N-cadherin stabilises neural identity by dampening anti-neural signals

K Punovuori, RP Migueles, M Malaguti, G Blin, KG Macleod, NO Carragher, T Pieters, F van Roy, MP Stemmler, S Lowell

Preprint posted on 16 July 2019 https://doi.org/10.1101/704817

Article now published in Development at http://dx.doi.org/10.1242/dev.183269

Signaling regulation by Cadherin switching, a mechanism to coordinate cell fate decisions across tissues

Selected by Claire Simon & Sophie Morgani

Categories: cell biology, developmental biology

Background

When building an organism, cell types are simultaneously specified and spatially organized, requiring the coordination of both signaling and morphogenesis. A prime example is the formation and patterning of the embryonic germ layers during gastrulation. The Cadherin family of proteins mediates cell-cell adhesion and regulates a number of signaling pathways, hence sits at the interface of these processes.

The clearest relationship between these adhesion molecules and signaling is the interaction of Cadherins with the transcriptional effector of the Wnt pathway, β-CATENIN, at cell junctions. A simple model proposes that E-CADHERIN sequesters β-CATENIN at the membrane, eliminating it from the cytoplasmic signaling pool, leading to low Wnt signaling activity. E-CADHERIN downregulation then releases β-CATENIN from the membrane, elevating Wnt signaling capacity. However, Wnt signaling is not elevated when Cadherins are lost prior to ligand exposure, leading to the suggestion that the initial Cadherin interaction primes β-CATENIN for signaling, which is supported by the finding that membrane and cytoplasmic β-CATENIN are molecularly distinct.

In the gastrulating embryo there is switch from E- to N- CADHERIN in cells of the prospective neurectoderm at the anterior side of the embryo and the primitive streak at the posterior. Consistent with the proposed model, downregulation of E-CADHERIN at the primitive streak during epithelial-to-mesenchymal transition (EMT) correlates with increased Wnt signaling. However, both E- and N- CADHERIN bind β-CATENIN, suggesting that the regulation of Wnt signaling during germ-layer specification is more complex.

In this pre-print the authors show that, during anterior neural differentiation, the switch from E-CADHERIN to N-CADHERIN has the opposite effect and in fact results in a reduction in Wnt and FGF signaling. Intriguingly, when N-CADHERIN expression is forced, increased neural differentiation is observed prior to the downregulation of E-CADHERIN. Hence N-CADHERIN or the interaction between E- and N- CADHERIN is more critical than E-CADHERIN downregulation for the modulation of signaling activity.

What we like about this pre-print

While the association between adhesion and signaling, especially during EMT, has been long established, we still do not fully understand the mechanisms that link these properties and how they regulate cell-fate decisions. This study investigates how Cadherin switching affects signaling activity and transcription. The authors show that the logical model i.e. low E-CADHERIN, high Wnt signaling, is not true in all contexts. Moreover, although the most established role of Cadherins is to regulate adhesion and Wnt signaling, the authors show that Cadherin switching can also dampen FGF signaling.

We love that the authors compare their in vitro findings to the in vivo situation to ask whether similar mechanisms regulate embryonic development – always important! Through this comparison the authors suggest that, rather than being a driver of neural induction, in vivo this E- to N- switch facilitates robust, coordinated differentiation across a field of cells.

Open questions

What is the mechanism by which Cadherins regulate signaling activity and differentiation? Does this involve direct interactions with receptors, interaction with downstream signaling components or perhaps changes in signaling activity based on altered physical properties?

In vivo Wnt, Nodal, BMP and FGF signals in the posterior of the embryo induce the primitive streak, and subsequent mesoderm and endoderm specification. The anterior visceral endoderm (AVE) secretes antagonists of the Wnt, Nodal and BMP pathways, thought to block signaling within the anterior of the embryo, permitting neural differentiation. We wonder whether in vivo the Cadherin-mediated dampening of Wnt and FGF signals is required in addition to these inhibitors, or if this mechanism is more important during in vitro differentiation where there is no AVE equivalent.

Further reading

Malaguti M, Nistor P a, Blin G, Pegg A, Zhou X, Lowell S. Bone morphogenic protein signalling suppresses differentiation of pluripotent cells by maintaining expression of E-Cadherin. Elife. 2013 Jan;2:e01197.

