Abstract
Early mammalian development entails transit through naive pluripotency towards post-implantation epiblast, which subsequently gives rise to primordial germ cells (PGC), the founding germline population. To investigate these cell fate transitions, we developed a compound-reporter to track cellular identity in a model of PGC specification (PGC-like cells; PGCLC), and coupled it with genome-wide CRISPR screening. We identify key genes both for exit from pluripotency and for acquisition of PGC fate, and characterise a central role for the transcription regulators Nr5a2 and Zfp296 in germline ontogeny. Abrogation of these genes results in widespread activation (Nr5a2−/−) or inhibition (Zfp296−/−) of WNT pathway factors in PGCLC. This leads to aberrant upregulation of the somatic programme or failure to activate germline genes, respectively, and consequently loss of germ cell identity. Our study places Zfp296 and Nr5a2 as key components of an expanded PGC gene regulatory network, and outlines a transferable strategy for identifying critical regulators of complex cell fate decisions.
Highlights
Behaviour, National Institute for Physiological Sciences, 5-1 Higashiyama Myodaiji, Okazaki, Aichi 444-8787, Japan
PGC-like cells (PGCLC) are derived by inducing naive embryonic stem cells (ESC) that are equivalent to the inner cell mass (ICM) of blastocysts (E3.5–E4.5), towards competent epiblast-like cells (EpiLC), which closely resemble pre-gastrulation mouse epiblast (E5.5–E6.5)[14,15]
Single-cell RNA-seq data revealed that Stella is expressed in the naive pluripotent ICM but is rapidly downregulated in post-implanation epiblast, with re-activation occurring in nascent PGCs20
Summary
PGC specification follows WNT-dependent induction of the primitive streak/mesodermal gene Brachyury (T), which in mice promotes activation of key PGC specifiers; Blimp[1], Prdm[14], Cbfa2t2 and Ap2γ5–8 These transcription factors form a selfreinforcing network that feeds back to suppress other WNT/ BMP-induced mesodermal genes (including T), thereby repressing the ongoing somatic programme in early PGCs9. Specified PGCLC are equivalent to migratory PGCs in vivo (E8.5–E10.5)[12], and have the potential to develop to mature functional gametes[13,16] This model is appropriate to investigate the inherent regulatory mechanisms of nascent germ cells, and the preceding developmental transitions from ICM, through postimplantation development and PGC specification. We demonstrate that unbiased CRISPR screening can be adapted to probe the genetic basis of complex multi-step developmental processes, using the germline lineage as a paradigm
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