Loss of ppargc1a Results in Ciliogenesis Defects (A) Schematic indicating location of MCCs in the proximal portion and monociliated cells in the distal portion of the zebrafish pronephros at 24 hpf. (B) WT (top) and ppargc1asa13186/sa13186 mutant (bottom) zebrafish at 24 hpf stained via WISH for odf3b to mark MCCs. Scale bar, 90 μm. (C) Graph representing the number of MCCs present in WT and ppargc1asa13186/sa13186 zebrafish at 24 hpf. (D) Fluorescent in situ hybridization for MCC marker (odf3b, green) and transporter cell marker (trpm7, red) in WT and ppargc1a morphant zebrafish at 24 hpf. Scale bar, 10 μm. (E and F) Graphs representing the percentage of odf3b+ (E) or trpm7+ (F) cells in WT (black) and ppargc1a morphants (white) pronephros between somites 9 and 11. (G) Twenty-eight hours post-fertilization WT (top), ppargc1a MO-injected (middle), and ppargc1asa13186/sa13186 (bottom) zebrafish stained via whole-mount immunofluorescence for acetylated α-tubulin (cilia, green), γ-tubulin (basal bodies, red), and DAPI in the proximal pronephros. Scale bar, 10 μm. (H) Cilia length in micrometers for the proximal pronephros. (I) Graph of the percentage of ciliated basal bodies/total basal bodies per 100 μm in the proximal pronephros. (J) Twenty-eight hours post-fertilization WT (top) and ppargc1a MO-injected (middle) and ppargc1asa13186/sa13186 (bottom) zebrafish stained via whole-mount immunofluorescence for acetylated α-tubulin (cilia, green), γ-tubulin (basal bodies, red), and DAPI in the distal pronephros. Scale bar, 10 μm. (K) Cilia length in micrometers for the distal (right) pronephros. (L) Graph of the percentage of ciliated basal bodies/total basal bodies per 100 μm in the distal pronephros. (M and N) Fluorescent intensity plots of WT (black) and ppargc1a MO-injected (red) zebrafish in proximal (M) and distal (N) pronephros. Data are represented as mean ± SD. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 (one-way ANOVA or t test).

ppargc1a and Prostaglandin Pathway Component ptgs1 Act Together to Properly Form Ciliated Cells (A) Twenty-eight hours post-fertilization WT (top), ppargc1a suboptimal dose MO (SMO), ptgs1 SMO, ppargc1a + ptgs1 SMO, and ppargc1a + ptgs1 SMO + dmPGE2-treated zebrafish stained via whole-mount immunofluorescence for acetylated α-tubulin (cilia, green), γ-tubulin (basal bodies, red), and DAPI in the proximal pronephros. Scale bar, 7 μm. (B) Cilia length in micrometers for the proximal pronephros. (C) Graph of the percentage of ciliated basal bodies/total basal bodies per 100 μm in the proximal pronephros. (D) Twenty-eight hours post-fertilization WT (top), ppargc1a SMO, ptgs1 SMO, ppargc1a + ptgs1 SMO, and ppargc1a + ptgs1 SMO + dmPGE2-treated zebrafish stained via whole-mount immunofluorescence for acetylated α-tubulin (cilia, green), γ-tubulin (basal bodies, red), and DAPI in the distal pronephros. Scale bar, 7 μm. (E) Cilia length in micrometers for the distal pronephros. (F) Graph of the percentage of ciliated basal bodies/total basal bodies per 100 μm in the distal pronephros. (G) MCCs stained via WISH for odf3b at 24 hpf in WT, ppargc1a SMO, ptgs1 SMO, ppargc1a + ptgs1 SMO, and ppargc1a + ptgs1 SMO + dmPGE2-treated zebrafish. Scale bar, 50 μm. (H) Graph depicting the number of MCCs present. (I) Forty-eight hours post-fertilization zebrafish injected with ppargc1a SMO, ptgs1 SMO, ppargc1a + ptgs1 SMO, and ppargc1a + ptgs1 SMO + dmPGE2-treated displaying either a WT phenotype (left) or an affected phenotype (right). The respective penetrance is listed within each image area. Scale bar, 100 μm. Data are represented as mean ± SD. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 (one-way ANOVA); n.s., not significant.

Characterization of yulink-MO KD zebrafish morphants. a Expression of yulink in zebrafish using whole-mount in situ hybridization. Embryos were fixed overnight at 4 °C in 4% paraformaldehyde buffered with 1 × phosphate-buffered saline (PFA/PBS). After hybridization with Dig-labeled antisense or control sense RNA probes of yulink, embryos were incubated with anti-Dig antibody conjugated to AP and developed with NBT-BCIP reagents. The yulink was expressed in whole zebrafish embryo ubiquitously from 0.5 hpf (zygote stage) to 3 dpf (larval stage) with lateral overview. The black arrows point to heart regions at 24–30 hpf and 3 dpf with ventral overview. The black arrowheads point to yolk of embryos, hour post-fertilization (hpf); day post-fertilization (dpf). b After injection with yulink-MO to knockdown yulink expression, the yulink KD morphants presented with small eyes, a small head, abnormal blood circulation, and pericardial edema at 3 dpf. Increasing the yulink-MO dosage from 2.3 ng (n = 119) to 4.6 ng (n = 119 or 9.2 ng (n = 156) caused the proportion of severely affected embryos with pericardial edema to increase from 6 to 54% (purple). c Hemodynamic changes. Yulink KD morphants had a slower heart rate and reduced cardiac output, as compared to WT embryos at 2 dpf (** p < 0.01, Student’s t test). d Diagram of the EYFP-fusion construct with the yulink 5′-UTR region and partial coding region (amino acids 1–79). The position of the corresponding MO binding site is indicated by a bar. To demonstrate the specificity of the yulink-MO, embryos were injected with pYulink-EYFP plasmid (100 pg/embryo) alone, or together with yulink-MO or the mismatch control, yulink-5mmMO (4.6 ng/embryo). About 58% of embryos injected with pYulink-EYFP plasmid alone exhibited fluorescence at 1 dpf, while fluorescence was absent in embryos co-injected with yulink-MO. In contrast, co-injection with yulink-5mmMO did not affect EYFP expression

Figure 1. Excessive exercise leads to pathological cardiac hypertrophy. (A,B) Representative images from control (A) and excessively-exercised zebrafish hearts (B) after 4 weeks of static and excessive-exercise treatment. (C–F) Representative pictures of Masson’s trichrome-stained ventricular tissues of control (C,D) and excessively-exercised zebrafish hearts (E,F). Blue area indicates fibrosis. (D,F) Are enlargements of the rectangle area in (C,E), respectively. Scale bar = 100 μm. (G) Quantitative analysis of Masson staining positive myocardial fibrosis area using Image J. ∗p < 0.05 by unpaired Student’s t-test. n = 3. (H,I) TEM images of control (H) and excessively exercised heart tissue (I). Scale bar = 5 μm. (J) RT-qPCR analysis of the expression of mitochondrial functional markers. ∗p < 0.05, ∗∗p < 0.01 by unpaired Student’s t-test. (K) Echocardiographic analysis of zebrafish after excessive exercise and in the static control (LVPW, LV posterior wall thickness). Error bars indicate SEM. n = 4. (L) The maximal oxygen consumption (MO2) of zebrafish after excessive exercise and in the control group was analyzed. n = 8. (M) RT-qPCR analysis of the expression of pathologic and physiological hypotrophy-related marker genes. ∗p < 0.05, ∗∗p < 0.01 by unpaired Student’s t-test.