Genomic Fate Maps

描述（由申请人提供）：最近令人惊讶且有争议的发现挑战了有关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，并强调与更简单的模型生物体相比，人们对哺乳动物的发育知之甚少。以透明线虫为例，Sulston 及其同事使用显微镜记录了其一生中每个细胞的诞生和死亡，而这张命运图谱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谱更为复杂，因为它们不透明，需要很长时间才能成熟，并且具有大量细胞。因此，命运图谱实验依赖于用染料或遗传标记标记祖细胞，以便随后识别其后代。然而，这种方法提取的信息很少，因为它只揭示了一群细胞，全部继承相同的标签，共享一个共同的祖先，但没有告诉每个细胞如何彼此相关。为了避免这种情况以及当前细胞命运图谱方法的技术限制，我们提出了一种新策略，通过使用系统发育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出现的顺序，从而回顾性地得出细胞命运图谱。每次细胞分裂时，DNA 复制都会引入突变，尤其是重复序列。在初步研究中，我们证明，对影响多鸟嘌呤束长度的频繁突变进行编目可以推断小鼠细胞在培养物中传代或从单个个体的各种组织中取样的历史。为了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传统命运图谱进行比较。 2. 推进该方法的技术。 3. 相继构建肝细胞命运图谱，以解决有关肝干细胞在发育和疾病过程中的克隆、胚胎和解剖起源的争议。生物学的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。命运图描述了个体内的细胞如何相互关联，可以比作一棵生命树，其中特定细胞的起源可以通过其祖细胞追溯到受精卵。我们提出了一种探索发育生物学的新方法，通过基于每次细胞分裂时不可避免地在基因组中积累的独特突变构建命运图谱。我们首先将研究重点放在肝脏如何发育以及对损伤的反应。

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.