Ciruna, B. and J. Rossant, FGF signaling regulates mesoderm cell fate specification and morphogenetic movement at the primitive streak. Developmental Cell, 2001. 1(1): p. 37-49.

Howard, S., et al., A positive role of cadherin in Wnt/beta-catenin signalling during epithelial-mesenchymal transition. PLoS One, 2011. 6(8): p. e23899.

IMAGE CREDIT: Dr Guillaume Blin: Image illustrates cell-cell interactions that may be critical to regulate signaling and coordinate cell fate choices.

Posted on: 13 August 2019 , updated on: 14 August 2019

doi: https://doi.org/10.1242/prelights.13290

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Author's response

Karolina Punovuori and Sally Lowell shared

Questions for the authors

What is the most important message that you would like readers to take home from your paper?

We like the idea that cells make decisions as cooperatives rather than as a group of individuals, but mechanisms for local cooperation are not well understood in the context of neural commitment. The idea we’d like people to take away is that changes in adhesion could contribute part of the information that makes up this local communication network.

It is interesting that, in the absence of E-CADHERIN and forced N-CADHERIN, you lose the functional capacity of Wnt to block neural differentiation but still see a Wnt transcriptional response in a subset of genes. Have you looked further into the differential response of Wnt transcriptional targets?

We were really excited when we saw that cells in which we’d forced a Cadherin switch were able to ignore the anti-neural effects of Wnt, and that they also had lower levels of nuclear Beta-catenin. At that point we thought that effects on Wnt signalling would neatly explain everything. So, it was big surprise that at least part of the transcriptional response to Wnt seemed to be intact in these cells. It could be that a subset of “anti-neural” Wnt transcriptional targets are differentially affected. We’ve not directly investigated this, but one argument against it is that one of the targets we did look at, Brachyury, is thought to be at least partly responsible for the anti-neural effects of Wnt. Another possibility is that the “Wnt resistance” of these cells is secondary to effects on other anti-neural pathways, such as FGF.

In vivo neural specification occurs in different regions of the embryo with distinct signaling environments – e.g. posterior NMPs vs. anterior neural plate. Do you think that the E- to N- CADHERIN switch and modulation of signaling is similar in both regions?

We like this question! We were intrigued when Val Wilson and James Briscoe’s labs reported a striking upregulation of N-Cadherin within NMPs as they mature during axis elongation (Gouti et al. 2017). We’ve started to look into this and Karolina has some interesting preliminary data suggesting that N-Cadherin does preserve neural potency in NMPs in vitro. This fits nicely with our data on anterior neural differentiation, but at the same time it is a curious result given that N-Cadherin is expressed in both neurectoderm and mesoderm. Lots left to explore there.

In vitro, the forced expression of N-CADHERIN increases neural differentiation suggesting that it drives this process. However, in vivo, N-CADHERIN is expressed after neural fate is established. Do you think that, while N-CADHERIN may not be driving neural differentiation during normal development that it has the capacity to i.e. does overexpression of N-CADHERIN within the epiblast ectopically induce neural fates?

We would love to know the answer to this. We could use either chimera experiments or in-vivo electroporation to force a cadherin switch in a subset of epiblast cells to see if this can induce ectopic neural fates. We’d be particularly excited about looking at non-autonomous effects: does N-Cadherin propagate neural commitment to surrounding cells in vivo? We’ve just published a paper reporting image analysis tools that will make it possible to do this (Blin et al. 2019) so it is definitely in our future plans, although there remain some technical hurdles to overcome before we can do this experiment in a properly controlled way.

What is your lab tradition for celebrating an accepted paper? Did you have a pre-print celebration?

Of course, pre-prints should be celebrated just like any other paper! Our paper celebrations always include home-baked cakes – the first-author of this preprint Karolina is just one of several amazing bakers in the lab. We have even sent a picture of one of the lab bakes as a candidate cover image for a recently accepted paper. If it is chosen, we’ll fall over in shock and then celebrate with yet more cakes!

References

Blin, G. et al., 2019. Nessys: A new set of tools for the automated detection of nuclei within intact tissues and dense 3D cultures. K. G. Storey, ed. PLoS Biol, 17(8), p.e3000388.

Gouti, M. et al., 2017. A Gene Regulatory Network Balances Neural and Mesoderm Specification during Vertebrate Trunk Development. Dev Cell, 41(3), pp.243–261.e7.

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N-cadherin stabilises neural identity by dampening anti-neural signals

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