Decoding cellular signals: SMAD signalling and commitment during human embryonic stem cell differentiation

Abstract

During early development, environmental triggers prompt a collection of pluripotent cells to begin the dramatic process of differentiation giving rise to the three germ layers. How and when cells decode information from their environment to bring about commitment is a fundamental, yet unanswered, question. Poised to differentiate, human embryonic stem (hES) cells provide an invaluable model for addressing this question. Bone morphogenic protein 4 (BMP4) triggers hES cell differentiation towards mesoderm, via SMAD signal transduction pathways. In this thesis, we find that commitment to BMP4-driven differentiation is a surprisingly early event. We investigated how BMP4 signals are first decoded by SMAD signalling networks leading to differentiation. Quantitative imaging of SMAD activation in hES cells showed that, in single cells, activation of SMAD1 is ‘switch-like’ and sustained after BMP4 stimulation. We found that SMAD activation is bistable and irreversible, both properties characteristic of signalling networks embedded in positive feedback, and suggest that positive feedback regulation involving expression of the SMAD1 activator, BMPR1A, contributes to the sustained SMAD1 activation. Using a genomics approach, we shed light on gene expression dynamics at the immediate onset of differentiation and reveal that BMP4-induced target genes show distinctive behaviours. We have identified a novel set of SMAD responsive genes, upregulated immediately following commitment, capable of mirroring SMAD activation dynamics with ‘switch-like’, irreversible expression. Focusing on one such gene, GATA3, we find its expression is necessary and sufficient to drive differentiation. We have deemed this novel set of genes early commitment genes (ECGs) and propose that ECG expression provides a crucial link between SMAD activation dynamics and early, irreversible commitment to differentiation. Together these studies shed light on how cells decode signals and irreversibly commit to differentiation, and highlight the usefulness of using hES cells as a model system to understand fate decisions in early development.