Morpholino (MO)‐based knockdown and rescue of zebrafish tnnt1. (A) Representative zebrafish (3 days postfertilization [dpf]) injected with tnnt1 SP8‐MO (top middle and right) display prominent truncal curvature and reduced birefringence under plane polarized light compared to wild–type (WT) larvae (top left). Overexpression of human WT TNNT1 mRNA in tnnt1 SP8‐MO morphants restores the curvature and the birefringence (bottom left). In contrast, human TNNT1 mRNA encoding the p.Leu96Pro mutation (p.L96P) fails to rescue the tnnt1 SP8‐MO morphant phenotype (bottom middle and right). Matched pairs of images illustrate appearance under light microscopy above the same field showing birefringence under plane polarized light. (B) Actin (green) and nuclear (blue) stains of 3‐dpf tnnt1 SP8‐MO morphants show decreased numbers and disorganization of muscle fibers (right) compared to WT larvae (left). (C) Microinjection of either tnnt1 TR‐MO or tnnt1 SP8‐MO morpholinos leads to significant reduction in numbers of morphologically normal “straight” embryos. Coinjection of human WT TNNT1 mRNA in SP8‐MO morphants significantly increases the number of straight embryos. In contrast, overexpression of mRNA with the p.Leu96Pro mutation with tnnt1 SP8‐MO fails to rescue the truncal curvature. (D) Similarly, reduced birefringence in the tnnt1 SP8‐MO morphants is restored upon overexpressing the human WT mRNA, whereas mRNA with the p.Leu96Pro mutation fails to rescue the birefringence. For each experimental condition, between 130 and 232 embryos from 3 to 8 different clutches were scored. Error bars in D reflect standard deviation between clutches. Numbers for uninjected WT controls were pooled across experiments, totaling 605 embryos from 20 clutches. *p < 0.00001 for each indicated comparison for numbers of “straight” (C) or normally birefringent (D) embryos; ns indicates p > 0.05.
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