Early exercise disrupts a pro-repair extracellular matrix program during zebrafish fin regeneration

Reviewer #1 (Public review):

Summary:

The goal of the manuscript was to determine if strenuous exercise negatively impacted regeneration. Indeed, the major conclusion of the manuscript is that elevated exercise during the early stages of regeneration compromises the regenerative process. The authors further conclude that regeneration is disrupted due to defects in blastema formation, which is caused by impaired HA deposition and reduced active (nuclear) Yap.

Strengths:

(1) The paradigm of elevated exercise disrupting ECM and regeneration is significant, and provides an experimental model to better understand connections between the ECM and cell/tissue activities.

(2) The conclusion that exercise intensity correlates with defects in regeneration is supported.

(3) The demonstration for the requirement for HA is well supported via transcriptomics and multiple independent strategies to manipulate HA levels.

(4) The demonstration that nuclear Yap depends on the amount of HA is well-supported.

Weaknesses:

(1) The authors conclude throughout the manuscript that "blastema formation" is disrupted, but they do not provide any insights into how blastema formation is disrupted (reduced de-differentiation? reduced cell migration? both?). While they show that there are fewer dividing cells, the timing of exercise is prior to outgrowth. So, the effect of dividing cells is likely secondary, which is not considered (or not clearly explained).

(2) The authors conclude that patterning is affected, but their analyses of patterns (bifurcations) are very limited. It is also not clear if patterning is believed to be affected by a common exercise-induced mechanism or a different exercise-induced mechanism (or by a secondary mechanism).

(3) The significance of HA in regeneration has been shown before in zebrafish fins, as well as in a handful of other models of regeneration. Although largely cited, explaining some of this work in more detail would give the reader a better picture of how HA is believed to promote regeneration. It may also highlight some emerging questions about the role of HA in regeneration that would permit a richer story and specific future directions.

(4) In general, parts of the text lack specificity/clarity, and in other cases, there seems to be contradictory information.

(5) Overall, many of the conclusions were well supported by the data, and this study is likely to provide a foundation for future research on the role of the ECM in tissue repair and regeneration. The main limitations were in connecting the experimental details with the specific processes required for regeneration, and in clearly explaining the findings.

Reviewer #2 (Public review):

In this study, Lewis et al. established a forced swimming paradigm to investigate how mechanical loading influences caudal fin regeneration. They found that forced exercise impaired the normally robust regeneration process, particularly in the peripheral/lateral ray regions. Transcriptomic profiling of exercised fish further revealed that extracellular matrix (ECM) gene programs were affected, and the authors provided evidence that disruption of hyaluronic acid (HA) synthesis may underlie this impairment. While the question of how mechanical loading impacts tissue regeneration is rather intriguing and the study nicely demonstrates a role for HA in fin regeneration, I have some concerns regarding the specificity of forced exercise as a model for mechanical loading, and thus the causal link between mechanical loading and HA synthesis disruption.

Major concerns:

(1) Forced exercise as a model for mechanical loading.

Is it possible that the forced exercise paradigm imposes greater shear stress on the peripheral/lateral ray regions, thereby disrupting the fragile wound epidermis at this early stage and consequently affecting the regeneration program and phenotypes? The wound epidermis appears visibly torn or disrupted (Figure 1A, right panel, 2 dpa image). Given the critical role of the wound epidermis in blastema establishment and fin regeneration (PMID: 11002347; PMID: 34038742; PMID: 26305099), could this be a simpler explanation to consider, instead of the proposed role of mechanical loading and cryptic mechanical sensors?

(2) The general effect of HA on fin regeneration.

While the authors convincingly show that exogenous HA can ameliorate fin regeneration defects caused by forced exercise (Figure S7), it would be important to include a control examining the effect of HA supplementation in non-exercised animals. Does HA act as a general enhancer of fin regeneration even in the absence of forced exercise? Additionally, please consider merging Figure S7 (HA supplement) with Figure 5 (HA depletion) to improve clarity for readers.

(3) Proper annotation of the investigated ray regions.

As the authors clearly demonstrate that peripheral and central rays respond differently to forced exercise, it is important to explicitly define the regions corresponding to these rays. Do the peripheral rays refer to the dorsal-most and ventral-most rays among the 18-20 rays across the amputation plane? Which rays are considered central? Please clarify.

Reviewer #3 (Public review):

Summary:

In the submitted article by Lewis et al., the authors investigate how mechanical stimulation influences organ regeneration using the well-characterized zebrafish caudal fin regeneration model. Using a swim flume and a 30min/day exercise regime, the authors found that exercise during the establishment of the blastema reduced regeneration and led to skeletal deformations. Transcriptional profiling of regenerated caudal fin tissue revealed reduced expression of extracellular matrix-associated genes, which were found to be expressed by blastemal fibroblast and osteoblast lineage cells.

