objective:

Morphogen gradients are used repeatedly during development to pattern embryonic tissues. Absolute concentration, duration or even temporal derivative of morphogen concentration have all been proposed to carry positional information depending on the context. However, establishing the causal relationship between the spatio-temporal profile of the gradient and the resulting cellular diversity and tissue patterning is difficult to address in live embryo because of lack of tools to take control of those variables. This is especially true during mammalian gastrulation, where the primitive streak is patterned by a complex, time evolving signaling landscape of the BMP, WNT and NODAL pathways

Methods

To be able to control precisely the spatio-temporal profile of morphogens applied on a developing tissue in vitro, we have developed a set of microfluidics devices able to apply well defined morphogen landscapes on the basal side of human embryonic stem cells colonies, thus mimicking how BMP4 is delivered to the pluripotent epiblast during mouse gastrulation.

results

Using this unique devices, we observed that the cell types emerging in a hESC colony upon differentiation depends on the shape of the morphogen gradient. In a parabolic gradient of BMP4, we have observed a sharp and reproducible pattern of 3 cell identities (undifferentiated hESC, mesoderm, amnion-like extra-embryonic tissue). Varying maximum concentration of the gradient we could show that fate transitions happen at constant BMP concentration, thus behaving exactly like Wolpert’s French flag model. This capability of hESC to interpret BMP as a morphogen had not been observed so far in other experimental settings, showing the importance of basal delivery of BMP and precise control of its concentration. Endoderm cells, by contrast emerged only in sharper, step like gradients. Using cell lines engineered with fluorescent reporters of BMP, WNT and NODAL signaling and tracking of single cells movements, we have shown that the appearance of endoderm is due to radial movements of cells, resulting in transient exposure to BMP. A mathematical model recapitulating these observations predicted a bistability in WNT signaling which we have verified experimentally.

As morphogen gradients and signaling centers are ubiquitous during development, our toolbox provides powerful mean to dissect the logic of patterning at all developmental stages and to engineer tissue precisely.