Bilateral JNK activation is a hallmark of interface surveillance and promotes elimination of aberrant cells

  1. Deepti Prasad
  2. Katharina Illek
  3. Friedericke Fischer
  4. Katrin Holstein
  5. Anne-Kathrin Classen

  • Curated by eLife

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    This is an interesting study investigating a mechanism for the elimination of aberrant cells from epithelial tissues dependent on the contractility of the interface between cells with different fates regulated by JNK activity. This work offers insights into robustness and error correction mechanisms that help understand cell-cell competition and the origin of tumors. The study should be relevant for cell, developmental and cancer biologists.

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Abstract

Tissue-intrinsic defense mechanisms eliminate aberrant cells from epithelia and thereby maintain the health of developing tissues or adult organisms. ‘Interface surveillance’ comprises one such distinct mechanism that specifically guards against aberrant cells which undergo inappropriate cell fate and differentiation programs. The cellular mechanisms which facilitate detection and elimination of these aberrant cells are currently unknown. We find that in Drosophila imaginal discs, clones of cells with inappropriate activation of cell fate programs induce bilateral JNK activation at clonal interfaces, where wild type and aberrant cells make contact. JNK activation is required to drive apoptotic elimination of interface cells. Importantly, JNK activity and apoptosis are highest in interface cells within small aberrant clones, which likely supports the successful elimination of aberrant cells when they arise. Our findings are consistent with a model where clone size affects the topology of interface contacts and thereby the strength of JNK activation in wild type and aberrant interface cells. Bilateral JNK activation is unique to ‘interface surveillance’ and is not observed in other tissue-intrinsic defense mechanisms, such as classical ‘cell-cell competition’. Thus, bilateral JNK interface signaling provides an independent tissue-level mechanism to eliminate cells with inappropriate developmental fate but normal cellular fitness. Finally, oncogenic Ras-expressing clones activate ‘interface surveillance’ but evade elimination by bilateral JNK activation. Combined, our work establishes bilateral JNK interface signaling and interface apoptosis as a new hallmark of interface surveillance and highlights how oncogenic mutations evade tumor suppressor function encoded by this tissue-intrinsic surveillance system.

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  1. Version published to 10.7554/elife.80809 on eLife
  2. eLife

    eLife assessment

    This is an interesting study investigating a mechanism for the elimination of aberrant cells from epithelial tissues dependent on the contractility of the interface between cells with different fates regulated by JNK activity. This work offers insights into robustness and error correction mechanisms that help understand cell-cell competition and the origin of tumors. The study should be relevant for cell, developmental and cancer biologists.

  3. eLife

    Reviewer #1 (Public Review):

    In this article, Prassad and colleagues describe a new mechanism involved in the elimination of misspecified/mislocated cells in the wing imaginal disc. This study follows a previous study from the same group (Bilmeier et al. Curr Biol 2016) which showed that a large panel of genetic backgrounds changing locally cell fate can trigger aberrant sorting of the misspecified cells triggered by the increased of contractility at clone interfaces. This process was suggested to directly participate to clone elimination below a certain clone size. However, the mechanism involved in apoptosis induction was not really studied per se. Here, they use similar genetic backgrounds and showed that JNK activation occurs specifically at the interface of the misspecified clones on both side (inside and outside the clone) hence leading to a local increase of cell death both in the WT and misspecified cells. This local activation of cell death participates to clone elimination, although the authors also delineate an alternative mechanism of death induction in the center of the clone that may correlate with the local buckling and the deformation. Importantly, this mechanism seems quite specific of these misspecified backgrounds and is unrelated to other more classical cell competition scenarios which trigger the elimination of Minute mutant (affecting ribosomes) or based on differential levels of Myc.
    The model proposed is interesting and clearly delineate a distinctive feature of this quality control mechanism which triggers local JNK activation. It is based on solid genetic evidences and use a large panel of genetic backgrounds and careful quantifications. The demonstration is overall very convincing. Moreover, these results provide a novel perspective for the field of cell competition and quality control mechanism which has been dominated by the concept of absolute fitness, which is not at all required in this context (where both WT or altered cells can be eliminated provided they are in minority in the tissue).

    Admittedly, the unicity and novelty is bit tuned down by former studies showing similar patterns of JNK activity upon local distortion of morphogens (so called morphogenetic apoptosis, Adachi-Yamada and O'Connor Dev Biol 2002), or the pattern of JNK activation observed near polarity mutant clones (Ohsawa et al, Dev Cell 2011) suggesting that this bilateral JNK activation might not be completely unique to these contexts. But non of these studies characterised such large range of genetic backgrounds and this study clearly provide new mechanistic insights.

    It is important to note that at this stage, it is not clear whether there is any link between the sorting behaviour and the activation of JNK (they could be both activated by unknown upstream factors), while the terminology "interfacial contractility" used to define this type of clone elimination may convey the idea that this is the most upstream factor in the process. Also further quantifications may be required to see to which extend JNK activation is indeed restricted to cell directly contacting clone border and also to support the final proposed model suggesting that the number of contact could influence the levels of JNK (actually alternative models could also explain why smaller clones get eliminated). Finally, while the JNK levels clearly influence death in the clone, further experiments may be required to test how the line of JNK activation in WT cells contribute to their death and their elimination similar to mispecified cells, specially in the context where the majority of tissue is covered by mispecified clones.

