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Article Figures & Data

Figures

  • Fig. 1.

    Developmental anemia in Stat5a−/−5b−/−mice.

    Hematocrit measurement on Stat5a−/−5b−/−, Stat5a+/−5b+/− or wild-type littermates at the ages indicated. The same hematocrit data are tabulated in Table 1. Some of the embryo hematocrit data were published previously.11

  • Fig. 2.

    Stat5a−/−5b−/−-mice with near-normal hematocrit have a sluggish response to erythropoietic stress.

    Six adult Stat5a−/−5b−/− mice with initial hematocrit more than 35% and 6 wild-type controls were injected with phenylhydrazine on days 0, 1, and 3 (indicated by small arrows). The corrected reticulocyte count (also known as reticulocyte index21) allows an assessment of erythropoietic rate, and was calculated assuming a normal hematocrit of 45%, as follows: Corrected reticulocyte count (%) = reticulocyte count (%) × (hematocrit/45).

  • Fig. 3.

    Splenomegaly in Stat5a−/−5b−/− mice correlates with their degree of anemia.

    (A) Hematoxylin and eosin-stained sections of adult spleen from either wild-type or Stat5a−/−5b−/− mice. Thick arrow points at a region of red pulp. Thin arrow points at a region of white pulp. Photographs were taken at an original magnification of × 25. (B) Splenomegaly in Stat5a−/−5b−/−mice inversely correlates with both the hematocrit and hemoglobin concentration. Correlation curve was fitted using Microsoft Excel Trendline tool to the Stat5a−/−5b−/− adult data, excluding one data point from an extremely anemic mouse (in brackets). The curves drawn fit the equations y = 3487 × −2.4 (R2 = 0.91) for the hemoglobin data, and y = 12 089 × −2.0(R2 = 0.72) for the hematocrit data.

  • Fig. 4.

    Flow cytometry assessment of spleen erythroblast maturation.

    Freshly dissociated wild-type mouse spleen cells were labeled with a biotin-conjugated monoclonal antibody (mAb) to CD71 and a PE-conjugated anti-Ter119 mAb, followed by APC-conjugated streptavidin. Dead cells (staining positive with propidium iodide) and anucleated red cells (with low forward scatter) were excluded from analysis. The left-hand panel illustrates a density plot of all viable spleen cells; axes indicate relative fluorescence units for PE (x-axis) and APC (y-axis). Regions I to IV were selected as indicated. The right-hand panels show May-Grunwald Giemsa–stained cytospin preparations of cells sorted from each of regions I to IV. Representative cells from 2 to 3 fields are shown for each region. These are predominantly proerythroblasts in region I, basophilic erythroblasts in region II, late basophilic and chromatophilic erythroblasts in region III, and orthochromatophilic erythroblasts in region IV. The photographs were taken at an original magnification of × 400.

  • Fig. 5.

    Massive increase in the ratio of early to late erythroblasts in spleens of anemic Stat5a−/−5b−/− mice.

    (A) Flow cytometry density plots of spleen cells from 3 representative wild-type mice (numbers 1-3, mean hematocrit = 48.5%) and 3 anemic Stat5a−/−5b−/− mice (numbers 4-6, mean heamtocrit = 23%) from the same mouse colony. Cells were labeled for Ter119 and CD71 as described in Figure 4. The relative number of cells in each of regions I to IV as a percent of all viable, nucleated erythroid cells is indicated on each plot. Erythroid cells (defined as all cells in regions I to IV) in each spleen constituted 61.7%, 53%, and 37.4% of all spleen cells for the wild-type mice (numbers 1-3, respectively); and 87.7%, 93.2% and 91.1% of all spleen cells for Stat5a−/− 5b−/− mice (numbers 4-6, respectively). (B) Flow cytometry forward-scatter distribution histograms of wild-type and Stat5a−/−5b−/−mice (corresponding to mice numbers 1 and 4, respectively, in panel A). Left-hand panel shows data for all Ter119high cells. Middle and right-hand panels show data for Ter119highCD71high (region II) cells and Ter119highCD71low (region IV) cells, respectively. (C) Flow cytometry density plots of spleen cells from a representative wild-type mouse (right-hand panel, hematocrit = 42%) and a representative mouse with a tissue-specific deletion of VHL (VHL−/−, left-hand panel; see “Materials and methods”). The VHL−/− mouse shown had an enlarged spleen and a hematocrit of 84%.

  • Fig. 6.

    Increased apoptosis in Stat5a−/−5b−/− bone marrow erythroblasts.

