miR-145 overexpression affects gut and heart development. (A–C) Overexpression of miR-145 causes mild pericardial edema (arrowhead) in 24 hpf embryos, (D–F) which then develops into severe pericardial edema accompanied by a unlooped gut at 96 hpf. The control mimic RNA injection has no effect. (G) Overexpression of miR-145 leads to strong down-regulation of sm22α-b but does not affect αsma levels in 48 hpf embryos as measured by qPCR. Arrows, gut. (Scale bar, 200 μm.)

Normalization of gata6 levels rescues loss of miR-145. (A–C) Knockdown of miR-145 results in increased expression of gata6 in zebrafish gut at 48 hpf. (D–F) Overexpression of miR-145 leads to decreased gata6 expression in the gut of 96 hpf embryos. Pericardial edema (arrowhead) is also found in miR-145 morphant. (G–I) gata6 knockdown normalizes the gut morphology (arrow) of miR-145 morphants. (J) qPCR shows strong up-regulation of gata6 in miR-145 morphants of 48 hpf. (K) qPCR indicates significantly decreased expression of gata6 in 48 hpf miR-145 mimic treated embryos. Arrows, gut; bars, mean ± SEM. *, P < 0.01. (L) The number of embryos exhibiting normal gut morphology in the rescue was analyzed by using Student's t test (P < 0.05, n = 286). (Scale bar, 200 μm.)

miR-145 directly targets gata6 in vitro and in vivo. (A) Schematic diagram of a luciferase construct with or without the gata6 3′UTR for the in vitro assay. (B) Luciferase reporter activity shows the interaction between miR-145 and gata6 3′UTR. Luciferase activity was normalized to β-galactosidase activity and expressed relative to the gata6 3′UTR conjugated luciferase vector-only transfection. (C) Schematic of EGFP reporters for the in vivo assay which were constructed with or without the gata6 3′UTR. (D) EGFP-gata6-3′UTR sensor assays demonstrating specific regulation of the gata6 3′UTR in vivo by miR-145. EGFP reporter expression (green) and control mCherry expression (red) are shown at 25–28 hpf as modulated by the addition of miR-145 mimic with or without the Gata6-TP^miR145 or control target protector. mCherry mRNA is an injection control. (E) For each experimental group in D, EGFP-gata6-3′UTR (sensor) or EGFP-3′UTR fluorescence is expressed as a percentage of fluorescence observed from the sensor injected alone group (n = 10 embryos per group). Bars, mean ± SEM. *, P < 0.01.

miR-145 expression pattern and loss of function phenotype. (A) Lateral view of whole-mount in situ expression of miR-145 at 16–19S shows ubiquitous expression. (B) Later at 96 hpf, miR-145 is expressed in the gut (g) and swimbladder (sb) in wild-type embryos. (C) Longitudinal section of 96 hpf embryo shows miR-145 expressed strongly in the gut smooth muscle cells (white arrowhead) and weakly in gut epithelium. (D–F) Animals injected with miR-145 MO have hydrocephalus in the hind brain ventricle (*) at 48 hpf. (G–I) At 96 hpf, miR-145 morphants have severe pericardial edema (black arrowhead), and an underdeveloped gut and swimbladder (black arrow). In situ hybridization staining of αsma (J–L) and sm22α-b (M–O) in 96 hpf embryos shows an increase of sm22α-b expression but no change of αsma in the miR-145 morphant gut. Gut morphology in the miR-145 morphant is unlooped as compared to the broader tube-like morphology found in UIC and miR-145 control MO embryos. Arrows mark the gut. (P) qPCR of αsma and sm22α-b in miR-145 morphants at 48 hpf. (Q and R) Histology of 96 hpf zebrafish gut. miR-145 morphant shows a thinner layer of SMCs (SM) and lack of villi in the epithelial layer as compared to UIC embryos. (Scale bars, 200 μm except for C, Q, and R, which are 50 μm.)