Molecular and functional dissection of the zebrafish hematopoietic stem cell nich

描述（由申请人提供）：在发育过程中指定四种独立的造血前体细胞类型，最终指定造血干细胞（hsc），其自我更新并在成年生物体的一生中提供所有主要的血液谱系。前三种前体细胞类型是短暂的祖细胞，被认为暂时为胚胎提供血液和免疫细胞，直到造血干细胞最终出现。直到最近，造血干细胞的具体位置和时间一直存在高度争议，但最近的研究已经明确表明，它们起源于造血内皮，这是原始背主动脉腹壁上的一种特殊内皮细胞群，可转分化为造血干细胞。在我们第一个资助期进行的研究中，我们首次对腹主动脉内皮产生的HSC进行了直接成像。这些造血干细胞建立者的互补谱系追踪表明，它们提供了所有成年造血细胞，因此是所有造血干细胞的唯一来源。造血内皮发育规范背后的信号事件仍然知之甚少。在这项应用中，我们将利用斑马鱼胚胎提供的独特实验优势来测试和完善一种新的HSC诱导模型。我们的初步结果表明Notch信号至少需要两次来指定造血内皮。第一个条件发生在体细胞发生的早期，并且相对于造血干细胞前体是非细胞自主的。我们已经发现Wnt信号通过调节两个Notch配体基因deltaC和deltaD的体细胞表达而位于Notch需求的上游。这些体细胞信号事件与HSC前体规范的关系目前尚不清楚，并形成了该应用的主要研究方向。我们的研究将从机制上开始，彻底剖析Wnt16信号是如何被接收和转导的，以及这些事件如何与deltaC和deltaD的调节联系起来。接下来，我们将确定哪些Notch受体位于DeltaC和DeltaD的下游，以及哪些细胞类型接收该信号并将指导线索传递给主动脉内皮。同样，我们将进一步剖析和区分指定造血干细胞所需的环境和内在Notch信号的作用。最后，我们将补充我们的遗传方法，努力更好地理解细胞相互作用和迁移事件，传递Wnt16-Notch依赖信号来塑造胚胎HSC生态位。我们发现的Wnt16-Notch通路是已知最早的HSC命运的环境调节因子之一。随着这一信号轴及其控制的细胞行为的阐明，我们的工作将最终使离体方法能够指导患者特异性iPS细胞走向HSC的命运，从而用于细胞替代疗法。

DESCRIPTION (provided by applicant): Four independent hematopoietic precursor cell types are specified during development, culminating in specification of hematopoietic stem cells (HSCs), which self-renew and provide all of the major blood lineages over the lifetime of an adult organism. The first three precursor cell types are transient progenitors believed to temporarily provide blood and immune cells to the embryo until HSCs finally emerge. Precisely where and when HSCs are specified has until recently been highly controversial, but recent studies have conclusively demonstrated that they arise from hemogenic endothelium, a special population of endothelial cells within the ventral wall of the primitive dorsal aorta that transdifferentiate into HSCs. In studies performed during our first funding period, we have directly imaged HSC birth from ventral aortic endothelium for the first time. Complementary lineage tracing of these HSC founders indicate that they provide all adult hematopoietic cells, and are thus the unique source of all HSCs. The signaling events underlying the developmental specification of hemogenic endothelium remain poorly understood. In this application, we will utilize the unique experimental advantages aforded by the zebrafish embryo to test and refine a novel model of HSC induction. Our preliminary results suggest that Notch signaling is required at least twice to specify hemogenic endothelium. A first requirement occurs early during somitogenesis and is non-cell autonomous with respect to HSC precursors. We have discovered that Wnt signaling lies upstream of this Notch requirement by regulating the somitic expression of two Notch ligand genes, deltaC and deltaD. How these somitic signaling events relate to the specification of HSC precursors is presently unclear, and form a major research direction of this application. Our studies will begin mechanistically, with a thorough dissection of how the Wnt16 signal is received and transduced, and how these events connect to the regulation of deltaC and deltaD. Next, we will determine which Notch receptor(s) are downstream of DeltaC and DeltaD, and which cell types receive this signal to relay instructive cues to aortic endothelium. Similarly, we will work to further dissect and distinguish the roles of environmental and intrinsic Notch signaling required to specify HSCs. Finally, we will complement our genetic approaches with efforts to better understand the cellular interactions and migration events that relay the Wnt16-Notch dependent signals to pattern the embryonic HSC niche. Our discovery of the Wnt16-Notch pathway represents one of the earliest known environmental regulators of HSC fate. With the elucidation of this signaling axis, and the cellular behaviors it controls, our work will ultimately enable ex vivo approaches to direct patient-specific iPS cells towards the HSC fate for cellular replacement therapies. PUBLIC HEALTH RELEVANCE: Hematopoietic stem cells (HSCs) are rare cells within human bone marrow that are responsible both for the life-long replenishment of all blood cell lineages and for the curative effects of bone marrow transplantation. The creation of human induced pluripotent cells holds great promise for cellular regeneration therapies, but we cannot currently instruct these cells to specifically generate HSCs in vitro. The overall goal of this application is to determine the molecular cues that instruct HSC fate in the vertebrate embryo such that these events may ultimately be replicated in vitro for clinical utility.