Signalling and transcriptional regulation of early developmental lineage decisions

Embryonic stem (ES) cells are cell lines isolated from the embryo at a time just prior to implantation into the uterus. In the right cocktail of medium and cytokines, these cell lines can be maintained indefinitely in vitro in a self-renewing state. Initially it was assumed that these cells represented a homogeneous population however, more recently it has been shown that there are a great number of genes that are expressed heterogeneously. ES cell cultures are therefore a mix of different subpopulations, some of which have distinct functional properties including a bias or ‘lineage priming’ towards a particular cell fate. These populations are also dynamic in nature, converting from one state to another with fairly rapid kinetics. The main focus of this thesis was to gain a more in depth understanding of the mechanisms regulating heterogeneity and lineage priming in murine ES cells by asking which signalling pathways play a role in this phenomenon and how the switch between states is regulated at a transcriptional level. These questions were asked using an ES cell line containing a sensitive reporter for the endoderm marker Hex. This reporter, developed by a previous lab member, allowed the identification and separation of a population of ES cells primed towards a primitive endoderm fate. Primarily, I assessed the effect of a defined culture system (2i) on the Hex-expressing population. This culture system contains inhibitors that block FGF signalling and the Wnt pathway component GSK3. Culturing ES cells in 2i has been suggested to generate a more homogeneous culture. Here, I have shown that culturing ES cells or pre-implantation embryos in 2i did not eliminate heterogeneity but maintained them in an early state prior to lineage segregation. When ES cells were cultured in standard serum-containing medium, Hex was expressed in a mutually exclusive manner with the embryonic marker NANOG, while in 2i a subpopulation of cells coexpressed both Hex and NANOG. This population was functionally primed towards extraembryonic endoderm and trophoblast. Furthermore, these ES cells could efficiently contribute to 2-cell embryos in chimaera assays. LIF signalling promoted this population through the JAK/STAT pathway. I then asked how transcription was regulated during the switch between unprimed ES cells to those primed towards a primitive endoderm fate, as well as how regulation changes during further differentiation. To ask this, Hex positive (primed) and negative (unprimed) ES cell populations were sorted as well as a Hex positive differentiated sample. These samples were analysed by GRO-seq to determine the location, density and orientation of RNA-polymerase throughout the genome. Changes in gene expression between primed and unprimed states were regulated primarily through elongation whereas genes upregulated during differentiation were regulated at the point of de novo initiation