Implication of nanos2-3'UTR in the expression and function of nanos2

Translational control of gene expression is an important component of the regulation of cellular differentiation and development. To elucidate the function of the 3'untranslated region (UTR) of the nanos2 gene in mice, we compared the phenotypes of lacZ knock-in mice with or without a native nanos2 3'UTR and found that this region of the nanos2 gene has a potential role during translational regulation in germ cells. The nanos2-3'UTR functions to repress the translation of mRNA in oocytes, but enhances the production of protein in the male gonads. To further understand the significance of the nanos2 3'UTR in vivo, we generated the mouse line nanos2pA/pA, which lacks this region endogenously. In nanos2(-/pA) mice, the number of germ cell-depleted seminiferous tubules was increased when compared with that of nanos2pA/pA mice, indicating a dose-dependent defect in spermatogenesis. These results suggest that the level of nanos2 protein is critical for normal spermatogenesis, and that this pathway may be regulated through the nanos2-3'UTR. We found that the defects in nanos2pA/pA and nanos2(-/pA) mice were caused by apoptosis of gonocytes in the embryonic gonads and gonocyte/spermatogonia in neonatal testes. In addition, it was noted that the nanos2 expression was restricted to a particular subset of spermatogonia after birth, which indicates that nanos2 plays a role in the maintenance and differentiation of gonocytes/spermatogonia in neonatal testes.     

Le 斑马鱼nanos1基因在配子发生中的原位杂交研究

利用组织原位杂交技术,以地高辛标记的反义RNA为探针,检测了斑马鱼(Danio rerio) nanos1基因在卵子发生及精子发生中的表达分布特点,初步探讨了该基因在斑马鱼配子发生中可能的功能。结果表明：在斑马鱼卵子发生中,nanos1 mRNA均匀分布于卵原细胞和各时期卵母细胞的胞质中;在卵原细胞和Ⅰ、Ⅱ期卵母细胞中,nanos1 mRNA的杂交信号十分强烈,而较晚期卵母细胞中信号明显减弱。在斑马鱼精子发生中, nanos1 mRNA可在精原细胞和初级精母细胞中检测到。nanos1 mRNA的阳性信号在精原细胞中极为强烈,在初级精母细胞中较为微弱,而精子细胞中没有阳性信号。本研究结果初步表明,斑马鱼nanos1基因对生殖干细胞-卵原细胞和精原细胞的维持和正常功能可能起着重要作用。     

Germ cell specification and early embryonic patterning in Bombyx mori as revealed by nanos orthologues

In the silkmoth Bombyx mori, the germ cells first appear from the posterior ventral side of the egg (from within the mesodermal primordium) after blastoderm formation. This is in contrast to Drosophila, where germ cells appear at the posterior pole before cellular blastoderm formation. To date, germ plasm has not been found in B. mori. In this study, we describe the identification and expression pattern of nanos from B. mori, in which we recovered four nanos orthologues. One orthologue showed strong expression in embryonic germ cells, which was traced back to periplasmic granules dispersed on the ventral midline of the egg from the posterior-ventral focus of preblastoderm embryos. This suggests that, in B. mori, as in dipterans, germ cell formation depends on a localized determinant in the egg. The expression of another orthologue was observed in the posterior of the germ band. We speculate that nanos has dual functions; one in germ cell formation and the other in posterior body patterning, which is conferred by one nanos gene in Drosophila, but is assigned to different genes in B. mori.     

The nanos gene of Bombyx mori and its expression patterns in developmental embryos and larvae tissues

The nanos gene encodes a zinc-finger protein which is required for the migration and differentiation of primordial germ cells as well as for their fate maintenance. In this study, a 1913 bp nanos gene was cloned and characterized in silkworm (Bombyx mori). RT-PCR and Western blot analysis showed that the nanos was expressed in developing embryos and various silkworm larval tissues. The expression patterns of Nanos and Vasa in silkworm larval gonads were analyzed using immunohistochemistry. It was found that, in silkworm larval ovaries, the Nanos and Vasa proteins were expressed in oocytes. While in testes, high expression of Nanos and Vasa was detected in spermatogonia and relatively weaker expression was found in spermatocytes at latter stages.     

