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ES-mediated chimera analysis revealed requirement of DDX6 for NANOS2 localization and function in mouse germ cells.

ABSTRACT: In embryonic male germ cells, the RNA-binding protein NANOS2 recruits its target RNAs to processing bodies (P-bodies), where they are repressed. This process is necessary to promote male-type germ cell differentiation. However, it remains unclear whether all NANOS2 functions depend on P-bodies. To address this question, we established ES cell lines containing a germ cell-specific inducible Cre and reporter together with the floxed Ddx6 allele. We deleted the Ddx6 gene by administering tamoxifen to chimeric embryos containing germ cells derived from recombinant ES cells. DDX6-null germ cells exhibited both similar and distinct defects from those observed in NANOS2-null germ cells. These results demonstrate that NANOS2 function is carried out via both P-body-dependent and -independent mechanisms. RNA-seq analyses further supported the phenotypic differences between DDX6-null and NANOS2-null germ cells, and indicated distinct molecular cascades involved in NANOS2-mediated gene regulation.

SUBMITTER: Shimada R

PROVIDER: S-EPMC6345806 | biostudies-other | 2019 Jan

REPOSITORIES: biostudies-other

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ES-mediated chimera analysis revealed requirement of DDX6 for NANOS2 localization and function in mouse germ cells.

Shimada Ryuki R Kiso Makoto M Saga Yumiko Y

Scientific reports 20190124 1

In embryonic male germ cells, the RNA-binding protein NANOS2 recruits its target RNAs to processing bodies (P-bodies), where they are repressed. This process is necessary to promote male-type germ cell differentiation. However, it remains unclear whether all NANOS2 functions depend on P-bodies. To address this question, we established ES cell lines containing a germ cell-specific inducible Cre and reporter together with the floxed Ddx6 allele. We deleted the Ddx6 gene by administering tamoxifen ...[more]

PMID: 30679547

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Project description:BackgroundThe Houbara bustard (Chlamydotis undulata) is a wild seasonal breeding bird populating arid sandy semi-desert habitats in North Africa and the Middle East. Its population has declined drastically during the last two decades and it is classified as vulnerable. Captive breeding programmes have, hitherto, been unsuccessful in reviving population numbers and thus radical technological solutions are essential for the long term survival of this species. The purpose of this study was to investigate the use of primordial germ cell-mediated chimera technology to produce viable Houbara bustard offspring.Methodology/principal findingsEmbryonic gonadal tissue was dissected from Houbara bustard embryos at eight days post-incubation. Subsequently, Houbara tissue containing gonadal primordial germ cells (gPGCs) was injected into White Leghorn chicken (Gallus gallus domesticus) embryos, producing 83/138 surviving male chimeric embryos, of which 35 chimeric roosters reached sexual maturity after 5 months. The incorporation and differentiation of Houbara gPGCs in chimeric chicken testis were assessed by PCR with Houbara-specific primers and 31.3% (5/16) gonads collected from the injected chicken embryos showed the presence of donor Houbara cells. A total of 302 semen samples from 34 chimeric roosters were analyzed and eight were confirmed as germline chimeras. Semen samples from these eight roosters were used to artificially inseminate three female Houbara bustards. Subsequently, 45 Houbara eggs were obtained and incubated, two of which were fertile. One egg hatched as a male live born Houbara; the other was female but died before hatching. Genotyping confirmed that the male chick was a pure-line Houbara derived from a chimeric rooster.ConclusionThis study demonstrates for the first time that Houbara gPGCs can migrate, differentiate and eventually give rise to functional sperm in the chimeric chicken testis. This approach may provide a promising tool for propagation and conservation of endangered avian species that cannot breed in captivity.

Project description:The gene expression and protein synthesis of small leucine-rich proteoglycans (SLRPs), including decorin, biglycan, fibromodulin, and lumican, was analyzed in the context of the hypothesis that they are closely related to tooth formation. In situ hybridization, immunohistochemistry, and organ culture with metabolic labeling of [35S] were carried out in mouse first molar tooth germs of different developmental stages using ICR mice at embryonic day (E) 13.5 to postnatal day (P) 7.0. At the bud and cap stage, decorin mRNA was expressed only in the surrounding mesenchyme, but not within the tooth germ. Biglycan mRNA was then expressed in the condensing mesenchyme and the dental papilla of the tooth germ. At the apposition stage (late bell stage), both decorin and biglycan mRNA were expressed in odontoblasts, resulting in a switch of the pattern of expression within the different stages of odontoblast differentiation. Decorin mRNA was expressed earlier in newly differentiating odontoblasts than biglycan. With odontoblast maturation and dentin formation, decorin mRNA expression was diminished and localized to the newly differentiating odontoblasts at the cervical region. Simultaneously, biglycan mRNA took over and extended its expression throughout the new and mature odontoblasts. Both mRNAs were expressed in the dental pulp underlying the respective odontoblasts. At P7.0, both mRNAs were weakly expressed but maintained their spatial expression patterns. Immunostaining showed that biglycan was localized in the dental papillae and pulp. In addition, all four SLRPs showed clear immunostaining in predentin, although the expressions of fibromodulin and lumican mRNAs were not identified in the tooth germs examined. The organ culture data obtained supported the histological findings that biglycan is more predominant than decorin at the apposition stage. These results were used to identify biglycan as the principal molecule among the SLRPs investigated. Our findings indicate that decorin and biglycan show spatial and temporal differential expressions and play their own tissue-specific roles in tooth development.

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ES-mediated chimera analysis revealed requirement of DDX6 for NANOS2 localization and function in mouse germ cells.

Publications

ES-mediated chimera analysis revealed requirement of DDX6 for NANOS2 localization and function in mouse germ cells.

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