dead end, a novel vertebrate germ plasm component, is required for zebrafish primordial germ cell migration and survival. Weidinger G et al. (2003) In most animals, primordial germ cell (PGC) specification and development depend on maternally provided cytoplasmic determinants that constitute the so-called germ plasm. Little is known about the role of germ plasm in vertebrate germ cell development, and its molecular mode of action remains elusive. While PGC specification in mammals occurs via different mechanisms, several germ plasm components required for early PGC development in lower organisms are expressed in mammalian germ cells after their migration to the gonad and are involved in gametogenesis. Here we show that the RNA of dead end, encoding a novel putative RNA binding protein, is a component of the germ plasm in zebrafish and is specifically expressed in PGCs throughout embryogenesis; Dead End protein is localized to perinuclear germ granules within PGCs. Knockdown of dead end blocks confinement of PGCs to the deep blastoderm shortly after their specification and results in failure of PGCs to exhibit motile behavior and to actively migrate thereafter. PGCs subsequently die, while somatic development is not effected. We have identified dead end orthologs in other vertebrates including Xenopus, mouse, and chick, where they are expressed in germ plasm and germ-line cells, suggesting a role in germ-line development in these organisms as well.//////////////////DND1 maintains germline stem cells via recruitment of the CCR4-NOT complex to target mRNAs. Yamaji M et al. (2017) The vertebrate-conserved RNA-binding protein DND1 is required for the survival of primordial germ cells (PGCs), as well as the suppression of germ cell tumours in mice. Here we show that in mice DND1 binds a UU(A/U) trinucleotide motif predominantly in the 3' untranslated regions of mRNA, and destabilizes target mRNAs through direct recruitment of the CCR4-NOT deadenylase complex. Transcriptomic analysis reveals that the extent of suppression is dependent on the number of DND1-binding sites. This DND1-dependent mRNA destabilization is required for the survival of mouse PGCs and spermatogonial stem cells by suppressing apoptosis. The spectrum of target RNAs includes positive regulators of apoptosis and inflammation, and modulators of signalling pathways that regulate stem-cell pluripotency, including the TGFβ superfamily, all of which are aberrantly elevated in DND1-deficient PGCs. We propose that the induction of the post-transcriptional suppressor DND1 synergizes with concurrent transcriptional changes to ensure precise developmental transitions during cellular differentiation and maintenance of the germ line.//////////////////
RNA-binding protein Dnd1 inhibits microRNA access to target mRNA. Kedde M et al. MicroRNAs (miRNAs) are inhibitors of gene expression capable of controlling processes in normal development and cancer. In mammals, miRNAs use a seed sequence of 6-8 nucleotides (nt) to associate with 3' untranslated regions (3'UTRs) of mRNAs and inhibit their expression. Intriguingly, occasionally not only the miRNA-targeting site but also sequences in its vicinity are highly conserved throughout evolution. We therefore hypothesized that conserved regions in mRNAs may serve as docking platforms for modulators of miRNA activity. Here we demonstrate that the expression of dead end 1 (Dnd1), an evolutionary conserved RNA-binding protein (RBP), counteracts the function of several miRNAs in human cells and in primordial germ cells of zebrafish by binding mRNAs and prohibiting miRNAs from associating with their target sites. These effects of Dnd1 are mediated through uridine-rich regions present in the miRNA-targeted mRNAs. Thus, our data unravel a novel role of Dnd1 in protecting certain mRNAs from miRNA-mediated repression.
