Fig 1.
Gata1cKOMK mice display severe macrothrombocytopenia and platelet dysfuncion while other blood cell counts are unaffected.
(a) Gata1 and PU.1 (Spi1) mRNA expression levels in Gata1cKOMK and WTlox cultured megakaryocytes. PU.1 (Spi1) levels are normal in Gata1cKOMK mice. RFE, relative fold enrichment. (b) Blood parameters of Gata1cKOMK and WTlox mice at 8–12 weeks of age. PLT, platelets; MPV, mean platelet volume; RBC, red blood cells; WBC, white blood cells. (c) Platelet adhesion to collagen under physiological shear rate. Whole blood from WTlox and Gata1cKOMK mice was perfused over collagen-coated slides. Adhered platelets were visualized with CD61-FITC conjugated antibody, and immunofluorescence coverage of slides was quantified, and is represented.
Fig 2.
Functional analysis of platelets from Gata1cKOMK and SykcKOMK mice show overlapping defects.
(a) Flow cytometry-based platelet aggregation assay (FCA) shows the aggregation capacity of platelets when stimulated with different agonists. Gata1cKOMK and WTlox platelets were studied. PMA, phorbol myristate acid; Agg, aggretin; Botro. Botrocetin; Coll, collagen; CVX, convulxin. (b) MFI of receptors expressed on Gata1cKOMKplatelets, relative expression of a given receptor in WTlox platelets was set to 100. For clarification: CD61 (Itgb3), CD41 (Itga2b), CD42a (GPIX), CD42b (Gp1ba), CD42c (Gp1bb), CD49b (Itga2). (c) Flow cytometry-based platelet aggregation assay (FCA) shows the aggregation capacity of SykcKOMK and WTlox platelets when stimulated with various agonists as described above.
Fig 3.
Gata1 regulates Syk expression.
(a) Syk mRNA expression levels in Gata1cKOMK and WTlox cultured megakaryocytes measured by qRT-PCR. (b) Syk protein levels in Gata1cKOMK and WTlox platelets, analyzed by Western blotting. Syk expression level normalized to loading control Gapdh is indicated, setting the average expression levels of Syk in WTlox platelets to 100. (c) Chromatin immunoprecipitation (ChIP) assay showsGata1 binding to the Syk promoter in WTlox compared to Gata1cKOMK (background control) cultured megakaryocytes. A GATA positive (+) site on the promoter of the known target Gp1ba (CD42b) was used as positive control and a GATA negative (−) site on the promoter of Cd9 was used as negative control.
Fig 4.
Gata1cKOMKmice have a defect in megakaryopoiesis.
(a) Gating strategy to identify megakaryocytes at consecutive stages of differentiation in the bone marrow and the spleen based on surface marker expression CD31, CD61, CD41 and CD42b. The dot plot depicts the extra population, named II+ found exclusively in Gata1cKOMK bone marrow. On the right we show the ploidy status of the individual subpopulations, thereby justifying our gating strategy. (b) Percentage of megakaryocytes at consecutive stages of differentiation (I-V) of nucleated bone marrow and spleen cells. (c) Ploidy staining of CD41-positive bone marrow and spleen megakaryocytes. The right bar graph depicts ploidy staining of CD41+ in bone marrow. (d) Ploidy status of gated II+ megakaryocyte differentiation stage vs total CD41+ cells in two representative Gata1cKOMK bone marrow samples.
Fig 5.
Gata1cKOMK mice have a defect in the hematopoietic early precursor compartment
(a) Flow cytometry analysis of the stem cell and committed progenitor compartment. LSK, Lin-|Sca-1+|Kit+ cells; MP (Lin-|Sca-1-|Kit+), multipotent progenitors; CMP (MP gate—CD34+|CD16/CD32mid), common myeloid progenitor; GMP (MP gate—CD34-|CD16/CD32+), granulocyte-monocyte progenitor; MEP (MP gate—CD34-|CD16/CD32-), megakaryocyte-erythroid progenitor. (b) Percentage of the different hematopoietic progenitors. Absolute cell number of bone marrow megakaryocytes at consecutive stages of differentiation [31]. The dot plot depicts the extra population, named II+ found exclusively in Gata1cKOMK bone marrow. (c) Whisker/Box plot depicts plasma TPO levels from Gata1cKOMK and WTlox blood samples, as measured by ELISA. At least 5 mice were analyzed per genotype. (d) qPCR analysis of Pf4 mRNA expression levels in cultured bone marrow derived Gata1cKOMK and WTlox megakaryocytes.
Fig 6.
Gata1cKOMKmice show alterations in the erythroid compartment.
(a) Gating strategy to identify erythrocytes at consecutive stages of differentiation in the bone marrow and the spleen based on surface marker expression KIT, CD71 and Ter119. (b) Percentage of reticulocytes at consecutive stages of differentiation of live cells. Left graph depicts the bone marrow compartment, right the splenic compartment. (c) Photograph of representative spleens from Gata1cKOMK and control mice shows the splenomegaly that Gata1cKOMKdevelop.