Cdx2 maintains stem-cell identity in the mouse trophoblast lineage by preventing default differentiation as trophoblast giant cells

Student thesis: Doctoral ThesisDoctor of Philosophy


The transcription factor Cdx2 is required for the formation of several developmental lineages in the mouse embryo, and is thought to play different roles within the same lineage at different stages. The role of Cdx2 in establishing trophoblast fate has been extensively studied, but the reason for its continued expression in established trophoblast cells is less well understood. 
To explore this question, I generated ATAC-seq and RNA-seq libraries from wild-type and Cdx2 knockdown trophoblast stem cells (TSCs) in vitro. ATAC-seq experiments show, as expected, that loss of Cdx2 causes decreased accessibility at CDX2 consensus binding sites. However, of the regions showing differential chromatin accessibility in Cdx2 knockdown cells, two thirds have increased accessibility. These sites are enriched for the TFAP2C consensus motif and footprint; Tfap2c is a TSC marker that plays a part in trophoblast differentiation by driving genome-wide increases in accessibility. 
Gene ontology analysis of sites with increased accessibility in Cdx2 knockdown cells suggests that these regulatory regions are associated with trophoblast giant cell (TGC) differentiation. Consistent with this observation, continued Cdx2 knockdown drives homogenous differentiation of trophoblast stem cells into trophoblast giant cells within days. Similarly, homozygous Cdx2 knockout TSCs are unstable and spontaneously differentiate into TGCs, although in this context cells appear to retain some ability to compensate. 
TSC differentiation can also be initiated in vitro by growth factor withdrawal, albeit in a heterogeneous manner. However, the sites of increased chromatin accessibility observed under these circumstances differ from those observed in Cdx2 knockdown cells. Moreover, although RNA-seq analysis shows that 87% of genes whose expression changes in Cdx2 knockdown cells are also mis-regulated during conventional differentiation, 31% of these transcripts change in opposite directions. 
This work suggests that Cdx2 normally maintains ‘stemness’ in TSCs by preventing them from differentiating directly and homogeneously into TGCs. This gatekeeping role of Cdx2 differs from its earlier function in repressing pluripotency genes and suggests that Cdx2 is repurposed to play different roles within the trophoblast lineage. Furthermore, our work may finally provide a plausible mechanism by which half of non-progenitor derived parietal TGCs are generated.
Date of Award1 Nov 2019
Original languageEnglish
Awarding Institution
  • King's College London
SupervisorJack Price (Supervisor)

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