ZFIN ID: ZDB-FIG-181012-30
Kustermann et al., 2018 - Loss of the novel Vcp (valosin containing protein) interactor Washc4 interferes with autophagy-mediated proteostasis in striated muscle and leads to myopathy in vivo. Autophagy   14(11):1911-1927 Full text @ Autophagy
ADDITIONAL FIGURES
EXPRESSION / LABELING:
Gene:
Fish:
Knockdown Reagents:
Anatomical Term:
Stage: Long-pec
PHENOTYPE:
Fish:
Knockdown Reagents:
Observed In:
Stage Range: Prim-5 to Day 4

Fig. 5

Phenotypic analysis washc4 morpholino-mediated knockdown in zebrafish embryos. (a to d) Lateral view of embryos at 48-hpf injected with washc4 5 bp mismatch start or splice morpholino (MO1/2-control; a, c) or washc4 start or splice MO (MO1/2-washc4; b, d). MO1/2- washc4 injection leads to a myopathic phenotype (b, d). scale bar: 450 μm. (e) Quantification of myopathic embryos after MO1/2-control or MO1/2- washc4 injection. N = 3/4 Experiments with in total n = 290 (MO1-control), n = 238 (MO2-control) n = 226 (MO1- washc4), n = 259 (MO2- washc4). The data were analyzed via N two-sided Fisher exact tests combined with a modified Fisher combination. The ratios of N experiments are shown. Western blot analysis shows reduction of Washc4 protein level in MO1-washc4-injected embryos. Actb1/β-actin was used as loading control. MO2-washc4 injection effects on mRNA splicing shown by RT-PCR. (f) Ventricular fractional shortening (FS) measurements at 48, 72 and 96 hpf of control and washc4 morphants. n = 30 (MO1-control), n = 30 (MO1-washc4); The individual measurements are shown (two-sided Wilcoxon rank-sum test). (g) Heart beat per minute of MO1-control- or MO1-washc4-injected embryos at 48, 72 and 96 hpf. n = 30 (MO1-control), n = 30 (MO1- washc4). The individual measurements are shown (two-sided Wilcoxon rank-sum test). (h, i) Spontaneous movement assay shown by false-colored superimposed pictures of control embryos (h) or Washc4 morphants (i) at 24 hpf; red pictures = 0 s; green pictures = 10 s; yellow = merge. scale bar: 1 mm. (j) Statistical analysis of spontaneous movement assay; N = 3 experiments with in total n = 56 (MO1-control), n = 60 (MO1-washc4) were performed (N two-sided Fisher exact tests combined with a modified Fisher method). The ratios of N experiments are shown. (k) Quantification of touch-evoke flight response assay of MO1-control- or MO1-washc4-injected embryos at 60 hpf. n = 40, N = 3 (MO1-control), n = 40, N = 3 (MO1-washc4); data represent means.

Gene Expression Details
Gene Antibody Fish Conditions Stage Qualifier Anatomy Assay
washc4 WT control Long-pec whole organism RTPCR
Long-pec whole organism WB
WT + MO1-washc4 standard conditions Long-pec whole organism WB
WT + MO2-washc4 standard conditions Long-pec Not Detected whole organism RTPCR
Antibody Labeling Details No data available
Phenotype Details
Fish Conditions Stage Phenotype
WT + MO1-washc4 standard conditions Prim-5 involuntary skeletal muscle contraction decreased occurrence, abnormal
Long-pec blood circulation decreased magnitude, abnormal
Long-pec heart contraction decreased frequency, abnormal
Long-pec heart contraction magnitude, normal
Long-pec heart edematous, abnormal
Long-pec whole organism washc4 expression decreased amount, abnormal
Pec-fin thigmotaxis decreased occurrence, abnormal
Protruding-mouth heart contraction decreased frequency, abnormal
Day 4 heart contraction decreased frequency, abnormal
WT + MO2-washc4 standard conditions Long-pec blood circulation decreased magnitude, abnormal
Long-pec heart edematous, abnormal
Long-pec whole organism washc4 expression absent, abnormal
Acknowledgments:
ZFIN wishes to thank the journal Autophagy for permission to reproduce figures from this article. Please note that this material may be protected by copyright. Full text @ Autophagy