WNT signalling in the patterning of human gastruloids
The study of early human development poses a challenge due to technical and ethical limitations, particularly during the initial four weeks following fertilisation. During this critical period, the fundamental aspects of the body plan are established through a process known as gastrulation, which begins 14 days post fertilisation. Because of the 14-day rule imposed on human embryo research, no experimental studies have been conducted beyond this date. The derivation of human embryonic stem cells (hESCs) has opened up numerous opportunities for studying early cell fate decisions and signals involved in this process. In particular, these cells have enabled the development of embryo models that allow the exploration of various aspects of early development. One of these models is the ‘gastruloid’ which results from the aggregation of small numbers of hESCs and leads to the formation of the body plan. WNT signalling, an evolutionary conserved pathway, is known to play a crucial role during gastrulation with its dysregulation leading to various developmental abnormalities and diseases. This thesis examines the role of WNT signalling in the patterning of human gastruloids. By applying different concentrations of WNT agonists, it is shown that the levels of WNT signalling affect cell differentiation as well as elongation and morphology of the gastruloids. Higher WNT levels induce Brachyury expression, whereas lower favour SOX17 expression, triggering the morphogenesis of SOX17+ tube-like structures. Through these experiments, it is further revealed that human gastruloids might recapitulate the mode of endoderm specification recently observed in mouse embryos. Additionally, this thesis indicates that specifically non-canonical WNT signalling affects the elongation of gastruloids. These responses highlight the high sensitivity of the system to the signalling environment. In addition to WNT signalling, BMP and NODAL also act as critical molecular pathways during gastrulation. This thesis shows that BMP and NODAL can affect cell differentiation and morphology in gastruloids and suggest that human gastruloids require a balance of BMP, WNT and NODAL signalling for their generation. Due to the tractable nature of the gastruloid in vitro model, more factors apart from signalling molecules can be manipulated to study the effects of various stimuli on cell behaviour. One such factor includes the initial cell density. In this thesis, it is suggested that small variations in the standard size of the human gastruloids result in aggregates that exhibit scalable self-organisation, an ancestral feature present in most embryos. By increasing the variation in aggregate size, there was also a concomitant increase in the variation of gene expression. The robust recapitulation of gastrulation-like events in combination with the easy manipulation of the gastruloid system, make it also a powerful tool for teratogenicity assessment. In a first proof-of-concept study presented finally in this thesis, application of a reference panel of seven different compounds results in gastruloid morphological and gene expression changes dependent on the compound and its concentration. Human gastruloids present a powerful system. They can be utilised to explore the mechanisms of human embryo patterning and discern unique features of early development that would otherwise be impossible due to limitations associated with human embryo research. Human gastruloids further have fundamental implications in biomedical research.
Developmental biology, Embryology, Human gastrulation, Human gastruloids, Stem cell models, Teratogenicity
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