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（血小板减少症）和增加（血小板增多症）分别导致严重的并发症，如出血和栓塞。因此，了解巨核细胞和血小板生成的机制具有重要的临床和治疗意义。出版物表明，β-连环蛋白信号传导在骨髓增生性肿瘤的形成中起决定性作用，其特征在于MK增加，初步数据显示，Cre/loxP诱导的β-连环蛋白在“血小板衍生生长因子B”启动子下活化，（Ctnnb 1BM-GOF），导致MK中的β-连环蛋白活化和致死小鼠表型。突变体的骨髓和脾脏中的MK显著增加，而红细胞压积降低，导致低级别正常红细胞性贫血。基于Wnt/β-catenin在造血中的激活在巨核细胞生成中起作用的假设，我们计划利用野生型和转基因小鼠模型在体内验证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处理。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3D重建来分析报告小鼠造血中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（Ctnnb 1BM-GOF/-LOF）中激活或抑制它。为了排除造血非依赖性效应，我们将用Ctnnb 1BM-GOF/-LOF BM移植野生型小鼠。连续移植将阐明转基因骨髓重建永久性多系造血的潜力。第二部分通过RNA-Seq分析Ctnnb 1BM-GOF/-LOF小鼠巨核-红系祖细胞（MEPs）和巨核-红系祖细胞（MK）的靶基因调控，以及β-catenin对MEPs和MK转录启动子的调控，评价β-catenin在巨核细胞生成中的激活作用。随后，我们将探索Wnt/β-连环蛋白信号传导与已知的巨核细胞和红细胞生成调节因子的相互作用，如体外人K562或CD 34+祖细胞中的Runx 1，以及通过Chip-Seq分析β-连环蛋白与MK相关启动子的结合。此外，我们将进行“集落形成单位”（CFU）测定MK（CFU-MK）和红细胞（ERCs）（CFU-E/BFU-E）从野生型和转基因BM。拟议的项目将有助于了解Wnt/β-catenin信号转导的MK和ERCs分化的BM，及其治疗潜力的贡献。

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.