Stem cell regulation during development and whole-body regeneration - PROJECT SUMMARY Although the early embryos of humans and other vertebrates have pluripotent cells that can differentiate into all cell types of the animal, these flexible cells are absent in adults. In contrast, many invertebrate animals maintain pluripotency beyond embryogenesis, and harbor adult pluripotent stem cells (aPSCs) that enable whole-body regeneration. The long-term goal of the PI’s research program is to obtain a mechanistic understanding of how pluripotent stem cells are made, retained, and regulated in vivo such that they can regenerate any missing cell type in an adult animal. Across 750 million years of animal evolution, cells operate on conserved principles, thus invertebrate species that maintain pluripotent stem cells in adult animals can serve as informative model systems. The overall objective in this application is to identify the molecular and cellular mechanisms that regulate aPSCs called “neoblasts” in the acoel Hofstenia miamia. Hofstenia can regenerate any missing cell type and is amenable to high-throughput functional studies of regeneration. The rationale for choosing a new model system over planarians, the more established system for studying neoblasts, is that Hofstenia produces manipulable embryos in large numbers, allowing the use of methods such as transgenesis, currently unavailable in planarians, to answer outstanding questions about neoblast biology. The experiments proposed here will combine lineage-tracing methods with functional genomics approaches to uncover and characterize critical regulatory control of pluripotent cells, asking three major questions: 1) Which genome-wide chromatin regulatory landscapes and transcriptional programs control the establishment and retention of pluripotent cells in embryos? 2) Which chromatin regulatory mechanisms and cellular dynamics enable neoblasts to achieve pluripotency during regeneration? 3) Which cellular mechanisms allow neoblast progeny to assemble into functional tissues? Isolation of the neoblast lineage followed by transcriptome and chromatin profiling in Hofstenia embryos, techniques that have been feasible in the applicants’ hands, will be utilized to identify chromatin landscapes and their regulators. Transgenesis, a technique recently developed in Hofstenia by the PI, will be used to specifically label neoblast subpopulations to trace their fates. Live imaging of differentiating neoblast progeny and functional studies of cell adhesion genes will be used to study how fate decisions are integrated with cell adhesion to enable assembly of newly regenerated tissues. In all research directions, RNA interference or CRISPR-Cas9 gene editing, two approaches that are feasible in this system, will be used to study gene function. This proposal is innovative in its use of a novel model system that has enabled new approaches, including the labeling and isolation of specific cell lineages, for studying long-standing questions about stem cell biology and regeneration. This project will reveal basic molecular and cellular principles for the regulation of pluripotent cells that have the potential to inform the development of new applications in human regenerative medicine.
