Activation of WNT signaling restores the facial defects present in the Vu57 allele. (a) Experimental design schematic with onset of treatment with WNT‐Agonist I at 30 hours post fertilization (HPF) and removal of treatment at 54 HPF. Alcian Blue was performed at 4 days post fertilization (DPF) in treated and untreated individuals that were incubated in embryo media from 54–96 HPF following treatment. (b–e) Alcian blue staining was performed at 4 DPF in homozygous carriers of the Vu57 allele (Vu57−/−) and wild type siblings (Sibling) according to the treatment schematic in (a). Total numbers of animals reflected in Table 1. *p = .0001 using a Fisher's Exact test. (f). Homozygous carriers of the Vu57 allele (Vu57−/−) or wild type siblings (Sibling) were treated with vehicle control (DMSO) or WNT‐agonist I at either 0.1 or 0.2 μM concentration according to the schematic in (a). Total RNA was isolated from a pool of embryos and the expression of sox10, col2a1a, or axin2 was measured by quantitative PCR (qPCR). Error bars represent SD of biological replicates. N = 11/group except for 0.2 μM concentration where N = 9. *p = 4.07457E‐05, **1.14579E‐06, ***p = 3.80572E‐07, #2.45716E‐05, ##p = 7.28315E‐08, ###p = .000716073, &p = 3.22846E‐05, &&p = 7.2866E‐07, &&&p = 8.45279E‐07

Notochord collagen markers col2a1a and col9a2 affected by dietary treatment. At 12 hpf, col2a1a expression was localized in the developing notochord of E+ (A, n = 5/5) and E− (B, n = 6/6) embryos. Arrow indicates dorsal region of the embryo. At 24 hpf, the notochord sheath in E+ and E− embryos showed col2a1a (C, D) and col9a2 (G, H) expression with a wavy notochord phenotype observed in both groups. The wavy notochord was more apparent when viewing dorsally (E, F, I, J) relative to lateral view (C, D, G, H). At 24 hpf, ~ 25% of E+ embryos (E, n = 4/12) showed a wavy col2a1a expression, while ~ 66% of E− embryos (F, n = 6/9) were obviously wavy; similarly a wavy col9a2 expression was observed in 33% of E+ embryos (I, n = 3/10), while ~ 60% of E− embryos had a wavy expression (J, n = 5/8). Scale bar represents 500 μm (C–J) and 100 μm (A, B); representative embryos are shown. Figure panels were generated with the BZ- × 700 microscope, processed with BZ-X Analyzer Software with image adjustments equally applied across time points in Adobe Photoshop. This figure was created with Adobe Photoshop v21.2.1.

Knockdown of mondoa affects microtubule stability.(A) Confocal image of the blastoderm margin of an uninjected embryo at sphere stage; microtubules are stained with a mouse α-tubulin antibody followed by an anti-mouse Alexa Fluor 488-coupled antibody. DEL; deep cell layer; MT, microtubules; MTOC, microtubule organization center; YCL, yolk cytoplasmic layer; YSL, yolk syncytial layer; YSN, yolk syncytial layer nuclei. (B) Quantification of effects on YSL microtubule organization in uninjected (n = 9), mondoa-mo injected (n = 16), and mondoa-mo injected + pregnenolone (P5) treated (n = 9) embryos. Presence of stellar structures as shown in (F) = ‘affected’, normal appearance as in (A) = ‘unaffected’. (C–H) Overview (C, E, G) and close-up (D, F, H) images of uninjected embryos (C, D), mondoa-mo morphants (E, F) and P5 treated mondoa-mo morphants (G, H). Arrowheads indicate long parallel MT in uninjected and P5 rescued embryos (D, H) and short MT asters in untreated morphants (F). Scale bar: 90 µm; for higher magnifications, 30 µm.

Ttpa signal localized throughout early embryo and brain ventricle borders regardless of VitE status. Ttpa expression in E+ and E− embryos is indicated with red fluorescence; dorsal direction is indicated by arrow (A–D). At 6 hpf (dorsal shield stage, A, B), Ttpa expression was present throughout the animal poles [E+ embryos, n = 5/5 (n = number of animals with the observed defect/total number of animals observed)], E− embryos, n = 6/6). At 12 hpf (90% epiboly, C, D), ttpa expression was present both in the embryo and in the yolk syncytial layer (E+ embryos, n = 6/6; E− embryos, n = 7/8 ), arrow indicates anterior region of the embryo. At 24 hpf (E, F), Ttpa expression was localized in the brain ventricle borders and within cells of the fore (f), mid- (m), and hindbrain (h). Arrows indicate the midbrain-hindbrain boundary where diencephalic ventricle expansion was altered; *Represent inflation in E+ embryos (E, n = 6/6) or lack thereof in E− embryos (F, n = 3/7). Scale bar represents 500 μm (A–D) and 50 μm (E, F); representative embryos are shown. Figure panels were generated with the BZ- × 700 microscope, processed with BZ-X Analyzer Software with image adjustments equally applied across time points in Adobe Photoshop v21.2.1.

