E(spl)m4 Directly Antagonizes Traf4 to Inhibit JNK Signaling in Drosophila

Reviewer #1 (Public review):

Summary:

The authors investigate how the Drosophila TNF receptor-associated factor Traf4 - a multifunctional adaptor protein with potential E3 ubiquitin ligase activity - regulates JNK signaling and adherens junctions (AJs) in wing disc epithelium. When they overexpress Traf4 in the posterior compartment of the wing disc, many posterior cells express the JNK target gene puckered (puc), apoptose, and are basally extruded from the epithelium. The authors term this process "delamination", but I think that this is an inaccurate description, especially since they can suppress the "delamination" by blocking programmed cell death (by concomitantly overexpressing p35). Through Y2H assays using Traf4 as a bait, they identified the Bearded family proteins E(spl)m4 (and to a lesser extent E(spl)m2), as Traf4 interactors. They use Alphafold to model computationally the interaction between Traf4 and E(spl)m4. They show that co-overexpression of Traf4 with E(spl)m4 in the posterior domain of the wing disc reduces death of posterior cells. They generate a new, weaker hypomorphic allele of Traf4 that is viable (as opposed to the homozygous lethality of null Traf4 alleles). There is some effect of these mutations on wing margin bristles; fewer wing margin bristle defects are seen when E(spl)m4 is overexpressed, suggesting opposite effects of Traf4 and E(spl)m4. Finally, they use the Minute model of cell competition to show that Rp/+ loser clones have greater clone area (indicating increased survival) when they are depleted for Traf4 or when they overexpress E(spl)m4. Only the cell competition results are quantified. Because most of the data in the preprint are not quantified, it is impossible to know how penetrant the phenotypes are. The authors conclude that E(spl)m4 binds the Traf4 MATH/TRAF domain, disrupts Traf4 trimerization, and selectively suppresses Traf4-mediated JNK and caspase activation without affecting its role in AJ destabilization. However, I believe that this is an overstatement. First, there is no biochemical evidence showing that Traf4 binds E(spl)m4 and that E(spl)m4 disrupts Traf4 trimerization. Second, the data on AJs is weak and not quantified; additionally, cells that are being basally extruded lose contact with neighboring cells, hence changes in adhesion proteins. Related to this, the authors, in my opinion, inaccurately describe basal extrusion of dying cells from the wing disc epithelium as delamination.

Strengths:

(1) The authors use multiple approaches to test the model that overexpressed E(spl)m4 inhibits Traf4, including genetics, cell biological imaging, yeast two-hybrid assays, and molecular modeling.

(2) The authors generate a new Traf4 hypomorphic mutant and use this mutant in cell competition studies, which supports the concept that E(spl)m4 (when overexpressed) can antagonize Traf4.

Weaknesses:

(1) Conflation of "delamination" with "basal extrusion of apoptotic cells": Over-expression of Traf4 causes apoptosis in wing disc cells, and this is a distinct process from delamination of viable cells from an epithelium. However, the two processes are conflated by the authors, and this weakens the premise of the paper.

(2) Dependence on overexpression: The conclusions rely heavily on ectopic expression of Traf4 and E(spl)m4. Thus, the physiological relevance of the interaction remains inferred rather than demonstrated.

(3) Lack of quantitative rigor: Except for the cell competition studies, phenotypic descriptions (e.g., number of apoptotic cells, puc-LacZ intensity) are qualitative; additional quantification, inclusion of sample size, and statistical testing would strengthen the conclusions.

(4) Limited biochemical validation: The Traf4-E(spl)m4 binding is inferred from Y2H and in silico models, but no co-immunoprecipitation or in vitro binding assays confirm direct interaction or the predicted disruption of trimerization.

(5) Specificity within the Bearded family: While E(spl)m2 shows partial binding and Tom shows none, the mechanistic basis for this selectivity is not deeply explored experimentally, leaving questions about motif-context contributions unresolved.

Reviewer #2 (Public review):

Summary:

This manuscript analyzes the contribution of Traf4 to the fate of epithelial cells in the developing wing imaginal disc tissue. The manuscript is direct and concise and suggests an interesting and valuable hypothesis with dual functions of Traf4 in JNK pathway activation and cell delamination. However, the text is partially speculative, and the evidence is incomplete as the main claims are only partially supported. Some results require validation to support the conclusions.

Strengths:

(1) The manuscript is direct and concise, with a well-written and precise introduction.

