Robustness of epithelial sealing is an emerging property of local ERK networks driven by cell elimination

Léo Valon, Florence Levillayer, Andela Davidović, Mathilde Chouly, Fabiana Cerqueira-Campos, Romain Levayer

Posted on: 19 April 2020 , updated on: 22 April 2020

Preprint posted on 17 March 2020

Article now published in Developmental Cell at http://dx.doi.org/10.1016/j.devcel.2021.05.006

How do epithelia regulate cell elimination to remain intact? Using recently developed in vivo tools, Valon et al. find that extruding cells protect their immediate neighbors from elimination via an EGFR-dependent process.

Selected by Sophia Friesen

Categories: cell biology, developmental biology

Background:

From the skin to the renal tubules, epithelia act as impermeable or selectively permeable barriers that separate different environments, often protecting other cells from a harsh environment. Somehow, epithelia maintain this crucial barrier function during development and homeostasis, despite frequent cell turnover. The intestinal epithelium, for instance, turns over at a high enough rate that the entire surface is replaced about once a week, but it remains able to insulate the circulation from bacteria and toxins in the intestinal lumen.

To maintain barrier function in the presence of cell extrusion, the rate of extrusion must be tightly regulated, and the authors intuit that extrusion may be spatially regulated as well. While elimination of one cell at a time can be compensated for by remodeling of the surrounding cells, elimination of a cluster of multiple adjacent cells might create a hole in the epithelium, breaking the barrier. By live imaging the Drosophila pupal notum, an epithelium with a high level of cell turnover, the authors determine that this kind of clustered extrusion almost never happens in the tissue, suggesting a mechanism that protects the neighbors of extruding cells from also being lost. This local inhibition of cell elimination is dependent on ERK signaling, and is required to prevent clustered cell extrusion, which impairs epithelial barrier function.

Key findings:

To see whether clustered cell extrusion impairs the epithelium’s ability to form a seal, the researchers developed an optogenetic tool to induce cell elimination when cells are exposed to blue light. This tool, optoDronc, fuses Drosophila Caspase9 to a protein that clusters when illuminated with blue light, which activates the caspase domains and initiates cell elimination. By switching on extrusion in small clones or single cells, then injecting the pupa with a fluorescent label, the authors could see how the spatial distribution of extrusion affected barrier integrity. Sure enough, although extrusion of single cells leaves the barrier intact, simultaneous extrusion of clusters of three or more cells allowed fluorescence to leak from one side of the epithelium to the other, indicating loss of barrier function.

Because loss of barrier function can be so detrimental, the authors wondered if there were mechanisms to prevent clusters of cells from extruding simultaneously in vivo. They live imaged the developing pupal notum as cell turnover occurred and saw that, despite frequent cell elimination, clusters of extruding cells were almost never seen. By comparing the spatial distribution of cell extrusion to what would be expected by random chance, the authors determined that the cells immediately neighboring an extruding cell had a greatly reduced chance of elimination. This “window of protection” lasted for about an hour, preventing clusters of cells from extruding simultaneously.

To investigate the mechanism of this pro-survival effect, the researchers imaged the developing notum using an in vivo sensor of caspase activity, GC3Ai, which becomes fluorescent upon cleavage by effector caspases. Surprisingly, the neighbors of extruding cells frequently initiated caspase activity, indicating that they were undergoing the first steps of apoptosis which would lead to their extrusion from the tissue – but they then reverted the caspase activity and survived. This suggests that a signal from the extruding cell prevented neighbor cells from completing the extrusion process.

The authors suspected that this protective effect might be mediated by EGFR and ERK signaling, which they already knew inhibited cell elimination in the pupal notum. The neighbors of extruding cells indeed had elevated ERK signaling. Furthermore, overall depletion of EGFR randomized the location of extruding cells, and frequently resulted in clustered extrusion of cells. This indicates that EGFR/ERK signaling is required for neighbor protection. Intriguingly, neighbor protection didn’t require EGF secretion from extruding cells, and could be induced by laser-induced wounds, leading the authors to speculate that ERK activation in neighbors may be induced by mechanical, rather than chemical, signals.

What I liked about this paper:

This paper stood out to me because the problem the authors address – how epithelia remain intact despite cell extrusion – is at once very simple and very broadly relevant. At the level of basic research, epithelia are a key building block of animal tissues, and must maintain stable barriers while undergoing constant turnover. Learning how cell elimination is regulated to maintain barrier function may shed light on disorders where that function is lost, as in inflammatory bowel disease resulting from excessive shedding of epithelial cells.