Downregulated genes included hyaluronic acid synthases 1 and 2; accordingly, hyaluronic acid levels were found to be reduced in regenerating fins exposed to exercise. The link between regeneration and HA was further confirmed through HA depletion and HA overexpression experiments, which showed a reduction in blastema size and partial rescue of blastema formation, respectively. The authors further show that HA levels, as well as the extent of mechanical loading correlate with nuclear localization of the mechanotransducer Yap and conclude that biomechanical forces play a significant role during regeneration through regulation of HA levels in the ECM and therewith regulation of YAP downstream signaling.

This work expands our understanding of the biochemical signaling connecting biomechanical forces with tissue regeneration. The conclusions are well supported by the data.

Strengths:

(1) Analysis is performed in multiple replicate experimental groups and shows the robust response to the experimental conditions.

(2) The link of HA levels to blastema formation was confirmed through HA overexpression and two different HA depletion experiments.

(3) The use of a previously established fin regeneration single cell dataset does elegantly show the correlation of changes in gene expression levels and specific tissue types, which was further confirmed by in vivo imaging of cell type-specific transgenic lines.

Weaknesses:

Tissue sections stained with hematoxylin and eosin would be helpful to show the changes in tissue architecture more clearly.

Author response:

Reviewer #1

We agree that further clarification how elevated exercise disrupts blastema formation would strengthen the manuscript. Our data suggests a major contribution of proliferation. Exercise reduced the fraction of proliferative cells at 3 dpa, consistent with disrupted HA production and downstream Yap signaling. This interpretation aligns with prior studies showing that proliferation contributes to blastema establishment and is not restricted to the outgrowth phase of fin regeneration (Poleo et al, 2001; Poss et al, 2002; Wang et al, 2019; Pfefferli et al, 2014; Hou et al, 2020). We will explore additional experiments to reinforce these insights into the cellular mechanisms underlying exercise-disrupted blastema formation.

We acknowledge that our analysis of ray branching abnormalities is limited in the current manuscript. We focus our study on introducing the zebrafish swimming and regeneration model and then characterizing ECM and signaling changes accounting for disrupted blastema establishment. For completeness, we included the observation of skeletal patterning defects (branching delays and bone fusions) but without detailed analysis. We note that decreased expression of shha and Shh-pathway components following early exercise corresponds with the branching defects. However, we recognize exercise could have additional effects during the outgrowth[KS1] phase when branching morphogenesis actively occurs. Therefore, we will expand our discussion to outline future research directions related to exercise impacts on regenerative skeletal patterning.

We will expand the Introduction and/or Discussion sections to provide more context on known HA roles across regeneration contexts, including in zebrafish fins. Finally, we will improve the text’s clarity and specificity throughout the manuscript, including to resolve or explain any apparent contradictions.

Reviewer #2

We appreciate the Reviewer's concern regarding the specificity of forced exercise as a model for mechanical loading. Forced exercise has been widely used in vivo to induce mechanical loading without the requirement for specialized implants or animal restraint, including in mouse (Wallace et al, 2015; Bomer et al, 2016), rat (Honda et al, 2003; Boerckel et al, 2011; Boerckel et al, 2012), and, most relevant to our study, zebrafish models (Fiaz et al, 2012; Fiaz et al, 2014; Suniaga et al, 2018). However, we will expand our discussion of this approach and ensure precise language distinguishing exercise from mechanical loading.

We acknowledge the possibility that early shear stress disrupts the wound epidermis, which we will elaborate on in a revised Discussion. However, exercise-induced disruptions to the fin epidermis of early regenerates (1–2 dpa; Figure 2) typically resolve within one day, whereas fibroblast lineage cells still fail to establish a robust blastema. Therefore, sustained effects of mechanical loading and/or mechanosensation are likely major contributors to the observed regeneration phenotypes.

We will explore whether HA acts as a general enhancer of fin regeneration by comparing blastemal HA supplementation vs. controls in non-exercised regenerating animals, if technically feasible. We will merge Figure S7 (HA supplementation) with Figure 5 (HA depletion) for clarity, as suggested.

We will include a schematic and clear definitions for 'peripheral' and 'central' rays in a revised manuscript.

Reviewer #3

We included Hoechst and eosin fluorescent staining in the manuscript to show changes in tissue architecture following swimming exercise (Supplemental Figure 4). We will extend this histological analysis to include hematoxylin and eosin staining to provide additional tissue visualization.

References

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