- Title
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Generation of a Triadin KnockOut Syndrome Zebrafish Model
- Authors
- Vecchi, V.M., Spreafico, M., Brix, A., Santoni, A., Sala, S., Pistocchi, A., Marozzi, A., Di Resta, C.
- Source
- Full text @ Int. J. Mol. Sci.
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Expression analysis of trdn in zebrafish embryo. (A) RT-PCR performed on RNA isolated from zebrafish embryos at different developmental stages. β-actin was used as the loading control. (B–F) WISH analyses with a trdn-specific probe in zebrafish embryos. trdn is expressed in skeletal muscles and heart. (B–D) Lateral view of a 13 somite (B, anterior to the left), 24 hpf (C) and 48 hpf (D) embryos. (E,F) Dorsal (E) and frontal (F) view of a 48 hpf embryo. Scale bars indicate 100 μm. sk, skeletal muscle; h, heart. EXPRESSION / LABELING:
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Phenotypical analysis of embryos with trdn loss-of-function. (A) Schematic representation of Triadin interactions network and trdn knockdown strategy. RyR: ryanodine receptor; DHPR: dihydropyridine receptor; SERCA: sarco-endoplasmic reticulum calcium ATPase (B–E) Representative bright field images of the phenotype of 48 hpf embryos injected with std-MO and trdn-MOs. (F) Classification of the phenotypes obtained with trdn-MOs injection. Values indicate the number of embryos for each phenotype. Scale bars indicate 100 μm. e, edema; ctt, curved trunk/tail. PHENOTYPE:
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Skeletal muscle morphology evaluation. (A–C) Representative images of WISH analysis using a ckma probe in std-MO (A), class 1 wild-type-like (B) and class 3 affected (C) trdn-MO-injected embryos at 48 hpf. (D–F) Representative birefringence images of std-MO (D), class 1 wild-type-like (E) and class 3 affected (F) trdn-MO-injected embryos at 3 dpf. (G–I) Representative MF20 immunofluorescence images of std-MO (G), class 1 wild-type-like (H) and class 3 affected (I) trdn-MO-injected embryos. (A–F) Scale bars indicate 100 µm. (G–I) Scale bars indicate 50 μm. |
Heart morphology and function evaluation. (A,B) Frontal view of 48 hpf std-MO and trdn-MOs embryos hybridized with cmlc2 probe using WISH technique. Scale bars indicate 100 μm. (C,D) Semithin section of std-MO and trdn-MOs embryos in the heart region. Scale bars indicate 100 μm. (E) Quantification of heartrate in embryos at 48 hpf injected with std-MO and trdn-MOs. (F) Percentage of embryos with a heartbeat superior to the mean of the heartbeat (bpm) of trdn-MOs. (G) Heartbeat count in 3 dpf std-MO and trdn-MOs untreated or adrenaline-, isoprenaline- and atropine-treated embryos. (H) Heartbeat count in 3 dpf std-MO and trdn-MOs untreated or treated with metoprolol or flecainide. (F–H) At least 30 embryos were analyzed for each group. Values are expressed as mean ± SEM. *** p < 0.001, ** p < 0.01, n.s. = not significant, Mann–Whitney test (E) and one-way ANOVA followed by Tukey post-hoc correction (G,H). Adr, adrenaline; Iso, isoprenaline; Atr, atropine. EXPRESSION / LABELING:
PHENOTYPE:
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