Molecular and functional dissection of the zebrafish hematopoietic stem cell nich

描述(申请人提供)：在发育过程中指定了四种独立的造血祖细胞类型，最终指定了造血干细胞(HSCs)，它可以自我更新，并在成年生物体的一生中提供所有主要的血统。前三种前体细胞类型是暂时性前体细胞，被认为是暂时为胚胎提供血液和免疫细胞，直到HSCs最终出现。直到最近，HSCs的具体位置和时间一直存在很大的争议，但最近的研究已经确凿地证明，它们起源于血源性内皮细胞，这是原始背主动脉腹壁中的一种特殊的内皮细胞，可以转分化为HSCs。在我们的第一个资助期进行的研究中，我们第一次直接从腹主动脉内皮细胞成像了HSC的出生。对这些HSC创建者的互补谱系追踪表明，他们提供所有成人造血细胞，因此是所有HSC的唯一来源。血源性内皮发育规范背后的信号事件仍然知之甚少。在这一应用中，我们将利用斑马鱼胚胎所具有的独特的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，至少需要两次Notch信号才能指定血源性内皮细胞。第一个要求发生在体细胞发生的早期，对于HSC前体来说是非细胞自主的。我们发现，Wnt信号位于Notch要求的上游，通过调节两个Notch配体基因deltaC和deltaD的体细胞表达。这些体细胞信号事件如何与HSC前体的特性有关目前还不清楚，这构成了这一应用的一个主要研究方向。我们的研究将从机制上开始，彻底剖析WNT16信号是如何接收和转导的，以及这些事件如何与DeltaC和DeltaD的调节有关。接下来，我们将确定哪些Notch受体(S)位于DeltaC和DeltaD的下游，以及哪些细胞类型接收到此信号以将指示信号传递给主动脉内皮细胞。同样，我们将致力于进一步剖析和区分指定HSC所需的环境和内在Notch信号的作用。最后，我们将通过努力更好地了解细胞相互作用和迁移事件来补充我们的遗传方法，这些细胞相互作用和迁移事件传递依赖于Wnt16-Notch的信号来形成胚胎HSC生态位。我们对Wnt16-Notch通路的发现代表了已知的对HSC命运最早的环境调节因子之一。随着这个信号轴及其控制的细胞行为的阐明，我们的工作最终将使体外方法能够指导患者特定的iPS细胞走向HSC的命运，用于细胞替代治疗。

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.