Fig. 6.

miR-205 is regulated by Arf and enhances ExEn formation from WT ES progenitors. (A Left) Arf154 ES cells expressing a Dox-inducible Arf shRNA were cultured to form EBs in the absence of LIF and presence of Dox, resulting in a small decrease (P < 0.01) in miR-205 mRNA expression, quantified by PCR. WT ES cells were unaffected by Dox. Error bars, mean ± SEM (n = 3 experiments). (Right) Arf-null ES cells transduced with Arf–exon-1β–ER^TAM were induced to form EBs and simultaneously treated with tamoxifen (TAM) for 4 d. (B) The V1 vector transcribes miR-205 (hairpin) embedded in a miR-30 backbone, whereas the V2 vector expresses the complete pre-miRNA sequence (gray rectangle). Vectors included a PGK promoter-driven cassette and encoded neomycin resistance (Neo^r); GFP was translated from an internal ribosome entry site (IRES). Long terminal repeat (LTR) sequences required for viral integration and viral promoter-driven mRNA expression and a psi-2 (φ) virion packaging sequence are indicated. (C) WT ES cells infected with a control vector (Ctl GFP; Left) or with miR-205 vectors (Center and Right) were maintained in LIF to retard differentiation. Nonetheless, enforced miR-205 expression generated cells with altered morphology reminiscent of ExEn cells. (Magnification: 100×.) (D) Cells shown in C down-regulated the pluripotency marker Nanog and up-regulated the ExEn marker Dab2. (E) A more comprehensive quantitative RT-PCR analysis of infected WT iPS cells (left two bars) revealed up-regulation of other ExEn markers, Gata4 and Sox7, but insignificant alterations in the expression of markers of other germ-cell layers (mesoderm T, ectoderm Fgf5, and trophectoderm Cdx2). As expected, p19^Arf, p53, and the p53-responsive gene p21^Cip1 were induced as cells assumed the ExEn fate. By contrast, enforced expression of miR-205 in established ExEn cell lines (right two bars) did not affect the mRNA expression of the above genes.