The ability of organisms to pass their genetic information onwards to subsequent generations is crucial for survival and propagation of a species. In mice, primordial germ cells (PGCs) are set aside very early in development to become the germline lineage. PGCs are first distinguishable as a group of 45 cells in the epiblast of 6-6.5 day old embryos (E6-6.5). These cells then simultaneously migrate and proliferate to the genital ridges (the location of the future gonads) at E10.5 and by E13.5 the population of PGCs reaches its ultimate peak of approximately 25,000 cells.
Because DNA replication associated with rapid PGC proliferation is subject to spontaneous errors, and because PGCs carry the genetic information that will be passed down to the next generation, mechanisms exist to avoid the propagation of these mutations. In accordance with the desire to maintain genomic integrity in the germline, studies have revealed that PGCs are, to a greater extent than somatic cells, highly sensitive to genetic defects and environmental perturbations affecting DNA. While studies of cultured somatic cells and single-celled eukaryotes such as yeast have elucidated DNA damage response pathways, cell cycle checkpoints, and DNA repair mechanisms, our understanding of these processes in the mammalian germline is much less clear. To investigate how genomic integrity is maintained in the mammalian germline, we are characterizing DNA damage response mechanisms in primordial germ cells. We found that mutations affecting two different DNA damage response pathways: Fanconi Anemia that respond to errors in DNA replication and MCM9 being involved in double strand break repair, trigger cell cycle slowdown rather than apoptosis. Double mutant analyses suggested that canonical checkpoint pathways are not involved in responding to these defects in PGCs. To further explore these findings, we are conducting studies to examine mutational burden in mice defective for certain checkpoint pathways, as well as using CRISPR/Cas9 genome editing to develop in vivo DNA damage pathway sensors.