    Bone marrow from either wild-type or Stat5a−/−5b−/− mice was labeled with CD71 (APC channel) and Ter119 (PE channel) as described in Figure 4. Cells were then incubated with FITC-conjugated annexin V and propidium iodide and analyzed by flow cytometry. Cells positive for propidium iodide were excluded from analysis. The top and bottom left-hand panels show flow cytometry density plots for CD71 and Ter119 similar to those shown for spleen cells in Figures 4 and 5. The right-hand panels show density plots for annexin V binding on Ter119high cells only. The region of increased annexin V binding is indicated and contains 0.4% of wild-type Ter119high cells (top right-hand panel) and 8% of Stat5a−/−5b−/−Ter119high cells (bottom right-hand panel). Data shown are representative of 2 experiments.

  • Fig. 7.

    Decreased bcl-xLexpression in Stat5a−/−5b−/− early erythroblasts correlates with anemia.

    Panels A-B show evaluation of a flow cytometry assay for bcl-xL. (A) Histogram of HCD-57 cells labeled for bcl-xL (x-axis: relative fluorescence). HCD-57 cells were cultured in Epo (1 U/mL) or starved of Epo for 18 hours in the presence of serum. Cells were then fixed, permeabilized, and labeled for bcl-xL using a rabbit polyclonal antiserum (Pharmingen, see “Materials and methods,” here labeled as Pharm556361) and Alexa Fluor 488 goat anti–rabbit IgG. In the presence of Epo, 83% of cells label positive for bcl-xL (marked by “M”); after 18 hours of starvation, this declines to 24%. (B) Bcl-xLlevels in HCD-57 cells growing either in Epo (1 U/mL) or in SCF (100 ng/mL) assessed by flow cytometry or by Western blot analysis. Flow cytometry as described in panel A; 75% of cells are positive for bcl-xL when cultured in Epo, but only 20% of cells express bcl-xL when cultured in SCF. Western blot analysis was carried out either with a polyclonal rabbit IgG bcl-xL(SC-634, Santa-Cruz Biotechnology, Santa Cruz, CA) or with the Pharm556361 antibody, as indicated. (C) A representative flow cytometry measurement of bcl-xL in spleen erythroblasts. Spleen cells from one wild-type and one anemic Stat5a−/−5b−/− mouse were labeled with CD71 (APC channel) and Ter119 (PE channel). Cells were then fixed, permeabilized, and labeled for bcl-xL (Alexa 488 channel). Bcl-xL expression in CD71high cells (which are >90% Ter119+ erythroblasts) is clearly reduced in Stat5a−/−5b−/− cells when compared with wild-type cells (right-hand panel). (D) Bcl-xL expression in CD71high cells was measured as described in panel C, in 5 wild-type mice and 6 Stat5a−/−5b−/− mice in the same experiment. Correlation line was fitted to the Stat5a−/−5b−/− data points (R2 = 0.64). Representative of 3 similar experiments.

  • Fig. 8.

    Increased apoptosis in Stat5a−/−5b−/−neonatal spleens.

    (A) Hematoxylin and eosin-stained sections of neonatal spleen from either wild-type or Stat5a−/−5b−/− mice. Original magnification at the time of photography was × 250. (B) Flow cytometry density plots of wild-type and Stat5a−/−5b−/−neonatal spleen cells simultaneously labeled for Ter119, CD71, and annexin V binding. Regions II to IV are indicated and correspond to the analysis of annexin V binding on these cells shown in panel C. Ter119 expression was variably reduced in Stat5a−/−5b−/− spleens. In the representative plots shown, the geometric mean fluorescence for Ter119 in the Ter119-positive populations is 73 and 68 for wild-type spleens 1 and 2, and 14 and 50 for Stat5a−/−5b−/−spleens 1 and 2, respectively. (C) Annexin V binding of Ter119high erythroblasts in regions II to IV for the spleen cells analyzed in panel B. Inset shows an example of annexin V binding histogram for region II. Cells within the marker M1′ were considered annexin V–postiive.

  • Fig. 9.

    Decreased expression of bcl-xL and increased erythroblast apoptosis correlate with anemia in Stat5a−/−5b−/− neonates.

    (A) Flow cytometry histograms for bcl-xL expression in neonatal spleens. The left-hand histogram contains data from all spleen cells of one representative wild-type neonate and one Stat5a−/−5b−/− neonate. The right-hand histogram contains data from CD71high (early erythroblasts) cells only for the same spleens. In wild-type spleens, 65% of all cells and 94% of CD71high cells express bcl-xL(within the marker “M1”). In Stat5a−/−5b−/−spleens, 11% of all cells and 9% of CD71high cells express bcl-xL. (B) Summary of bcl-xL and annexin V binding data on representative neonates from 2 litters analyzed in the same experiment. Bcl-xL was measured as in panel A; annexin V was measured as described in Figure 8. Correlation lines were fitted to all the data points.

  • Fig. 10.

