Fig. S9

Fig. S9

miR-27amutants have pharyngeal cartilage defects upon partial knock-down ofmiR-27b. (A) Schematic representation of the miR-27a precursor and generation of miR-27a mutants by CRISPR/Cas9. The blue box on the miRNA precursor represents the gRNA target region corresponding to the mature miR-27a seed. (B) The region of the miR-27a gene sequence that shows the CRISPR/Cas9 targeted region. miR-27a-5p and miR-27a-3p are shown in red. The seed region of mature miR-27a is shown in the grey box. The target region for the designed sgRNA is underlined in green. Arrow points to the PAM sequence. (C) Alcian blue staining of 4 dpf miR-27a^Δ/Δ and WT siblings injected with 1.5 ng of MO-27b loop at the one-cell stage. (D) Phenotypic categorization of the pharyngeal cartilage in 4dpf miR-27a^Δ/Δ, miR-27a^+/Δ and WT siblings injected with 1.5 ng of MO-27b loop at the one-cell stage. n=20 for WTs, n=36 for miR-27a^+/Δ and n=20 in miR-27a^Δ/Δ. (E) Analysis of cartilage positions. Ventral view of head cartilage in wild-type embryos stained with alcian blue. A represents the distance between the palataquadrate (pq) and ceratohyal (ch) cartilage joints. B represents the distance from the anterior end of Meckel's cartilage (m) to the baseline. (F) The B/A ratio was calculated for quantitative analysis of anterior-posterior extension of the Meckel's cartilage. Error bars represent SEM. Statistical analysis was done using one-way ANOVA with Fisher's Least Significant Difference (LSD) test, *p<0.05.