Roles of transcription factor eomesodermin in the trophectoderm and definitive endoderm lineages of the murine embryo

Eomesodermin (Eomes), a member of the T-box family of transcription factors, is required for the specification of a multitude of lineages during mouse gastrulation. Initially it is expressed in the trophectoderm (TE) of the pre-implantation blastocyst, where it is essential for peri-implantation development. Eomes is also expressed in the post- implantation embryo, where it has a critical role in the specification of several lineages during gastrulation, including the definitive endoderm (DE). The DE is fated to give rise to the gut tube and all gut-associated organs of the developing embryo. Although Eomes is necessary in both, the molecular mechanisms by which it acts to maintain or specify these two diverse cell lineages remain largely unknown. The aim of my thesis was to investigate how Eomes functions at a cellular level to generate these distinct lineages.
In this thesis, I describe the generation of an Eomes-degron allele that permits the inducible depletion of Eomes protein in Eomes-expressing cells, and the subsequent generation of a mouse line harbouring this allele. I have taken advantage of this allele, in combination with genome-wide RNA and chromatin accessibility techniques, to examine the functions of Eomes protein within the TE and DE lineages in a finely time- resolved manner.
Eomesdeg/deg mice are viable and fertile. Although the Eomes-degron system is functional and highly efficient in vitro, we encountered setbacks in its implementation in vivo, resulting in inconsistent depletion of Eomes protein in post-implantation embryos. We suggest that degron-tagged mouse lines may therefore not always be the best approach for the study of protein functionality, and rigorous validation of the system should always be carried out when it is adapted to a gene or protein of interest.
In trophoblast stem cells (TSCs), the in vitro equivalent of the TE, Eomes depletion followed by RNA-seq suggests that in addition to regulating components of the core TSC gene regulatory network, Eomes appears to promote the expression of a suite of genes associated with cell motility, extracellular matrix components, and cell-cell interactions. Eomes may therefore have an important role in modulating the interactions of the TE with its environment, thus promoting successful attachment and invasion of the uterus by the blastocyst.
Using an in vitro embryonic stem cell-derived embryoid body model of anterior primitive streak (APS) development, which includes the DE and node/notochord lineages, I demonstrate that Eomes depletion affects the expression of many known DE markers, including the DE master regulators FoxA2 and Sox17. Eomes protein loss also results in altered chromatin accessibility, suggesting a possible mechanism for the regulation of DE genes by Eomes. In collaboration with the Göttgens group at the University of Cambridge, we have profiled at single-cell resolution the transcriptome and chromatin accessibility of APS-directed EBs. Ongoing analyses of this data set which will further elucidate the roles of Eomes in the specification of the APS progenitors and the resulting daughter lineages.

Keywords: mouse
eomes
definitive endoderm
eomesodermin
transcription factor
gastrulation
degron
T-box
trophectoderm