Despite the simplicity of the authors’ question, investigating the dynamic and semi-random processes of extrusion and neighbor protection is a tricky task. The authors made good use of a variety of recently developed genetic tools that allowed them to perturb and observe cell extrusion, barrier function, and signaling processes in a living epithelium. In addition to addressing an important question, I think this paper showcases the amazing power of in vivo tools and imaging to observe dynamic processes.

Questions for the researchers:

How does the level of cell elimination induced by your optogenetic tool compare to endogenous rates of extrusion in the notum? Are there any concerns related to inducing a higher level of extrusion notum-wide, for instance, by elevated levels of diffusible signals?
When cells are eliminated or ablated, how far do the changes in mechanical tension spread? Do direct neighbor cells dissipate all of the tension or is some of it passed to farther cells?
Do you anticipate that this signaling might be conserved in other highly dynamic epithelia?

Tags: cell death, drosophila, epithelia, erk, extrusion, fly

doi: https://doi.org/10.1242/prelights.18841

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Author's response

The author team shared

How does the level of cell elimination induced by your optogenetic tool compare to endogenous rates of extrusion in the notum? Are there any concerns related to inducing a higher level of extrusion notum-wide, for instance, by elevated levels of diffusible signals?

Let’s discuss first the level of cell elimination induced by our optogenetic tool. The level of cell elimination in the pupal notum is relatively high (~750 cell deaths within 24 hours, so roughly 15 deaths per hour). When we trigger death in clones with optoDronc, we are therefore not so far from this rate (10-70 deaths/hour), but we obviously trigger also a lot of ectopic cell eliminations. We did not worry so much about the putative effects notum wide (such as diffusive factors like the ones described for compensatory proliferation, the induction of proliferation by massive cell death) as we were focusing on very short timescales (less than one hour), which were sufficient to observe cell extrusion and potential loss of epithelial sealing.

Having said that, it could be indeed very interesting to investigate what are the effects of these ectopic deaths on tissue wide mechanics, cell rearrangements and proliferations, which could all participate to compensatory effects and tissue homeostasis, but that was not the focus of this experiment and this study (maybe a future project?). Lastly, we should also mention that cell eliminations induced by optoDronc can be massive and very fast. Indeed, if we express optoDronc ubiquitously and expose tissue to blue light, the full tissue will collapse in less than 1 hour (not shown in this study). That is why we mostly use it after induction in small clones. The fast and efficient induction of cell death was actually essential for our work, otherwise it would have been difficult to trigger simultaneous extrusion of neigbours in a controlled manner.

When cells are eliminated or ablated, how far do the changes in mechanical tension spread? Do direct neighbor cells dissipate all of the tension or is some of it passed to farther cells?

Regarding this question, we do not have good ways to probe tension on the timescale of extrusion (~30 minutes), but we can use the change of cell shape as a proxy. So far, both for eliminated and ablated cells, we mostly see changes of apical area in the first row of cells next to the extruding cells, while the changes get rapidly very small in the second and not visible in the third row of cells. Indeed, since cell number increases from one row to the next one, the mechanical perturbation (i.e. cell stretching) gets more and more “diluted”. Accordingly, we mostly see ERK activation in the direct neighbours, while we do not see significant activation in the other cell rows.

Do you anticipate that this signaling might be conserved in other highly dynamic epithelia?

This is obviously a very important question, and we believe that this is the case. There are several recent reports showing that ERK waves are observed near extruding cells in human cell lines (see for instance https://doi.org/10.1101/826917, Aikin et al. bioRXiv 2019). A similar process has also been observed in human cell lines affecting the rate of cell elimination (unpublished work from O. Pertz, as mentioned in our manuscript). In our study we also observed that this process also occurs during the elimination of the larval cells of the pupal abdomen. Altogether we believe that this mechanism is conserved and fairly ubiquitous. Yet, the dynamics and ranges of ERK activation are different between systems, which may be explained by differences in the EGFR/ERK networks and/or differences in the mechanical properties. So far, the other studies have been performed ex vivo and there is to our knowledge no other examples of such ERK feedbacks in vivo. It would be very interesting to look at other tissues in Vertebrates with high cell turnover rates in which tissue sealing is important, such as the midgut.

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Robustness of epithelial sealing is an emerging property of local ERK networks driven by cell elimination

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