Characterization of PIWI+ stem cells in Hydractinia

Hydractinia is a colonial marine invertebrate and a member of the phylum Cnidaria. It represents a very powerful tool for studying basic developmental biology and differentiation due to the presence of a population of stem cells (termed i-cells) capable of giving rise to all somatic and germ cell populations. The genes believed to be involved in the regulation of stem cells and their lineages, such as PIWI, are conserved in Hydractinia. PIWI proteins are classical stem and germ cell markers in virtually all studied animals. The Hydractinia genome encodes two PIWI genes, Piwi1 and Piwi2. Phylogenetic analysis placed them as orthologues of other cnidarian PIWI proteins. I show that the genes are co-expressed, at least in adults, exclusively detectable in i-cells and germ cells, and that both PIWI proteins are cytoplasmic. Piwi1 is ubiquitous in early embryos but segregates into embryonic i-cells that remain in the endoderm/gastroderm during later embryonic and larval stages. All Piwi1+ i-cells migrated to the epidermis during metamorphosis and could only be found in the gastroderm in the context of sexual development. Ectopic expression of Piwi1, or its mosaic knockout by CRISPR/Cas9 mediated mutagenesis caused no obvious phenotype. However, transgene expression was silenced in post metamorphic life, and cells with mutated Piwi1 were eliminated in the animals, suggesting that Piwi1 does have an essential role in i-cells. Using EdU/BrdU pulse-chase experiments I identified a rare, slow-cycling Piwi1+ i-cell sub-population that was found exclusively in some areas in the stolons, away from the growing tips. By contrast, all Piwi1+ i-cells in polyps were fast cycling. Stolonal slow-cycling i-cells did not incorporate EdU for at least 12 days. Injuring the colonies resulted in S-phase entry of these cells within 24 hours, suggesting that slow-cycling i-cells represent a reserve stem cell population that is reactivated under stressful situations. PIWI interacting RNAs (piRNAs) have been identified by small RNA IP by Piwi1 protein immunoprecipitation, but their sequence analysis is pending. I suggest that Piwi genes/proteins in Hydractinia are reliable i-cell markers, but i-cells constitute at least two sub-populations with distinct cell cycle characteristics. The developmental potential of these i-cell subsets at single cell level remains to be studied.

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University of Galway Theses (PhD Theses)