Endomitosis in megakaryocytes (MKs) involves repeated DNA replication in the absence of cytokinesis and is a crucial part of MK development. However, chromosomal dynamics have never been observed in living MKs. We developed a new transgenic mouse model in which the expression of human histone H2B fused in-frame to green fluorescent protein is targeted to MKs. Ex vivo time-lapse microscopy analysis indicated that chromosomal condensation occurs at early mitosis in all MKs. In high ploidy MKs (≥8N), late anaphase was marked by a ring-type alignment of chromosomes with multiple territories formed between them. By contrast, in low ploidy MKs mitotic chromosomes segregated to form two groups separated by a clear space before rejoining to one cluster. This is the first study to document chromosomal segregation patterns during endomitosis ex vivo and to indicate their potential differential regulation in low and high ploidy cells.

IntroductionThe megakaryocyte (MK) is a unique hematopoietic cell that undergoes multiple rounds of polyploidization in which the cellular DNA content can reach up to 128N, with 2N being normal diploid content. The process of polyploidization in MKs is thought to play an important role in supporting the large increase in size of mature MKs, which are capable of producing thousands of platelets upon fragmentation, 1 however, much of this process remains enigmatic.Recent studies have implicated a member of the chromosome passenger complex, survivin, as being mislocalized or downregulated at the protein or mRNA level during mitosis of murine MKs of high ploidy. 2,3 In contrast, protein levels and localization of survivin was reported to be typical during mitosis of human megakaryocytes (which, in culture, are typically of low ploidy). 4 Retroviral overexpression of survivin in vitro decreased murine MK ploidy level and number of MK colonies in MK colony assays in comparison to untreated MKs. 3 Similarly, primary vascular smooth muscle cells, which naturally undergo polyploidization in culture, were also shown to decrease their ploidy level upon survivin overexpression. 5 Lately, an increasing number of studies have identified survivin expression in normal adult cells. 3,6,7 Leung et al 7 recently reported on the requirement of survivin in terminal differentiation of erythroid cells and maintenance of hematopoietic stem and progenitor cells. While survivin expression has been demonstrated to be essential in various hematopoietic cell types, the effect of its ectopic expression has not yet been analyzed in vivo. We report the establishment of 2 tissue-specific hematopoietic survivin transgenic models, one overexpressing survivin in both the erythroid and megakaryocyte lineages, and the other solely in the megakaryocytic lineage. MethodsTransgenic mouse production, MK purification, flow cytometry, and MK colony assay Detailed procedures are outlined in Document S1 (available on the Blood website; see the Supplemental Materials link at the top of the online article). Reverse transcriptase-polymerase chain reaction of transgenic mRNATotal RNA was harvested from mouse bone marrow cells using TRIzol reagent, according to the manufacturer's instructions (Invitrogen, Carlsbad, CA). Transgenic mRNA in total bone marrow was detected via reversetranscriptase polymerase chain reaction (RT-PCR) as previously described. 8 ␤-galactosidase assayVisualization of ␤-galactosidase transgene activity in MKs was performed as previously described 8 using freshly isolated bone marrow (BM) from wild-type and PF4-surv mice. Western blot analysisWestern blotting was performed as previously described. 8 Primary antibodies used were anti-survivin, anti-CD41, anti-HSC70 (Santa Cruz Biotechnology, Santa Cruz, CA; sc-10811, sc-15328, and sc-7298, respectively), and anti-␤-actin (Sigma-Aldrich, St Louis, MO; A5441). Acetylcholinesterase assayMK number was calculated based on a positive signal resulting from MK-specific acetylcholinesterase activity...

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