Oct4 and Sox2 are transcription factors required for pluripotency during early embryogenesis and for the maintenance of embryonic stem cell (ESC) identity. Functional mechanisms contributing to pluripotency are expected to be associated with genes transcriptionally activated by these factors. Here, we show that Oct4 and Sox2 bind to a conserved promoter region of miR-302, a cluster of eight microRNAs expressed specifically in ESCs and pluripotent cells. The expression of miR-302a is dependent on Oct4/Sox2 in human ESCs (hESCs), and miR-302a is expressed at the same developmental stages and in the same tissues as Oct4 during embryogenesis. miR-302a is predicted to target many cell cycle regulators, and the expression of miR-302a in primary and transformed cell lines promotes an increase in S-phase and a decrease in G 1 -phase cells, reminiscent of an ESC-like cell cycle profile. Correspondingly, the inhibition of miR-302 causes hESCs to accumulate in G 1 phase. Moreover, we show that miR-302a represses the productive translation of an important G 1 regulator, cyclin D1, in hESCs. The transcriptional activation of miR-302 and the translational repression of its targets, such as cyclin D1, may provide a link between Oct4/Sox2 and cell cycle regulation in pluripotent cells.Pluripotent stem cells preserve their identity by promoting self-renewal and preventing differentiation. Transcription factors expressed early in development play a key role in regulating these processes. The first cell fate decision in the preimplantation embryo requires Oct4, a transcription factor expressed in the blastocyst that represses differentiation in the inner cell mass (28). Oct4 works in concert with a transcription factor binding partner, Sox2, and the pair is known to activate genes essential for early development (5,29,32,36,41,42). Upon the formation of the late blastocyst, a third factor, Nanog, is required to repress the differentiation of pluripotent cells to visceral and parietal endoderm (26), highlighting the importance of these transcription factors in maintaining pluripotency at early developmental phases. All three of these factors are also expressed in human embryonic stem cells (hESCs) and mouse ESCs (mESCs), and the transcriptional programs orchestrated by the coordinated efforts of these factors are key mechanisms of maintaining pluripotency. Recent studies of ESCs have revealed a considerable number of genomic regions with overlapping Oct4, Sox2, and Nanog binding sites adjacent to genes that are likely important for pluripotency (2, 24). Functional analysis of the genes expressed and regulated by these transcription factors will further elucidate new mechanisms associated with the maintenance of pluripotency.Included in this candidate set of factors are microRNAs (miRNAs) (2, 24). Like Oct4, Sox2, and Nanog, miRNAs have also been implicated in the maintenance of cell fate and the regulation of stem cell differentiation. miRNAs regulate their targets posttranscriptionally by pairing with a short antisense region of t...

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BMP signaling plays pleiotropic roles in various tissues. Transgenic mouse lines that overexpress BMP signaling in a tissue-specific manner would be beneficial; however, production of each tissue-specific transgenic mouse line is labor-intensive. Here, using a Cre-loxP system, we generated a conditionally overexpressing mouse line for BMP signaling through the type I receptor ALK2 (alternatively known as AVCRI, ActRI, or ActRIA). By mating this line with Cre-expression mouse lines, Cre-mediated recombination removes an intervening floxed lacZ expression cassette and thereby permits the expression of a constitutively active form of Alk2 (caAlk2) driven by a ubiquitous promoter, CAG. Tissue specificity of Cre recombination was monitored by a bicistronically expressed EGFP following Alk2 cDNA. Increased BMP signaling was confirmed by ectopic phosphorylation of SMAD1/5/8 in the areas where Cre recombination had occurred. The conditional overexpression system described here provides versatility in investigating gene functions in a tissue-specific manner without having to generate independent tissue-specific transgenic lines.

Craniosynostosis describes conditions in which one or more sutures of the infant skull are prematurely fused, resulting in facial deformity and delayed brain development. Approximately 20% of human craniosynostoses are thought to result from gene mutations altering growth factor signaling; however, the molecular mechanisms by which these mutations cause craniosynostosis are incompletely characterized, and the causative genes for diverse types of syndromic craniosynostosis have yet to be identified. Here, we show that enhanced bone morphogenetic protein (BMP) signaling through the BMP type IA receptor (BMPR1A) in cranial neural crest cells, but not in osteoblasts, causes premature suture fusion in mice. In support of a requirement for precisely regulated BMP signaling, this defect was rescued on a Bmpr1a haploinsufficient background, with corresponding normalization of Smad phosphorylation. Moreover, in vivo treatment with LDN-193189, a selective chemical inhibitor of BMP type I receptor kinases resulted in partial rescue of craniosynostosis. Enhanced signaling of the fibroblast growth factor (FGF) pathway, which has been implicated in craniosynostosis, was observed in both mutant and rescued mice, suggesting that augmentation of FGF signaling is not the sole cause of premature fusion found in this model. The finding that relatively modest augmentation of Smad-dependent BMP signaling leads to premature cranial suture fusion suggests an important contribution of dysregulated BMP signaling to syndromic craniosynostoses, and potential strategies for early intervention.