NCBI Summary:
This gene encodes a protein that binds to microRNA-targeting sequences of mRNAs, inhibiting microRNA-mediated repression. Reduced expression of this gene has been implicated in tongue squamous cell carcinoma. Two pseudogenes of this gene are located on the long arm of chromosome 17. [provided by RefSeq, Dec 2010]
General function
Cell proliferation, RNA processing, RNA binding
Comment
Cellular localization
Cytoplasmic
Comment
Ovarian function
Early embryo development
Comment
Expression regulated by
Comment
Ovarian localization
Primordial Germ Cell, Oocyte
Comment
Expression of RNA binding proteins DND1 and FXR1 in the porcine ovary, and during oocyte maturation and early embryo development. Yang CX et al. The porcine oocyte and early embryo are transcriptionally quiescent following germinal vesicle breakdown in the oocyte and prior to activation of the embryonic genome, at approximately the 4-cell stage of development. Despite a lack of new transcription, mRNA and protein repertoires are subject to regulation during this time. One potential mechanism of regulation is through the functional activity of miRNAs and/or the presence of specific RNA binding proteins. Both DND1 (dead end homolog 1) and FXR1 (fragile-X-mental retardation related protein 1) are RNA binding proteins that have been demonstrated to impact miRNA-mediated, post-transcriptional gene regulation. The objective was to characterize the presence and the expression changes in DND1 and FXR1 during pig oocyte maturation and early embryo development. DND1 and FXR1 expression were evaluated in oocytes and cumulus cells during meiotic progression and in 4-cell stage embryos using quantitative RT-PCR, Western blot analysis, and immunostaining. These data demonstrate DND1 and FXR1 mRNA are expressed in the maturing oocyte and early in vitro-fertilized embryos, with significantly less DND1 in 4-cell stage embryos as compared to germinal vesicle and metaphase II-arrested oocytes. Based on immunohistochemistry, DND1 protein abundance is greater in secondary follicles in comparison to primary and tertiary follicles. Using ribonucleoprotein immunoprecipitation from germinal vesicle-stage oocyte, DND1 was demonstrated to interact with several mRNAs associated with pluripotency. This work provides a better understanding of the biological relevance of DND1 and FXR1 during female gametogenesis and embryo development in pigs. Mol. Reprod. Dev. 2012 Wiley Periodicals, Inc.
SSR 2010 abstract 323. Expression of microRNAs and RNA-Binding Protein DND1
During Oocyte Maturation and Early Embryonic Development in the Pig.
Cai-Xia Yang, Elane C. Wright, Robyn Scanlon, Ben Selman, Randall S. Prather, and
Jason W. Ross. Iowa State University, Ames, IA, USA; University of Missouri,
Columbia, Missouri, USA
Following germinal vesicle breakdown (GVBD), the genome of an immature
oocyte is transcriptionally inactive and any changes in mRNA and protein repertoire
until the maternal to zygote transition (MZT) are controlled through interactions with
the surrounding cumulus oophorus and post transcriptional events occurring within
the oocyte and early embryo. MicroRNAs have a demonstrated ability to regulate
both mRNA and protein repertoires through their ability to confer post transcriptional
gene regulation through interactions with the 3? untranslated region (UTR) of mRNA
molecules. The outcome following an miRNA:mRNA interaction can depend largely
on the expression of several critical RNA binding proteins, such as dead end homolog
1 (DND1). DND1 is critical for germ cell development and has more recently been
demonstrated to prevent miRNA mediated transcript degradation through interactions
with the 3? UTR of miRNA targeted transcripts. Our working hypothesis is that
numerous miRNAs are expressed during oocyte maturation and that the presence of
RNA binding proteins such as DND1 influences miRNA impact on mRNA stability
and translation during oocyte maturation and prior to MZT following in vitro
fertilization. The objective of the current study was to determine the expression of
miRNAs in the oocyte during in vitro maturation and the presence of DND1 during
oocyte maturation and early embryo development following in vitro fertilization. We
utilized one microgram of total RNA from germinal vesicle (GV) stage oocytes and
metaphase II (MII) arrested oocytes for miRNA microarray analysis and identified
several hundred mature miRNAs are expressed in the oocyte, including miR-21, miR-
23, miR-24, miR-34, miR-483-5p, and numerous others consistent with those
expressed in mice oocytes during meiotic progression. We then utilized pools of 25
GV, MII and 4- to 8-cell stage embryos (n=4 per stage) for quantitative real-time PCR
and pools of 100 GV and MII oocytes (n=3 per stage) for western blot analysis of
DND1. Absolute quantification of DND1 mRNA indicated that abundance was
greatest in GV stage oocytes and was approximately 2-fold (P . 0.05) less in MII
arrested oocytes following in vitro maturation. Embryos in vitro fertilized and
cultured to the 4- to 8-cell stage demonstrated an approximate 7.9 fold (P , 0.05)
reduction in DND1 mRNA compared to MII arrested oocytes. Based on western blot
analysis DND1 (~34 kDa) was present in both GV stage and MII arrested oocytes and
was not significantly different (P . 0.05) between the two stages. Collectively, these
data demonstrate the presence of numerous mature miRNAs in the pig oocyte during
maturation temporal to the relative abundance of DND1 mRNA and protein
expression. The presence of numerous miRNAs in the maturing oocyte suggests post
transcriptional gene regulation may be partially controlled by DND1 interactions with mRNAs possessing the appropriate RNA recognition motif in the 3?UTR.
