Phylogenetic Fate Mapping: Following Cellular Lineages in Embryogenesis and Aging

描述（由申请人提供）：细胞谱系的绘制，也称为细胞命运绘图，在胚胎发生和衰老的研究中具有多种应用，并导致对健康和疾病的更全面的理解。然而，唯一一个已经构建了完整细胞命运图的生物体是简单、透明的蛔虫C。elegans，其中可以显微镜下观察每个细胞分裂。高等生物的命运映射依赖于间接方法，其中细胞被视觉或遗传标记，以便以后识别其后代，这种方法比直接观察更具限制性且信息量少得多。然而，在哺乳动物中，几乎每次有丝分裂都会出现新的突变，这意味着大多数细胞获得独特的基因组。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，其中在发育过程中发生突变的顺序反映了细胞的祖先谱系关系。我们提出了一种系统发育的方法来构建命运图，在该方法中，我们采用系统发育方法来研究进化和微生物生物学中的种群结构，以回顾性地追踪基于细胞体细胞突变模式的谱系关系。初步研究表明，聚鸟嘌呤重复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。我们已经基于影响聚鸟嘌呤标记物长度的这种突变，通过遗传学方法重建了培养的小鼠NIH 3T3细胞的人工细胞谱系树的历史。然后，我们采用了全基因组扩增的基因型多鸟嘌呤标记的单细胞取自成年小鼠，并已使用遗传学构建了一个证明的原则命运图的采样组织。 本项目的目的是完善和验证“系统发育命运图谱”技术，并制作一系列小鼠肝脏的第二代系统发育命运图谱，以解决有关肝脏胚胎起源和衰老动物肝细胞产生的几个悬而未决的争议。 作为一种技术，系统发育命运作图有可能为癌症研究、哺乳动物胚胎发生研究以及干细胞在衰老生物体中的持续作用做出广泛贡献。我们希望，拟议的研究将作为基础，检查在更异质和结构复杂的器官的器官发生和衰老的过程。

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.