Temporal complexity within a translational control element in the nanos mRNA

Translational control of gene expression plays a fundamental role in the early development of many organisms. In Drosophila, selective translation of nanos mRNA localized to the germ plasm at the posterior of the embryo, together with translational repression of nanos in the bulk cytoplasm, is essential for development of the anteroposterior body pattern. We show that both components to spatial control of nanos translation initiate during oogenesis and that translational repression is initially independent of Smaug, an embryonic repressor of nanos. Repression during oogenesis and embryogenesis are mediated by distinct stem loops within the nanos 3' untranslated region; the Smaug-binding stem-loop acts strictly in the embryo, whereas a second stem-loop functions in the oocyte. Thus, independent regulatory modules with temporally distinct activities contribute to spatial regulation of nanos translation. We propose that nanos evolved to exploit two different stage-specific translational regulatory mechanisms.     

Pharyngeal endoderm expression of nanos1 is dispensable for craniofacial development

Nanos proteins are essential for developing primordial germ cells (PGCs) in both invertebrates and vertebrates. In invertebrates, also contribute to the patterning of the anterior-posterior axis of the embryo and the neural development. In vertebrates, however, besides the role of Nanos proteins in PGC development, the biological functions of the proteins in normal development have not yet been identified. Here, we analyzed the expression and function of nanos1 during craniofacial development in zebrafish. nanos1 was expressed in the pharyngeal endoderm and endodermal pouches essential for the development of facial skeletons and endocrine glands in the vertebrate head. However, no craniofacial defects, such as abnormal pouches, hypoplasia of the thymus, malformed facial skeletons, have been found in nanos1 knockout animals. The normal craniofacial development of nanos1 knockout animals is unlikely a consequence of the genetic redundancy of Nanos1 with Nanos2 or Nanos3 or a result of the genetic compensation for the loss of Nanos1 by Nanos2 or Nanos3 because the expression of nanos2 and nanos3 was rarely seen in the pharyngeal endoderm and endodermal pouches in wild-type and nanos1 mutant animals during craniofacial development. Our findings suggest that nanos1 expression in the pharyngeal endoderm might be dispensable for craniofacial development in zebrafish.     

家蚕生殖细胞相关基因nanos的克隆表达及在胚胎发育中的表达模式

【目的】探讨nanos基因在家蚕Bombyx mori胚胎发育中的表达模式,为进一步研究该基因在家蚕胚胎发育中的功能奠定基础。【方法】根据本实验室提交到GenBank中家蚕nanos的cDNA序列(登录号EF647589)设计引物,扩增出了一条长684 bp的编码片段,对该片段进行了克隆和表达,亲和纯化表达的蛋白并免疫新西兰大白兔制备抗体。Western blot检测家蚕早期胚胎nanos的表达情况,荧光定量PCR检测nanos在整个家蚕胚胎发育中的表达情况。【结果】克隆并表达了一条长684 bp的编码片段,得到了分子量约33 kD的融合蛋白。用制备的抗血清对家蚕早期胚胎蛋白的Western blot检测表明,nanos在此阶段基本是恒定表达。荧光定量PCR结果显示刚产的卵中nanos的表达量最大,第2天开始急剧下降,此后到第10天表达量几乎没有变化。【结论】本实验克隆的nanos是家蚕中的一个同源物,该基因在家蚕胚胎发育中的表达模式与蜜蜂等有很大的不同,反映了昆虫生殖细胞形成机制的多样性。     

Localization of nanos RNA controls embryonic polarity.

