5B); however, mir20a showed no targeting effect on the Kat2b 3′UT

5B); however, mir20a showed no targeting effect on the Kat2b 3′UTR (Fig. 5B). Neither mir302b overexpression nor mir20a knockdown significantly affected the luciferase activity of Camk2n1 3′UTR reporter vector (Fig. S7B). Together, these data demonstrate that both mir302b and mir20a are able to regulate Tgfbr2 expression, while only mir302b can target Kat2b. Since both Tgfbr2 and Kat2b are associated with TGFβ signaling, we tested whether mir302b and mir20a can affect TGFβ signal transduction. We employed a reporter assay, 3TP-lux, in which a TGFβ-responsive promoter drives the expression of luciferase.16 Irrespective of addition

of TGFβ, the promoter activity was reduced in cells expressing mir302b but increased in cells with mir20a knockdown (Fig. 6). Notably, mir302b cannot repress TGFβ signaling when Tgfbr2, lacking the 3′UTR, is overexpressed, Endocrinology antagonist and knockdown of mir20a does not increase the signal with dominant-negative Tgfbr2 (Tgfbr2(DN)) (Fig. 6), demonstrating that both mir302b and mir20a are able to suppress TGFβ signal transduction by targeting Tgfbr2. Mice heterozygous for both smad2 and smad3 die at midgestation small molecule library screening with liver hypoplasia and anemia.27 To investigate whether inhibition of TGFβ signaling affects hepatoblast development, we used a stepwise hepatoblast differentiation protocol in ESCs (Fig. S9A). ESC-derived endoderm, expressing Foxa2, Gsc, and

Sox17 (Fig. S9B), was generated with medium containing Activin

Sclareol A and exposed to liver specification factors of bone morphogenetic protein 4 (BMP4), beta fibroblast growth factor (bFGF), Activin A, and vascular endothelial growth factor (VEGF). Cells were further cultured in hepatoblast expansion medium with growth factor cocktails. The hepatic markers Alb, AFP, Hnf4α, transthyretin (Ttr), hemopexin (Hpx), and Serpina1a were induced during differentiation (Fig. S9C). Of note, both mir20a and mir302b showed dynamic expression (Fig. 7) with mir302b expression highest at the endoderm stage. Forced expression of mir302b through lentiviral vector during hepatoblast expansion resulted in decreased expression of Tgfbr2 and liver markers, compared to control cells (Figs. 7, 8). A similar reduction of liver markers, but not Tgfbr2, was observed with the TGFβ inhibitor, SB505124 (Fig. S9D). These results demonstrate that mir302b represses liver development during ESC differentiation and suggests that de-repressing TGFβ signaling by down-regulation of mir302b provides a favorable environment for hepatoblast development. Little is known about miRNA expression during early liver development due to the difficulty in isolating specific embryonic tissues. Here, we describe the first miRNA libraries from dissected E8.5 foregut and E14.5 Dlk1+ hepatoblasts. Our data illustrate the dynamic patterns of miRNA expression that occur during liver development.

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