Synergy with TGFβ ligands switches WNT pathway dynamics from transient to sustained during human pluripotent cell differentiation
Preprint posted on September 22, 2018 https://www.biorxiv.org/content/early/2018/09/22/406306
Article now published in Proceedings of the National Academy of Sciences at http://dx.doi.org/10.1073/pnas.1815363116
Cell specification in the embryo or during embryonic stem cell differentiation requires fine tuning of signalling pathways such as WNT and TGFβ. The canonical WNT pathway acts through dephosphorylation and liberation of β-Catenin (βCat) – from the Axin/GSK3β destruction complex – which becomes available for nuclear translocation. Once in the nucleus, βCat binds TCF/LEF partners to activate its target genes such as WNT3, Brachyury (T), EOMES and LEF1. Despite many years of study across multiple species, many aspects of the WNT signalling pathway remain to be discovered, such as the link between the dynamics of its expression (including ligand concentration dependence), the timing of activation and the persistence of the signalling, and cell fate during differentiation. This study attempts to model WNT regulation in the embryo by using reporter human embryonic stem cell lines followed by fine-tuning of multiple signalling pathways. The authors bring new insights on how WNT pathway may act in synergy with Activin A and BMP4 both members of the TGFβ signalling pathway.
The study started by engineering a βCat GFP fusion protein hESC reporter line to track the translocation of nuclear βCat by time-lapse microscopy upon different WNT condition. After characterising the new transgenic lines, the authors studied the effect of WNT3A addition at different concentrations. They were able to observe an adaptative response of βCat translocation to the nucleus up to a saturating point (at 300ng/mL): the WNT pathway activation peaked between 2 and 8 hours post addition but went down after 10hrs, then plateaued. Moreover, above 30ng/mL the plateau expression of nuclear βCat was at a higher level than that of unstimulated cells, suggesting that the WNT signalling remained activated for 24hrs post stimulation when high concentrations of WNT3A are added. This result was confirmed by using multiple hESC lines. Of particular note, stimulating WNT pathway by adding CHIR099 resulted in a different signaling dynamics: nuclear βCat increased upon addition of the chemical but, instead of dropping like in the WNT3A condition, it plateaued with a mildly increasing slope for at least 24hrs post-simulation. This result has important implications for developmental biologists using CHIR099 as a WNT agonist.
In the gastrulating embryo, WNT and TGFβ signalling activity overlap, suggesting a possible effect of TGFβ signalling on nuclear βCat levels. To test this hypothesis, cells were grown in the presence of either Activin A or BMP4 with WNT signalling inhibitors (either DKK1 or IWP2 or both). Activin A alone was able to trigger WNT signalling but this signal was shut down by the addition of IWP2. Conversely, BMP4 action on nuclear βCat was not completely shut down even when the medium was supplemented with both IWP2 and DKK1, suggesting an independent role on βCat. Adding Activin A or BMP4 on top of WNT3A increased the total amount of nuclear βCat 20hrs post-stimulation. These results are critical for understanding gastrulating events in the primitive streak where both signalling pathways intersect. It is therefore possible that Massey and colleagues have identified the mechanism that lead to extra-embryonic ectoderm BMP signalling triggering WNT. Once WNT is activated it might be self-maintaining.
Altogether, this study brought new evidence on cross-talking effects of the main signalling pathways involved in the gastrulation process. It becomes clearer that not only Wnt ligands are triggering the increase in nuclear βCat.
Why did I choose this article?
I chose this article because of my interest in understanding WNT activity in gastrulating cells (embryo and stem cells). The authors caught my attention by studying the synergy between multiple signalling involved in this important stage of development. I have always been convinced that the cascade of signalling is not a simple arrow going down on a diagram but rather a massive furball of intricated signalling pathways.
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Posted on: 9th October 2018Read preprint
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