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

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Project Summary Sexual reproduction requires a germline, a lineage of cells formed in early embryos that ultimately develops into eggs or sperm in the adult. Lore has it that when you lose your germline in development, you become a sterile adult. In many animals, especially in the current favorites of lab animals, that is largely true. We use “rule breakers” though and find such lore unfounded. Our work focuses on the biology of primordial germ cells, 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, morpholinoantisense 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. We interrogate all levels of gene expression for this work, from chromatin modification to post- transcriptional processing and post-translational networks, because that is what the embryos are “telling” us is needed to understand these complex, and deeply rooted events in sexual reproduction. Our work emphasizes longitudinal, in vivo analysis using high resolution optical imaging coupled with genomic perturbations, signal pathway manipulations and manual transplantations and expirations to leverage contrasting mechanisms in germ cell formation between closely related organisms. Sea urchins and sea stars have historically not been genetically manipulated, and this reason is precisely how germ line regeneration has been discovered in this and other animals seen to bear this trait. Relying on manual manipulations meant the genes needed for regeneration were not disturbed, revealing their germ cell regenerative abilities. With new state-of-the-art technologies, these animals can now be exploited with transgenerational analysis. Overall, our work interrogates important biological questions from unique experimental perspectives using rule-breaking models for innovation in the pursuit of new knowledge.