFU-E colonies per 2 × 10⁴ BM cells. (E) Splenic CFU-C were calculated as the number of CFU-C colonies per 1 × 10⁵ spleen cells and each bar shows both red colonies in black (BFU-E) and non-red colonies in white (all other CFU-C colony types: CFU-G, CFU-M, CFU-GEMM, CFU-GM).

Megakaryocyte ploidy analysis. An experimental cohort is represented by two groups of three C57BL/6J female mice. Each group was treated at 5 weeks old with 5FU or phosphate-buffered saline (PBS). One experimental cohort was used for each time point of 18 hours, 4 days, 7 days and 14 days post-treatment. Data in each fluorescence-activated cell sorting plot is representative from one animal from each group. Megakaryocytes were identified by positive CD41 expression and the DNA content (ploidy) of each megakaryocyte was measured by propidium iodide (PI) intensity. PI intensity doubling was set at 2N, 4N, 8N, 16N, 32N, and 64N on CD41⁺ cells. Table 1 displays the average and standard deviation for each group as well as statistical analysis. Cells positive for CD41 expression were divided into two categories: region one (R1) DNA content of 2N, 4N, or 8N and region two (R2) DNA content of 16N, 32N, and 64N. (A) Ploidy analysis of CD41⁺ cell at 18 hours, 4 days, 7 days, and 14 days after 5FU or PBS treatment. (B) The percentage of CD41-expressing cells in the BM for control or 5FU group 18 hours, 4, 7, and 14 days animals after treatment. Statistically significant differences (p < 0.05) from baseline (D0) are indicated by a line over each group and an asterisk.

ENU breeding scheme and selection of optimized 5FU testing days. (A) Our dominant ENU breeding scheme used for the 5FU screen. The mutagenized mouse strain was 129S1/SVImJ and the backcross strain was C57BL/6J. (B) The recovery kinetics for G1 hybrid (B6:129) control males of RBC, PLT, and WBC are shown in blue. Four independent groups of F1 control mice were used to create a 5FU recovery curve (Group1: D0, D1, D5, D9; Group 2: D0, D2, D6, D8, D12, D16, D20; Group 3: D0, D3, D7, D11; Group 4: D0, D4, D8, D12, D16, D20). The testing days chosen for the 5FU screen to identify defects in multiple lineages in G1 hybrid males are indicated by the black squares.

Analysis of G1 for defects in PLT recovery after 5FU administration. (A) The RBC, PLT, and WBC recovery is shown for all G1 males tested in the 5FU screen. The horizontal red line in each graph indicates no change occurred, and points above the line indicate that the values were increasing between two time points, while points below the line indicate that values were decreasing between the two time points. Platelet overshoot was analyzed on D13 and all G1 mice tested are displayed in the graphs in (B) (females) and (C) (males). Values above the black line in panels (B) and (C) represent outliers (z-score >2.75). The value for each G1 is expressed as a black dot, except outliers that are shown in color. Delayed PLT recovery was assessed on D6 and D8, where PLT values are decreasing or negative and all G1 mice were displayed in the graphs in panel (D) (females) and (E) (males). Values below the black line in panel (D) and (E) represent outliers or PLT values are decreasing between D6 and D8.

Heritability assessment of PLT recovery mutants. In each graph, the black line represents the average of control mice and the error bars represents 1 SD. Outlier mice are shown in red. (A) The G1 founder for the 7321 line, presented with increased PLT overshoot. The 7321 line is heritable, with 15% penetrance. The graph only displays G2 males. (B) The G1 for the 7324 phenotype originally presented with delayed PLT recovery. The 7324 line is heritable with 20% penetrance. Only G2 females are displayed in graph. (C) The 7323 phenotype displays high platelets on D0, but normal PLT recovery. 7323 is heritable with a 30% penetrance. Only G2 males are shown. (D) 7325 phenotype has PLT overshoot on D13. 7325 is heritable with 5/54 female mice affected (18.5% penetrance).