Host-defense mechanisms against transposable elements are critical to protect the genome information. Here we show that tudor-domain containing 9 (Tdrd9) is essential for silencing Line-1 retrotransposon in the mouse male germline. Tdrd9 encodes an ATPase/DExH-type helicase, and its mutation causes male sterility showing meiotic failure. In Tdrd9 mutants, Line-1 was highly activated and piwi-interacting small RNAs (piRNAs) corresponding to Line-1 were increased, suggesting that feedforward amplification operates in the mutant. In fetal testes, Tdrd9 mutation causes Line-1 desilencing and an aberrant piRNA profile in prospermatogonia, followed by cognate DNA demethylation. TDRD9 complexes with MIWI2 with distinct compartmentalization in processing bodies, and this TDRD9-MIWI2 localization is regulated by MILI and TDRD1 residing at intermitochondrial cement. Our results identify TDRD9 as a functional partner of MIWI2 and indicate that the tudor-piwi association is a conserved feature, while two separate axes, TDRD9-MIWI2 and TDRD1-MILI, cooperate nonredundantly in the piwi-small RNA pathway in the mouse male germline.

Human induced pluripotent stem (hiPS) cells have potential uses for drug discovery and cell therapy, including generation of pancreatic β-cells for diabetes research and treatment. In this study, we developed a simple protocol for generating insulin-producing cells from hiPS cells. Treatment with activin A and a GSK3β inhibitor enhanced efficient endodermal differentiation, and then combined treatment with retinoic acid, a bone morphogenic protein inhibitor, and a transforming growth factor-β (TGF-β) inhibitor induced efficient differentiation of pancreatic progenitor cells from definitive endoderm. Expression of the pancreatic progenitor markers PDX1 and NGN3 was significantly increased at this step and most cells were positive for anti-PDX1 antibody. Moreover, several compounds, including forskolin, dexamethasone, and a TGF-β inhibitor, were found to induce the differentiation of insulin-producing cells from pancreatic progenitor cells. By combined treatment with these compounds, more than 10% of the cells became insulin positive. The differentiated cells secreted human c-peptide in response to various insulin secretagogues. In addition, all five hiPS cell lines that we examined showed efficient differentiation into insulin-producing cells with this protocol.

The germ-line cells of many animals possess a characteristic cytoplasmic structure termed nuage or germinal granules. In mice, nuage that is prominent in postnatal male germ cells is also called intermitochondrial cement or chromatoid bodies. TDRD1/MTR-1, which contains Tudor domain repeats, is a specific component of the mouse nuage, analogously to Drosophila Tudor, a constituent of polar granules/nuage in oocytes and embryos. We show that TDRD6 and TDRD7/TRAP, which also contain multiple Tudor domains, specifically localize to nuage and form a ribonucleoprotein complex together with TDRD1/MTR-1. The characteristic co-localization of TDRD1, 6 and 7 was disrupted in a mutant of mouse vasa homologue/DEAD box polypeptide 4 (Mvh/Ddx4), which encodes another evolutionarily conserved component of nuage. In vivo over-expression experiments of the TDRD proteins and truncated forms during male germ cell differentiation showed that a single Tudor domain is a structural unit that localizes or accumulates to nuage, but the expression of the truncated, putative dominant negative forms is detrimental to meiotic spermatocytes. These results indicate that the Tudor-related proteins, which contain multiple repeats of the Tudor domain, constitute an evolutionarily conserved class of nuage components in the germ-line, and their localization or accumulation to nuage is likely conferred by a Tudor domain structure and downstream of Mvh, while the characteristic repeated architecture of the domain is functionally essential for the differentiation of germ cells.

BackgroundHuman pluripotent stem cells (hPSCs) are being applied in regenerative medicine and for the in vitro modeling of human intractable disorders. In particular, neural cells derived from disease-specific human induced pluripotent stem cells (hiPSCs) established from patients with neurological disorders have been used as in vitro disease models to recapitulate in vivo pathogenesis because neural cells cannot be usually obtained from patients themselves.ResultsIn this study, we established a rapid, efficient, and simple method for efficiently deriving motor neurons from hPSCs that is useful for pathophysiological analysis and the development of drugs to treat motor neuron diseases. Treatment with GSK3β inhibitors during the initial phase of differentiation in combination with dual SMAD inhibition was sufficient to induce PAX6+ and SOX1+ neural progenitors within 1 week, and subsequent treatment with retinoic acid (RA) and purmorphamine, which activates sonic hedgehog (SHH) signaling, resulted in the highly efficient induction of HB9+ and ISL-1+ motor neurons within 2 weeks. After 4 weeks of monolayer differentiation in motor neuron maturation medium, hPSC-derived motor neurons were shown to mature, displaying larger somas and clearer staining for the mature motor neuron marker choline acetyltransferase (ChAT). Moreover, hPSC-derived motor neurons were able to form neuromuscular junctions with human myotubes in vitro and induced acetylcholine receptor (AChR) clustering, as detected by Alexa 555-conjugated α-Bungarotoxin (α-BTX), suggesting that these hPSC-derived motor neurons formed functional contacts with skeletal muscles. This differentiation system is simple and is reproducible in several hiPSC clones, thereby minimizing clonal variation among hPSC clones. We also established a system for visualizing motor neurons with a lentiviral reporter for HB9 (HB9e438::Venus). The specificity of this reporter was confirmed through immunocytochemistry and quantitative RT-PCR analysis of high-positive fractions obtained via fluorescence-activated cell sorting (FACS), suggesting its applicability for motor neuron-specific analysis.ConclusionsOur motor neuron differentiation system and lentivirus-based reporter system for motor neurons facilitate the analysis of disease-specific hiPSCs for motor neuron diseases.Electronic supplementary materialThe online version of this article (doi:10.1186/s13041-015-0172-4) contains supplementary material, which is available to authorized users.

SummaryPelizaeus-Merzbacher disease (PMD) is a form of X-linked leukodystrophy caused by mutations in the proteolipid protein 1 (PLP1) gene. Although PLP1 proteins with missense mutations have been shown to accumulate in the rough endoplasmic reticulum (ER) in disease model animals and cell lines transfected with mutant PLP1 genes, the exact pathogenetic mechanism of PMD has not previously been clarified. In this study, we established induced pluripotent stem cells (iPSCs) from two PMD patients carrying missense mutation and differentiated them into oligodendrocytes in vitro. In the PMD iPSC-derived oligodendrocytes, mislocalization of mutant PLP1 proteins to the ER and an association between increased susceptibility to ER stress and increased numbers of apoptotic oligodendrocytes were observed. Moreover, electron microscopic analysis demonstrated drastically reduced myelin formation accompanied by abnormal ER morphology. Thus, this study demonstrates the involvement of ER stress in pathogenic dysmyelination in the oligodendrocytes of PMD patients with the PLP1 missense mutation.