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

骨髓（BM）中巨核细胞（mk）释放的血小板减少（血小板减少）和血小板增多（血小板增多）分别导致严重的并发症，如出血和栓塞。因此，了解巨核和血小板形成的机制具有重要的临床和治疗意义。出版物表明，β -连环蛋白信号在骨髓增生性肿瘤的形成中起决定性作用，其特征是mk增加，如原发性血小板增多症和真性红细胞增多症。初步数据显示，Cre/ loxp在“血小板源性生长因子B”启动子（Ctnnb1BM-GOF）下诱导β -catenin的激活，导致mk中β -catenin的激活，并导致小鼠的致死表型。在突变体中，骨髓和脾脏中的mk显著增加，而红细胞压积减少，导致低级别、正红细胞贫血。骨髓造血干细胞未发生改变，可排除致死性的血管原因。基于Wnt/ β -catenin在造血过程中的激活在巨核生成中发挥作用的假设，我们计划利用野生型和转基因小鼠模型在体内表征其在正常造血中的功能，并在体外研究其分子机制。这些任务将在两个独立但互补的研究项目中进行。项目第一部分将通过荧光激活流式细胞术（FACS）和体内免疫标记的三维重建来分析报告小鼠造血过程中β -连环蛋白的基础活性。为了研究调节β -连环蛋白转录对造血系统的影响，我们将在转基因小鼠（Ctnnb1BM-GOF/-LOF）中激活或抑制它。为了排除与造血无关的影响，我们将用Ctnnb1BM-GOF/-LOF骨髓移植野生型小鼠。连续移植将阐明转基因骨髓重建确定的多系造血的潜力。此外，我们将从药理学上激活/抑制β -连环蛋白，并分析巨核生成和红细胞生成。项目第二部分将评估β -catenin在巨核生成中的激活作用，通过分析FACS分类的巨核细胞-红细胞祖细胞（MEPs）和MKs的靶基因调控，以及β -catenin对其转录启动子的调控。随后，我们将通过Chip-Seq分析探索Wnt/ β -catenin信号与已知的巨核生成和红细胞生成调节因子（如体外人K562或CD34+祖细胞中的Runx1）的相互作用，以及β -catenin与mk相关启动子的结合。此外，我们将对野生型和转基因BM的mk （CFU- mk）和红细胞（ERCs） （CFU- e /BFU-E）进行“菌落形成单位”（CFU）检测。该项目将有助于了解Wnt/ β -catenin信号传导对BM中mk和ERCs分化的贡献及其治疗潜力。

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.