Mechanisms of specification, quiescence, and regeneration of primordial germ cells

Project Summary We have been successful at isolating primordial germ cells (PGCs) from echinoderms at different times in their developmental progression. We are now in a position to test the interactions of PGCs with specific elements of their environment to determine how they become the gametogenic stem cells. A strength of this model system is an ability to analyze PGC in vivo, and to compare the different mechanisms between sea urchins (an acquired system of specification) and sea stars (an induced system of specification). Culturing them both gives us strong leverage in analysis. We have attempted benchtop culture, manipulations, and passages, with little success. These cultures quickly become contaminated and the expensive media and isolation success is for naught. The research supported by this request is focused on understanding the mechanisms of formation of primordial germ cells during embryogenesis, how they form during early development, and how they regenerate when the originals are removed. Our work leverages embryos from a sister group to chordates – the sea star and sea urchin. While not common organisms for biomedical research, these echinoderms have many strategic benefits for revealing unique perspectives in the biology of germline formation and regeneration. Millions of synchronous embryos from a single male/female cross allow biochemical and metabolic analysis of the germline, the resultant embryos have ideal transparency for in vivo longitudinal imaging, they develop rapidly, are easy to manipulate (single cell drop-mRNA-seq, optogenetics, cell and tissue transplantations) and they are well suited to complementary gene perturbation approaches (CRISPR/Cas9, morpholino antisense oligonucleotides, MASO), and small molecule perturbations. The existing deep genomic and reagent resources for these animals, coupled with their tractable experimental characteristics, yields a unique system for understanding primordial germ cell biology with defined molecular and morphological endpoints, in live embryos with longitudinal analysis, distinct metrics of quantitation, and transgenerational evaluations.