Phylogenetic Fate Mapping: Following Cellular Lineages in Embryogenesis and Aging

描述（由申请人提供）：细胞谱系图，也被称为细胞命运图，在胚胎发生和衰老的研究中有多种应用，并导致对健康和疾病的更全面的理解。然而，唯一构建了完整细胞命运图的生物是简单的、透明的秀丽隐杆线虫，它可以在显微镜下观察到每个细胞的分裂。高等生物的命运图谱依赖于间接方法，即对细胞进行视觉或遗传标记，以便以后识别其后代，这种方法比直接观察更有局限性，而且信息量也少得多。然而，在哺乳动物中，几乎每次有丝分裂都会产生新的突变，这意味着大多数细胞获得了独特的基因组。子细胞对这种自发体细胞突变的遗传可以被认为是细胞分裂的自然发生记录，其中突变在发育过程中出现的顺序反映了细胞的祖先谱系关系。我们提出了一种构建命运图的系统发育方法，其中我们采用了进化和微生物生物学中用于研究群体结构的系统发育方法，根据细胞的体细胞突变模式回顾性地追踪谱系关系。初步研究表明，多鸟嘌呤重复DNA序列作为高度可变的遗传标记是有价值的，在有丝分裂过程中经常改变长度。基于这些影响多鸟嘌呤标记长度的突变，我们从系统发育上重建了培养小鼠NIH3T3细胞的人工细胞谱系树的历史。然后，我们利用全基因组扩增对取自成年小鼠的单细胞中的多鸟嘌呤标记物进行基因型分析，并利用系统遗传学构建了样本组织的原理命运图。

DESCRIPTION (provided by applicant): The charting of cellular lineages, also referred to as cell fate mapping, has diverse applications in the study of embryogenesis and aging, and has led to a more comprehensive understanding of health and disease. However, the only organism for which a complete cell fate map has been constructed is the simple, transparent roundworm C. elegans, for which it is possible to microscopically observe each cell division. Fate mapping in higher organisms has relied on indirect methods where a cell is visually or genetically tagged in order to later identify its descendants, an approach that is more limiting and substantially less informative than direct observation. In mammals, however, new mutations arise with almost every mitosis, implying that most cells acquire unique genomes. The inheritance of such spontaneous somatic mutations by daughter cells can be thought of as a naturally occurring record of cell divisions, in which the order that mutations have arisen during development reflects cells' ancestral lineage relationships. We propose a phylogenetic approach to constructing fate maps, in which we adapt phylogenetic methods developed to study population structure in evolutionary and microbial biology to retrospectively trace lineage relationships based on cells' patterns of somatic mutations. Preliminary studies have shown that polyguanine repeat DNA sequences are valuable as highly mutable genetic markers, frequently changing length during mitosis. We have phylogenetically reconstructed the history of an artificial cell lineage tree of cultured mouse NIH3T3 cells based on such mutations affecting the length of polyguanine markers. We then have employed whole genome amplification to genotype polyguanine markers in single cells taken from an adult mouse and have used phylogenetics to construct a proof-of principle fate map of the sampled tissues. The aims of this project are to refine and validate the technology of "phylogenetic fate mapping", and to produce a series of second generation phylogenetic fate maps of the mouse liver in order to resolve several outstanding controversies regarding the embryonic origins of the liver, and the production of hepatocytes in the aging animal. As a technology, phylogenetic fate mapping has the potential to make broad contributions to cancer research, the study of mammalian embryogenesis, and the ongoing role of stem cells in the aging organism. We hope that the proposed studies will serve as the basis for examining the processes of organogenesis and aging in more heterogeneous and structurally complex organs.