Fig. S1

mir92a sequence conservation and its knockdown effect on cartilage development of pectoral fins, related to Figure 1. (A) Conservation plots of mir-17-92 (top) and mir-106a-92 (bottom) paralogous clusters in vertebrates. (B) Sequence alignment of mature mir92a from different species. (C) Embryos were injected with 4 ng of 92a-MO at the one-cell stage and collected at 36 hpf for whole-mount in situ hybridization using LNA antisense mir92a probe. UIC, uninjected control. (D) Ventral views showing live embryos at 96 hpf. Embryos were injected with 4 ng 92a-MO alone or together with 4 ng p53-MO at the one-cell stage. The pectoral fins were indicated by arrowheads. Scale bar, 200 µm. (E) Staining of fin skeletons with Alcian blue at 80 hpf. The right-side pectoral fin skeleton was shown with anterior to the top. Embryos were injected as in (D). Note the absence of cleithrum (cl) and scapulocoracoid (sc) cartilages and postcoracoid process (pop) of pectoral fins as well as smaller endoskeletal disc cartilage (ed) in mir92a morphants. Scale bar, 100 µm. (F) 92a-MO injection effectively blocked amplification of mir92a expression in embryos at 42 hpf. Embryos were injected with 4 ng of the corresponding morpholino and collected at 42 hpf for detection of mir92a by semi-quantitative stem-loop RT-PCR. ***, p < 0.001 (by Student’s t-test); error bars indicated SD. (G-H) The head cartilage formation and pectoral fin development were unaffected by 92a-rMO injection. Head cartilages (G) and fin skeletons (H) were stained with Alcian blue at 80 hpf. The live injected embryo at 96 hpf (H, right panel) also showed a pair of normal fins. All images were dorsal views with anterior to the left. Scale bars, 200 µm for the left panel and 100 µm for the right panel. (I-J) Co-injection with 1 nl of 25 µM mir92a duplex rescued 92a-MO-induced loss of pectoral fins. Live embryos at 96 hpf were shown in (I) with the pectoral fins indicated by arrowheads. Fin cartilages stained with Alcian blue at 80 hpf were shown in (J). The ratio of affected embryos to the total number of embryos was indicated. All were dorsally viewed. Scale bars, 200 µm (I) or 100 µm (J).