Unraveling Fundamental Aspects of Preimplantation Development using Single Cell Genomics

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Embryonic development during the first week is arguably the most critical period of human development, however, information pertaining to fundamental aspects of embryogenesis (lineage segregation and X-chromosome inactivation (XCI)) during this window are still lacking. Current knowledge has almost exclusively been determined using mouse models, yet caution is warranted when extrapolating from the mouse, given the emerging discrepancies, thus highlighting the need for human specific studies. We have recently demonstrated that during embryonic day (E) 5, the first lineages are established: 1) trophectoderm (TE), 2) primitive endoderm (PE), and 3) pluripotent epiblast cells (EPI), which is in contrast to the mouse. Another striking difference I identified is a novel mechanism of XCI in the human embryo; activity from both X-chrs is gradually reduced to levels observed in the male by E7. What drives lineage segregation and mechanism(s) of blastocyst development is undetermined. Our preliminary data demonstrate the presence of signalling components of both WNT and TGF-ß pathways in the individual lineages at embryonic day (E)5 and that the majority of genes involved in these pathways are turned on/off during E5; suggesting that they play an important role in lineage specification. The role(s) of WNT and TGF-ß signalling in human preimplantation development remains unclear. Further, PORCN (regulator of WNT pathway) is an X-linked gene. Given the regulatory function of PORCN on WNT signalling, we would also now like to examine whether there is a link between X-chr dosage compensation and WNT signalling; which may explain the human preimplantation embryo approach to XCI. Thus, to gain a better understanding of the underlying mechanism(s) involved in lineage segregation and potentially XCI in the human embryo, I now aim to explore the functional role(s) of WNT and TGF-ß signalling using small molecule agonists/antagonist. Immunohistochemistry will measure changes in components (translocation, phosphorylation, cellular location) and downstream targets at the protein level in addition to obtaining a spatial overview of the embryo and lineages formed. Single-cell RNA sequencing will measure the impact of modulating these pathways, at lineage specific resolution across multiple developmental timepoints and also determine relationship with XCI together with smFISH. These pioneering studies will provide first detailed mechanistic insights into the gene regulatory mechanisms underlying cell fate specification, XCI and blastocyst formation, providing fundamental knowledge toward early human embryogenesis. Overall, understanding what regulates early human development is of great importance for enhancing the fields of Reproductive Biology and Development. Further, results from this proposal may lead to improved culture conditions or derivation protocols in application to stem cell biology (naïve and primed conditions) and In Vitro Fertilization (IVF).