Evaluation of the actin architecture in dysplastic megakaryocytes expressing the NUP98-HOXD13 leukemic fusion gene

Some myelodysplastic syndrome (MDS) patients present with macrothrombocytopenia due to impaired megakaryocyte (MK) differentiation. Transgenic mice that express the NUP98-HOXD13 (NHD13) fusion gene is a model for MDS and recapitulates the key features of MDS. The study investigated the hypothesis that expression of NHD13 disrupts actin architecture during MK differentiation leading to macrothrombocytopenia. To test the hypothesis, sternums were stained with hematoxylin and eosin, and evaluated by light microscopy to analyze MK morphology in vivo. NHD13 bone marrow (BM) contained many dysplastic MK. BM from wild type (WT) and NHD13 mice were also flushed, cultured in media supplemented with thrombopoietin only or with estrogen to induce proplatelet formation, and MK harvested after 5 days. Harvested MK and BM cores were processed and analyzed by transmission electron microscopy (TEM) to detail the ultrastructural features. TEM of MK revealed that NHD13 leads to formation of an irregular demarcation membrane system and fewer proplatelets. Cultured WT and NHD13 MK were also cytospun onto glass slides, labeled with fluorescent-tagged F-actin, α/β-tubulin and myosin IIa, and their cytoskeleton compared. Interestingly WT MK had actin either distributed evenly or predominantly in the periphery of the cytoplasm, NHD13 MK displayed only the former phenotype. Additionally, proplatelets lacked actin cytoplasmic extensions. The results from the present thesis demonstrate actin expression and architecture are impaired in dysplastic MK expressing the NHD13 leukemic fusion gene and leads to macrothromcytopenia. Understanding the molecular mechanisms of abnormal MK differentiation in MDS is important as many MDS patients die of hemorrhagic complications.