Abstract
dc:description.abstractIn mammals, successful pre-implantation development leads to the formation of a tri-lineage structure known as the blastocyst, consisting of the epiblast, primitive endoderm and trophectoderm, which will give rise to the new organism, the yolk sac and placenta respectively. These three lineages must be established from the totipotent zygote via two successive cell fate decisions in the appropriate sequence, position and proportion, to generate a blastocyst capable of implantation and further development. In mammals this process has long been thought to be regulative, with cell-cell interactions flexibly determining the eventual fate of cell. This is in contrast to commonly studied non-mammalian embryos in which pre-patterning of the embryo, driven by spatially localised factors, is a common feature. However, early blastomeres of mouse embryos have been reported to have distinct developmental fates, potential and heterogeneous abundance of certain transcripts, prior to the first cell fate decision. Nevertheless, the extent of the earliest intra-embryo differences remains unclear and controversial. Utilizing single-cell proteomics by mass-spectrometry I show that 2-cell mouse and human embryos contain an alpha and a beta blastomere as defined by differential abundance of hundreds of proteins. Such asymmetrically distributed proteins include Gps1 and Nedd8, depletion or overexpression of which in one blastomere of the 2-cell embryo impacts lineage segregation. Fascinatingly, halved mouse zygotes already display protein asymmetries, which resembles alpha and beta blastomeres, suggesting differential proteome localisation already within zygotes. I also find that beta blastomeres may have a greater developmental potential, and give rise to a blastocyst with a higher proportion of epiblast cells than alpha blastomeres. Human 2-cell blastomeres also partition into two clusters sharing strong concordance with clusters found in mouse, in terms of differentially abundant proteins and functional enrichment. This provides the first demonstration of intra-zygotic and inter-blastomere proteomic asymmetry in mammals that has a role in lineage segregation. In humans, this early period of development is prone to failure, with a third of human pregnancies estimated as being lost prior to implantation. A high incidence of aneuploidy is thought to be a major driver of pregnancy failure and understanding the behaviour of aneuploid cells is of great interest. As the blastocyst forms and implants, aneuploid cells may be eliminated through cell competition with diploid cells or show differences in their lineage segregation, impacting the composition and proportion of each lineage in the blastocyst, The mouse embryo does not have similar high intrinsic rates of aneuploidy, but reversine, a spindle assembly checkpoint inhibitor, can be used to recapitulate the human aneuploid embryo to some extent, and to observe the elimination of aneuploid cells. Here, I return to the human, utilising human embryonic stem cells and new integrated stem cell based embryo models, to characterise conserved aneuploid cell depletion in mouse and human stem cell co-cultures of diploid and aneuploid cells. Furthermore, I use stem cell lines harbouring specific aneuploidies to determine if specific aneuploidies confer differential ‘fitness’ and elimination rates. Overall my PhD has examined two questions regarding early mammalian development: 1) when do the cells of the embryo first become different to each other and how does this interplay with lineage segregation and 2) can a model of the mosaic aneuploid human embryo be generated to better understand the fate of aneuploid cells within the three lineages of the blastocyst.
Degree
thesis:*- Name dc:type.qualificationname
- Doctor of Philosophy (PhD)
- Level dc:type.qualificationlevel
- Doctoral
- Grantor dc:publisher.institution
- University of Cambridge
- Year dc:date.issued
- 2023
Author and committee
dc:creator, dc:contributor.*- Author dc:creator
-
- Iwamoto-Stohl, Lisa
- Advisors dc:contributor.advisor
-
- Zernicka-Goetz, Magdalena
- Paluch, Ewa
Subjects
dc:subject × 4Rights
dc:rightsIdentifiers
dc:identifier.*- DOI dc:identifier.doi
- https://doi.org/10.17863/CAM.106137
- OAI identifier oai:identifier
- oai:www.repository.cam.ac.uk:1810/364503