Abstract
ABSTRACTDiffusible signals are known to orchestrate patterning during embryogenesis, yet diffusion is sensitive to noise. The fact that embryogenesis is remarkably robust suggests that additional layers of regulation reinforce patterning. Here, we demonstrate that geometrical confinement orchestrates the spatial organisation of initially randomly positioned subpopulations of spontaneously differentiating mouse embryonic stem cells. We use micropatterning in combination with pharmacological manipulations and quantitative imaging to dissociate the multiple effects of geometry. We show that the positioning of a pre-streak-like population marked by brachyury (T) is decoupled from the size of its population, and that breaking radial symmetry of patterns imposes polarised patterning. We provide evidence for a model in which the overall level of diffusible signals together with the history of the cell culture define the number of T+ cells, whereas geometrical constraints guide patterning in a multi-step process involving a differential response of the cells to multicellular spatial organisation. Our work provides a framework for investigating robustness of patterning and provides insights into how to guide symmetry-breaking events in aggregates of pluripotent cells.
Highlights
Developmental patterning is the process through which spatially defined regions of distinct cell types emerge from a group of cells that initially appear to be equivalent
This effect is shown by the binned density map (BDM) of T− cells, which represented the majority of the population (Fig. 2A-D and Fig. S3) and by the z projection of 3D confocal images (Fig. 2E-G)
We found that conditioned media (CM) from cells grown at high density had a dosedependent effect on the expression of T, Wnt3 and cerberus 1 in cells cultured at low density, indicating that diffusible signalling molecules contribute to regulation of these genes
Summary
Developmental patterning is the process through which spatially defined regions of distinct cell types emerge from a group of cells that initially appear to be equivalent. Wnt expression emerges in the proximo-posterior side of the embryo (Rivera-Pérez and Magnuson, 2005) and engages in a signalling autoregulatory loop involving Nodal from the epiblast and BMP4 from the extra-embryonic ectoderm (ExE) The PS is characterised by the expression of early mesendodermal markers such as brachyury (T) (Beddington et al, 1992; Wilkinson et al, 1990; loss of epithelial characteristics reviewed by Morali et al, 2013) and an inversion of polarity prior to migration of ingressing cells (Burute et al, 2017; Stern, 1982)
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