David Andrew Turner | Search bioRxiv

bioRxiv2016

Interactions between Nodal and Wnt signalling Drive Robust Symmetry Breaking and Axial Organisation in Gastruloids (Embryonic Organoids)

Generation of asymmetry within the early embryo is a critical step in the establishment of the three body axes, providing a reference for the patterning of the organism. To study the establishment of asymmetry and the development of the anteroposterior axis (AP) in culture, we utilised our Gastruloid model system. Gastruloids, highly reproducible embryonic organoids formed from aggregates of mouse embryonic stem cells, display symmetry-breaking, polarised gene expression and axial development, mirroring the processes on a time-scale similar to that of the mouse embyro. Using Gastruloids formed from mouse ESCs containing reporters for Wnt, FGF and Nodal signalling, we were able to quantitatively assess the contribution of these signalling pathways to the establishment of asymmetry through single time-point and live-cell fluorescence microscopy.\n\nDuring the first 24-48h of culture, interactions between the Wnt/{beta}-Catenin and Nodal/TGF{beta} signalling pathways promote the initial symmetry-breaking event, manifested through polarised Brachyury (T/Bra) expression. Neither BMP nor FGF signalling is required for the establishment of asymmetry, however Wnt signalling is essential for the amplification and stability of the initial patterning event. Additionally, low, endogenous levels of FGF (24-48h) has a role in the amplification of the established pattern at later time-points.\n\nOur results confirm that Gastruloids behave like epiblast cells in the embryo, leading us to translate the processes and signalling involved in pattern formation of Gastruloids in culture to the development of the embryo, firmly establishing Gastruloids as a highly reproducible, robust model system for studying cell fate decisions and early pattern formation in culture.

Developmental Biology↗

bioRxiv2014

Brachyury cooperates with Wnt/β-Catenin signalling to elicit Primitive Streak like behaviour in differentiating mouse ES cells.

The formation of the Primitive Streak is the first visible sign of gastrulation, the process by which the three germ layers are formed from a single epithelium during early development. Embryonic Stem Cells (ESCs) provide a good system to understand the molecular and cellular events associated with these processes. Previous work, both in embryos and in culture, has shown how converging signals from both Nodal/TGF{beta}R and Wnt/{beta}-Catenin signalling pathways specify cells to adopt a Primitive Streak like fate and direct them to undertake an epithelial to mesenchymal transition (EMT). However, many of these approaches have relied on genetic analyses without taking into account the temporal progression of events within single cells. In addition, it is still unclear as to what extent events in the embryo are able to be reproduced in culture. Here, we combine flow-cytometry and a quantitative live single-cell imaging approach to demonstrate how the controlled differentiation of mouse ESCs (mESCs) towards a Primitive Streak fate in culture results in cells displaying many of the characteristics observed during early mouse development including transient Brachyury expression, EMT and increased motility. We also find that the EMT initiates the process, and this is both fuelled and terminated by the action of Bra, whose expression is dependent on the EMT and {beta}-Catenin activity. As a consequence of our analysis, we propose that a major output of Brachyury expression is in controlling the velocity of the cells that are transiting out of the Primitive Streak.

Developmental Biology↗