Anterior-posterior polarity of the Drosophila embryo is initiated during oogenesis through differential maternal RNA localization. The RNA of the anterior morphogen bicoid is localized to the anterior pole of the embryo, where bicoid protein controls head and thorax development. The RNA of the posterior morphogen nanos is localized to the posterior pole, where nanos protein is required for abdomen formation. Here we show that the nanos 3' untranslated region, like that of the bicoid RNA, is sufficient for RNA localization. We have used the bicoid RNA localization signal to mislocalize nanos, producing embryos with two sources of nanos protein. Such embryos form two abdomens with mirror image symmetry. Embryos with nanos RNA localized only to the anterior have greater nanos gene activity than embryos with nanos RNA localized posteriorly. We propose a role for RNA localization in regulating nanos activity.     

Cloning and characterization of nanos gene in silkworm Bombyx mori

Gene nanos is a maternal posterior group gene required for normal development of abdominal segments and the germ line in Drosophila. Expression of nanos-related genes is associated with the germ line in a broad variety of other taxa. In this study, the 5＇-RACE method and the in silico cloning method are used to isolate the new nanos-like gene of Bombyx mor/and the gene obtained is analyzed with bioinformatics tools. The putative protein is expressed in Escherichia coli and the antiserum has been produced in New Zealand white rabbits. The result shows that the nanos cDNA is 1,913 bp in full length and contains a 954 bp open reading frame. The deduced protein has 317 amino acid residues, with a predicted molecular weight of 35 kDa, isoelectric point of 5.38, and contains a conserved nanos RNA binding domain. The conserved region of the deduced protein shares 73% homology with the nanos protein conserved region of Honeybee （Apis mellifera）. This gene has been registered in the GenBank under the accession number EF647589. One encoding sequence of the nanos fragment has been successfully expressed in E. coli. Western blotting analysis indicates that homemade antiserum can specifically detect nanos protein expressed in prokaryotic cells.     

Posterior expression of nanos orthologs during embryonic and larval development of the anthozoan Nematostella vectensis

Cnidarians are primitive animals located in a basal position in the phylogenetic tree of the Animal Kingdom, as an outgroup of the Bilaterians. Therefore, studies on cnidarian developmental biology may illustrate how fundamental developmental processes have originated and changed during animal evolution. A particular example of this is the establishment of polarity along the body axes, which is under the control of a number of developmental genes, most of them conserved in evolution and playing similar roles in diverged species. Concerning the anterior-posterior axis, genetic and molecular studies on Drosophila have shown that the nanos gene plays an essential role in defining posterior structures during early embryonic development. Here we report the isolation of two nanos orthologs in the anthozoan Nematostella vectensis. We show that nanos mRNA is asymmetrically distributed in the fertilized egg and this asymmetry is maintained during embryonic development. At gastrula and planula larva stages, nanos expression is permanently associated with posterior body regions. These results, together with our previous analysis in the hydrozoan Podocoryne carnea, indicate that posterior nanos expression during development is a conserved feature among cnidarians. Therefore, the potential role of cnidarian nanos in defining axial polarity as a posterior determinant would represent an ancestral trait in the Animal Kingdom.     

Conserved Mechanisms for Germ Cell-Specific Localization of nanos3 Transcripts in Teleost Species with Aquaculture Significance

The importance of the aquaculture production is increasing with the declining global fish stocks, but early sexual maturation in several farmed species reduces muscle growth and quality, and escapees could have a negative impact on wild populations. A possible solution to these problems is the production of sterile fish by ablation of the embryonic primordial germ cells (PGCs), a technique developed in zebrafish. Cell-specific regulation of mRNA stability is crucial for proper specification of the germ cell lineage and commonly involves microRNA (miRNA)-mediated degradation of targeted mRNAs in somatic cells. This study reports on the functional roles of conserved motifs in the 3′ untranslated region (UTR) of the miRNA target gene nanos3 identified in Atlantic cod, Atlantic salmon, and zebrafish. The 3′UTR of cod nanos3 was sufficient for targeting the expression of green fluorescent protein (GFP) to the presumptive PGCs in injected embryos of the three phylogenetically distant species. 3′UTR elements of importance for PGC-specific expression were further examined by fusing truncated 3′UTR variants of cod nanos3 to GFP followed by injections in zebrafish embryos. The expression patterns of the GFP constructs in PGCs and somatic cells suggested that the proximal U-rich region is responsible for the PGC-specific stabilization of the endogenous nanos3 mRNA. Morpholino-mediated downregulation of the RNA-binding protein Dead end (DnD), a PGC-specific inhibitor of miRNA action, abolished the fluorescence of the PGCs in cod and zebrafish embryos, suggesting a conserved DnD-dependent mechanism for germ cell survival and migration.     

