Implantation is the hallmark event during early pregnancy where a connection between the embryo and mother is formed. During the following phase of early postimplantation development, the pluripotent cells of the embryonic disc, the embryo proper, undergo gastrulation. This key process establishes the three germ layers, endoderm, mesoderm and ectoderm, which set the basis for all tissues emerging in the human embryo.

Since postimplantation human embryos cannot be accessed routinely due to ethical and legal restrictions, early postimplantation development has been studied so far using stem cell-based in vitro models or embryo culture systems that provided valuable insights into early embryo development. However, the developmental relevance of these system could not be validated due to a lack of an early postimplantation, spatially defined in vivo reference.

In my PhD project, I aimed to delineate the signalling environment of spatially defined tissues of early gastrulating embryos, using the marmoset, an established primate model, to study conserved human postimplantation embryo developmental processes.

I first characterised marmoset embryos of three early postimplantation developmental stages on a morphological and molecular level, which allowed me to then perform spatially defined transcriptome profiling. Preserving the spatial coordinates of each sample processed for transcriptome analysis enabled me to integrate the transcriptome information of each sample individually into 3D embryo reconstructions. A collaborator applied gaussian process regression to establish 3D embryo transcriptomes models with continuous gene expression patterns.

Using these 3D embryo transcriptomes, I identified the earliest timepoint of symmetry breaking and the onset of gastrulation, and analysed gene expression patterns associated with anterior-posterior axis formation. I defined genetic marker profiles for extraembryonic lineages such as the visceral endoderm, specifically defining markers for the anterior visceral endoderm (AVE), a signalling centre which drives symmetry breaking in the mouse.

Furthermore, I defined marker profiles and analysed signalling pathways involved in the formation of the secondary yolk sac, and I explored the molecular framework and the potential origin of the extraembryonic mesoderm, which presumably forms the connecting stalk of the embryo with its mother. I further described the migration of primordial germ cells (PGCs), the precursor cells of germ cells, validating previous studies in primates, and I investigated involved signalling pathways and established novel marker profiles in primates. I analysed the formation of the primitive streak, a structure which defines the embryo midline in the posterior of the embryo proper and is established following the onset of gastrulation. I investigated signalling pathways which I extrapolated from mouse and primate literature i.e., NODAL, FGF, WNT, and BMP, and I analysed their expression patterns within the embryonic disc and its surrounding tissues.

To test the signalling environment hypotheses derived from said analyses, I established in vitro marmoset embryonic stem cell-based models of the postimplantation embryonic disc and amnion, an extraembryonic membrane derived from the embryonic disc, and I tested the effects of NODAL, FGF, WNT and BMP on the in vitro models. Taken together, I found that FGF, together with NODAL, maintains pluripotency in the embryonic disc, similar to BMP and WNT inhibition, that I found in the anterior embryonic disc. WNT and BMP signalling drives mesoderm formation in the posterior, gastrulating embryonic disc. I was further able to delineate the role of NODAL, BMP and WNT in the formation of amnion. Given the versatility of the established in vitro systems, I tested the effects of these signalling pathways on a human stem cell-based system using a similar set-up, and characterised similarities and differences in amnion and mesendoderm formation.

Lastly, I provided an outlook of potential future applications of my established 3D embryo transcriptomes, including as a validation tool for in vitro models, stem cell lines, or in vivo datasets.