Brumm, Anna Sophie; (2022) Investigating the role of TGFβ signalling in early human development. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Approximately 6 days post fertilization, the human embryo forms a blastocyst comprised of three distinct cell types: the extraembryonic trophectoderm and primitive endoderm, and the pluripotent epiblast, which gives rise to the embryo proper. While mammalian preimplantation development is morphologically highly convergent, the signalling pathways underlying epiblast specification in a widely used model organism, the mouse, may not be conserved in human and the mechanisms that regulate human epiblast development are currently unclear. ACTIVIN/NODAL signalling is required for maintenance of human pluripotency in vitro, through regulation of pluripotency-associated transcription factor NANOG, which is the earliest known molecular marker with restricted expression in the human epiblast. Here, we investigate the requirement for NODAL signalling activity in initiation and maintenance of NANOG expression during human preimplantation development. Our detailed characterisation in human blastocysts reveals that NANOG expression is detectable before NODAL signalling is active in the inner cell mass. This suggests initiation of NANOG expression is independent of NODAL signalling in human embryos. With progressing development, NODAL signalling activity increases throughout the embryonic pole of the blastocyst, and is eventually active in most NANOG expressing cells. Surprisingly, inhibition of NODAL signalling activity in human blastocysts does not lead to loss of NANOG protein expression, contrary to what has been described in human pluripotent stem cells. Future experiments will address whether the transcription of NANOG is affected by NODAL signalling inhibition. Altogether, these results thus far suggest that NODAL signalling activity is not directly required for NANOG regulation in human blastocysts, indicating that other signalling pathways underlie this cell fate specification. This points to a possible intriguing difference in the regulation of NANOG expression between the human epiblast and in vitro pluripotent stem cell lines, underscoring the importance of functional investigation directly in the human embryo.

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