The Role of Dazl in Maintenance of Pluripotency and Murine Germ Cell Development in vivo and in vitro

Mammalian germ cells progress through a unique developmental program that encompasses proliferation and migration of the nascent primordial germ cell (PGC) population, reprogramming of nuclear DNA to reset imprinted gene expression, and differentiation of mature gametes. Little is known of the genes that regulate quantitative and qualitative aspects of early mammalian germ cell development both in vivo, and during differentiation of germ cells from mouse embryonic stem cells (mESCs) in vitro. Genetic and epigenetic programs that direct cellular identity are essential to embryonic development and the establishment of different cell types and patterning. Cells designated to become part of the germline face a diverse set of challenges during intertwined steps of embryonic development that encompasses specification, commitment, survival, migration, and sex-specific differentiation. There appears to be a core conservation of components across diverse metazoan species, including RNA binding proteins such as Deleted in Azoospermia (Dazl), involved in primordial germ cell development. PGCs in diverse organisms appear to use these conserved components in slightly different ways, however, in each they are utilized to create and maintain a germ cell-specific identity that is dependent on cytoplasmic translational regulatory machinery. How and when Dazl functions during development of mouse PGCs is not fully understood. We used a transgenic mouse system that enabled isolation of small numbers of Oct4ΔPE:GFP-positive germ cells in vivo, and following differentiation from mESCs in vitro, to uncover quantitative and qualitative phenotypes associated with the disruption of the translational regulator, Dazl. We demonstrate that disruption of Dazl results in a post-migratory, pre-meiotic reduction in PGC number accompanied by aberrant expression of pluripotency genes and failure to erase and re-establish genomic imprints in isolated male and female PGCs, as well as subsequent defect in progression through meiosis. Moreover, the phenotypes observed in vivo were mirrored by those in vitro, with inability of isolated mutant PGCs to establish pluripotent EG (embryonic germ) cell lines and few residual Oct-4-expressing cells remaining after somatic differentiation of mESCs carrying a Dazl null mutation. Finally, we observed that even within undifferentiated mESCs, a nascent germ cell subpopulation exists that was effectively eliminated with ablation of Dazl. This work establishes the translational regulator Dazl as a molecular linker between pluripotency, genetic, and epigenetic programs at multiple time points of germ cell development in vivo and in vitro, and validates use of the ESC system to model and explore germ cell biology. Furthermore, elucidation of processes in germ cell development is relevant to continuing studies of human infertility, a common health problem in men and women