Roles of the transcriptional regulator Id1 in pluripotency and differentiation

Abstract

The transition from pluripotency to differentiation is a key event in the life of all complex multicellular organisms. In the development of the mouse, the pluripotent epiblast undergoes gastrulation and gives rise to three multipotent germ layers, which will in turn form the tissues of the adult body. The events leading up to gastrulation have been extensively studied in vivo in developing embryos, and modelled in vitro making use of embryonic stem (ES) cells.

Bone morphogenic protein (BMP) signalling plays a key role in these processes. BMP can in fact maintain ES cells in a self-renewing state by inhibiting their differentiation into neural ectoderm, whilst at the same time being required for the specification of mesoderm in the developing embryo (Winnier et al. 1995, Ying et al. 2003a). A key intracellular target of BMP is the transcriptional regulator Id1, which can recapitulate the effects of BMP in the preservation of ES cell pluripotency and in the inhibition of neural specification from pluripotent cells (Ying et al. 2003a).

This thesis will focus on understanding the roles of this molecule in the early decisions affecting the transition from pluripotency to differentiation. In particular, I aim to study the expression pattern of Id1 in cultures of pluripotent cells, and to clarify which extracellular and intracellular molecules regulate the expression of the factor; I aim to understand how forced Id1 expression inhibits the differentiation of pluripotent cells, and whether Id1 may play a similar role in the regulation of the asynchronous exit from pluripotency observed in differentiating wild-type cells; finally, I aim to characterise the expression pattern of Id1 in the early stages of post-implantation development at the single-cell resolution, and to understand how the expression of the molecule correlates with the previously characterised expression patterns of key signalling molecules and transcription factors.

The generation of a reporter ES cell line expressing the yellow fluorescent protein Venus fused to the C-terminus of Id1 allowed me to assess the expression of the factor in culture on a single-cell basis, making use of immunofluorescence and flow cytometry. I observed that expression of Id1 is reliant on active BMP signalling and low Activin/Nodal signalling, and I characterised the combinatory effects of the two pathways on Id1 expression. Furthermore, I demonstrated that high Nanog expression is incompatible with high Id1 expression in ES cell cultured in the presence of LIF and serum, which raises the possibility that Nanog may be affecting the expression of Id1 in vivo, both in pre-implantation and in post-implantation embryos.

I generated ES cell lines overexpressing Id1 and observed that the factor inhibits differentiation of pluripotent cells into neural ectoderm by delaying their exit from a post-implantation epiblast-like pluripotent state, and ultimately favouring mesodermal specification. This suggests that Id1 is acting at a specific stage of differentiation and that the differentiation process itself is following a similar developmental pathway to what is observed in the peri-gastrulation stage embryo. I performed single-cell transcriptional analysis on differentiating wild-type ES cells and observed that Id1 is not expressed at an appropriate point in time to affect the asynchronous the exit from pluripotency observed in neural adherent monolayer differentiation, which suggests that other factors must be responsible for this phenomenon.

Finally, I addressed the expression pattern of Id1 protein in the embryonic tissue of gastrulating mouse embryos by imaging chimaeric embryos generated using the Id1- Venus reporter ES cells. I observed that Id1 is expressed in the proximal regions of streak stage embryos; in the epiblast and migrating mesendoderm of bud stage embryos; in cardiac, lateral and allantoic mesoderm and in foregut endoderm in headfold stage embryos. These expression patterns fit with the reported expression of BMP molecules at these stages of development, and suggest that Id1 expression is primarily dependent on BMP expression in early post-implantation embryos.

However, I also observed Id1 expression in a ring of cells surrounding the node in headfold stage embryos, a previously uncharacterised expression pattern not directly attributable to BMP expression. This raises the intriguing question of what is regulating Id1 expression and what roles Id1 may be playing in this key embryonic structure.