    Model for Stat5a−/−5b−/− erythropoiesis.

    Erythroid progenitors vary in their sensitivity to apoptosis and in their requirements for antiapoptotic signaling from EpoR.34 Under conditions of maximal erythropoietic rate, during rapid growth or in response to stress, all erythroid progenitors are rescued from apoptosis and give rise to red cells (left-hand panel). Impairment of EpoR antiapoptotic signaling in Stat5a−/−5b−/− neonates under rapid growth conditions will lead to apoptosis of the more sensitive progenitors and result in developmental anemia (middle panel). Surviving Stat5a−/−5b−/− adults are able to partly compensate for the deficit in EpoR antiapoptotic signaling by increasing the size of the early erythroblast population. This may result in improved rates of red cell production at the expense of expanded erythropoietic tissue and increased apoptosis (right-hand panel).

Tables

  • Table 1.

    Hematologic parameters of Stat5a−/−5b−/− mice

    AgeGenotype
    (no. of animals)
    Mean ± SEMP
    Hematocrit (%)Neonates5a−/−5b−/−(65)28.9 ± 0.9*, * P< .0001
    P < .0001
    5a+/−5b+/−(88)41.3 ± 0.5* * P = .0014
    Wild-type (12)47.6 ± 1.6
    3 wk5a−/−5b−/−(12)32.6 ± 1.0*, * P< .0001
    P = .03
    5a+/−5b+/−(23)36.5 ± 1.2* * P = .0006
    Wild-type (5)45.7 ± 1.2
    5-12 wk5a−/−5b−/−(26)27.9 ± 2.0* * P < .0001
    5a+/−5b+/−(3)29.5 ± 6.2* * P = .008
    Wild-type (15)45.2 ± 0.9
    3-6 mo5a−/−5b−/−(21)41.1 ± 1.9* * P = .0014
    5a+/−5b+/−(5)44.0 ± 2.6
    Wild-type (15)49.2 ± 1.0
    MCV (μ3)Neonates5a−/−5b−/−(14)89.7 ± 1.6*, * P< .0001
    P = .0003
    5a+/−5b+/−(25)97.3 ± 1.25
    Wild-type (9)102.8 ± 1.5
    3 wk5a−/−5b−/−(10)56.1 ± 0.73*, * P= .004
    P = .002
    5a+/−5b+/−(18)59.1 ± 0.45
    Wild-type (5)60.0 ± 1.47
    5 wk5a−/−5b−/−(10)51.8 ± 2.75
    5a+/−5b+/−(18)54.6 ± 1.1
    Wild-type (5)54.6 ± 1.3
    Total bilirubin (mg/dL)>6 wk5a−/−5b−/−(9)0.46 ± 0.19
    Wild-type (6)0.12 ± 0.02
    LDH (IU/L)5a−/−5b−/−(9)715 ± 372
    Wild-type (6)282 ± 120
    Serum iron (μg/dL)5a−/−5b−/−(6)161.3 ± 15.1
    Wild-type (6)152.7 ± 17.9
    TIBC (μg/dL)5a−/−5b−/−(6)617 ± 40.0* * P < .0001
    Wild-type (6)427 ± 18.8
    • All measurements were carried out on Stat5a−/−5b−/− mice of the ages indicated, and on Stat5a−/−5b−/−, Stat5a+/−5b+/− or wild-type controls from the same mouse colony. Hematocrit data corresponds to Figure 1.P values are indicated only when P < .05. The increase in total iron-binding capacity (TIBC) in Stat5a−/−5b−/− mice is consistent with ineffective erythropoiesis. Red cell indices in Stat5a−/−5b−/− mice other than MCV were not significantly different than control values. P was calculated using Fisher protected least significant difference (PLSD) post hoc analysis.

    • * Significantly different from wild-type value.

    • Significantly different from Stat5a+/−5b+/− value.

  • Table 2.

    Raised serum Epo in anemic Stat5a−/−5b−/− mice

    Mouse no.GenotypeHematocrit
    (%)
    Hemoglobin
    (g/dL)
    Serum Epo (mU/mL)
    4355a−/−5b−/− 239.5360
    4315a−/−5b−/− 208.4720
    1815a−/−5b−/− 16ND4 000
    4365a−/−5b−/− 41.012 000
    5735a−/−5b−/− 3611.9< 100
    5755a−/−5b−/− 4513.6< 100
    449Wild-type4517.9< 100
    557Wild-type4615< 100
    • Epo was measured using a sandwich ELISA in several anemic and nonanemic Stat5a−/−5b−/− mice and in wild-type littermate controls. Serum Epo below 100 mU/mL could not be reliably measured by this assay. Normal mouse serum Epo is in the range of 10 to 20 mU/mL.22

    • ND indicates not done.