Fig. 4

Quantification of the effect of MO-based knockdown (without/with mRNA rescue) and CRISPR/Cas9-knockout of TPC2 on the organization of the trunk musculature and the formation of the sarcomeres. (Ai) The number of SMCs in each somite was determined via immunolabelling embryos fixed at ~24 hpf with an anti-prox1 antibody (a representative example of an untreated control is shown). Scale bar, 50 µm. (Aii) Schematic to show the various dimensions of the trunk musculature and SMC myofibers that were measured using series of optical sections projected as single images from the fluorescently-labeled embryos at ~25 hpf (for representative examples, see Fig. 3), in order to determine the level of disruption on SMC development following TPC2 knockdown or knockout. (B-F) Bar charts to show the mean ±SEM: (B) number of SMCs; (C) myotome width, and (D) somite angle, (all n≥12, from 3 somites in ≥4 embryos); (E) myofiber length: somite length ratio (these lengths were measured in n≥72 myofibers and n≥24 somites, respectively ≥4 embryos); and (F) myofiber width (n≥40, from 3 somites in ≥4 embryos). The dashed black line in panel (E) indicates a myofiber: somite length ratio of 1. Statistical analysis was carried out using one-way ANOVA and significance differences (at p<0.05) as shown by the different letters, a-d between any pair of groups was determined using the Tukey's post hoc method. Thus, significant differences between groups were denoted by different letters whereas groups where no significant differences were observed displayed the same letter.