Stephen Salipante

Joined Program: 2005 (from UW Medical Scientist Training Program)
Previous Degree: B.S. Microbiology, University of Rochester
Horwitz Lab
stevesal (at) u.washington.edu

Research:
Cell fate maps describe how the sequence of cell division, migration, and apoptosis transform a zygote into an adult. Yet, it is only in C. elegans where microscopic observation of each cell division has allowed for construction of a complete fate map. More complex—and opaque—animals prove less yielding. DNA replication, however, generates somatic mutations. Consequently, multicellular organisms comprise mosaics where most cells acquire unique genomes that are potentially capable of delineating their ancestry.

We have taken a phylogenetic approach to passively retrace embryonic relationships by deducing the order in which mutations have arisen during development. We have demonstrated that polyguanine repeat DNA sequences are particularly useful genetic markers, because they frequently change length during mitosis.

To demonstrate feasibility, we phylogenetically reconstructed the lineage of
cultured mouse NIH3T3 cells based on mutations affecting the length of polyguanine markers. We then employed whole genome amplification to genotype polyguanine markers in single cells taken from a mouse and use phylogenetics to infer the developmental relationships of the sampled tissues. The result was consistent with the present understanding of embryogenesis and demonstrates the large scale potential of this method for producing a complete mammalian cell fate map at the resolution of a single cell.

We are presently focusing our efforts on improving throughput and fidelity of the system, and are beginning to produce second-generation fate maps of the mouse which will hopefully shed light on several outstanding questions of embryogenesis.