Early swimming exercise disrupts robust regeneration of the zebrafish caudal fin.

(A) Quantification of regenerated area (mm2) at different percentages of maximum swimming speed (static, 12.5%, 25%, 37.5%, 50%) at 3 days post-amputation (dpa) (n= 5 fish/trial and 2 replicate trials). Representative whole-mount images are shown on the right for each swimming speed condition. (B) Quantification of regenerated area (mm2) at 28 dpa when exercise is initiated at different days post-amputation (1, 2, 3, and 5 dpa) and unexercised controls (n= minimum of 3 fish/trial and 2 replicate trials). Representative whole-mount images of fins at 4 dpa, 7 dpa, and 28 dpa are shown on the right for each exercise initiation condition. The timeline above each panel represents the exercise regimen (30 minutes/day) over the experimental period. White dashed lines show amputation plane. Scale bars represent 1 mm. Significance determined by one-way ANOVA with Dunnett’s post-hoc tests. Error bars represent standard deviation.

Early exercise initiation inhibits blastemal fibroblast establishment.

(A) Representative 1-week time series of whole mount regenerating caudal fin overlay images in Tg(sost:nlsEos) blastemal fibroblast labeled animals (n=6). White arrowheads and arrows highlight either a delay or the complete loss of regeneration in peripheral regions, respectively, in the example shown. (B) Animal in (A) following release from exercise regimen showing robust re-establishment of fibroblast lineage cells. Dashed white lines indicate amputation plane. The scale bar shows 1 mm. The timeline above each panel represents the exercise regimen (30 minutes/day) over the experimental period.

Transcriptomic profiling identifies an extracellular matrix (ECM) program downregulated in exercised animals.

(A) Gene Ontogeny analysis showing the most significantly enriched biological processes from genes differentially downregulated in transcriptomic comparison. (B) Heatmap and hierarchical clustering of RNA-Seq replicates showing the expression profiles of genes identified within the top two ECM-related GO terms. (C) UMAP of previously published 3 and 7 dpa scRNA-Seq dataset, subset to clusters representing superficial epidermis, basal epidermis, and the combined blastemal fibroblast and osteoblast-lineage cells. (D) Bubble plot showing over-representation of genes identified within the top two ECM-related GO terms within the fibroblast and osteoblast lineage cluster.

Exercise disrupts blastema-enriched hyaluronic acid production.

(A) Representative confocal maximum intensity projection of a 3 dpa regenerating caudal fin paraffin section stained for bHABP (green) (n=3 sections; 1 animal). (B) Transverse paraffin section showing bHABP staining (green) within unamputated caudal fin tissue (n=3 sections; 1 animal). (C-C’’) Cryosection of a 3 dpa regenerating caudal fin from a Tg(sost:nlsEos) fibroblast lineage reporter fish showing co-staining of bHABP (green), Zn5 (cyan; osteoblasts), and Eos (magenta). Panels showing bHABP with Zn5 (C’) and bHABP with Eos (C’’) highlight cellular localization (n=6 sections; 2 animals). (D-F) Representative images of wildtype regenerating caudal fin paraffin sections at 3 dpa stained for bHABP (magenta) in control (D, D’) peripheral ray (E, E’) and central ray (F, F’) (n=9 sections; 3 animals). The boxed areas in panels D-F are shown 3x enlarged in panels D’-F’ to highlight bHABP staining. (G) Quantification of bHABP fluorescence intensity (AU) in control, peripheral ray, and central ray conditions from D-F (n= 3 sections/replicate and 3 replicate animals). Dashed white lines indicate the amputation plane. Scale bars represent 100 µm in panel A, 50 µm in panels B-F. Significance determined by one-way ANOVA with Dunnett’s post-hoc test. Error bars represent standard deviation.

Hyaluronic acid supports robust blastema establishment during fin regeneration.

(A, B) Representative regenerating caudal fins following 3 days treatment with 150 µM 4-MU (B) or DMSO controls (A) (n=6 animals/trial, 2 replicate trials). (A’, B’) Fluorescent overlay from A and B showing decreased fibroblast-lineage blastema establishment following 4-MU treatment in 3 dpa Tg(tph1b:mCherry) reporter animals. (C, D) Quantification of regenerated area (C) and blastema area (D) following 150 µM 4-MU from 0-3 dpa (E, F). Representative whole-mount fins after intra-blastemal injection of 15 mg/mL Hyaluronidase (HAse) (n=6 animals/trial, 2 replicate trials). (F’, F’’) Fluorescent overlay from F showing decreased fibroblast establishment relative to uninjected control tissue following blastemal HAse injection in 3 dpa Tg(sost:nlsEos) animals. (G,H) Quantification of regenerated (C) and blastema area (D) following HAse injection. Fluorescent lineage-reporter images are confocal maximum intensity projection overlays. Dashed white lines indicate amputation plane. Scale bars show 1 mm. Significance in (C, D) determined by unpaired two-tailed Student’s t-test. Significance in (G, H) determined by paired two-tailed Student’s t-test. Error bars represent standard deviation. Figure legend on next page.

Hyaluronic acid supports Yap nuclear localization during fin regeneration.

(A–E) Representative images of 3 days post amputation (dpa) regenerating fin sections stained for Yap (green) under different hyaluronic acid (HA)-altered conditions: (A) Unexercised DMSO controls. (B) Swim exercise for 3 days at 50% maximum swim speed. (C) 3-day treatment with 150 µM 4-methylumbelliferone (4-MU). (D) Intra-blastemal injection of 15 mg/mL hyaluronidase (HAse) at 60-hour post amputation. (E) Intra-blastemal injection of 1.25 ng/nL HA at 36 hpa followed by 2 swim exercise sessions at 50% maximum swim speed. The boxed areas in panels A-E indicate 3x enlarged in panels A’-E’ to highlight Yap colocalization with nuclei (n=3 sections; 3 animals). (F-H) Quantification of Yap-Hoechst colocalization (Pearson correlation coefficient, PCC) following (F) DMSO, exercised or 150 μM 4-MU treatment, (G) mock or intra-blastemal injection of 15 ng/nL HAse, and (H) with and without intra-blastemal injection of 1.25 ng/nL HA prior to exercise (n= 3 sections/replicate and 3 replicate animals). (I) Representative immunofluorescence images of primary fin blastema-derived fibroblasts cultured overnight on 0.1% collagen alone (0% HA) or supplemented with 1% hyaluronic acid (HA) (n=9 over 3 replicate trials). Cells were stained for GFP (green; marking tph1b:GCaMP6s⁺ fibroblasts), Yap (magenta), and nuclei (blue; Hoechst). Cells treated with 1% HA display enhanced nuclear Yap localization (arrowheads) (J) Quantification of Yap nuclear localization by Pearson’s correlation coefficient (PCC) of Yap and Hoechst signal across increasing HA concentrations (0%, 0.1%, 0.5%, and 1%) on 0.1% collagen. Each dot represents a single image; colored dots represent independent replicates. ns = not significant. Dashed white lines indicate amputation plane. Scale bars are as labeled. Significance in (F, J) determined by one-way ANOVA with Dunnett’s post-hoc test. Significance in (G, H) determined by paired two-tailed Student’s t-tests. Error bars show standard deviation.