Smg9-deficient zebrafish display brain malformations.

a Genomic locus diagram depicting the zebrafish smg9 gene and its 10-nucleotide deletion using CRISPR/Cas9. The red box indicates the mutated region in smg9^oi7/oi7 zebrafish. b Western blot analyses of lysates from smg9^+/+ and smg9^oi7/oi7 larvae at 2 dpf to determine the Smg9 protein levels. c The percentage of smg9^+/+, smg9^oi7/+, and smg9^oi7/oi7 zebrafish were determined from 2 to 14 dpf. d Representative images of smg9^+/+ and smg9^oi7/oi7 larvae at 14 dpf. Scale bar: 1 mm. e Body length of smg9^+/+ and smg9^oi7/oi7 larvae at 14 dpf. f Representative images of smg9^+/+ and smg9^oi7/oi7 fish at 3 mpf. Scale bar: 1 cm. g Body length of smg9^+/+ and smg9^oi7/oi7 zebrafish at 3 mpf. h Representative images of H&E staining of transverse and coronal sections of smg9^+/+ and smg9^oi7/oi7 larvae at 14 dpf. Scale bar: 50 µm in the upper panel and 100 µm in the lower panel. i Telencephalon areas of smg9^+/+ and smg9^oi7/oi7 larvae at 14 dpf were measured using transverse sections of H&E images. j Representative images of immunostaining of 14 dpf larval brain tissue with PCNA (red), S100β (green), and DAPI (blue). Scale bar: 100 µm. k Graph shows PCNA/S100β double-positive radial glial cells among DAPI-positive cells in the telencephalon of smg9^+/+ and smg9^oi7/oi7 larvae at 14 dpf. l Telencephalon size of 14 dpf smg9^+/+ and smg9^oi7/oi7 larvae injected with control (con-in) or zebrafish smg9 mRNA (smg9-in). m Representative images of the dissected brains of smg9^+/+ and smg9^oi7/oi7 zebrafish at 3 mpf. Scale bar: 1 mm. n Quantification of the areas of the telencephalon, optic tectum, and cerebellum in smg9^+/+ and smg9^oi7/oi7 zebrafish at 3 mpf. o Representative images of dissected brains of smg9^+/+ and smg9^oi7/oi7 zebrafish at 6 mpf. Scale bar: 1 mm. p Quantification of the areas of the telencephalon, optic tectum, and cerebellum in smg9^+/+ and smg9^oi7/oi7 zebrafish at 6 mpf. Error bars indicate SD. *P < 0.05, **P < 0.01, and ***P < 0.001. ns: not significant.

Impaired cardiac contractions in Smg9-deficient zebrafish.

a Representative image of the heart of cmlc2:EGFP zebrafish. The red and white dotted lines indicate the atrium and ventricle, respectively. b Quantification of the atrium and ventricle areas in smg9^+/+ and smg9^oi7/oi7 zebrafish at 6 dpf. c Representative images of hematoxylin and eosin staining of smg9^+/+ and smg9^oi7/oi7 hearts at 6 dpf. d Representative dynamic pixel change patterns of the heart in cmlc2:EGFP zebrafish, showing the heartbeat and cardiac contraction of smg9^+/+ and smg9^oi7/oi7 zebrafish. e Quantification of cardiac contractions in smg9^+/+ and smg9^oi7/oi7 zebrafish larvae at 6 dpf. f Quantification of the heart rates of smg9^+/+ and smg9^oi7/oi7 zebrafish larvae at 6 dpf. g Quantification of cardiac contractions of 6 dpf smg9^+/+ and smg9^oi7/oi7 larvae injected with control (con-in) or zebrafish smg9 mRNA (smg9-in). Error bars indicate SD. bpm: beats per minute. *P < 0.05, **P < 0.01, and ***P < 0.001. ns: not significant.

Premature aging phenotype in various tissues in Smg9-deficient zebrafish.

