NIH Director's Pioneer Award

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Abstract: Cell fate maps describe how the sequence of cell division, migration, and apoptosis transform a zygote into an adult and are fundamental to understanding stem cell biology. Yet, it is only in the transparent worm C. elegans where tedious microscopic observation of each cell division has allowed for construction of a complete cell fate map. More complexand opaqueanimals prove less yielding. DNA replication, however, inevitably generates somatic mutations. Consequently, multicellular organisms comprise mosaics where most cells acquire unique genomes that are potentially capable of delineating their ancestry. We propose to construct mammalian cell fate maps using a phylogenetic approach to passively retrace embryonic relationships by deducing the order in which mutations have arisen during development. We have found that polyguanine repeat DNA sequences are particularly useful genetic markers, because they frequently change length during mitosis. To demonstrate feasibility, we have used phylogenetics to reconstruct the lineage of cultured mouse NIH3T3 fibroblasts based on mutations affecting the length of polyguanine markers. We have then employed whole genome amplification to genotype polyguanine markers in single cells taken from a mouse and used phylogenetics to infer the developmental relationships of the sampled tissues. Our preliminary results demonstrate the potential of this approach for retrospectively producing a complete mammalian cell fate that, in principle, could describe the developmental lineage of any cell and resolve outstanding questions relevant to stem cell biology.