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Fig. 4. miR-30a-5p knockdown and global inhibition of miRNA biogenesis have very similar kidney phenotypes. (A) To determine the specificity of miR-30a5p-MO, three consecutive miR-30a-5p binding sites (BS) were introduced in the 3′UTR of a lacZ reporter. Xenopus embryos were injected with the reporter in the presence or absence of miR-30a-5p duplex and miR-30a5p-MO and processed for lacZ staining at gastrula stage. Injections with miR-34b-MO served as a specificity control. (A′) Quantification of the lacZ staining in embryos: white, no staining; gray, partial staining; black, strong staining. The number of embryos analyzed in three different experiments is indicated above the bars. (B-H′) Xenopus embryos injected with miR-30a5p-MO and uninjected controls were analyzed by morphology (B,B′), histology (C,C′), immunohistochemistry with 3G8 and 4A6 (D-E′), and whole-mount in situ hybridization for Cadherin-16 (Cad-16; F,F′), ClC-K (G,G′) and NKCC2 (H,H′). Arrowheads indicate the presence of edema (B′), loss of 4A6 staining in the pronephric duct (E′), and the shortening of the tubule segments IT1, IT2 and DT1 (F′,G′,H′). en, endoderm; no, notochord; nt, neural tube; pn, pronephros; s, somites. |
