Fig. 2.
miR-1/206 regulate the expression of vegfaa. (A) The zebrafish vegfaa 3′UTR, indicating the sequence of each miR-1/206 target site (TS1-3), the alignment of each TS with miR-1, and the sequence of the mutant reporter in which the seed sequence complementary to miR-1/206 has been mutated from ACAUUCCU to ACUAACCU (mut). (B) The luciferase assay to test the functionality of the TS in the vegfaa 3′UTR. Embryos were co-injected with a firefly luciferase reporter containing the 3′UTR of vegfaa and a control Renilla luciferase reporter, in the presence (+) or absence of miR-1 and miR-206 duplex miRNA. (C) The TSs in vegfaa are functional and necessary for miR-1/206-mediated repression. miR-1/206 downregulate the luciferase activity expressed from the luciferase-vegfaa 3′UTR reporter (vegfaa 3′UTR) compared with the Renilla control. A luciferase reporter construct containing a 3′UTR with mutated miR-1/206 TS (vegfaa 3′UTR mut miR-1 TS) is not significantly repressed by the miR-1/206 duplex. As a positive control for repression, a luciferase reporter containing three partially complementary TSs for miR-1/206 (3×IPT miR-1) was downregulated in the presence of miR-1/206 duplex. Mean ± s.d.; *P<0.01, Student's t-test. (D-G) In situ hybridization to detect vegfaa expression at 24 hpf in embryos injected at the one-cell stage with control MO (D), miR-1/206 MO (E), vegfaa-TPmiR-1 (F) or dominant-active rat Smo mRNA (rSmoM2) (G). Note that blocking miR-1/206 function with miR-1/206 MO (E), miR-1/206 activity on vegfaa using target protectors (F), or upregulating the Shh pathway (G) increases the levels of vegfaa mRNA.