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.