a Kaplan–Meier survival curve analysis showing the lifespan of smg9^+/+ (n = 20), smg9^oi7/+ (n = 20), and smg9^oi7/oi7 (n = 20) zebrafish. b, c Representative images of gross appearance of smg9^+/+ and smg9^oi7/oi7 zebrafish at 3 mpf and 6 mpf. Scale bar: 5 mm. d Representative H&E images of the intestine showing tissue morphology in smg9^+/+ and smg9^oi7/oi7 zebrafish at 6 mpf. Scale bar: 50 μm. e–g Representative images of immunofluorescence staining for PCNA (e), γH2AX (f), and cleaved caspase-3 (g) in the intestinal segments of smg9^+/+ and smg9^oi7/oi7 zebrafish at 6 mpf. Scale bar: 50 μm. h Representative H&E images of the kidney tissue morphology in smg9^+/+ and smg9^oi7/oi7 zebrafish at 6 mpf. Scale bar: 50 μm. i, j Representative images of Masson’s trichrome staining of smg9^+/+ and smg9^oi7/oi7 skin (i) and muscle (j) at 6 mpf. Scale bar: 10 μm (i). Scale bar: 20 μm (j). k Fertility rate of smg9^+/+ and smg9^oi7/oi7 zebrafish at 4 mpf and 7 mpf, as determined by mating smg9^+/+ or smg9^oi7/oi7 male zebrafish with smg9^+/+ female zebrafish and counting embryo survival after 12 hpf. l Representative images of the morphological features of testes in smg9^+/+ and smg9^oi7/oi7 zebrafish at 3 mpf and 6 mpf. Scale bar: 2.5 mm. m Representative image of H&E staining of testes tissue in smg9^+/+ and smg9^oi7/oi7 at 3 mpf and 6 mpf. Sperm (black arrow) was absent in the 6 mpf smg9^oi7/oi7 zebrafish. Scale bar: 50 μm. Error bars indicate SD. *P < 0.05, ** P < 0.01, and *** P < 0.001. ns: not significant.

Age-related staining and markers in Smg9-deficient zebrafish.

a, b SA-β-gal staining of the skin of smg9^+/+ and smg9^oi7/oi7 zebrafish at 1 mpf. Scale bar: 1 mm (a). Quantification of the percentage of positive areas in the trunk at 1 mpf (b). c, d SA-β-gal staining of the skin of smg9^+/+ and smg9^oi7/oi7 zebrafish at 3 mpf (c). Quantification of the percentage of positive areas in the trunk at 3 mpf. Scale bar: 3 mm (d). e, f SA-β-gal staining of the skin of smg9^+/+ and smg9^oi7/oi7 zebrafish at 6 mpf. Scale bar: 3 mm (e). Quantification of the percentage of positive areas in the trunk at 6 mpf (f). g, h SA-β-gal staining of the frozen brain sections of smg9^+/+ and smg9^oi7/oi7 zebrafish at 1 mpf. Scale bar: 50 μm (g). Quantification of the percentage of positive areas in the brain at 1 mpf (h). i, j SA-β-gal staining of the frozen brain sections of smg9^+/+ and smg9^oi7/oi7 zebrafish at 3 mpf. The arrows indicate positive regions. Scale bar: 100 μm (i). Quantification of the percentage of positive areas in the brain at 3 mpf (j). k, l SA-β-gal staining of the frozen brain sections of smg9^+/+ and smg9^oi7/oi7 zebrafish at 6 mpf. The arrows indicate positive regions. Scale bar: 100 μm (k). Quantification of the percentage of positive areas in the brain at 6 mpf (l). m Quantitative PCR analysis of p21, p53, p16, p27, and p57 expression in smg9^+/+ and smg9^oi7/oi7 zebrafish larvae at 14 dpf. n Quantitative PCR analysis of il-6, il-1β and tnfα expression in smg9^+/+ and smg9^oi7/oi7 zebrafish larvae at 14 dpf. Error bars indicate SD. *P < 0.05, ** P < 0.01, and *** P < 0.001. ns not significant.

Upregulation of endogenous targets of nonsense-mediated mRNA decay (NMD) in Smg9-deficient zebrafish.

a–j Quantitative PCR analysis of endogenous target genes of NMD in smg9^+/+ and smg9^oi7/oi7 larvae at 14 dpf. k Western blot analysis of lysates from smg9^+/+ and smg9^oi7/oi7 larvae at 2 dpf to determine the total FLAG-UPF1 (UPF1) and phosphorylated FLAG-UPF1(p-UPF1) levels. l Quantification of the total FLAG-UPF1 (UPF1) and phosphorylated FLAG-UPF1 (p-UPF1) levels in three independent experiments. Error bars indicate SD. *P < 0.05, ** P < 0.01, and *** P < 0.001. ns: not significant.

Smox overexpression is sufficient for brain and heart defects in Smg9-deficient zebrafish.

a Quantitative PCR analysis of endogenous target genes of nonsense-mediated mRNA decay in smg9^+/+ and smg9^oi7/oi7 larvae at 14 dpf. b–fSmox mRNA was injected into single-cell-stage fertilized eggs derived from the smg9^+/+ zebrafish. The telencephalon area at 14 dpf and the heart function at 6 dpf in zebrafish larvae were evaluated. b Representative images of H&E staining of smox mRNA-injected (smox-in) and control-injected (con-in) larvae at 14 dpf. Scale bar: 50 μm. c Quantification of the telencephalon area of smox-in and con-in larvae at 14 dpf was performed using hematoxylin and eosin staining. d Quantification of cardiac contractions in smox-in and con-in larvae at 6 dpf. e Quantification of the heart rate of smox-in and con-in larvae at 6 dpf. f SA-β-gal staining of the brain of smox-in and con-in zebrafish at 4 mpf. Error bars indicate SD. bpm: beats per minute. *P < 0.05, ** P < 0.01, and *** P < 0.001. ns: not significant.