Follicle stages
Comment
Phenotypes
Mutations
3 mutations
Species: mouse
Mutation name: type: null mutation fertility: infertile - ovarian defect Comment: CRISPR-Cas9-mediated base-editing screening in mice identifies DND1 amino acids that are critical for primordial germ cell development. Li Q et al. (2018) CRISPR-mediated base editing can introduce single-nucleotide changes in the DNA of living cells. One intriguing application of base editing is to screen pivotal amino acids for protein function in vivo; however, it has not been achieved. Here, we report an enhanced third-generation base-editing system with extra nuclear localization sequences that can efficiently introduce a homozygous base mutation in embryonic stem cells. Meanwhile, we establish a strategy to generate base-mutant mice by injection of haploid embryonic stem cells carrying a constitutively expressed enhanced third-generation base-editing system (4B2N1) and single guide RNA into oocytes. Moreover, transfection of 4B2N1 cells with a single guide RNA library targeting the Dnd1 gene allows one-step generation of mutant mice with a base mutation. This enables the identification of four missense mutations that completely deplete primordial germ cells through disruption of DND1 protein stability and protein-protein interaction. Thus, our strategy provides an effective tool for in vivo screening of amino acids that are crucial for protein function.////////////////// [
Species: mouse
Mutation name: type: naturally occurring fertility: infertile - ovarian defect Comment: The Ter mutation in the dead end gene causes germ cell loss and testicular germ cell tumours. Youngren KK et al. (2005) In mice, the Ter mutation causes primordial germ cell (PGC) loss in all genetic backgrounds. Ter is also a potent modifier of spontaneous testicular germ cell tumour (TGCT) susceptibility in the 129 family of inbred strains, and markedly increases TGCT incidence in 129-Ter/Ter males. In 129-Ter/Ter mice, some of the remaining PGCs transform into undifferentiated pluripotent embryonal carcinoma cells, and after birth differentiate into various cells and tissues that compose TGCTs. Here, we report the positional cloning of Ter, revealing a point mutation that introduces a termination codon in the mouse orthologue (Dnd1) of the zebrafish dead end (dnd) gene. PGC deficiency is corrected both with bacterial artificial chromosomes that contain Dnd1 and with a Dnd1-encoding transgene. Dnd1 is expressed in fetal gonads during the critical period when TGCTs originate. DND1 has an RNA recognition motif and is most similar to the apobec complementation factor, a component of the cytidine to uridine RNA-editing complex. These results suggest that Ter may adversely affect essential aspects of RNA biology during PGC development. DND1 is the first protein known to have an RNA recognition motif directly implicated as a heritable cause of spontaneous tumorigenesis. TGCT development in the 129-Ter mouse strain models paediatric TGCT in humans. This work will have important implications for our understanding of the genetic control of TGCT pathogenesis and PGC biology.//////////////////
Species: None
Mutation name: type: null mutation fertility: infertile - ovarian defect Comment: Application of dead end-knockout zebrafish as recipients of germ cell transplantation. Li Q et al. (2018) Germ cell transplantation is a promising technology for the propagation of endangered or valuable fishes. In this technique, sterile male and female recipient fish are injected with donor germ cells so they can produce viable gametes derived from the donor cells. The dead end (dnd) gene is involved in the migration of primordial germ cells; therefore, dnd-knockout zebrafish are expected to be germ-cell-free, making them suitable recipients for germ cell transplantation. dnd mutants were produced by microinjecting 2 nl of 10 ng/μl cRNAs encoding zinc finger nucleases against dnd into the blastodisc of zebrafish embryos before the cell- cleavage stage. One of the resulting founder males was mated with a wild-type female, and produced heterozygous mutants in the F1 generation. Mating of these F1 mutants produced an F2 generation with approximately 25% of the clutch being homozygous mutant (dnd-knockout) male, and lacking germ cells (as confirmed by expression analyses of vasa). The resulting dnd-knockout zebrafish males were tested for suitability as germ cell transplantation recipients by intraperitoneal transplantation of testicular cells prepared from vasa-gfp zebrafish. GFP-positive germ cells incorporated into the germ-cell-free gonads of the dnd-knockout recipients matured into functional sperm. Progeny tests revealed that the sperm from these dnd-knockout recipients were derived entirely from donor cells. Thus, we demonstrated that homozygous dnd mutants became germ-cell-free males that are able to nurse donor-derived germ cells.//////////////////