Uncovering novel regulators of neural fate commitment in embryonic stem cells
How do pluripotent stem cells reliably select the neural lineage during differentiation? When embryonic stem (ES) cells are exposed to a homogeneous signalling environment, differentiation remains variable and unpredictable. Whilst the key extrinsic signalling pathways that inhibit neural lineage commitment have long been established, much less progress has been made in identifying the downstream effectors of these pathways that could be mediating the differentiation response. BMP signalling is a potent inhibitor of neural induction both in vivo and in vitro. In ES cells, BMP blocks entry into the neural lineage via transcriptional upregulation of Inhibitor of Differentiation (Id) proteins, providing strong evidence that neural induction proceeds via derepression of an unknown positive-acting regulator. Previous work has identified that the major binding partner of Id proteins in ES cells is the basic helix-loop-helix transcription factor, E2A. In this thesis I hypothesise that dissolution of the Id1-E2A interaction represents an important first step towards neural lineage commitment. To explore a role for E2A in this process I first generated an endogenously-tagged ES cell line to characterise its expression during the transition from pluripotency to neural differentiation. Gain-of-function experiments revealed that overexpression of an E2A homodimer construct was able to drive neural differentiation in ES cells, even under self-renewal conditions, suggesting a key role for E2A in mediating this lineage decision. The generation and characterisation of a new E2A knockout ES cell line in this study provided further support to this, and I therefore propose a molecular mechanism whereby E2A homodimers regulate commitment to the neural lineage, downstream of BMP inhibition.