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Title: Signalling and transcriptional regulation of human primordial germ cell specification
Author: Sybirna, Anastasiya
ISNI:       0000 0004 8501 2165
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2020
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Primordial germ cells (PGCs), the precursors of gametes, are specified in early post-implantation human embryos, which are inaccessible for direct investigation. This process can however be modelled using human embryonic stem cells (hESCs) for in vitro derivation of human PGC-like cells (hPGCLCs). hESCs in conventional culture conditions have very low potential for germ cell fate; this increases significantly if the cells are first cultured in PGC competence-promoting media. To address how the PGC-competent state might be gained, I used a new computational tool - Branch-Recombinant Gaussian Processes (B-RGPs) - to identify potentially relevant pathways based on single-cell RNA sequencing of early human embryos, as well as gonadal PGCs and soma. I then tested these predictions experimentally by modulating the candidate signalling pathways in competent hESCs, followed by hPGCLC induction. I found that inhibition of FGF receptor (FGFR) or its downstream effectors (MEK and PI3K) enhanced competency for PGC fate. By contrast, premature MEK/ERK activation in hESCs resulted in aberrant expression of endoderm markers and loss of germ cell potential. Altogether the study showed that FGFR pathway is a negative regulator of cellular competence for human PGCLC fate. PGCLC specification from competent cells occurs in response to BMP signalling and is orchestrated by specific transcription factors (TFs). Emerging evidence points to many differences between TF networks in mouse and human PGCs. For example, SOX17 is essential for human germ cell fate, but is dispensable for mouse PGCs. On the other hand, a key pluripotency factor PRDM14 is crucial for mouse PGC specification, but its role in human PGC development is unclear. I designed genetic approaches to address PRDM14 involvement in human pluripotency versus PGC specification. To achieve inducible loss of function, I deleted endogenous PRDM14 in the background of doxycycline-repressible PRDM14 transgene expression. This approach, however, did not allow fast and homogenous control over protein levels. To circumvent this, I used CRISPR/Cas9 in conjunction with conditional degrons (auxin- and jasmonate-inducible degrons) for rapid and reversible degradation of the endogenous protein. Notably, PRDM14 depletion during differentiation significantly reduced the number of specified hPGCLCs, revealing its role in human PGC fate. Furthermore, RNA sequencing of mutant cells showed aberrant gene expression, with derepression of WNT signalling and somatic genes. Importantly, ectopic PRDM14 expression in mutant cells rescued hPGCLC specification and transcriptional changes, thus proving the specificity of the observed phenotype. Interestingly, loss of PRDM14 in hESCs had a distinct transcriptional effect, indicating a context-dependent role of PRDM14 in PGCs and pluripotent cells. ChIP-sequencing in hESCs and hPGCLCs identified PRDM14 binding to regulatory elements of key pluripotency and germ cell genes and motif analysis suggested its integration in the core TF circuitry in both cell types. Interestingly, the overlap between PRDM14 targets in mouse and human was limited. Altogether, this reveals a key role of PRDM14 in human germ cell fate and exemplifies the importance of complete and fast protein depletion in loss-of-function studies. Overall my research has furthered our understanding of signalling and transcriptional regulators controlling the segregation of human germ cells from soma. It also expanded the toolkit for protein studies in dynamic processes and uncovered hitherto unknown roles of PRDM14 in early human development.
Supervisor: Surani, Azim Sponsor: Wellcome Trust ; Cambridge International Trust
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
Keywords: human primordial germ cells (hPGCs) ; embryonic stem cells ; PRDM14 ; auxin-inducible degron (AID) ; jasmonate-inducible degron ; developmental competence ; human development ; FGFR signalling ; human primordial germ cell-like cells (hPGCLCs)