O-fucose Modified Notch as a Regulator of the Hematopoietic Stem Cell Homeostasis

描述（申请人提供）：在干细胞和造血稳态中起关键作用的信号通路之一是Notch信号级联。Notch在许多发育和病理生理过程中起着关键作用。Notch通路的突变或异常激活与多种人类发育障碍和癌症有关。Notch受体被糖分子修饰，聚焦，关键调控Notch受体与其对偶体Notch配体的相互作用，对Notch信号激活很重要。Notch受体的聚焦是由O-聚焦转移酶1 （Pofut1）介导的。使用两种小鼠模型，其中小鼠经过基因修饰，要么在所有携带焦点的糖蛋白或糖脂（FX-/-）的表达上有条件缺陷，要么在包括Notch在内的少数蛋白质的EGF重复序列上仅存在o -连接的焦点聚糖（条件Pofut1-/-），我们最近发现了O-聚焦修饰Notch信号的作用，它对造血稳态至关重要，也是造血干细胞（HSC）在骨髓微环境（也称为生态位）中正确定位的需要。这些发现揭示了聚焦修饰Notch在造血和HSC生物学中的独特作用。在本研究中，为了验证聚焦修饰的Notch对于依赖于Notch-配体相互作用的HSC稳态控制至关重要的假设，我们将首先通过使用转基因小鼠模型和小鼠胚胎干细胞来定义缺乏配体结合所需的Notch聚焦部分对HSC稳态和血液谱系分化的影响。我们还将通过使用尖端成像技术（双光子活体显微镜）来评估pofut1缺陷的造血干细胞，或缺乏Notch配体结合所必需的聚焦的造血干细胞，是否在适当的骨髓龛中移位。然后，我们将确定Notch O聚焦缺失导致异常HSC稳态的机制。我们将评估由于配体结合区域缺乏聚焦而导致Notch与其配体之间的破坏性相互作用是否是导致干细胞生态位异常和HSC功能异常的主要原因。此外，我们将评估Notch通路与其他主要粘附分子和细胞因子之间的相互作用，这些分子和细胞因子对干细胞骨髓归巢和骨髓生态位倒立很重要。最后，我们将确定Notch增强的聚焦修饰在HSC与配体结合和HSC骨髓归巢和沉积中的意义。在这些研究结束时，我们希望我们所获得的知识将提高我们对Notch糖基化在干细胞生物学中的作用的理解，并将形成基础，导致未来干细胞治疗和癌症医学的新治疗策略的研究和发展。

DESCRIPTION (provided by applicant): One of the signaling pathways that play pivotal roles in stem cell and hematopoietic homeostasis is the Notch signaling cascade. Notch plays a critical role in many developmental and pathophysiological processes. Mutation or abnormal activation of Notch pathway is linked to a variety of human development disorders and cancers. Notch receptor modification by a sugar molecule, fucose, critically regulates Notch receptor interaction with their counterparts, Notch ligands, and is important for Notch signaling activation. The addition of fucose to Notch receptors are mediated by protein O-fucosyltransferase 1 (Pofut1). Using two mouse models, in which mice are genetically modified to be conditionally deficient either in the expression of all fucose-carrying glycoproteins or glycolipids (FX-/-), or only O-linked fucosyl-glycans present on EGF repeats of a few proteins including Notch (conditional Pofut1-/-), we recently identified a role of O-fucose-modified Notch signaling that is essential for hematopoietic homeostasis as well as a requirement of fucose modification of hematopoietic stem cell (HSC) for their proper location in the bone marrow microenvironment, also called niche. These findings uncovered a unique role of fucose-modified Notch in hematopoiesis and HSC biology. In this study, in order to test the hypothesis that fucose-modified Notch is critical for Notch-ligand interaction- dependent control of HSC homeostasis, we will first define the impact of lack of Notch fucose moieties that are required for ligand binding on HSC homeostasis and blood lineage differentiation by using genetically modified mouse models and mouse embryonic stem cells. We will also assess if Pofut1-deficient HSCs, or HSCs that lack fucose known essential for Notch ligand binding, are displaced in the proper marrow niches by using the cutting edge imaging technology (2-photon intravital microscopy). Then, we will determine the mechanism(s) by which lack of Notch O-fucose causes aberrant HSC homeostasis. We will assess if a disruptive interaction between Notch and its ligand due to the lack of fucose in the ligand binding domain is the main reason that causes aberrant stem cell niche location and abnormal HSC function. Further, we will assess the interaction between Notch pathway with other major adhesion molecules and cytokines that are important for stem cell bone marrow homing and marrow niche lodging. Finally, we will determine the significance of the enhanced fucose modification of Notch in HSC binding with ligand and in HSC marrow homing and lodgment. At the end of these studies, we hope the knowledge we have gained will improve our understanding of the role of Notch glycosylation in stem cell biology, and will form the basis that leads to future studies and development of novel therapeutic maneuvers in stem cell therapy and cancer medicine.