Origin and segregation of the human germline

Acknowledgements This work was supported by the Wellcome Investigator Awards in Science (2094)75/Z/17/Z (to MA Surani), the Wellcome Investigator Awards in Science 096738/Z/11/Z (to MA Surani), the BBSRC research grant G103986 (to MA Surani), the Croucher Postdoctoral Research Fellowship (to WWC Tang), the Wellcome 4-Yr PhD Programme in Stem Cell Biology & Medicine (2038)31/Z/16/Z (to A Castillo-Venzor) and the Cambridge Commonwealth European and International Trust (to A Castillo-Venzor), the Isaac Newton Trust (to WWC Tang), the Butterfield Awards of Great Britain Sasakawa Foundation (to T Kobayashi and MA Surani), and the Astellas Foundation for Research on Metabolic Disorders (to T Kobayashi). The marmoset embryo research is generously supported by the Wellcome Trust (WT RG89228, WT RG9242), the Centre for Trophoblast Research, the Isaac Newton Trust, and JSPS KAKENHI 15H02360, 19H05759. TE Boroviak was supported by a Wellcome Sir Henry Dale Fellowship. JC Marioni acknowledges core support from EMBL and from Cancer Research UK (C9545/A29580), which supports MD Morgan. We would like to thank Roger Barker and Xiaoling He for providing human embryonic tissues and Charles Bradshaw for bioinformatics support. We also thank The Weizmann Institute of Science for the WIS2 human PSC line and the Genomics Core Facility of CRUK Cambridge Institute for sequencing services. We thank members of the Surani laboratory for insightful comments and critical reading of the manuscript.Peer reviewedPublisher PD

Genesis of the human germline

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

Tracing the emergence of primordial germ cells from bilaminar disc rabbit embryos and pluripotent stem cells.

Rabbit embryos develop as bilaminar discs at gastrulation as in humans and most other mammals, whereas rodents develop as egg cylinders. Primordial germ cells (PGCs) appear to originate during gastrulation according to many systematic studies on mammalian embryos. Here, we show that rabbit PGC (rbPGC) specification occurs at the posterior epiblast at the onset of gastrulation. Using newly derived rabbit pluripotent stem cells, we show robust and rapid induction of rbPGC-like cells in vitro with WNT and BMP morphogens, which reveals SOX17 as the critical regulator of rbPGC fate as in several non-rodent mammals. We posit that development as a bilaminar disc is a crucial determinant of the PGC regulators, regardless of the highly diverse development of extraembryonic tissues, including the amnion. We propose that investigations on rabbits with short gestation, large litters, and where gastrulation precedes implantation can contribute significantly to advances in early mammalian development

Origin and segregation of the human germline.

Funder: EMBL core supportFunder: Astellas Foundation for Research on Metabolic DisordersFunder: MEXT | Japan Society for the Promotion of ScienceFunder: Cambridge TrustFunder: Croucher FoundationFunder: Great Britain Sasakawa FoundationFunder: Isaac Newton TrustHuman germline-soma segregation occurs during weeks 2-3 in gastrulating embryos. Although direct studies are hindered, here, we investigate the dynamics of human primordial germ cell (PGCs) specification using in vitro models with temporally resolved single-cell transcriptomics and in-depth characterisation using in vivo datasets from human and nonhuman primates, including a 3D marmoset reference atlas. We elucidate the molecular signature for the transient gain of competence for germ cell fate during peri-implantation epiblast development. Furthermore, we show that both the PGCs and amnion arise from transcriptionally similar TFAP2A-positive progenitors at the posterior end of the embryo. Notably, genetic loss of function experiments shows that TFAP2A is crucial for initiating the PGC fate without detectably affecting the amnion and is subsequently replaced by TFAP2C as an essential component of the genetic network for PGC fate. Accordingly, amniotic cells continue to emerge from the progenitors in the posterior epiblast, but importantly, this is also a source of nascent PGCs

Origin and segregation of the human germline.

Human germline-soma segregation occurs during weeks 2-3 in gastrulating embryos. Although direct studies are hindered, here, we investigate the dynamics of human primordial germ cell (PGCs) specification using in vitro models with temporally resolved single-cell transcriptomics and in-depth characterisation using in vivo datasets from human and nonhuman primates, including a 3D marmoset reference atlas. We elucidate the molecular signature for the transient gain of competence for germ cell fate during peri-implantation epiblast development. Furthermore, we show that both the PGCs and amnion arise from transcriptionally similar TFAP2A-positive progenitors at the posterior end of the embryo. Notably, genetic loss of function experiments shows that TFAP2A is crucial for initiating the PGC fate without detectably affecting the amnion and is subsequently replaced by TFAP2C as an essential component of the genetic network for PGC fate. Accordingly, amniotic cells continue to emerge from the progenitors in the posterior epiblast, but importantly, this is also a source of nascent PGCs

Mutational synergy during leukemia induction remodels chromatin accessibility, histone modifications and three-dimensional DNA topology to alter gene expression.

Altered transcription is a cardinal feature of acute myeloid leukemia (AML); however, exactly how mutations synergize to remodel the epigenetic landscape and rewire three-dimensional DNA topology is unknown. Here, we apply an integrated genomic approach to a murine allelic series that models the two most common mutations in AML: Flt3-ITD and Npm1c. We then deconvolute the contribution of each mutation to alterations of the epigenetic landscape and genome organization, and infer how mutations synergize in the induction of AML. Our studies demonstrate that Flt3-ITD signals to chromatin to alter the epigenetic environment and synergizes with mutations in Npm1c to alter gene expression and drive leukemia induction. These analyses also allow the identification of long-range cis-regulatory circuits, including a previously unknown superenhancer of Hoxa locus, as well as larger and more detailed gene-regulatory networks, driven by transcription factors including PU.1 and IRF8, whose importance we demonstrate through perturbation of network members.Cancer Research UK, European Research Council, Wellcome Trust, Kay Kendall Leukaemia Fund, American Society of Haematolog