THE IMPACT OF GENOMIC INSTABILITY ON GERMLINE DEVELOPMENT AND MUTATION ACCUMULATION IN MICE

Abstract

The ability of organisms to pass their genetic information onwards to subsequent generations is crucial for survival and propagation of a species. In mice, embryonic germ cells are set aside very early in development to become the germline lineage. During development, these germ cells rapidly migrate and proliferate to the location of the future gonads over the course of only a few days. Importantly, while DNA replication associated with rapid cell proliferation is often subject to spontaneous errors, the germline has been shown to be highly refractory to mutation accumulation in comparison to somatic cells. To begin understanding the extent to which primordial germ cells (PGCs) are similar, or dissimilar, to other well-studied cell types in their response to altered genomic integrity, I developed a transgenic mouse strain which expresses a DNA double strand break (DSB) sensor specifically in PGCs. This strain provided me with a tool to monitor DNA DSB repair dynamics in vivo. Using this strain, I assessed the impact of ionizing radiation-induced DNA damage on PGCs and examined the impact of this damage on the downstream post-natal germ cell reserve (Chapter 2). Additionally, to better understand the DNA damage response (DDR) in these cells, I exposed pregnant mice to ionizing radiation (IR) at specific gestational time points and assessed the DDR. Our results show that PGCs prior to sex determination lack a G1 cell cycle checkpoint. Subsequent to sex determination, the response to IR-induced DNA damage differs between female and male PGCs. IR of female PGCs caused uncoupling of germ cell differentiation and meiotic initiation, while male PGCs exhibited repression of piRNA metabolism and transposon de-repression (Chapter 3). I also used whole genome single-cell DNA sequencing to assess whether genetic rescue of DNA repair-deficient germ cells leads to increased mutation incidence and biases. I generated Fancm and p21 null mutations on an isogenic strain background and observed that loss of p21 in Fancm null mutants leads to a partial, but significant rescue of germ cells in males. With this Fancm-/- PGC-proliferation defective mutant, I examined how mutation burden is impacted when DNA damage checkpoints are abrogated. Importantly, I observed an increase in the incidence of complex mutations in double mutants, highlighting that rescuing germ cell quantity through checkpoint bypass leads to a decrease in germline genome quality. Lastly, in Chapter 4, I examined how genome integrity is maintained in mouse meiocytes which either have unrepaired meiotic DSBs (Trip13Gt/Gt) or unsynapsed chromosomes (Spo11-/-). I show that signaling through p53 and TAp63, is responsible for elimination of oocytes with asynapsis or unrepaired DSBs. I also show that checkpoint kinase I (CHK1) becomes activated by persistent DSBs in oocytes and to an increased degree when CHK2 is absent. Taken together, the work described uncovers novel insights into how germ cells with DNA damage can become developmentally defective, leaving only those genetically fit cells to establish the adult germline.