Oxamflatin and salermide have dose-dependent effects, independent of dystrophin expression. a Graph of average normalized pixel intensities for treatments of dmd mutants with doses of oxamflatin and salermide. Control treatment is 1% DMSO. Chemicals were used between 0.5 μM and 4 μM, over two separate experiments. For each treatment condition, n = 4 replicates, with 2-11 dmd−/− embryos in each replicate. Plot shows the average normalized pixel intensity for each of the 4 replicate pools for each treatment. The vertical line separates the treatment conditions from the two experiments, each of which has its own WT + DMSO and dmd + DMSO controls. The dashed lines represent the average normalized pixel intensity for all of the DMSO-treated dmd animals (n = 26 and n = 30). Error bars represent standard error. Significance was determined using a one-way ANOVA test comparing each treatment group to the dmd DMSO control group with Dunnett’s correction for multiple comparisons. *p ≤ 0.029, **p = 0.0029, ***p = 0.0007 compared to dmd DMSO control. b-e Confocal images of anti-dystrophin staining in the trunk musculature of 4 dpf b WT + DMSO, c WT + oxamflatin and salermide, d dmd + DMSO, and e dmd + oxamflatin and salermide larvae. Lateral views, anterior to the left. Arrow points to dystrophin expression in the vertical myoseptum. All dmd+/+ animals showed normal dystrophin expression (WT + DMSO, n = 16; WT + ox+sal, n = 14) and all dmd−/− animals lacked detectable dystrophin expression (dmd−/− + DMSO, n = 23; dmd−/− + ox+sal, n = 18). Scale bar = 50 μm. f-i Confocal images of anti-β-dystroglycan (βDG) and phalloidin staining in the trunk musculature of 4 dpf f WT + DMSO, g WT + oxamflatin and salermide, h dmd + DMSO, i dmd + oxamflatin and salermide. Lateral views, anterior to the left. Arrow points to βDG expression (white) in the vertical myoseptum. Phalloidin staining of filamentous actin (magenta) shows the disrupted muscle structure in dmd mutants (* in h). All wild type animals (+/+ and +/−) showed normal β-dystroglycan expression (WT + DMSO, n = 27; WT + ox+sal, n = 26), and dmd−/− animals showed largely maintained β-dystroglycan expression (dmd−/− + DMSO, n = 9; dmd−/− + ox+sal, n = 14). Scale bar = 50 μm

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.

ppargc1a-Deficient Zebrafish Display Phenotypes Commonly Associated with Cilia Defects (A) WT sibling (top) and a ppargc1asa13186/sa13186 mutant (bottom) zebrafish with a curly tail, pronephric cyst (blue arrow, inset), and pericardial edema at 3 dpf. Heterozygous crosses resulted in the expected Mendelian ratio shown at the bottom right of the respective panels (WT group includes homozygous WT and ppargc1a+/sa13186 combined). Scale bar, 90 μm. (B) JB4 transverse section of a 4 dpf WT sibling (left) and ppargc1asa13186/sa13186 mutant (right) zebrafish (n, notochord; tubule outlined in green, cysts outlined in red). Scale bar, 90 μm. (C) Three days post-fertilization WT sibling (left) and ppargc1asa13186/sa13186 mutant (right) otoliths. Arrowheads indicate abnormal otoliths present in the mutant. Scale bar, 90 μm. (D) Four days post-fertilization otic cilia (green, acetylated α-tubulin) of the cristae in WT (left) and ppargc1asa13186/sa13186 mutant (right) zebrafish. Scale bar, 10 μm. (E) Dorsal view of 3 dpf WT sibling (top) and ppargc1asa13186/sa13186 mutant (bottom) approximately 24 h after being injected with 40 kDa dextran-FITC to assay for pronephric function. White arrowheads indicate PCT fluorescence in WT. White box highlights the approximate area of the PCT with no fluorescence. Scale bar, 90 μm. (F) Three days post-fertilization WT sibling (top) and ppargc1asa13186/sa13186 mutant (bottom) stained via WISH for slc20a1a to mark PCT. Scale bar, 90 μm. (G) Fifty-five hours post-fertilization WT and ppargc1a MO injected zebrafish stained via WISH for the heart marker myl7. Penetrance graph of atypical heart looping occurring in WT and ppargc1a morphants. Chi-square analysis indicates significance. n = 128 (WT), n = 103 (MO), p (H) Whole-mount immunofluorescence of the Kupffer’s vesicle (KV) at the 10 ss stained for acetylated α-tubulin (cilia, green), anti-PKC (membrane boundary, red). Scale bar, 8 μm. (I) Cilia length in micrometers of KV cilia. (J) The number of cilia present in the KV. (K) Twenty-four hours post-fertilization WT zebrafish stained via WISH to illustrate ppargc1a mRNA expression. Box is of approximate area of inset showing Ppargc1a (red) expression and the cilia marker acetylated α-tubulin (green). Scale bars, 65 μm for the whole embryo and 10 μm for the inset. Data are represented as mean ± SD; ∗∗p < 0.01 and ∗∗∗p < 0.001 (t test).

