MicroRNA 21 expression and knockdown. (A,B) In situ hybridization with mature miR-21 locked nucleic acid (LNA) probe at 48 (A) and 72 (B) hpf demonstrates expression in zebrafish atrioventricular canal (AVC) at 48 hours (A, inset) and staining of atrioventricular valve (AV) and outflow tract (B, inset). (C) miR-21 knockdown does not perturb the normal body plan or axis at 72 hpf. Control, top; morphant, bottom. (D,E) Mismatch MO control-injected embryos show normal AV ring constriction (D, arrow) at 48 hpf, whereas miR-21 knockdown results in loss of the normal AV constriction (E, arrow). There is also failure of normal cardiac looping, as well as pericardial edema. (F,G) Hematoxylin and Eosin stained sections at day 5 post-fertilization demonstrate normal AV in controls (F, arrow) but failure of AV development in miR-21 knockdown embryos (G, arrow). In all images, the atrial chamber (a) is below and to the right and the ventricle (v) is above and to the left.

miR-21 knockdown affects both endothelial and myocardial AV ring markers. (A,B) Confocal microscopy of control transgenic embryos (cMLC2:dsRed/Tie2:EGFP) show GFP expression (green) in pre-valve AV ring endothelium at 48 hpf (A), whereas miR-21 knockdown embryos do not (B). (C-P) In situ images are oriented with atrium at bottom and ventricle at top. The probe is indicated bottom left and the experimental condition [wild-type control (wt) versus miR-21 MO injected (MO)] bottom right. The percentage of embryos that display the representative in situ pattern is indicated top right (n=10-20). (C-H) Endocardial markers; (I-P) myocardial markers. White arrowheads indicate AV ring staining, where present. Black arrows (E,F) highlight spp1 expression in the fin bud. The majority of the AV ring markers are unchanged upon knockdown of miR-21. However, the myocardial marker tbx2b is absent from the AV ring after miR-21 knockdown (I,J), whereas bmp4 is expanded throughout the ventricle (L), as compared with control embryos (K).

pdcd4b is a target of miR-21 during zebrafish valve development. (A) Luciferase assays of candidate miR-21 targets. ΔPDCD4b refers to the pdcd4b 3′ UTR with the miR-21 binding site deleted. Data are mean ± s.d.; ^*P<0.05. (B) Western blot of whole zebrafish lysates injected at the single-cell stage with mRNA encoding GFP:Pdcd4b, co-injected with miR-21 or with miR-21 plus an miR-21 MO. Tubulin staining serves as a loading control. (C) Representative western blot of endogenous zebrafish Pdcd4 in uninjected and miR-21 morphants. (D) Quantification of zebrafish Pdcd4 western blots demonstrating a significant increase in Pdcd4 when miR-21 is knocked down. Data are mean ± s.d.; ^*P<0.01. (E-H) Injection of a PDCD4b target protector MO (TPMO) does not perturb the body plan or axis at 72 hpf (E, compared with control F), but does cause pericardial edema, loss of the normal cardiac looping, and AV constriction (G and H, arrow) at 48 hpf. (I,J) Confocal microscopy of the cMLC2:dsRed/Tie2:GFP line shows loss of upregulation of the Tie2:GFP marker (green) in the AV ring in pdcd4-TPmiR-21-injected fish (I, atrium at top, ventricle at bottom) compared with controls (J) at 48 hpf. A, atrium; V, ventricle.

miR-21 knockdown results in accumulation of miR-21 precursor and loss of mature miR-21. (A) Northern blot for short RNA molecules from control, miR-21-1 MO-injected, and miR-21-2 MO-injected embryos. Injection of miR-21-2 MO results in accumulation of the pre-miR-21 precursor (top band), which may be detected faintly in the miR-21-1 lane as well, but is absent from the control lane. (B) Injection of miR-21-2 MO causes a 90% reduction in the amount of mature miR-21 detectable by qPCR.

Rescue of miR-21 phenotypes. (A) The miR-21 MO AVC defect is rescued by co-injecting mature miR-21 RNA into embryos. *P=6×10^–12. (B) The PDCD4 target protector (TP) MO AVC defect is rescued by co-injecting a splice-acceptor PDCD4 MO. *P=5×10–⁸. (C) The miR-21 MO AVC defect is rescued by co-injecting a splice-acceptor PDCD4 MO into embryos. *P=0.0005. (DF) Representative embryos demonstrating recovery of in situ expression patterns of the indicated genes after the rescue experiments performed above. The AVC is indicated by white arrowheads. (G) Representative embryonic heart demonstrating recovery of AV constriction and looping after the rescue experiments performed above.

In situ expression pattern of pdcd4b. Shown are the expression patterns of pdcd4b under control and miR-21 knockdown conditions.