Fig. 9

Fig. 9 Tecpr2 depletion in zebrafish results in reduced survival, hatching defects, impaired motility and altered neuromuscular synaptic morphology.

A Yeast two-hybrid assay of the zebrafish ortholog of TECPR2 and its (R1234W) mutant with the Rab5 zebrafish ortholog. Co-transformants were spotted on -Leu/-Trp and -Leu/-Trp/-His to confirm viability and detect interactions, respectively. B The graph represents tecpr2 gene expression in zebrafish embryos at the indicated dpfs normalized to the β-actin gene expression. The normalized data of tecpr2 expression at different dpfs is shown relative to the expression at 0 dpf. In each experiment, 20 embryos were analyzed. The error bar represents mean ± SD from three independent experiments, where each dot indicates the mean value. C Representative brightfield images of zebrafish embryos showing spatial expression of tecpr2 transcripts at different developmental stages. The labeled sense RNA probe was used as a negative control. Scale bar: 200 µm. D The graph represents dose-dependent survival of zebrafish embryos at 24 hpf upon injection with the indicated morpholino. The total number of embryos analyzed was 329 for MO-control, 213 for MO-tecpr2 (201 + 202) (0.125 mM), 197 for MO-tecpr2 (201 + 202) (0.25 mM), and 207 for MO-tecpr2 (201 + 202) (0.5 mM). The error bar represents the mean ± SD from three independent experiments, where each dot indicates the mean value (^****p < 0.0001; ^**p = 0.0025 unpaired two-tailed Student’s t-test). E The graph represents the percentage of hatched embryos after 72 hpf upon injection with the indicated morpholino. The total number of embryos analyzed was 181 for MO-control (0.5 mM), 63 for MO-tecpr2 (201 + 202) (0.25 mM), 85 for MO-tecpr2 (201 + 202) (0.25 mM) + tecpr2 (rescue) WT mRNA, and 43 for MO-tecpr2 (201 + 202) (0.25 mM) + tecpr2 (rescue) (R1234W) mRNA. The error bar represents the mean ± SD from three independent experiments, where each dot indicates the mean value (^****p < 0.0001^{; **}p = 0.0012; ns=0.0866 unpaired two-tailed Student’s t-test). F Lateral view of 96 hpf zebrafish embryos injected with the indicated morpholino. The arrowheads are shown to indicate the tail curvature and pericardial edema phenotype in the tecpr2 morphant and tecpr2 morphant injected with tecpr2 (R1234W) mRNA. Scale bar: 200 µm. G Representative confocal micrographs showing embryo sections from 96 hpf control morphants, Tecpr2 morphants and Tecpr2 morphants co-injected with tecpr2 (WT) mRNA or with tecpr2 (R1234W) mRNA, immunostained with Alexa 488-conjugated α-BTX (green) and anti-SV2 antibodies (magenta). Arrowheads indicate the elongated and branched neuromuscular junctions. Scale bar: 10 µm. H, I Quantification of the percentage area of α-BTX (H) and SV2 (I) from experiments such as that shown in panel (G). The n represents the number of sections taken from 10 embryos for each sample. The error bar represents the mean ± SD of three independent experiments (^****p < 0.0001; ^*p = 0.0171; ns=0.5637; unpaired two-tailed Student’s t-test). Source data are provided as a Source Data file.