(2) It presents an interesting and valuable hypothesis regarding the dual role of Traf4 in JNK pathway activation and cell delamination.

(3) The study addresses a relevant biological question in epithelial tissue development using a genetically tractable model.

(4) The use of newly generated Traf4 mutants adds novelty to the experimental approach.

(5) The manuscript includes multiple experimental strategies, such as genetic manipulation and imaging, to explore Traf4 function.

Weaknesses:

(1) The evidence supporting key claims is incomplete, and some conclusions are speculative.

(2) The use of GFP-tagged Traf4 lacks validation regarding its functional integrity.

(3) Orthogonal views and additional imaging data are needed to confirm changes in apicobasal localization and cell delamination.

(4) Experimental conditions and additional methods should be further detailed.

(5) The interaction between Traf4 and E(spl)m4 remains speculative in Drosophila.

(6) New mutants require deeper analysis and validation.

(7) The elimination of Traf4 mutant clones may be due to cell competition, which requires further experimental clarification.

(8) The role of Traf4 in cell competition is contradictory and needs to be resolved.

Reviewer #3 (Public review):

Summary:

This is an important and well-conceived study that identifies the Bearded-type small protein E(spl)m4 as a physical and genetic interactor of TRAF4 in Drosophila. By combining classical genetics, yeast two-hybrid assays, and AlphaFold in silico modeling, the authors convincingly demonstrate that E(spl)m4 acts as an inhibitor of TRAF4-mediated induction of JNK-driven apoptosis in developing larval imaginal wing discs, while not affecting TRAF4's role in adherence junction remodeling.

Based primarily on modeling, the authors propose that the specificity of E(spl)m4 towards TRAF4-mediated signaling arises from its interference with TRAF4 trimerization, which is likely required for the activation of the JNK signaling arm but not for the maintenance of adherence junctions and stability of E-cadherin/β-catenin complex.

Overall, this study is of broad interest to cell and developmental biologists. It also holds potential biomedical relevance, particularly for strategies aimed at modulating TRAF protein activities to dissect and modulate canonical versus non-canonical signaling functions.

Strengths:

(1) The work identifies the Bearded-type small protein E(spl)m4 as a physical and genetic interactor of TRAF4 in Drosophila, extending the understanding of E(spl)m4 beyond its established functions in Notch signaling.

(2) The study is experimentally solid, well-executed, and written, combining classical genetics with protein-protein interaction assays and modeling to reveal E(spl)m4 as a new regulator of TRAF4 signaling.

(3) The genetic and biochemical data convincingly show the ability of E(spl)m4 overexpression to inhibit TRAF4-induced JNK-dependent apoptosis, while leaving the TRAF4 role in adherens junction remodeling unaffected.

(4) The findings have important implications for the regulation of cell signaling and apoptosis and may guide pharmacological targeting of TRAF proteins.

Weaknesses:

The study is overall strong; however, several aspects could be clarified or expanded to strengthen the proposed mechanism and data presentation:

(1) The proposed mechanism that E(spl)m4 inhibits TRAF4 activation of JNK signaling by affecting TRAF4 trimerization relies mainly on modeling. Experimental evidence would strengthen this claim. For example, a native or non-denaturing SDS-PAGE could be used to assess TRAF4 oligomerization states in the absence or presence of E(spl)m4 overexpression, testing whether E(spl)m4 interferes with high-molecular-weight TRAF4 assemblies.

(2) The study depends largely on E(spl)m4 overexpression, which may not reflect physiological conditions. It would be valuable to test, or at least discuss, whether loss-of-function or knockdown of E(spl)m4 modulates the strength or duration of JNK-mediated signaling, potentially accelerating apoptosis. Such data would reinforce the model that E(spl)m4 acts as a physiological modulator of TRAF4-JNK signaling in vivo.

(3) The authors initially identify both E(spl)m4 and E(spl)m2 as TRAF4 interactions, but subsequently focus on E(spl)m4. It would be helpful to clarify or discuss the rationale for prioritizing E(spl)m4 for detailed functional analysis.

(4) E(spl)m4 overexpression appears to protect RpS3 loser clones (Figure 6H-K), yet caspase-3-positive cells are still visible in mosaic wing discs. Please comment on the nature of these Caspase 3-positive cells, whether they are cell-autonomous to the clone or non-autonomous (Figure 6K)?

(5) This is a clear, well-executed, and conceptually strong study that significantly advances understanding of TRAF4 signaling specificity and its modulation by the Bearded-type protein E(spl)m4.