Role of multicellular organisation in mesoderm differentiation

Abstract

What cues and signals are important for the differentiation of pluripotent cells in vitro and in vivo? During development, pluripotent cell fate decisions are spatiotemporally coordinated with changing tissue architecture. Typically, changes in tissue architecture are thought to be a downstream consequence of changes in cell fate, which in turn are caused by change in exposure to secreted extracellular signalling molecules. However, recent evidence in multiple vertebrate systems has shown that tissue architecture and tissue mechanics can influence and causally feedback into cell fate decision-making processes. During gastrulation in the mouse embryo the pseudostratified epithelium of the proximal epiblast undergoes an epithelial to mesenchymal transition (EMT), forming the primitive streak. This is accompanied by a number of changes in tissue architecture including the loss of apical-basal polarity and cell-cell adhesion, an increase in cell clustering and migratory behaviour, and changes in cell shape associated with differentiation into mesodermal and endodermal derivatives. Primitive streak formation and gastrulation are orchestrated by Nodal, BMP, Wnt and Fgf signalling pathway activity. However, it is unclear if and how changes in tissue architecture influence the differentiation responses in the context of this signalling environment.

Here I set out to ask which aspects of changing tissue architecture can provide regulatory feedback to influence cell fate decisions during gastrulation in the mouse, and to explore the underlying mechanisms. Decoupling the contributions of tissue architecture and the chemical signalling environment in cell fate regulatory feedback is technically challenging in vivo. Therefore, I developed a more controllable in vitro ECM-based mESC model in which I could manipulate tissue architecture. Qualitative and quantitative analysis between in vitro and in vivo cellular organisation confirmed that in vitro ECM-based models recapitulated features of primitive streak morphogenesis. Further, I find that this ECM-based culture model displays increased mesodermal differentiation and demonstrates a more coherent patterning of cell fates, when compared with monolayer cultures exposed to the same exogenous signals.

Using quantitative image analysis and further experimental manipulation of in vitro tissue architecture, I identify a role for local cell density in promoting mesodermal fates in ECM-based models. Finally, I demonstrate that active Nodal and Wnt signalling are crucial in mediating enhanced mesodermal differentiation induced by increased local cell density in ECM-based cultures. These results highlight a potential positive regulatory feedback role for tissue architecture, in the form of increased local cell density, during mesodermal differentiation in an in vitro primitive streak model. We propose that the data suggests the requirement of a critical population size for robust and coherent mesodermal differentiation and that this mechanism is reminiscent of a community effect.