Fig. S7

zeb1a and foxo1a are miR-145 targets. a) Northern blot analyses of mature miR-145 levels in embryos injected with miR-145 morpholinos or miR-145 mimic to downregulate or overexpress miR-145, respectively. U6 snRNA was used for normalization. b) miR-145 expression can be modulated in vivo. Histograms show miR-145 levels measured by qRT-PCR in embryos injected with a morpholino blocking the mature miR-145 (miR-145 KD) and with amiR-145 mimic to overexpress miR-145 at 72 and 96 hpf. c) Knockdown of miR-145 induces a peculiar iSMC phenotype in zebrafish embryos. Bright-field and fluorescent images of double Tg(Xia.Eef1a:GFP)^s854 Tg(acta2:mCherry)^uto5 embryos injected with miR-145 morpholino in zebrafish embryos. While miR-145 KD did not alter embryonic development, miR-145-deficient embryos display reduced SMC-specific Tg expression around the gut and swim bladder (sb), as shown in Tg(acta2:mCherry)uto5 injected embryos (arrow). Scale bar, 200 μm. Notochord: n; swim bladder: sb; gut: g. d) Histograms show acta2, tagln, myh11, and foxa3 mRNA levels measured in the trunk of 96 hpf embryos by qRT-PCR after miR-145 KD. The iSMC markers acta2, tagln and myh11 were partially downregulated, whereas the endodermal compartment (foxa3) was normal. Stars represent the results of unpaired t-tests of mean difference = 0 (*p<0.05, **p<0.01, ***p<0.001). e) zeb1a and foxo1a 3' UTRs contain two miR-145 binding sites. Schematic illustration of the two putative binding sites of miR-145 in zeb1a and foxo1a 3′ UTRs. The position of the last base of the stop codon was numbered 0. Mutated (mBSs) miR-145 binding sites are indicated with stars. f) Disruption of miR-145 binding to zeb1a and foxo1a phenocopy miR-145 KD embryos. Confocal maximum projection of iSMCs covering the gut after Tagln staining of embryos co-injected with zeb1a and foxo1a TPs. Similar to miR-145 KD embryos, zeb1a+foxo1a TPs showed less iSMCs displaying an immature morphology. Scale bar, 25 μm.