Genomic Fate Maps

描述（由申请人提供）：最近，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的传统概念，以及它们对组织再生的贡献，并强调了与更简单的模式生物相比，我们对哺乳动物的发育知之甚少。以透明线虫秀丽隐杆线虫为例，萨尔斯顿和同事们用显微镜记录了每个细胞在其生命过程中的出生和死亡，这种命运图的编制代表了发育生物学的里程碑式成就。确定哺乳动物或其他高等生物的命运图更为复杂，因为它们是不透明的，需要很长时间才能成熟，并且有大量的细胞。因此，命运定位实验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，因为它只揭示了所有继承相同标签的细胞群体有一个共同的祖先，而没有说明每个细胞之间是如何相互关联的。为了避免这种情况，以及当前绘制细胞命运图方法的技术限制，我们提出了一种新的策略，通过使用系统遗传学来推断多细胞生物发育过程中单个细胞基因组中体细胞突变发生的顺序，从而回顾性地得出细胞命运图。每次细胞分裂时，DNA复制都会引入突变，尤其是在重复序列中。在初步研究中，我们证明了对影响多鸟嘌呤束长度的频繁突变进行编目，可以推断出小鼠细胞的历史，无论是传代培养还是从单个个体的各种组织中取样。为了实现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：将系统发育命运图与遗传标记小鼠的传统命运图进行比较。2. 推进这种方法的技术。3. 相继建立肝细胞命运图，以解决关于肝干细胞在发育和疾病中的克隆、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产生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关联的，可以比作生命之树，其中一个特定细胞的起源可以追溯到它的祖先到受精卵。我们提出了一种探索发育生物学的新方法，即基于每次细胞分裂时基因组中不可避免地积累的独特突变构建命运图。我们将首先集中研究肝脏的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.