miR-34/449 miRNAs are required for motile ciliogenesis by repressing cp110.

The mir-34/449 family consists of six homologous miRNAs at three genomic loci. Redundancy of miR-34/449 miRNAs and their dominant expression in multiciliated epithelia suggest a functional significance in ciliogenesis. Here we report that mice deficient for all miR-34/449 miRNAs exhibited postnatal mortality, infertility and strong respiratory dysfunction caused by defective mucociliary clearance. In both mouse and Xenopus, miR-34/449-deficient multiciliated cells (MCCs) exhibited a significant decrease in cilia length and number, due to defective basal body maturation and apical docking. The effect of miR-34/449 on ciliogenesis was mediated, at least in part, by post-transcriptional repression of Cp110, a centriolar protein suppressing cilia assembly. Consistent with this, cp110 knockdown in miR-34/449-deficient MCCs restored ciliogenesis by rescuing basal body maturation and docking. Altogether, our findings elucidate conserved cellular and molecular mechanisms through which miR-34/449 regulate motile ciliogenesis.

Use of CRISPR/Cas9 for the Modification of the Mouse Genome.

The use of CRISPR/Cas9 to modify the mouse genome has gained immense interest in the past few years since it allows the direct modification of embryos, bypassing the need of labor-intensive procedures for the manipulation of embryonic stem cells. By shortening the overall timelines and reducing the costs for the generation of new genetically modified mouse lines (Li et al., Nat Biotechnol 31: 681-683, 2013), this technology has rapidly become a major tool for in vivo drug discovery applications.

The reciprocal relationship between primordial germ cells and pluripotent stem cells.

Primordial germ cells (PGCs) are induced in the epiblast early in mammalian development. They develop their specific fate separate from somatic cells by the generation of a unique transcriptional profile and by epigenetic modifications of histones and DNA. PGCs are related to pluripotent cells in many respects, both on a molecular and a cell biological level. Mimicking their in vivo development, PGCs can be derived in culture from pluripotent cells. Vice versa, PGCs can be converted in vitro into pluripotent embryonic germ cells. Recent evidence indicates that the derivation of pluripotent embryonic stem cells from explanted inner cell mass cells may pass through a germ cell-like state, but that this intermediate is not obligatory. In this review, we discuss PGC development and its relevance to pluripotency in mammalian embryos. We outline possibilities and problems connected to the application of in vitro-derived germ cells in reproductive medicine.

MicroRNA-449 in cell fate determination.

The microRNAs 449a, b, and c (miR-449) are potent inducers of cell death, cell cycle arrest, and/or cell differentiation. They belong to the same family as the p53-responsive microRNAs miR-34. Instead of p53, however, the cell cycle regulatory transcription factor E2F1 induces miR-449. All members of this microRNA family are capable of mediating cell cycle arrest and apoptosis and might thereby contribute to tumor suppression. Underlying mechanisms include the downregulation of histone acetyl transferases and consecutive activation of p53, but also the targeting of cyclin dependent kinases and their association partners. Thus, miR-34 and miR-449 provide an asymmetric feedback loop to balance E2F and p53 activities. More recently, it was discovered that miR-449 displays strong tissue specificity, with high levels in lung and testes. Two model systems (Xenopus embryos and cultured human cells) revealed that miR-449 is essential for the development of ciliated epithelia, and this appears to depend on miR-449-mediated modulation of the Notch signaling pathway. Here we summarize our current knowledge on cell fate determination by miR-449, and we propose future directions to explore the function of miR-449 in cell regulation and organismal development. MiR-449 helps to ensure proper cell function but also to avoid cancer, marking a close link between cell differentiation and tumor suppression.

MicroRNA-449a levels increase by several orders of magnitude during mucociliary differentiation of airway epithelia.

MicroRNAs of the miR-34/449 family mediate cell cycle arrest and tumor suppression.  Here we show that the expression of microRNA miR-449a, unlike its paralogue miR-34a, is highly tissue specific and largely restricted to pulmonary and testicular tissue. MiR-449a levels in the murine lung are particularly high shortly before and after birth, coinciding with terminal differentiation of lung epithelia. Strikingly, miR-449a is upregulated by more than 1000-fold when epithelial cells from human airways are lifted from a liquid environment to air, allowing them to undergo mucociliary differentiation. The induction of miR-449a occurs in parallel to its host gene CDC20B and the transcription factor FoxJ1. Exposure to tobacco smoke induces a moderate further increase in the levels of miR-449a, and also miR-34a, in differentiated airway epithelia. We propose that miR-449a can serve as an exquisitely sensitive and specific biomarker for the differentiation of bronchial epithelia. Moreover, miR-449a may actively promote mucociliary differentiation through its ability to block cell cycle progression, and it may conribute to a first line of defence against genotoxic stress by its proapoptotic functions.