The primary aim of this thesis was to examine the specification of human primordial germ cells (PGCs) and the mechanism regulating the transient gain and loss of competence for germ cell fate. Specification of PGCs in humans occurs during weeks 2 to 3 in gastrulating embryos, which eventually develop into sperm and ova, generate totipotency at fertilisation, and transmit genetic and epigenetic information for development. Since direct studies on early human embryos are not feasible, I used an established human pluripotent stem cell-based in vitro model for early human development. Accordingly, I used human pluripotent stem cells (PSCs) to induce mesendoderm (ME), which at midpoint progresses through a pre-mesendoderm (PreME) state that transiently acquires competence for PGC fate and commits to PGC-like cells (PGCLCs) in response to BMP signalling. To address these questions, I performed highly-resolved transcriptomic profiling and careful epigenomic characterisation of cis-regulatory regions at key steps during the in vitro model. To determine the regulation of the transient gain and loss of competence for germ cell fate, I re-engineered a CRISPR toolkit for activation and inactivation to test candidates for germ cell competence. I found the inhibition of OTX2 increased the efficiency of human PGCLC specification, which is consistent with a transient reduction in OTX2 in the germline competent PreME against rising levels of EOMES. The OTX2 levels continue to increase in ME cells that gain competence for the mesoderm and endoderm fates but are incompetent for germ cell fate. Using the CRISPR toolkit, I further show that SOX17 enhancers are the critical targets of the regulatory circuitry of germline competence. SOX17 and TFAP2C initiate a core germline program, including induction of PRDM1 by SOX17 and maintenance of pluripotency factors, POU5F1 and NANOG. I also explored PGC competence and specification using highly resolved single-cell transcriptomic profiling of our in vitro PGC-like cells (PGCLC). Using an integrative analysis with existing human and primate embryonic datasets demonstrates that our model represents the lineages at the caudal region of the embryo, including mesoderm, endoderm, PGCs and amnion. I found a high degree of fidelity between in vitro PGCLC and bona fide early embryonic PGCs, which map spatially to a transition region in primate embryos in proximity to the amnion. I further identified TFAP2A as a critical early regulator of human PGC fate, with a potential role in suppressing SOX2, which might contribute to initiating the combinatorial role of OCT4-SOX17 for the PGC program. This differs from mouse PGC specification, where no functional relevance for TFAP2A or SOX17 has been described. Finally, to test if SOX17 is a critical regulator of PGC fate in another mammal that develops as a bilaminar disc at the time of PGC specification, I examined early development and PGC specification in bilaminar disc rabbit embryos (in collaboration with Toshihiro Kobayashi). Indeed, SOX17 is a crucial determinant of rabbit PGCs, which are transcriptionally similar to their human PGC counterparts. Accordingly, rabbits are a suitable animal surrogate for investigating early human development and the origin of the germline. This study provides further evidence that development via embryonic disc morphology is correlated with a SOX17-centric PGC network topology, in stark contrast to the PGC network in cup-shaped embryos such as in mice. In this thesis, I revealed several new facets about the origin of the human germline. I demonstrated for the first time that OTX2 has a role in human germline competence, discovered a novel role for TFAP2A during germline formation, and revealed an intricate enhancer-regulated transcription factor (TF) network underpinning human PGC specification and progression.
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