ptgs1 Functions Downstream of ppargc1a during Ciliogenesis (A) Schematic depicting several Ppargc1a consensus sites located within 1.5 kb of the ptgs1 open reading frame (ORF). (B) Twenty-four hours post-fertilization WT (top) and ppargc1asa13186/sa13186 (bottom) zebrafish stained via WISH for ptgs1 (n > 60). Scale bar, 100 μm. (C) Relative ptgs1 mRNA expression in WT and ppargc1a morphants. (D) Whole-mount immunofluorescence for acetylated α-tubulin (cilia, green), γ-tubulin (basal bodies, red), and DAPI in the proximal pronephros of WT (top), ppargc1a morphants (middle), and ppargc1a morphants injected with ptgs1 cRNA (bottom). Scale bar, 7 μm. (E) Cilia length for the proximal pronephros treatment groups shown in (D). (F) Graph of the percentage of ciliated basal bodies/total basal bodies per 100 μm in the proximal pronephros. (G) Whole-mount immunofluorescence for acetylated α-tubulin (cilia, green), γ-tubulin (basal bodies, red), and DAPI in the distal pronephros of WT (top), ppargc1a morphants (middle), and ppargc1a morphants injected with ptgs1 cRNA (bottom). Scale bar, 7 μm. (H) Cilia length for distal pronephros treatment groups shown in (G). (I) Graph of the percentage of ciliated basal bodies/total basal bodies per 100 μm in the distal pronephros. (J) WISH for the MCC marker odf3b in WT (top), ppargc1a morphants, and ppargc1a morphants injected with ptgs1 cRNA (bottom). Scale bar, 90 μm. (K) Number of MCCs per nephron. (L) Proposed mechanism by which ppargc1a regulates MCC development and ciliogenesis via PGE2 levels by controlling ptgs1 expression. Data are represented as mean ± SD. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 (one-way ANOVA or t test); n.s., not significant.

ppargc1a Loss of Function Phenotypes Can Be Rescued by Supplementing with PGE2 (A) Relative concentration of PGE2 in WT and ppargc1a morphants. (B) MCCs stained via WISH for odf3b at 24 hpf in WT (left), ppargc1a MO (middle), and ppargc1a morphants treated with dmPGE2 (right). Scale bar, 90 μm. (C) Graph depicting the number of MCCs present in WT, ppargc1a MO, and ppargc1a MO treated with dmPGE2. (D) Twenty-eight hours post-fertilization WT (top), ppargc1a MO-injected (middle), and ppargc1a MO-injected + dmPGE2 (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. (E) Cilia length in micrometers for the proximal pronephros of WT, ppargc1a morphants, and ppargc1a morphants treated with dmPGE2. (F) Graph of the percentage of ciliated basal bodies/total basal bodies per 100 μm in the proximal pronephros. (G) Twenty-eight hours post-fertilization WT (top), ppargc1a MO-injected (middle), and ppargc1a MO-injected + dmPGE2 (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. (H) Cilia length in micrometers for the distal pronephros of WT, ppargc1a morphants, and ppargc1a morphants treated with dmPGE2. (I) Graph of the percentage of ciliated basal bodies/total basal bodies per 100 μm in the distal pronephros. (J and K) Scatterplots illustrating the difference in fluorescent intensity signals of WT, ppargc1a MO, and ppargc1a MO + dmPGE2 in proximal (J) and distal (K) cilia. (L) Representative images of ppargc1a+/sa13186 in crosses shown at 48 hpf WT zebrafish treated with DMSO (top), curly ppargc1asa13186/sa13186 treated with DMSO (middle), and a WT-appearing, genotype-confirmed ppargc1asa13186/sa13186 rescued with dmPGE2 (bottom). Scale bar, 90 μm. (M) The percentage of zebrafish with WT (black) or curly (white) phenotypes at 48 hpf after treatment with DMSO or dmPGE2. Chi-square test indicates significance. Data are represented as mean ± SD. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 (one-way ANOVA or t test); n.s., not significant.