Wnt Signaling in Hematopoietic Stem Cell Specification and Leukemia

项目总结/摘要 造血干细胞（HSC）是具有自我更新和再生能力的祖细胞， 成熟的血细胞类型，包括红细胞和免疫细胞。hsc是 在治疗上用于治疗包括白血病和先天性血液病在内的多种疾病， 但是获得合适数量的组织相容性细胞用于移植仍然是一个问题。确定 胚胎干细胞（ESC）或诱导多能干细胞（iPSC）是如何定向成为组织的 特定的干细胞如HSC是再生医学的关键目标。最明显的方法来定义 所需的调控网络是确定胚胎发育过程中使用的内源性机制， 发展通常，还观察到所涉及的信号转导途径在白血病中失调， 了解它们的基础生物学，进一步临床相关性。我最近发现，“非- 经典的”，ω-连环蛋白/Tcf-独立的信号传导的Wnt途径，这是以前不知道的是， 参与HSC特化，是形成HSC所必需的。Wnt配体Wnt 16是保守的， 在脊椎动物门中，最初被鉴定为在前B细胞急性白血病中异常上调的基因。 淋巴细胞白血病（ALL）是第一批HSC的特化所必需的。初步结果显示， Wnt 16激活两种Notch配体deltaC和deltaD的表达，并且这些配体依次 HSC规范所需的冗余。尽管在小区中Notch信号的小区自主接收 注定成为HSC是脊椎动物中的既定要求，Notch信号传导事件由 DeltaC和DeltaD似乎是非细胞自主的，因此代表了一种独特的，以前的 Notch信令的未被重视的需求。在这里提出的研究中，我将寻求确定 位于Wnt 16和deltaC的转录激活之间的精确信号转导途径， δ D，通过鉴定所需的共受体和细胞内信号转导蛋白。初步数据 这表明缺失DeltaC和DeltaD导致体节形成或行为缺陷 室，巩膜，这是邻近原始背主动脉，该组织产生的， 第一个脊椎动物的HSC。在背主动脉内皮细胞生血， 在HSC的命运中，来源于巩膜的细胞从体节迁移成为平滑肌细胞 在主动脉周围，也可能直接有助于“替代”内皮，这表明， HSC规范失败的原因在于缺陷。利用斑马鱼，在胚胎期是透明的 发育和独特的接受转基因，允许直接可视化的荧光标记 组织，我将用标记的硬组织产生转基因动物，以确定这种组织在 正常和Wnt 16/Notch缺陷动物。我将通过以下方式测试开发中对硬组织切片器的总体要求： 条件性消融，以及通过条件性表达 野生型和显性负因子。 非经典Wnt受体Ryk似乎参与Wnt 16信号转导，但不能参与Wnt 16信号转导。 解释了在Wnt 16缺陷动物中观察到的所有效应。这些结果表明， 额外的辅助受体。该共受体的最强候选者家族是Ror家族的非- 典型Wnt受体。梭Wnt 16的elegans直系同源物EGL-20在物理和功能上与 单蠕虫Ror直系同源物CAM-1。斑马鱼有三个Ror家族成员，MuSK，Ror 1和Ror 2。我 已经确定Ror 2不是所需的Wnt 16共受体，并且未插入突变体的表型， 携带麝香基因的无效等位基因，表明MuSK也不太可能导致Wnt 16 造血表型因此，Ror 1是Wnt 16共受体的最强候选者。有趣的是， ROR 1错误表达与慢性单核细胞白血病（CLL）以及某些ALL密切相关。 WNT 16也以ALL和CLL的形式错误表达。再加上组织培养 实验表明WNT 16与前B-ALL有因果关系，这些结果表明WNT 16和 ROR 1可以协同或独立地促进白血病的发生。为了测试这些可能性，我将 产生转基因动物，其中wnt 16，ror 1和癌基因E2 A-PBX 1，其先前已被 与前B-ALL中WNT 16介导的疾病进展相关，在B细胞中以多种表达水平表达。 成熟阶段由于CLL没有已知的起始病变，因此这些模型有可能成为 信息量很大最后，我将使用B细胞转基因动物作为一个平台，公正地发现 通过正向遗传学参与B细胞白血病的其他突变。

Project Summary/Abstract Hematopoietic stem cells (HSCs) are progenitor cells that have the ability to both self-renew and regenerate all mature blood cell types, including red blood cells and immune cells over the lifetime of an individual. HSCs are used therapeutically in the treatment of numerous diseases including leukemia and congenital blood disorders, but obtaining suitable numbers of histocompatible cells for transplantation remains a problem. Determining how embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) are directed to become tissue specific stem cells such HSCs is a key goal of regenerative medicine. The most obvious approach to defining required regulatory networks is to determine the endogenous mechanisms used during embryonic development. Often, involved signal transduction pathways are also observed to be dysregulated in leukemia, making an understanding of their basic biology of further clinical relevance. I have recently shown that "non- canonical", ¿-catenin/Tcf-independent signaling by the Wnt pathway, which was not previously known to be involved in HSC specification, is required for formation HSCs. The Wnt ligand, Wnt16, which is conserved across vertebrate phyla and was originally identified as a gene aberrantly upregulated in pre-B acute lymphocytic leukemia (ALL), is required for specification of the first HSCs. My preliminary results show that Wnt16 activates expression of two Notch ligands, deltaC and deltaD, and that these ligands are in turn required redundantly for HSC specification. Although cell-autonomous reception of a Notch signal in cells fated to become HSCs is an established requirement in vertebrates, the Notch