(A) Phylogenetic tree of ChREBP, MondoA and Mlx proteins. h, human, m, mouse, b, bovine, g, chicken, zf, zebrafish. Outgroup: zf Neurogenin 1 (Neurog 1). Scale bar: 0.1 estimated amino acid substitutions per site. (B) Amino acid identities in % between zebrafish and human domains of ChREBP, MondoA and Mlx: ‘Mondo conserved regions/glucose-sensing module’ (MCR/GSM); ‘low-glucose inhibitory domain’ (LID; light blue); ‘glucose-response activation conserved element’ (GRACE; dark blue); ‘basic-helix-loop-helix/leucine zipper’ (bHLH/ZIP; green); ‘dimerization and cytoplasmic localization domain’ (DCD; red). (C, D) Bioluminescence levels after 24 hr of glucose treatment of PAC2 cells transiently transfected with the 2xChoRE reporter consisting of a luciferase reporter gene (yellow) regulated by a minimal promoter (TATA; arrow) and two carbohydrate response elements (ChoREs; each with the sequence 5’-CACGCG-N5-CTCGTG-3’; pA for polyadenylation site; n = 4, (C) or with the constitutively expressed pGL3-Control reporter construct (D, n = 4). (E, F) Bioluminescence levels in 2xChoRE reporter expressing PAC2 cells upon 24 hr of treatment with 0.3 mM (white bars) or 12 mM (black bars) glucose after overexpression of MondoA and/or Mlx (E, n = 4) or transfection with mondoa-mo or mondoa-mis (F, n = 8). Data were normalized to Renilla luciferase activity (Norm. bioluminescence). (G–I) mRNA expression profiles of mondoa (G), chrebp (H) and mlx (I) during zebrafish developmental stages from zygote to larval stage five extracted from a published dataset (White et al., 2017). n = 20; FKPM, Fragments Per Kilobase of transcript per Million mapped reads. (J) WISH of mondoa transcripts at zygote (maternal), 50% epiboly (50% epi.) and 18-somite stages. as, antisense probe, ss, sense probe. Scale bar: 0.2 mm. (K) Epon sections of 50% epi. embryos showed mondoa expression in the enveloping layer (EVL), the deep cell layer (DEL) of the blastoderm (Bl) and the yolk syncyctial layer (YSL). Scale bar: 0.2 mm, for higher magnification 50 µm. (L–O) Glucose induction of Mondo pathway target gene expression in early zebrafish embryos. Embryos were injected with the glucose analog 2-deoxy-D-glucose (2-DG; black bars) or with water (white bars) as a control. RNA was extracted at the sphere stage to perform RT-qPCR of genes known to be Mondo pathway targets in mammals: hexokinase 2 (hk2, L), fatty acid synthase (fasn, M), thioredoxin-interacting protein a (txnipa, N), eukaryotic translation elongation factor 1 alpha 1 (ef1a, O) (n = 9). (P) Embryos (n ≥ 72) injected with the 2xChoRE reporter showed increased bioluminescence at sphere stage when co-injected with 2-DG. Error bars represent SEM; *, p≤0.05; **, p≤0.01; ***, p≤0.001.

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-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).

In situ hybridization of adamts9 (A–D) Lightly-stained staged series from WT fish showing adamts9 expression in the head, cloaca, and at the tips of growing fins rays. (E–G) Extended staining at 13 dpf highlights specific adamts9 expression in the head in bilateral strips in the brain (red arrows; E, F), staining of the melanocytes (yellow arrows), in the ciliated marginal zone of the eyes (F) and in the lateral line (G).

(A) Plot of the moderated log2 fold change (mondoa-mo vs. mondoa-mis injected embryos) over averaged normalized counts. The most downregulated gene nsdhl is indicated. (B) The main steps in the conversion of acetyl coenzyme A (acetyl-CoA) to cholesterol and pregnenolone. (C, D) Transcript levels of nsdhl (C) and ef1a (D) of embryos injected with water (white bars) as a control or with the glucose analog 2-deoxy-D-glucose (2-DG; black bars) in the presence (+) or absence (-) of mondoa and mlx mRNA (n = 9). (E) Embryos injected with a splice-site MO against nsdhl (nsdhl-mo) show a severe developmental delay and arrest at ~50% epiboly, when uninjected controls were at bud stage (~10 hpf). (F) Quantification of the experiments in (E). Percentages of ‘affected’ embryos: uninjected (0/84, 0%), nsdhl-mo injected (n = 136/141, 96.5%). (G) Injection of nsdhl-mo into 1 k cell stage embryos led to arrest at 95% epiboly (n = 11/11, 100%), when uninjected control embryos (n = 15/15, 100%) were at bud stage (10 hpf). (H,I) Percentage of epiboly progression under different treatments determined via no tail (ntl) WISH. (H) Uninjected control embryos (n = 30) were fixed at the 75% epiboly stage along with the treated embryos. Treatment with 20 µM pregnenolone (P5) led to a partial rescue of the mondoa morphants (n = 19), which achieved 48% epiboly (untreated morphants: 38% epiboly; n = 25). Control and mondoa-mo morphant results are reproduced from Figure 2E, as these experiments were carried out in parallel. (I) P5 treatment also led to a partial rescue of the nsdhl morphants (n = 30, 74.6% epiboly compared to 59.0% in untreated morphants, n = 50). Asterisks label the blastoderm margin. Scale bar: 0.2 mm. Error bars represent SEM; *, p≤0.05; **, p≤0.01; ***, p≤0.001.

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

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.

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

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).