Wnt Signaling in Hematopoietic Stem Cell Specification and Leukemia

描述（由申请人提供）：造血干细胞（HSC）是具有自我更新和再生所有成熟血细胞类型（包括红细胞和免疫细胞）能力的祖细胞。HSC在治疗上用于治疗许多疾病，包括白血病和先天性血液病，但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）如何定向成为组织特异性干细胞（如HSC）是再生医学的关键目标。确定所需调控网络的最明显方法是确定胚胎发育过程中使用的内源性机制。通常，还观察到相关的信号转导通路在白血病中失调，从而了解其进一步临床相关的基础生物学。我最近已经表明，通过Wnt途径的“非经典”、2-连环蛋白/Tcf-非依赖性信号传导是形成HSC所需的，所述Wnt途径先前不知道参与HSC特化。Wnt配体Wnt 16在脊椎动物门中是保守的，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，它是第一批HSC的特化所必需的。我的初步研究结果表明，Wnt 16激活两个Notch配体，deltaC和deltaD的表达，而这些配体反过来又是HSC特化所必需的。虽然注定成为HSC的细胞中Notch信号的细胞自主接收是脊椎动物中的既定要求，但由DeltaC和DeltaD调节的Notch信号传导事件似乎是非细胞自主的，因此代表了对Notch信号传导的独特的、先前未被认识的要求。在这里提出的研究中，我将通过鉴定所需的共受体和细胞内信号转导蛋白，寻求确定Wnt 16与deltaC和deltaD的转录激活之间的精确信号转导途径。初步数据表明，缺乏DeltaC和DeltaD导致体节隔室（与原始背主动脉相邻的硬结）的形成或行为缺陷，该组织在脊椎动物中产生第一批HSC。在背主动脉的内皮变得生血并致力于HSC命运的时间期间，源自巩膜的细胞从体节迁移以成为主动脉周围的平滑肌细胞，并且还可以直接贡献“替代”内皮，这表明巩膜缺陷是HSC特化失败的基础。使用斑马鱼，这是透明的胚胎发育过程中和独特的接受转基因，允许直接可视化的荧光标记的组织，我将产生转基因动物与标记的硬组织，以确定如何在正常和Wnt 16/缺口缺陷动物的组织行为。我将通过条件性消融来测试发育中对硬化节的总体需求，以及通过野生型和显性负性因子的条件性表达来测试硬化节细胞中对特定蛋白的需求。 非典型的Wnt受体Ryk似乎参与Wnt 16信号转导，但不能解释在Wnt 16缺陷动物中观察到的所有效应。这些结果表明存在额外的共受体。该共受体的最强候选家族是非典型Wnt受体的Ror家族。梭线虫Wnt 16的直系同源物EGL-20与单蠕虫Ror直系同源物CAM-1在物理和功能上相互作用。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辅助受体，并且携带musk基因的无效等位基因的未插入突变体的表型表明MuSK也不太可能有助于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）以及一些ALL密切相关。WNT 16也以ALL和CLL的形式错误表达。结合组织培养实验表明WNT 16与前B-ALL有因果关系的事实，这些结果表明WNT 16和ROR 1可能协同或独立地促进白血病发生。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（以前与前B-ALL中WNT 16指导的疾病进展相关）在各种成熟阶段的B细胞中表达。由于没有初始病变是已知的CLL，这些模型有可能是非常翔实的。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.