The identification of novel factors required for the development of primordial germ cells

DNA is constantly exposed to both exogenous and endogenous sources of DNA damage. Therefore, organisms evolved a plethora of DNA repair pathways to repair these distinct lesions and prevent mutations from arising. Mutations that occur in somatic cells can lead to disease, which may be detrimental to that single individual. However, germ line mutations can be passed on to the next generation. These mutations not only drive genome evolution but can also impair the fitness of the offspring. It has been shown that primordial germ cells (PGCs), the germ cells specified during embryonic development, have the highest mutation frequency compared to germ cells belonging to other stages of gametogenesis. Furthermore, mutations in PGCs are particularly important because mutations in these cells can affect multiple offspring. Evidence shows that during PGC development, which entails extensive genomic and epigenomic transactions, there is the generation of DNA damage. However, the DNA repair pathways used in PGCs and the nature of DNA damage encountered by these cells which can provide mechanistic insight into how mutagenesis is suppressed in the germ line remains largely unexplored. Here we describe the use of an in vitro system that generates PGC-like cells (PGCLCs) from ESCs to discover novel factors required for PGC development. Using this system, we have firstly shown that we can recapitulate the genetic requirement for DNA repair in PGCLCs in vitro. Secondly, we coupled this system with CRISPR/Cas9 screening technology, which permitted us to identify novel factors required during the development of PGCLCs. We identified the translesion synthesis (TLS) factor Rev1 as a key factor involved in PGCLC development. Furthermore, we validated this requirement in vivo and showed that absence of Rev1 causes a numeric reduction in PGCs during the migratory period of PGC development. We also identified additional factors, namely involved in homologous recombination, chromosome cohesion which will be investigated in the future. This screen permitted the identification for the first time of the requirement for TLS in PGCs suggesting that the germ cell lineage may employ this mutagenic pathway to promote genome evolution.PD/BD/105747/2014 - PhD fellowship provided by the Portuguese Foundation for Science and Technology Strauss Bequest Fun