Induction and Specification of Hematopoietic Mesoderm

描述(申请人提供)：中胚层形成和分化为血液和其他衍生物的分子和细胞事件至关重要。 对于我们理解正常发育和疾病的机制很重要。有关造血发育的知识极大地受益于能够分离和鉴定处于不同承诺阶段的细胞群体的方法。很少有表面标记可用于分离致力于造血系和血管系的非常早期的中胚层细胞群。已知的最早的中胚层细胞表面标记是Flk1。基于Flk1和PODXL蛋白的表达，我们成功地分离了ES细胞来源的类胚体中原始的(胚胎的，EryP的)和确定的(成人的)造血系和血管系，并证明了相同的亚群存在于小鼠胚胎中。我们开发了转基因小鼠系，其中荧光蛋白仅在EryP中表达，允许在怀孕期间对其进行标记和跟踪。我们能够分离和FAC纯净、同步的EryP群体，使我们能够确定从祖细胞(卵黄囊)到循环和胎儿六个不同发育阶段的全球基因表达谱。 肝脏分期。前体阶段的EryP表达一组与典型的Wnt/？-catenin途径有关的基因，已知这对正常的原肠形成和中胚层形成是必不可少的。通过分析双转基因小鼠的Wnt/β-catenin(GFP)和EryP(CFP或Cyan)报告基因，我们发现典型的Wnt/β-catenin在大多数EryP祖细胞中都是活跃的，并且存在Wnt活性的梯度，因此在Wnt途径活性最高的细胞中发现了最高的祖细胞活性。有趣的是，在胎儿肝脏中，正常的Wnt通路也活跃在未成熟的终止性红细胞中。了解个体发育过程中的Wnt通路与人类疾病有关，因为它在造血再生过程中被重新激活，并在一定程度上被颠覆 白血病，尽管它似乎不是HSC功能或谱系特异性所必需的 成体的造血分化。在这个方案中，我们将检验假设：()原始的和确定的造血和心血管血统起源于不同的中胚层祖细胞亚群；(2)Wnt/？-catenin通路调节红系发育；(3)规范的Wnt信号通路启动红系对转化生长因子？1的反应。

DESCRIPTION (provided by applicant): The molecular and cellular events underlying the formation and differentiation of mesoderm to blood and other derivatives are critically important for our understanding of mechanisms of normal development and of disease. Knowledge of hematopoietic development has benefitted enormously from methods that allow populations of cells at different stages of commitment to be isolated and characterized. Few surface markers are available for the isolation of very early mesodermal cell populations committed to the hematopoietic and vascular lineages. The earliest known cell surface marker of mesoderm is Flk1. Based on expression of Flk1 and Podocalyxin (Podxl) proteins, we succeeded in separating the primitive (embryonic, EryP) and definitive (adult) hematopoietic and vascular lineages in ES cell-derived embryoid bodies and demonstrated that the same subpopulations are present in mouse embryos. We developed transgenic mouse lines in which fluorescent proteins are expressed exclusively in EryP, permitting their tagging and tracking through gestation. Our ability to isolate and FACS puriy distinct, synchronized populations of EryP allowed us to define global gene expression profiles during six distinct stages of development, from progenitor (yolk sac) through circulating and fetal liver stages. Progenitor stage EryP expressed a set of genes involved in the canonical Wnt/¿-catenin pathway, known to be essential for normal gastrulation and mesoderm formation. By analyzing mice doubly transgenic for reporters for Wnt/¿-catenin (GFP) and EryP (CFP or cyan), we discovered that the canonical Wnt/¿-catenin is active in most EryP progenitors and that there is a gradient of Wnt activity such that the highest progenitor activity is found in cells with the highest Wnt pathway activity. Interestingly, the caonical Wnt pathway is also active in immature definitive erythroblasts in the fetal liver. An understanding of the Wnt pathway during ontogeny is pertinent to human disease because it is reactivated during hematopoietic regeneration and is subverted in certain leukemias, although it does not appear to be essential for HSC function or lineage specific hematopoietic differentiation in the adult. In this proposal, we will test the hypotheses that: () the primitive and definitive hematopoietic and cardiovascular lineages arise from distinct subsets of mesodermal progenitors; (2) the Wnt/¿-catenin pathway regulates erythroid development; and (3) the canonical Wnt signaling pathway primes the erythroid response to TGF¿1.