Gata1cKO^MK mice have a defect in the hematopoietic early precursor compartment

<p>(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/CD32^mid), 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 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154342#pone.0154342.ref031" target="_blank">31</a>]. The dot plot depicts the extra population, named II+ found exclusively in Gata1cKO^MK bone marrow. (c) Whisker/Box plot depicts plasma TPO levels from Gata1cKO^MK and WT^lox 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 Gata1cKO^MK and WT^lox megakaryocytes.</p

Functional analysis of platelets from Gata1cKO^MK and SykcKO^MK mice show overlapping defects.

<p>(a) Flow cytometry-based platelet aggregation assay (FCA) shows the aggregation capacity of platelets when stimulated with different agonists. Gata1cKO^MK and WT^lox platelets were studied. PMA, phorbol myristate acid; Agg, aggretin; Botro. Botrocetin; Coll, collagen; CVX, convulxin. (b) MFI of receptors expressed on Gata1cKO^MKplatelets, relative expression of a given receptor in WT^lox 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 SykcKO^MK and WT^lox platelets when stimulated with various agonists as described above.</p

Gata1 regulates Syk expression.

<p><b>(a)</b> Syk mRNA expression levels in Gata1cKO^MK and WT^lox cultured megakaryocytes measured by qRT-PCR. (b) Syk protein levels in Gata1cKO^MK and WT^lox platelets, analyzed by Western blotting. Syk expression level normalized to loading control Gapdh is indicated, setting the average expression levels of Syk in WT^lox platelets to 100. (c) Chromatin immunoprecipitation (ChIP) assay showsGata1 binding to the <i>Syk</i> promoter in WT^lox compared to Gata1cKO^MK (background control) cultured megakaryocytes. A GATA positive (+) site on the promoter of the known target <i>Gp1ba</i> (CD42b) was used as positive control and a GATA negative (−) site on the promoter of <i>Cd9</i> was used as negative control.</p

Gata1cKO^MKmice show alterations in the erythroid compartment.

<p>(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 Gata1cKO^MK and control mice shows the splenomegaly that Gata1cKO^MKdevelop.</p