  4. eLife

    Reviewer #2 (Public Review):

    Prasad et al investigate mechanisms of interface contractility that occur at borders between cells of different specification states. Cells of different specification states typically sort out, minimizing interface contact, in association with increased junctional contractility that can be visualized by phalloidin labeling. Here, Prasad et al show, for multiple different examples of specification and/or signaling states, that bilateral activation of JnK flanks these interfaces, which are associated with elevated rates of Jnk-dependent apoptosis. Blocking Jnk activity does not seem to affect phalloidin labeling, however, placing interphase contractility upstream or parallel to Jnk activity and apoptosis. Interestingly, activated Ras[V12] is an exceptional case where interphase contractility and bilateral Jnk activation occur without elevated apoptosis. Indeed, RasV12 can suppress apoptosis associates with interfaces between other distinct cell types. Prasad et al suggest that this property of Ras[V12] activated cells may underlie their oncogenic potential in mammals. These are potentially interesting observations that address what happens when cell of disparate signaling and/or specification states are opposed. In principle, they could be of interest both to developmental biologists, from the perspective of correction of developmental errors, and to cancer biologists, from the perspective of eliminating precancerous cells.

    It is not clear how much advance is represented over the prior description of 'morphogenetic apoptosis', in which bilateral Jnk activity was also an integral part (Adachi-Yamada and O'Connor, Devl Biol vol251 pp74-90 2002). There is little new mechanistic insight provided here. As such, the observations seem preliminary and to represent only a limited advance.

  5. eLife

    Reviewer #3 (Public Review):

    Elimination of aberrant cells from epithelial tissues is important for normal tissue physiology. Here the authors study a specific type of cell elimination that is dedicated to the removal of miss-specified cells. This type of elimination is dependent on interface contractility. The authors now identified an important role for JNK signaling, which is activated at this interface, where contractility is highest.

    Strength: The authors use a large variety of cell specification mutants and different drivers to manipulate cell specification. Together, this shows that the observed phenotypes are of a general nature and not dependent on single signaling pathways.
    Weakness: Quantitative characterization of much of the data is missing. Only single representative images are shown for many of the experiments. The manuscript would strengthen massively when these images are supported with a quantitative measurement. For example (but not limited to), TRE-GFP in correctly vs mis-specified clones in Figure 2K-L, TRE-GFP intensity in Figure 3, clonal analysis in Figure 5.

    Type of elimination:
    The authors describe a very distinct and specific phenotype of smooth rounded clones with high contractility. It is obvious that this is, on a phenotypic scale, different from other types of cell elimination, such as live extrusion and cell-cell competition. Throughout the manuscript the authors emphasize that the underlying nature of interface contractility is different to cell competition. Because cell competition "responds to a clearly defined fitness gradient between two neighbouring cells, which ensures that always the aberrant loser cell dies, independent of spatial context." And "linking apoptosis to a fixed loser genotype". However, this only holds true for the classical types of cell competition (e.g. Minute), while many examples of cell competition have been reported where elimination of cells is not set in stone, but also highly context dependent. For example, HRasV12 expressing cells are eliminated from epithelia in mice on a normal diet, while a high fat diet prevents their elimination (Sasaki et al, Cell Reports 2018). Without the experimental support that relative differences in cell specification do not cause a difference in cellular fitness it is hard to grasp the conceptual difference. Instead, the concept reported by the authors is better described as a variety of cell competition.

    Clone size
    The authors claim that remove aberrant cells by interface contractility is dependent on clone size and only occurs when aberrant cells are the minority compared to the surrounding tissue. Currently, there is no data in the manuscript that supports this claim. The only analysis of tissues containing a majority of miss-specified cells (Figures 2I-2J) shows a bilateral activation of JNK, similar to a minority of miss-specified cells. To support the claim that the phenotype is size dependent further analysis of clone size in relation to apoptosis and JNK activation is essential.

    JNK and cell autonomous regulation:
    The authors validate that expression of TRE-GFP is dependent on JNK signaling, through over-expression of a dominant negative variant of the JNK kinase (BSKDN) in clones of miss-specified cells (ey or tkv). This experiment nicely shows that activation of JNK in surrounding WT cells is not altered. This furthermore illustrates that JNK signaling in the miss-specified cells is not needed for activation of JNK in their neighbors. However, this does not support the conclusion that JNK is activated in a cell autonomous fashion in either of these populations. The interaction of the two cell types can still cause signaling, but through inhibition of one of the kinases within the pathway, this just does not lead to downstream activation of TRE-GFP. In fact, one could argue that the expression of TRE-GFP is not cell-autonomous, because tkvCA clones that are not mis-specified (within dad4-LacZ regions) do not show induction of TRE-GFP (Fig 2L). The only way to untangle cell autonomous vs non-autonomous effects is through manipulation of upstream communication between the different cell populations. Such experiments, for example manipulation of contractility, are likely beyond the scope of this study. Therefore, I would suggest rephrasing this paragraph.

    Apoptosis:
    A large part of the manuscript is dedicated to the characterization of elimination of miss-specified cells through apoptosis. This process is important for maintenance of tissue integrity and a crucial part of the manuscript. Some conclusions are not fully supported by the data represented in the current form of this manuscript;
    The authors claim that fkh- and ey-expressing cells are not eliminated when apoptosis is blocked by expression of p35. This is based on analysis of apical vs basal clone count (Figure 1T). This analysis reflects a combination of induction efficiency and clone retention. Therefore, information on the cellular behavior within clones is lacking and only provides information on survival of cells when complete clones are eliminated. The conclusion should be supported by additional analysis on clone size and total clone area, ideally based on cell number. In addition, statistical analysis of conditions with and without expression of p35 should be included.
    Furthermore, the analysis of apoptosis at clonal interfaces does not support the conclusion that "many, but not all apoptotic events occur at interfaces". Overall, there is increased apoptosis within clones compared to wild-type tissue. However, the rates of apoptosis are higher (ey, Fig S5B) or similar (fkh and tkvCA, Fig 5B-C) in clonal cells compared to clonal interface cells. The authors should revise these statements or provide more compelling analysis.

  6. Version published to 10.1101/2022.06.20.496791v1 on bioRxiv