Human pre-gastrulation development is a complex process where the single cell zygote transitions to the multilineage blastocyst, comprised of the epiblast (precursor of the fetus), hypoblast (precursor of the yolk sac) and trophoblast (precursor of the placenta). The blastocyst then implants into the maternal uterus and specifies body axes in preparation for gastrulation. It is thought that most pregnancies fail during the two-week period between fertilization and gastrulation. However, the inaccessibility of human embryos during implantation has limited our understanding of these stages.

In this Thesis, I interrogated multiple aspects of human peri-implantation development. Utilizing human embryos cultured through implantation in vitro, I first generated a single cell transcriptomic dataset of post-implantation development. Through analysis of this dataset together with immunofluorescence and functional experiments, I characterized key events during peri-implantation development, including pluripotent state transitions within the epiblast, the role of the growth factor FGF to ensure proliferation of embryonic and extraembryonic tissues, and the emergence of a group of asymmetrically positioned hypoblast cells that express inhibitors of BMP, NODAL, and WNT signaling pathways and act as the anterior signaling center of the embryo.

Next, I extended this work by generating an integrated single cell RNA sequencing atlas. Using this atlas as a guide, I characterized the role of several signaling pathways in the pre- to post-implantation transition. I find that both NODAL and BMP signaling are enriched in the hypoblast and essential to specify the anterior hypoblast. Further, NODAL and BMP are enriched in the human epiblast at pre-implantation compared to post-implantation stages, while NOTCH signaling is required for the epiblast only after implantation. These results demonstrate a crucial role for NODAL, BMP, and NOTCH in anterior hypoblast formation and imply an unanticipated switch in the roles of these pathways upon implantation in humans.

Given the ethical and technical challenges of human embryo research, I have utilized stem cells as a complementary tool to study peri-implantation development. I established a human post-implantation embryo model comprised of embryonic and extraembryonic-like tissues. Combining the two types of extraembryonic-like cells generated by transcription factor overexpression with embryonic stem cells generates embryoids that contain an epiblast-like domain surrounded by extraembryonic-like tissues. These inducible human embryoids robustly generate amnion, extraembryonic mesenchyme, and primordial germ cell-like cells in response to BMP signaling. Using the modularity of this model, I identified an inhibitory role for SOX17 in anterior hypoblast specification. Modulation of the subpopulations of hypoblast-like cells impacted epiblast-like domain differentiation, highlighting functional tissue crosstalk.

Overall, this Thesis begins to illuminate the ‘black box’ of human implantation.