The process by which a single cell, such as a fertilized egg, develops into a complex, multicellular animal is perhaps one of the most fascinating questions in modern biology. Because all animals share a common ancestry many aspects of their development are conserved, particularly with regard to the network of genes they employ during the developmental process. Nonetheless, animals appear morphologically very different from each other (e.g., compare a starfish with a human) despite having a strikingly conserved set of developmental genes. To address this disparity, we focus on an understudied yet experimentally elegant organism, the leech, which offers a unique system for dissecting gene expression patterns in the critical early stages of embryogenesis. Specifically, leech embryos are atypically large and divide in asymmetrical patterns such that embryonic stem cells and their precursor cells can be readily identified and physically separated from the embryo, a technique that is not currently possible in more conventional model organisms (e.g., mouse, fly, nematode). Using modern molecular and genomics approaches, we will reconstruct the detailed molecular events that occur inside the embryo that lead to the formation, specification and maintenance of embryonic stem cells in leech. Our hypotheses will be tested directly by manipulating target genes during development, utilizing leech embryos as an experimental platform. This information will contribute to a broader understanding of how ancestral genetic pathways have evolved to generate the remarkable diversity of animal life we currently observe.
The spatiotemporal precision by which totipotent zygotes reproducibly generate properly situated cells (including stem cells) with varying degrees of more restricted cell fate potentials remains mostly an untenable question in conventional model organisms. The project offers an alternative and modern approach to this problem by applying bioinformatic and molecular techniques to a classical system that is uniquely well-suited for such methodologies. Specifically, glossiphoniid leeches provide experimentally tractable embryos whose stereotyped cleavages yield five bilateral pairs of large, identifiable and accessible, lineage-restricted stem cells of four distinct potencies, from which segmental mesoderm and ectoderm arise. Thus, one can routinely collect convenient quantities of RNA from small numbers of identifiable cells at each developmental step, from the zygote up to functioning stem cells. This project will generate transcriptional profiles for a set of uniquely defined stem cells, non-stem cells and selected precursors from which they arise in the leech zygote. Illumina sequencing and proven bioinformatic pipelines will be used to assemble in-depth transcriptomes for stages leading from zygote through the formation of four distinct classes of lineage-restricted stem cells (the M, N, O/P and Q teloblasts). Coupled with the availability of whole genome sequence, this presents an opportunity to profile transcriptional dynamics leading to stem cell formation on a cell-by-cell basis with unprecedented detail and completeness. Since the leech represents an understudied yet evolutionarily strategic member of the super-phylum Lophotrochozoa, this work will provide a critical perspective on the range of molecular genetic pathways leading to what we recognize operationally as stem cells. The experiments are specifically designed to provide meaningful training activities to many undergraduate students.
This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.
