Morpholino-mediated pitpnaa knockdown in zebrafish elicits dose-dependent changes in morphology and signaling. (A) Phase contrast images of sapje larvae injected with pitpnaa MO or control MO at 1 and 4 dpf. Embryos injected with 3 ng of pitpnaa MO at the one-cell stage showed abnormal development and were not viable, whereas 1 ng of pitpnaa MO did not negatively impact development. (B) Western blot showing decreased Pitpna expression with increasing doses of pitpnaa MO injected into sapje embryos. Protein lysates were harvested from sapje larvae at 4 dpf. Experiment performed in triplicate, n = 300 per replicate.

Pitpnaa knockdown rescues sapje zebrafish muscle phenotype, improves swim velocity, and increases long-term survival. (A) Birefringence assay (Upper) and immunofluorescent staining for myosin heavy chain (Lower) showing the typical abnormal muscle phenotype of affected homozygous-null sapje fish at 4 dpf. Injection of pitpnaa MO at the one-cell stage prevented manifestation of the muscle phenotype in some homozygous-null fish, termed escaper fish, which showed healthy muscle morphology comparable to normal fish. [Scale bars: 1 mm (Upper), 100 μm (Lower).] (B) Percent of affected sapje fish as determined by birefringence assay at 4 dpf. Mating of heterozygous sapje fish normally yields ∼25% of affected embryos. Cohorts injected with 1 ng of pitpnaa MO at the one-cell stage showed significantly lower percentages of affected fish compared with control MO. Ten replicate experiments were performed, n = 200–300 per replicate. Bars represent means ± SDM. *P < 0.0001 by Student’s t test. (C) Swim velocity of sapje fish tracked on the DanioVision system. Injection with 1 ng of pitpnaa MO increased swim velocity of affected fish during a 15-min tracking period performed at 4 dpf. Bars represent means ± SEM; *P < 0.05 by two-way ANOVA and Bonferroni post hoc test. (D) Long-term survival assay showing increased survival of affected fish injected with 1 ng of pitpnaa MO at the one-cell stage. Affected fish were identified by birefringence assay at 4 dpf and followed until 30 dpf. Data represent means ± SDM. *P < 0.05, **P < 0.002 versus noninjected by Student’s t test.