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

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

Preprint posted on 18 March 2020 https://www.biorxiv.org/content/10.1101/2020.03.17.994921v1

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

Epithelium on damage control mode: a transitory local feedback loop in response to cell elimination prevents neighbor cell death to ensure tissue sealing.

Selected by Glò Casas Gimeno

Introduction

Cell elimination is a physiological process in epithelial tissues with important functions in size regulation during development and in cell turnover in adult tissues. At the cellular level, epithelial cell elimination is a stereotyped event where coordinated cell apoptosis and tissue remodeling result in the extrusion of dying cells from the epithelium without perturbing tissue integrity [1,2]. In this pre-print, Valon and colleagues discover that there is an active mechanism that prevents “clustered” cell extrusion in order to maintain tissue integrity.

The reason I chose to highlight this pre-print is because it describes an exciting new mechanism of epithelial tissue regulation, but mainly because it provides yet another an example of the most fascinating about cell biology: it never relinquishes control.

Graphic summary (from Figure 4M of the pre-print from Valon et al. )

Research summary

The observation that starts this story is that simultaneous extrusion of clustered neighbouring cells impairs homeostatic tissue sealing. The authors developed a light-activated Caspase9 to sparsely trigger apoptosis in the Drosophila pupal notum. The pupal notum (the dorsal side of the thoracic segment) is a single layer epithelium, where cell extrusion is induced by tissue crowding and is caspase-dependent [3]. By live imaging, the authors tracked epithelial remodeling following cell extrusion. Single cell extrusion or the simultaneous (<30 min) extrusion of adjacent cells in line led to seamless tissue sealing. In contrast, the extrusion of clustered cells (>3 cells in <30 min) was followed by aberrant tissue remodeling, which was characterized by temporal loss of adherens junctions integrity and wound healing-like tissue closure.

Considering the detrimental effect of these clustered-extrusion type of events in tissue integrity, they then interrogated the frequency in which they occur in physiological conditions by in vivo live imaging. Surprisingly, they observed is that these events are significantly less frequent than would be expected under the assumption that cell elimination is a Poisson process (i.e. cell elimination is a random event, independent from other such events). Much to the contrary, they could determine that there is a spatiotemporal refractory phase (~10um radius from the dying cell, ~1h) after cell extrusion when neighbouring cells are less likely to be eliminated. This led them to hypothesize that there might be a mechanism that actively prevents these clustered-extrusions from taking place, thus protecting tissue integrity.

The authors had previously identified EGFR/ERK signaling as a central regulator of cell elimination in the pupal notum through downregulation of the pro-apoptotic protein Hid [4]. Here, they used a fluorescent biosensor of ERK activity and they found that, following cell death, ERK activity transiently increases in the immediate neighbour cells. Notably, this ERK activity interval correlated with subsequent caspase inhibition. Further, RNAi depletion of EGFR uncoupled caspase activity in neighbour cells from cell extrusion, and eliminated the refractory phase after cell extrusion observed in wild-type embryos. Finally, the transient ERK activation depends on EGFR expression on the neighbour cells, but not on EGF secretion from the dying cell. EGFR/ERK is sensitive to cell stretching, the authors explain, so the stretch resulting from the need to fill the space from the extruded cell might be sufficient to trigger this protective mechanism.

Questions for the authors

  • In the text, it is mentioned that adjacent cells simultaneously activating caspase is a frequent event. Does this frequency reflect the epithelia response to crowding?
  • Do you believe that in this context, EGFR/ERK is acting mainly via Hid inhibition? Reportedly, Hid activates a Caspase-9 apoptotic pathway [5]. How does the optogenetically induced apoptosis, phenotypically equivalent to physiological cell extrusion, bypass this control mechanism?

References

[1]. Marinari E, Mehonic A, Curran S, Gale J, Duke T, Baum B. Live-cell delamination counterbalances epithelial growth to limit tissue overcrowding. Nature. 2012 Apr 15;484(7395):542-5. doi: 10.1038/nature10984.

[2]. Eisenhoffer GT, Loftus PD, Yoshigi M, Otsuna H, Chien CB, Morcos PA, Rosenblatt J.  Crowding induces live cell extrusion to maintain homeostatic cell numbers in epithelia. Nature. 2012 Apr 15;484(7395):546-9.

[3]. Levayer R, Dupont C, Moreno E. Tissue Crowding Induces Caspase-Dependent Competition for Space. Curr Biol. 2016 Mar 7;26(5):670-7.

[4]. Moreno E, Valon L, Levillayer F, Levayer R. Competition for Space Induces Cell Elimination through Compaction-Driven ERK Downregulation. Curr Biol. 2019 Jan 7;29(1):23-34.e8.

[5]. Haining WN, Carboy-Newcomb C, Wei CL, Steller H. The proapoptotic function of Drosophila Hid is conserved in mammalian cells. Proc Natl Acad Sci U S A. 1999 Apr 27;96(9):4936-41

 

Posted on: 19 April 2020 , updated on: 23 April 2020

doi: https://doi.org/10.1242/prelights.18896

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Author's response

The author team shared

  1. In the text, it is mentioned that adjacent cells simultaneously activating caspase is a frequent event. Does this frequency reflect the epithelia response to crowding?

Regarding the pattern of caspase activation and mechanics , it is true that the concomitant activation of caspase could be related to crowding/compaction.  At the tissue level, there is a clear pattern of caspase activation in the notum (which we do not describe in this manuscript) with a high number of positive cells in the midline and in a posterior band. This pattern is most likely multifactorial: it is probably related to underlying pattern that sensitizes for cell death (including low levels of ERK), but also tuned by local mechanical properties. Indeed, we previously showed that cell deformations could modulate ERK, which is an important regulator of caspases[1]. But this does not exclude that other factors modulate caspase activity. In summary, we do not have a clear answer to your question, this would require a systematic characterization of multiple parameters (mechanics, signaling pathways…) and their correlation with caspase activation. An exciting but very long term goal !

  1. Do you believe that in this context, EGFR/ERK is acting mainly via Hid inhibition? Reportedly, Hid activates a Caspase-9 apoptotic pathway [5]. How does the optogenetically induced apoptosis, phenotypically equivalent to physiological cell extrusion, bypass this control mechanism?

Regarding first the epistatic relationship between EGFR, Hid and Caspase9. We do not discuss this point in this article, but we previously showed that EGFR regulates caspase activation and cell elimination in the notum mostly through Hid regulation[1]. We expect the same in the context of the ERK local feedbacks. Thus, since the optoDronc tool activates directly Caspase 9 (downstream of ERK and Hid), it should completely bypass the ERK feedbacks. That is actually essential to trigger multiple cell death in clusters. Finally, are the extrusion that we trigger with optoDronc similar to physiological extrusions ? Most likely yes. Firstly, the cell deformations we measured (variation of apical area) are very similar for spontaneous extrusions and extrusions triggered by optoDronc (see for instance in figure S1). Secondly, the extrusions triggered by optoDronc are inhibited by p35 (an effector caspase inhibitor) just like physiological extrusions. It is true however that we observed slightly faster extrusions in optoDronc compared to the physiological ones, which may be caused by the high and fast activation of caspases. It would actually be very interesting for the future to evaluate how caspase dynamics may influence the speed of cell extrusion.

  1. Moreno, E. et al. (2019) Competition for Space Induces Cell Elimination through Compaction-Driven ERK Downregulation. Curr Biol 29 (1), 23-34 e8.

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