Responses to transposon activity and genome damage during germline development

Abstract

The germline is the cell lineage that is responsible for the inheritance of genetic information in multicellular eukaryotes. In most animal species, germ cells are set aside from somatic lineages during early embryonic development and follow a unique developmental programme that culminates in the production of haploid gametes carrying the genetic material that is passed to the next generation. Due to its central role in genetic inheritance, the germline is known to be the battleground where genetic conflicts between selfish genetic elements, like transposons, and the host genome take place. Excessive transposon activity fulfils the selfish drive of transposons to increase in copy number in the host genome but can impair genome integrity and functionality, thereby threatening the faithful transmission of genetic information. How transposon activity affects the genome and how this impacts germ cell functionality and viability remains poorly understood. This thesis examines responses to transposon activity and genome damage during germline development. I use a classic example of transposon-induced sterility in Drosophila, P-M hybrid dysgenesis, as a model. I show that excessive activity of the P-element transposon in embryonic primordial germ cells (PGCs) leads to the accumulation of DNA double strand breaks (DSBs) and sustained cell cycle arrest prior to fully penetrant germ cell loss during early larval stages. Using a genetic screen for suppressors of the dysgenic sterility phenotype, I identify factors involved in cell cycle regulation and DNA damage responses that play a role in the process leading to germ cell loss in dysgenesis. I then develop a novel approach to characterise new P-element transposition events in PGCs genome-wide and at single-cell resolution. Contrary to the prevailing model that germ cell death is caused by high numbers of new transposition events into coding regions, I demonstrate that dysgenic PGCs acquire few new P-element insertions in gene promoters and introns prior to germ cell loss. I then explore the alternative hypothesis that germ cells are sensitive to the genome damage caused by transposon activity. Using engineered, Cas9-based systems, I show that inducing DNA DSBs at endogenous, silenced P-elements or other, non-coding sequences is sufficient to induce complete germ cell loss during development independent of gene disruption. Indeed, I find that both PGCs and adult mitotic germ cells are sensitive to DSBs in a dosage-dependent manner. Following the mitotic-to-meiotic transition, however, germ cells become more tolerant to DSBs, completing oogenesis despite accumulated genome damage and adverse effects on the development of the next generation. Finally, I investigate tolerance to DSB dosage in somatic cellular domains. Collectively, the findings presented in this thesis demonstrate the existence of developmentally regulated, dosage-dependent DNA damage tolerance thresholds that, on the one hand, safeguard genome integrity during germline development, while on the other hand forming a selective barrier that may shape transposon proliferation strategies. This work serves as a foundation for further study of how responses to genome damage in the germline influence genetic conflicts.