During an inflammatory response, zebrafish tnfa and tnfb are expressed by different cell types and have distinct expression kinetics

Reviewer #1 (Public review):

Summary:

This study investigates the roles of the two tumor necrosis factor genes (tnfa and tnfb) in zebrafish during inflammatory responses. TNF is a central regulator of inflammation across vertebrates; however, while mammalian TNF signaling is well characterized, the functional divergence of duplicated TNF genes in teleosts remains less well understood. In this work, the authors generate novel zebrafish fluorescent reporter lines for tnfb and use them to perform comparative analyses of the spatial and temporal expression patterns of tnfa and tnfb during inflammation. They report that these paralogous genes are produced by distinct immune cell populations and exhibit different induction kinetics during inflammatory processes. Based on these observations, the authors propose that tnfa and tnfb may fulfill non-redundant roles in the zebrafish immune response.

Strengths:

The study addresses an important gap in understanding the functional divergence of TNF paralogs in teleosts. Given that gene duplication events are common in fish genomes, clarifying how duplicated cytokines partition their functions is valuable for both evolutionary immunology and zebrafish model research. The work makes effective use of the zebrafish model, which is particularly well suited for in vivo imaging of dynamic immune cell behaviors during inflammation. A key strength of the study is the integration of analyses of cell-type specificity, transcriptional regulation, and temporal expression dynamics. In particular, the live imaging experiments are compelling and provide clear visual evidence that tnfa and tnfb differ in both cellular sources and expression kinetics, which strengthens the claim that these paralogs may have diverged in their regulation and potentially their function. By distinguishing these aspects of the two cytokines, the study provides useful conceptual and methodological guidance for future investigations of inflammatory signaling in zebrafish.

Weaknesses:

(1) While the manuscript convincingly documents distinct expression patterns, the functional consequences of these differences remain unexplored. The conclusions regarding non-redundant roles would benefit from functional perturbation experiments. Relatedly, the authors propose that tnfa and tnfb may play different immunological roles, but the mechanistic basis underlying these differences is not addressed. For example, do the two cytokines engage different receptors or signaling pathways? Do they trigger distinct downstream transcriptional programs?

(2) Some imaging-based observations appear largely qualitative. Additional quantitative analyses, such as statistical comparisons of expression levels across time points or cell populations, would strengthen the robustness of the conclusions. For instance, in Figure 4, the expression levels of tnfa and tnfb reporter transgenes in immune cells should be quantitatively compared between control and amputated conditions.

(3) It would also be important to clarify whether the distinct maturation kinetics of the fluorescent reporters were taken into account when interpreting expression timing. Since GFP typically matures more rapidly than mCherry in vivo, the authors should comment on whether this difference could influence the apparent expression kinetics of tnfa versus tnfb.

Reviewer #2 (Public review):

Summary:

In this manuscript, van Dijk et al analyse the expression of the largely ignored paralogue of TNF in zebrafish, tnfb. They generate reporter transgenic lines and show that the reporter expression is consistent with endogenous mRNA expression in zebrafish larvae. Unlike its better-known paralogue tnfa, tnfb is constitutively expressed in mantle cells of neuromasts, and in a few leukocytes. It is also inducible in macrophages and some neutrophils upon wounding or detection of microbes, with faster kinetics than tnfa or il1b.

Strengths:

Generation and convincing validation of new transgenic reporter lines for tnfb with either green or red fluorescent proteins. Superb imaging and careful analysis of these lines crossed to complementary reporter transgenics, backed with in situ hybridization and qRT-PCR analysis of FACS-sorted cells. Excellent methods section.

Weaknesses:

Lack of functional analysis; these lines are a potentially valuable tool, but so far provide no clue regarding the role of tnfb. Is it a pro-inflammatory cytokine acting in synergy with tnfa, or is it an antagonist? What are its receptor(s)? What signalling pathways and downstream genes does it induce? Addressing at least some of these questions should greatly increase the impact of the paper.

Author response:

Reviewer #1 (Public review):

(1) While the manuscript convincingly documents distinct expression patterns, the functional consequences of these differences remain unexplored. The conclusions regarding non-redundant roles would benefit from functional perturbation experiments. Relatedly, the authors propose that tnfa and tnfb may play different immunological roles, but the mechanistic basis underlying these differences is not addressed. For example, do the two cytokines engage different receptors or signaling pathways? Do they trigger distinct downstream transcriptional programs?

We agree functional analysis on Tnfb is relevant to address, however, the focus of the current manuscript (Tools and Resources article type) was to report the generation and validation of the new tnfb-reporter line, we feel that functional data is better suited for a separate manuscripts. In fact, this will be part of a follow manuscript which will be forthcoming soon.

(2) Some imaging-based observations appear largely qualitative. Additional quantitative analyses, such as statistical comparisons of expression levels across time points or cell populations, would strengthen the robustness of the conclusions. For instance, in Figure 4, the expression levels of tnfa and tnfb reporter transgenes in immune cells should be quantitatively compared between control and amputated conditions.

In figure 4, we focus on which cells express either cytokine, not on when they express it nor whether the one cell expresses more or less eGFP/mCh. Also, tnfb:mCh-F and tnfa:eGFP-F expression is membrane-bound as these protein is farnesylated, whereas il1b:eGFP is not, and has a cytoplasmic distribution. Because of possible biases due to the different distribution or abundance of cytoplasmic vs farnesylated proteins within a cell, we never compared max eGFP to max mCherry within a treatment group.

(3) It would also be important to clarify whether the distinct maturation kinetics of the fluorescent reporters were taken into account when interpreting expression timing. Since GFP typically matures more rapidly than mCherry in vivo, the authors should comment on whether this difference could influence the apparent expression kinetics of tnfa versus tnfb.

In figure 5, we do count the cells expressing either of the cytokine, and use eGFP/mCherry signal to infer on how early these cells express the cytokine. We, however, do not directly compare maximum eGFP or mCherry fluorescence intensity per cell, which, especially in the early time points, could be biased by differences in protein maturation, we only score eGFP or mCherry presence in a cell. We could not really compare or account for differences in protein maturation as we do not possess Il1b and tnfa transgenic lines driving mCherry expression for comparison (and to our knowledge are not available in other laboratories). Based on the obtained results however, it appears that the earlier maturation of eGFP compared to mCherry may not influence the outcome of the analysis, as no single tnfa:eGFP-F+ cells were observed at any time point and single il1b:eGFP+ cells were observed only 6h after amputation, whereas eGFP/mCherry double positive cells could be observed as early as 2h after amputation. Any bias should influence the period between 1h and 2h, and we did not look at time lapses shorter than 1h.

Reviewer #2 (Public review):

(1) Lack of functional analysis; these lines are a potentially valuable tool, but so far provide no clue regarding the role of tnfb. Is it a pro-inflammatory cytokine acting in synergy with tnfa, or is it an antagonist? What are its receptor(s)? What signalling pathways and downstream genes does it induce? Addressing at least some of these questions should greatly increase the impact of the paper.

Please refer to response to Reviewer #1 point 1.

We will address the other recommendation to the authors as they will improve the manuscript.