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Fig. 6 Effects of miR30a on somitogenesis are rescued by non-targetable six1a. (A) Western blot analysis at 24hpf reveals that co-injection of six1a mRNA lacking its 32UTR in combination with miR30a restores Six1 protein expression compared to that observed with overexpression of miR30a alone. NIC, non-injected control; OE, overexpression. (B) Live images (control, n = 54/55; miR30a, n = 50/66; miR30a + six1a mRNA, n = 25/56) at 24 hpf, anterior to the left, reveal that restoration of Six1 levels in the background of miR30a overexpression rescues normal somite morphology. (C) Confocal imaging of MyHC with A4.1025 antibody at 24 hpf (control, n = 24/27; miR30a, n = 24/28; miR30a+six1a: n = 14/18), and (D) birefringence (mean±s.e.m.) at 48 hpf (NIC, n = 38 total embryos; miR30a, n = 44; miR30a+six1a, n = 48; *P<0.05, ANOVA with Bonferroni′s post-hoc test) reveals that the disarrayed muscle fiber organization resulting from miR30a overexpression is rescued by non-targetable Six1. (E) A quantification of cleaved caspase-3 (CC3) shows cell death returns to endogenous levels in rescued embryos in a dose-dependent manner (NIC, n = 11 total embryos; 1ng miR30a: n = 12, 1ng miR30a+six1a, n = 11; 3ng miR30a, n = 10; 3ng miR30a+six1a, n = 11). The line represents the mean. ***P<0.0001, *P<0.05, ANOVA with Bonferroni′s post-hoc test. Scale bar: 50µm. For B, C, n = the number of embryos represented by each image/the total number of embryos analyzed.