The role of Wnt/β-catenin signaling as a mediator of megakaryopoietic cell fate decision

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Decreased (thrombocytopenia) as well as increased (thombocytosis) blood thrombocytes that are released by megakaryocytes (MKs) in the bone marrow (BM), lead to severe complications such as haemorrhages and embolisms, respectively. Hence, understanding the mechanisms involved in megakaryo- and thrombopoiesis is of essential clinical and therapeutic relevance.Publications suggest that beta-catenin signaling plays a decisive role in the formation of myeloproliferative neoplasia, characterised by increased MKs, as essential thrombocythemia and polycythemia vera.Preliminary data show that Cre/loxP-induced activation of beta-catenin under the "platelet-derived growth factor B" promotor (Ctnnb1BM-GOF), leads to beta-catenin activation in MKs and to a lethal mouse phenotype. MKs in the BM and in the spleen were significantly increased in mutants, whereas the haematocrit was reduced, resulting in low-grade, normocytic anaemia. BM haematopoietic stem cells were unaltered and a vascular reason for the lethality could be excluded.Based on the hypothesis that Wnt/beta-catenin activation in haematopoiesis plays a role in megakaryopoiesis, we plan to characterise its function in normal haematopoiesis, using wild type and transgenic mouse models in vivo, and the molecular mechanism in vitro. The tasks will be handled in two separated, but complementing research projects. Project part one will analyse the basal activity of beta-catenin in haematopoiesis in reporter mice via fluorescent-activated flow cytometry (FACS) and 3D reconstruction of immuno labelling in vivo. To investigate the effects of modulating beta-catenin transcription on the haematopoietic systems, we will activate or inhibit it in transgenic mice (Ctnnb1BM-GOF/-LOF). To exclude haematopoiesis-independent effects, we will transplant wild type mice with Ctnnb1BM-GOF/-LOF BM. Serial transplantations will clarify the potential of the transgenic BM to reconstitute definitive, multi-lineage haematopoiesis. Moreover, we will activate/inhibit beta-catenin pharmacologically and analyse the megakaryopoiesis and erythropoiesis.Project part two will evaluate beta-catenin activation in megakaryopoiesis, by analysing target gene regulation by RNA-Seq of FACS sorted megakaryocyte-erythroid progenitors (MEPs) and MKs from Ctnnb1BM-GOF/-LOF mice, and their transcriptional promotor regulation by beta-catenin. Subsequently, we will explore the interaction of Wnt/beta-catenin signaling with known regulators of megakaryopoiesis and erythropoiesis, as Runx1 in vitro in human K562 or CD34+ progenitor cells, as well as the binding of beta-catenin with MK-relevant promotors by Chip-Seq analysis. Additionally, we will perform "colony forming unit" (CFU) assays for MKs (CFU-MK) and erythrocytes (ERCs) (CFU-E/BFU-E) from wild type and transgenic BM.The proposed project will help to understand the contribution of Wnt/beta-catenin signaling to MKs and ERCs differentiation in the BM, and its therapeutic potential.