miR-1 and miR-206 regulate developmental angiogenesis. (A) Schematic representation of the zebrafish vasculature. The intersegmental vessels (ISVs) form by sprouting endothelial cells from the dorsal aorta and are typically composed by three cells: the tip, the stalk, and the basal cell that connects to the aorta (inset). (B) ISVs labeled with cytoplasmic GFP [Tg(fli-EGFP)^y1] or nuclear GFP [Tg(fli-nGFP)^y7] after injection of control morpholino (MO) or miR-1/206 MO at the one-cell stage. (C,D) Quantification of the cross-sectional area (C) and average number of nuclei per ISV (D) in control MO-injected and miR-1/206 MO-injected embryos at 30 hpf. Note the increase in ISV size (cross-sectional area) and number of endothelial cells in embryos injected with miR-1/206 MO compared with control MO-injected embryos. Mean ± s.d.; Student′s t-test. (E) Individual frames of a time-lapse confocal analysis of the vascular phenotype in wild-type and miR-1/206 MO-injected embryos between 18 and 28 hpf. Endothelial cells migrating into the vessel are numbered (1, 2, 3, etc.) and their daughter cells are labeled (1.1, 1.2, 1.2.1, etc.). The wild-type ISV shows a highly stereotyped pattern, in which a single endothelial cell migrates to the trunk midline and divides, leaving one cell stationary while the tip cell continues to migrate dorsally. Endothelial cells in miR-1/206 MO-injected embryos show elevated proliferation within the ISV (left vessel), as well as an increase in migration from the aorta (right vessel).

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 32UTR 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 32UTR reporter (vegfaa 32UTR) compared with the Renilla control. A luciferase reporter construct containing a 32UTR with mutated miR-1/206 TS (vegfaa 32UTR 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-TP^miR-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.

Reducing translation of vegfaa rescues the increase in angiogenesis caused by miR-1/206 loss of function. (A,B) Tg(fli-nGFP)^y7 zebrafish embryos injected at the one-cell stage with control MO (A) or miR-1/206 MO (B) were also injected with increasing concentrations of a translation-blocking AUG MO against vegfaa (vegfaa MO). Increasing amounts of vegfaa MO reduced ISV formation and rescued the phenotypes observed when blocking miR-1/206 function (B). (C) Quantification of the average number of nuclei per ISV in control MO-injected and miR-1/206 MO-injected embryos co-injected with increasing concentrations of vegfaa MO at 30 hpf. Mean ± s.d.; Student′s t-test.

Target protectors relieve miR-1/206-mediated repression of the vegfaa 32UTR. (A) Sequence of each target site, the cognate target protector (TP, violet) and miR-1. (B) Luciferase assay to test the action of vegfaa TP as described in Fig. 2. (C) Luciferase reporter assay to determine the ability of the TPs to prevent miR-1/206-mediated repression of the luciferase-vegfaa 3′UTR. Repression of the luciferase-vegfaa 3′UTR is relieved in the presence of vegfaa TPs (+TP). (D) Confocal analysis of GFP expression in the ISV of Tg(fli-nGFP)^y7 zebrafish embryos injected with control MO or vegf-TP^miR-1 at the one-cell stage. (E) Quantitation of the number of nuclei per ISV of control MO-, miR-1/206 MO- or TP-injected embryos. Note the increase in endothelial cells in the ISV of embryos injected with TP against the miR-1/206 target sites in the vegfaa 3′UTR. (C,E) Mean ± s.d.; Student′s t-test.