The Drosophila hnRNP M homolog Rumpelstiltskin regulates nanos mRNA localization

Anterior-posterior axis patterning of the Drosophila embryo requires Nanos activity selectively in the posterior. This spatial asymmetry of Nanos is generated by the localization of nanos mRNA to the posterior pole of the embryo, where it is subsequently translated. Posterior localization of nanos is mediated by a complex cis-acting localization signal in its 3' untranslated region comprising several partially redundant localization elements. This localization signal redundancy has hampered the identification of trans-acting factors that act specifically to effect posterior localization of nanos. Here, we have used a biochemical approach to identify Rumpelstiltskin, a Drosophila heterogeneous nuclear ribonucleoprotein (hnRNP) M homolog, which binds directly to an individual nanos localization element. Rumpelstiltskin associates with nanos mRNA in vitro and in vivo, and binding by Rumpelstiltskin correlates with localization element function in vivo. Through analysis of a rumpelstiltskin null mutation by genetic strategies that circumvent redundancy, we demonstrate that Rumpelstiltskin regulates anterior-posterior axis patterning by functioning as a direct-acting nanos mRNA localization factor.     

nanos1 is required to maintain oocyte production in adult zebrafish

Development of the germline requires the specification and survival of primordial germ cells (PGCs) in the embryo as well as the maintenance of gamete production during the reproductive life of the adult. These processes appear to be fundamental to all Metazoans, and some components of the genetic pathway regulating germ cell development and function are evolutionarily conserved. In both vertebrates and invertebrates, nanos-related genes, which encode RNA-binding zinc finger proteins, have been shown to play essential and conserved roles during germ cell formation. In Drosophila, maternally supplied nanos is required for survival of PGCs in the embryo, while in adults, nanos is required for the continued production of oocytes by maintaining germline stem cells self-renewal. In mice and zebrafish, nanos orthologs are required for PGC survival during embryogenesis, but a role in adults has not been explored. We show here that nanos1 in zebrafish is expressed in early stage oocytes in the adult female germline. We have identified a mutation in nanos1 using a reverse genetics method and show that young female nanos mutants contain oocytes, but fail to maintain oocyte production. This progressive loss of fertility in homozygous females is not a phenotype that has been described previously in the zebrafish and underlines the value of a reverse genetics approach in this model system.     

Structure of the Drosophila BicaudalD protein and its role in localizing the the posterior determinant nanos

Mutations in the BicaudalD (BicD) gene lead to a global reorganization of the Drosophila body pattern such that the head, thoracic, and anterior abdominal segments are replaced by posterior abdominal segments and terminalia. We first provide evidence that the primary cause of this phenotype is the inhibition of two anterior factors, bicoid and hunchback, by mislocalized activity of the posterior determinant nanos. We then describe the isolation of the BicD gene and show that it encodes a coiled-coil protein similar to the carboxy-terminal portion of the myosin heavy chain. Finally, we find that BicD protein is uniformly distributed throughout wild-type oocytes but is concentrated at the anterior pole of BicD mutant oocytes together with ectopic nanos activity. Taken together, these results suggest that BicD encodes a cytoskeleton-like protein involved in transporting or anchoring the nanos morphogen within the oocyte cytoplasm.     

