Fig. S4
microRNA-30a targeting morpholino specificity confirmed by in situ hybridization and immunoblot analyses. In situ hybridization (ISH) analysis (A, control: n=17/17 total embryos, miR30a MO: n=14/18) for microRNA-30a (miR30a) at 48 hours post fertilization (hpf) reveals a decreased expression of miR30a upon injection of miR30a-targeting morpholino (MO). Transverse cross-sections at 18 µm of 48 hpf embryos stained by ISH for miR30a (B) confirm miR30a is expressed in the somites (S), (NT=neural tube, NC=notochord). Western blot analysis (C) quantified at 48 hpf following normalization to GAPDH demonstrates that miR30a-targeted MO injection into 1-4 cell stage embryos leads to an increase in Six1 protein expression, which is lost if miR30a is co-injected with miR30a MO. Further, six1a mRNA injection does result in increased levels of Six1 protein expression. |