signaling events regulated by DeltaC and DeltaD appear to be non-cell-autonomous and therefore represent a distinct, previously unappreciated requirement for Notch signaling. In the research proposed here, I will seek to determine the precise signal transduction pathway(s) that lie between Wnt16 and transcriptional activation of deltaC and deltaD, by identifying the required co-receptors and intracellular signal transduction proteins. Preliminary data suggest that absence of DeltaC and DeltaD leads to defects in the formation or behavior of a somite compartment, the sclerotome, which is adjacent to the primitive dorsal aorta, the tissue that gives rise to the first HSCs in vertebrates. During the time when endothelium of the dorsal aorta becomes hemogenic and commits to an HSC fate, sclerotomally derived cells emigrate from the somite to become smooth muscle cells surrounding the aorta and may also directly contribute "replacement" endothelium, suggesting that sclerotomal defects underlie failure of HSC specification. Using zebrafish, which are transparent during embryonic development and uniquely receptive to transgenesis, allowing direct visualization of fluorescently labeled tissues, I will generate transgenic animals with labeled sclerotome, to determine how this tissue behaves in normal and Wnt16/Notch-deficient animals. I will test the overall requirement for sclerotome in development by conditional ablation, and the requirement for specific proteins in sclerotomal cells by conditional expression of wild-type and dominant negative factors. The non-canonical Wnt receptor, Ryk appears to participate in Wnt16 signal transduction, but cannot explain all of the effects observed in Wnt16 deficient animals. These results suggest the presence of additional co-receptors. The strongest family of candidates for this co-receptor is the Ror family of non- canonical Wnt receptors. The C. elegans ortholog of Wnt16, EGL-20 interacts physically and functionally with the single worm Ror ortholog, CAM-1. Zebrafish have three Ror family members, MuSK, Ror1, and Ror2. I have determined that Ror2 is not the required Wnt16 co-receptor, and the phenotype of the unplugged mutant, which carries a null allele of the musk gene, suggests that MuSK is also unlikely to contribute to the Wnt16 hematopoietic phenotype. Thus, Ror1 is the strongest candidate for the Wnt16 co-receptor. Interestingly, ROR1 misexpression is strongly associated with chronic lympocytic leukemia (CLL) as well as some ALL. WNT16 is also misexpressed in forms of ALL and CLL. Taken together with the fact that tissue culture experiments suggest that WNT16 is causally involved in pre-B-ALL, these results suggest that WNT16 and ROR1 may cooperatively or independently contribute to leukemogenesis. To test these possibilities, I will generate transgenic animals in which wnt16, ror1, and the oncogene E2A-PBX1, which has previously been associated with WNT16-directed disease progression in pre-B-ALL, are expressed in B-cells at a variety of maturation stages. Since no initiating lesions are known for CLL, these models have the potential to be extremely informative. Finally, I will use the B-cell transgenic animals as a platform for unbiased discovery of additional mutations that are involved in B-cell leukemia by forward genetics.