Smaug, a novel and conserved protein, contributes to repression of nanos mRNA translation in vitro

Proper deployment of Nanos protein at the posterior of the Drosophila embryo, where it directs posterior development, requires a combination of RNA localization and translational controls. These controls ensure that only the posteriorly-localized nanos mRNA is translated, whereas unlocalized nanos mRNA is translationally repressed. Here we describe cloning of the gene encoding Smaug, an RNA-binding protein that interacts with the sequences, SREs, in the nanos mRNA that mediate translational repression. Using an in vitro translation assay, we demonstrate that SRE-dependent repression occurs in extracts from early stage embryos. Immunodepletion of Smaug from the extracts eliminates repression, consistent with the notion that Smaug is involved. Smaug is a novel gene and the existence of potential mammalian Smaug homologs raises the possibility that Smaug represents a new class of conserved translational repressor.     

The maternal gene nanos has a central role in posterior pattern formation of the Drosophila embryo

A group of maternal genes, the posterior group, is required for the development of the abdominal region in the Drosophila embryo. We have used genetic as well as cytoplasmic transfer experiments to order seven of the posterior group genes (nanos, pumilio, oskar, valois, vasa, staufen and tudor) into a functional pathway. An activity present in the posterior pole plasm of wild-type embryos can restore normal abdominal development in posterior group mutants. This activity is synthesized during oogenesis and the gene nanos most likely encodes this activity. The other posterior group genes have distinct accessory functions: pumilio acts downstream of nanos and is required for the distribution or stability of the nanos-dependent activity in the embryo. Staufen, oskar, vasa, valois and tudor act upstream of nanos. Embryos from females mutant for these genes lack the specialized posterior pole plasm and consequently fail to form germ-cell precursors. We suggest that the products of these genes provide the physical structure necessary for the localization of nanos-dependent activity and of germ line determinants.     

vasa and nanos expression patterns in a sea anemone and the evolution of bilaterian germ cell specification mechanisms

Most bilaterians specify primordial germ cells (PGCs) during early embryogenesis using either inherited cytoplasmic germ line determinants (preformation) or induction of germ cell fate through signaling pathways (epigenesis). However, data from nonbilaterian animals suggest that ancestral metazoans may have specified germ cells very differently from most extant bilaterians. Cnidarians and sponges have been reported to generate germ cells continuously throughout reproductive life, but previous studies on members of these basal phyla have not examined embryonic germ cell origin. To try to define the embryonic origin of PGCs in the sea anemone Nematostella vectensis, we examined the expression of members of the vasa and nanos gene families, which are critical genes in bilaterian germ cell specification and development. We found that vasa and nanos family genes are expressed not only in presumptive PGCs late in embryonic development, but also in multiple somatic cell types during early embryogenesis. These results suggest one way in which preformation in germ cell development might have evolved from the ancestral epigenetic mechanism that was probably used by a metazoan ancestor.     

Live imaging of endogenous RNA reveals a diffusion and entrapment mechanism for nanos mRNA localization in Drosophila

BACKGROUND: Localization of nanos mRNA to the posterior pole of the Drosophila embryo directs local synthesis of Nanos protein that is essential for patterning of the anterior-posterior body axis and germ cell function. While nanos RNA is synthesized by the ovarian nurse cells and appears at the posterior pole of the ooctye late in oogenesis, the mechanism by which this RNA is translocated to and anchored at the oocyte posterior is unknown. RESULTS: By labeling endogenous nanos RNA with GFP, we have been able to follow the dynamic pathway of nanos localization in living oocytes. We demonstrate that nanos localization initiates immediately upon nurse cell dumping, whereby diffusion, enhanced by microtubule-dependent cytoplasmic movements, translocates nanos RNA from the nurse cells to the ooctye posterior. At the posterior, nanos is trapped by association, in particles, with the posteriorly localized germ plasm. Actin-dependent anchoring of nanos RNA complexed to the germ plasm at the posterior maintains localization in the face of rapid cytoplasmic movements. CONCLUSIONS: These results reveal a diffusion-based, late-acting posterior localization mechanism for long-range transport of nanos mRNA. This mechanism differs from directed transport-based localization mechanisms in its reliance on bulk movement of RNA.