Dataset: Transcription profiling of mouse megakaryocyte differentiation
Megakaryocyte (MK) differentiation is well described in morphologic terms but its molecular counterparts and the basis for platelet...
Megakaryocyte (MK) differentiation is well described in morphologic terms but its molecular counterparts and the basis for platelet release are incompletely understood. We profiled mRNA expression in populations of primary mouse MKs representing successive differentiation stages. Genes associated with DNA replication are highly expressed in young MKs, in parallel with endomitosis. Intermediate stages are characterized by disproportionate expression of genes associated with the cytoskeleton, cell migration and G-protein signaling, whereas terminally mature MKs accumulate hemostatic factors, including many membrane proteins. We used these expression profiles to extract a reliable panel of molecular markers for MKs of early, intermediate or advanced differentiation, and establish its value using mouse models of defective thrombopoiesis resulting from absence of GATA-1, NF-E2 or tubulinß1. Computational analysis of the promoters of late-expressed MK genes identified new candidate targets for NF-E2, a critical transcriptional regulator of platelet release. One such gene encodes the kinase adaptor protein LIMS1/PINCH1, which is highly expressed in MKs and platelets and significantly reduced in NF-E2-deficient cells. Transactivation studies and chromatin immunoprecipitation implicate Lims1 as a direct target of NF-E2 regulation. Attribution of stagespecific genes, in combination with various applications, thus constitutes a powerful way to study MK differentiation and platelet biogenesis Experiment Overall Design: MK progenitors expand in mouse bone marrow or fetal liver cell preparations cultured with thrombopoietin, and mature over 5-6 days in vitro. After 3 days of culture, a significant fraction of cells shows features of committed MK progenitors, and numerous terminally mature, proplatelet-forming MKs appear by day 5. Because isolation of cell populations that correspond to sequential stages in MK differentiation is hindered by the lack of synchrony in primary MK cultures, we applied flow cytometry to harvest sub-populations that are substantially enriched for MKs with defined properties. High surface expression of the lineage marker CD41 identified MKs, whereas forward-scatter (FSC) properties distinguished cells on the basis of size. We collected thrombopoietic culture suspensions from days 3, 4 and 6, and sorted populations by flow cytometry. RNAs were prepared from sorted cells to probe Affymetrix MOE430A mouse oligonucleotide arrays.
- Dec.12, 2014
- Nov.24, 2014