Wnt Signaling in Hematopoietic Stem Cell Specification and Leukemia

描述(申请人提供)：造血干细胞(HSCs)是一种祖细胞，具有自我更新和再生所有成熟血细胞类型的能力，包括红细胞和免疫细胞。造血干细胞被用于多种疾病的治疗，包括白血病和先天性血液疾病，但获得合适数量的组织相容细胞用于移植仍然是一个问题。确定胚胎干细胞(ESCs)或诱导多能干细胞(IPSCs)如何定向成为组织特异性干细胞是再生医学的一个关键目标。确定必需的调控网络最明显的方法是确定胚胎发育过程中使用的内源性机制。通常，相关的信号转导通路在白血病中也被观察到是失调的，这使得对它们的基础生物学的了解具有进一步的临床意义。我最近的研究表明，在HSC的形成过程中，需要通过Wnt通路传递“非规范的”、非依赖于2-catenin/Tcf的信号，而这在HSC规范中是未知的。Wnt配体Wnt16在脊椎动物门中保守，最初被鉴定为在前B急性淋巴细胞白血病(ALL)中异常上调的基因，是确定第一批HSCs所必需的。我的初步结果表明，WNT16激活了两个Notch配体DeltaC和DeltaD的表达，而这两个配体又是HSC规范所必需的。尽管在脊椎动物中，细胞自主接收Notch信号是一种既定的要求，但由DeltaC和DeltaD调控的Notch信号事件似乎是非细胞自主的，因此代表着对Notch信号的一种独特的、以前未被认识的要求。在这里提出的研究中，我将通过确定所需的辅助受体和细胞内信号转导蛋白来确定位于WNT16和DeltaC和DeltaD转录激活之间的确切信号转导途径(S)。初步数据表明，DeltaC和DeltaD的缺失导致紧邻原始背主动脉的体节隔室的形成或行为出现缺陷，原始背主动脉是脊椎动物中产生第一批HSCs的组织。在背主动脉内皮细胞变得血源性并导致HSC命运的过程中，硬化体来源的细胞从体节迁徙成为环绕主动脉的平滑肌细胞，也可能直接贡献“替代”内皮细胞，这表明硬化体缺陷是HSC规格失败的原因。斑马鱼在胚胎发育期间是透明的，唯一可以接受转基因，允许直接观察荧光标记的组织，我将产生带有标记的硬化体的转基因动物，以确定这种组织在正常和Wnt16/Notch缺陷动物中的表现。我将通过条件消融来测试对菌核发育的总体需求，以及通过野生型和显性负性因子的条件表达来测试对菌核细胞中特定蛋白质的需求。非规范的Wnt受体Ryk似乎参与了Wnt16信号转导，但不能解释Wnt16缺陷动物观察到的所有影响。这些结果表明存在更多的辅助受体。这种共同受体的最强候选家族是非典范Wnt受体的Ror家族。线虫同源基因WNT16、EGL-20与单虫ROR同源基因CAM-1在物理和功能上相互作用。斑马鱼有三个Ror家族成员，穆斯克、Ror1和Ror2。我已经确定Ror2不是所需的Wnt16联合受体，而未堵塞的突变体的表型表明，麝香基因的零等位基因也不太可能对Wnt16的造血表型有贡献。因此，Ror1是Wnt16共同受体的最强候选者。有趣的是，ROR1的错误表达与慢性淋巴细胞白血病(CLL)以及一些ALL密切相关。WNT16也以ALL和CLL的形式错误表达。综上所述，组织培养实验表明WNT16与前B-ALL有因果关系，这些结果表明WNT16和ROR1可能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，在这些动物中，wnt16、ROR1和癌基因E2a-Pbx1在不同成熟阶段的B细胞中表达，其中E2a-Pbx1以前被认为与WNT16导致的前B-ALL疾病进展有关。由于目前尚无慢性淋巴细胞性白血病的起始病变，这些模型有可能提供极大的信息量。最后，我将使用B细胞转基因动物作为平台，通过正向遗传学无偏见地发现与B细胞白血病有关的其他突变。

DESCRIPTION (provided by applicant): 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", 2-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.