Genomic Fate Maps

描述(申请人提供)：最近的、令人惊讶的和有争议的发现挑战了关于干细胞的起源和可塑性及其对组织再生的贡献的传统概念，并突显了与更简单的模式生物相比，人们对哺乳动物的发育知之甚少。在透明蠕虫的例子中，秀丽线虫和他的同事用显微镜记录了每个细胞在其生命过程中的出生和死亡，这种命运地图的编制代表了发育生物学的一项里程碑式的成就。确定哺乳动物或其他高等生物的命运图更为复杂，因为它们不透明，需要很长时间才能成熟，而且有大量的细胞。因此，命运图谱实验一直依赖于用染料或遗传标记标记祖细胞，以便以后识别其后代。然而，这种方法提取的信息很少，因为它只揭示了一群继承相同标签的细胞共享一个共同的祖先，但没有说明每个细胞是如何相互关联的。为了避免这种情况以及目前绘制细胞命运图方法的技术局限性，我们提出了一种新的策略，通过使用系统发育学来推断在多细胞生物体的发育过程中个体细胞基因组中出现体细胞突变的顺序，从而追溯地推导出细胞命运图。每次细胞分裂时，DNA复制都会引入突变，特别是在重复序列上。在《初步研究》中，我们证明，对影响聚鸟苷束长度的频繁突变进行分类，可以推断出在培养中传代或从单个个体的不同组织中采集的小鼠细胞的历史。对于构建高分辨率哺乳动物细胞命运图谱的最终目标，我们计划了三个具体目标：1.将系统发育命运图谱与来自基因标记小鼠的常规命运图谱进行比较。2.推进该方法的技术进步。3.相继建立肝细胞命运图，以解决有关肝干细胞在发育和疾病过程中克隆、胚胎和解剖起源的争议。生物学的一个目标是了解单个细胞如何分裂并形成体内所有不同的组织和器官。命运图描述了一个人体内的细胞是如何相互联系的，可以比作一棵生命树，在生命树中，一个特定细胞的起源可以通过它的祖先追溯到受精卵。我们提出了一种探索发育生物学的新方法，即根据每次细胞分裂时不可避免地积累在基因组中的独特突变来构建命运图。我们最初将重点研究肝脏如何发育和对损伤的反应。

DESCRIPTION (provided by applicant): Recent, surprising, and controversial discoveries have challenged conventional concepts regarding the origins and plasticity of stem cells, and their contributions to tissue regeneration, and highlight just how little is known about mammalian development in comparison to simpler model organisms. In the case of the transparent worm, C. elegans, Sulston and colleagues used a microscope to record the birth and death of every cell during its life, and the compilation of this fate maps represents a milestone achievement of developmental biology. Determining a fate map for mammals or other higher organisms is more complicated, because they are opaque, take a long time to mature, and have a tremendous number of cells. Consequently, fate mapping experiments have relied on tagging a progenitor cell with a dye or genetic marker in order to later identify its descendants. This approach, however, extracts little information, because it only reveals that a population of cells, all inheriting the same label, share a common ancestor but tells nothing of how each cell is related to one another. To avoid that, as well as the technical limitations of current methods for mapping cell fate, we propose a new strategy for retrospectively deriving cell fate maps by using phylogenetics to infer the order in which somatic mutations have arisen in the genomes of individual cells during the development of multicellular organisms. DNA replication introduces mutations, particularly at repetitive sequences, every time a cell divides. In Preliminary Studies we demonstrate that cataloging the frequent mutations affecting the length of polyguanine tracts allows for deducing the history of mouse cells either passaged in culture or sampled from the various tissues of a single individual. Toward an ultimate goal of constructing a high resolution mammalian cell fate map, we plan three Specific Aims: 1. Compare phylogenetic fate maps to conventional fate maps derived from genetically marked mice. 2. Advance the technology of this approach. 3. Successively build a liver cell fate map in order to address controversies regarding the clonal, embryonic, and anatomic origins of hepatic stem cells during development and in disease.A goal of biology is to understand how a single cell divides and gives rise to all the different tissues and organs in the body. Fate maps describe how cells within an individual are related to one another and can be likened to a tree of life, in which the origin of a particular cell can be traced back through its progenitors to the fertilized egg. We propose a new method for exploring developmental biology by constructing fate maps based on distinctive mutations inevitably accumulating in the genome each time a cell divides. We will initially focus our studies on how the liver develops and responds to injury.