ROS and acrolein accumulation in Smg9-deficient zebrafish.

a, c Representative flow cytometry histograms of CellROX fluorescence in smg9^+/+ and smg9^oi7/oi7 zebrafish at 3 mpf (a) and 6 mpf (c). b, d Mean fluorescence intensity of CellROX Deep Red in the brains of smg9^+/+ and smg9^oi7/oi7 zebrafish at 3 mpf (b) and 6 mpf (d). e, g Representative images of AcroleinRED staining of smg9^+/+ and smg9^oi7/oi7 zebrafish brains at 3 mpf (e) and 6 mpf (g). f, h Relative mean fluorescence intensities of smg9^+/+ and smg9^oi7/oi7 zebrafish brains at 3 mpf (f) and 6 mpf (h). i, j Representative images of transmission electron microscopy of smg9^+/+ and smg9^oi7/oi7 zebrafish brains at 6 mpf. The red arrowhead and red arrow indicate normal and damaged mitochondria, respectively. N Nucleus. Scale bar: 5 µm in the upper panel and 1 µm in the lower panel (i). The density of damaged mitochondria within a defined area (0.25 mm²) in the telencephalon region of six individual zebrafish brains (j). Error bars indicate SD. *P < 0.05, ** P < 0.01, and *** P < 0.001. ns: not significant.

Smox is required for brain and heart defects in Smg9-deficient zebrafish.

a Representative image of hematoxylin and eosin staining of smg9^+/+ and smg9^oi7/oi7 larvae after treatment with or without MDL72527. Scale bar: 50 µm. b Quantification of the telencephalon area of smg9^+/+ and smg9^oi7/oi7 larvae after treatment with or without MDL72527. c Representative images of immunostaining for PCNA (red), S100β (green), and DAPI (blue) for smg9^+/+ and smg9^oi7/oi7 larvae after treatment with or without MDL72527. Scale bar: 50 µm. d Quantification of PCNA/S100β double-positive radial glial cells among DAPI-positive cells in the telencephalon of smg9^+/+ and smg9^oi7/oi7 larvae after treatment with or without MDL72527. e Representative images of hematoxylin and eosin staining of smg9^+/+ and smg9^oi7/oi7 larvae after treatment with or without N-acetylcysteine (NAC). Scale bar: 50 µm. f Quantification of the telencephalon area of smg9^+/+ and smg9^oi7/oi7 larvae after treatment with or without NAC. Scale bar: 50 µm. g Representative images of immunostaining for PCNA (red), S100β (green), and DAPI (blue) for smg9^+/+ and smg9^oi7/oi7 larvae after treatment with or without NAC. Scale bar: 50 µm. h Quantification of PCNA/S100β double-positive radial glial cells among DAPI-positive cells in the telencephalon of smg9^+/+ and smg9^oi7/oi7 larvae, after treatment with or without NAC. i Representative dynamic pixel change patterns of the heart showing cardiac contractions of smg9^+/+ and smg9^oi7/oi7 zebrafish after treatment with or without MDL72527. j Quantification of cardiac contractions in smg9^+/+ and smg9^oi7/oi7 zebrafish larvae after treatment with or without MDL72527. k Representative dynamic pixel change patterns of the heart showing cardiac contractions of smg9^+/+ and smg9^oi7/oi7 zebrafish after treatment with or without NAC. l Quantification of cardiac contractions in smg9^+/+ and smg9^oi7/oi7 zebrafish larvae after treatment with or without NAC. m Representative images of hematoxylin and eosin staining of smg9^+/+ and smg9^oi7/oi7 larvae injected with control (con-in) or smox morpholino (MO-in). Scale bar: 50 µm. n Quantification of the telencephalon area of smg9^+/+ and smg9^oi7/oi7 larvae injected with control (con-in) or smox morpholinos (MO-in). o Representative images of immunostaining with PCNA (red), S100β (green), and DAPI (blue) for smg9^+/+ and smg9^oi7/oi7 larvae injected with control (con-in) or smox morpholino (MO-in). Scale bar: 50 µm. p Quantification of PCNA/S100β double-positive radial glial cells among DAPI-positive cells in the telencephalon of smg9^+/+ and smg9^oi7/oi7 larvae injected with control (con-in) or smox morpholino (MO-in). q Representative dynamic pixel change patterns of the heart showing cardiac contraction of smg9^+/+ and smg9^oi7/oi7 zebrafish injected with control (con-in) or smox morpholino (MO-in). r Quantification of cardiac contractions in smg9^+/+ and smg9^oi7/oi7 zebrafish larvae injected with control (con-in) or smox morpholino (MO-in). Error bars indicate SD. *P < 0.05, ** P < 0.01, and *** P < 0.001. ns: not significant.