Nanos Gene Research Articles

Germ cell specification and early embryonic patterning inBombyx morias revealed bynanosorthologues

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.

Vasa and nanos are coexpressed in somatic and germ line tissue from early embryonic cleavage stages through adulthood in the polychaete Capitella sp. I

Members of the vasa and nanos gene families are involved in germ line development in a number of diverse animals. As a polychaete annelid model for studies of the germ line, Capitella sp. I has several advantages including the presence of dedicated gonads, individuals that reproduce multiple times, and the presence of males, females, and hermaphrodites. Germ line development has not been characterized in Capitella sp. I, nor is the mechanism of germ line specification generally well understood in annelids. We have cloned vasa and nanos orthologues from Capitella sp. I and found that both CapI-vasa and CapI-nanos transcripts are expressed in developing gametes of sexually mature adults. Characterization of both these genes during embryonic, larval, and juveniles stages reveals expression in multiple somatic tissues for CapI-vasa and CapI-nanos with largely overlapping but not identical expression patterns. In early cleavage stages, both transcripts are broadly expressed; following gastrulation, expression is observed in the presumptive brain, mesodermal bands, and developing foregut. Using CapI-nanos and CapI-vasa as markers, we have identified putative primordial germ cells (PGCs) in larvae, which are initially present as small bilateral clusters in segment 4 and as a single cluster at late larval stages. In adults, a single large cluster of putative PGCs is present in segments 5 and 6. In addition to highlighting differences in expression profiles for these two genes among lophotrochozoans, we present a hypothesis concerning the origin and development of PGCs in Capitella sp. I.

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 mori 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.

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.

Potential role of Nanos3 in maintaining the undifferentiated spermatogonia population

Nanos gene encodes for zinc-finger protein with putative RNA-binding activity which shows an evolutionary conserved function in germ cell development. In the mouse, three Nanos homologs have been identified: Nanos1, Nanos2 and Nanos3. The Nanos3 ortholog is expressed in both male and female gonads of early embryo and, after birth, it is found only in the testis. Nanos3 targeted disruption results in the complete loss of germ cells in both sexes; however the role of Nanos3 in the testis during the postnatal period has not been explored yet. In this study, we show that, in prepuberal testis, Nanos3 is expressed in undifferentiated spermatogonia and that its up-regulation causes accumulation of cells in the G1 phase, indicating that this protein is able to delay the cell cycle progression of spermatogonial cells. This is in line with the observation that the cell cycle length of the undifferentiated germ cells is longer than in differentiating spermatogonia. We also demonstrate a conserved mechanism of action of Nanos3, involving the interaction with the murine RNA-binding protein Pumilio2 and consisting of a potential translational repressor activity. According to the possible role of Nanos3 in inhibiting spermatogonia cell differentiation, we show that treatment with the differentiating factor all-trans retinoic acid induces a dramatic down-regulation of its expression. These results allow to conclude that, in the prepuberal testis, Nanos3 is important to maintain undifferentiated spermatogonia via the regulation of their cell cycle.

Dispensability of nanos mRNA localization for abdominal patterning but not for germ cell development

The development of a functional germline is essential for species propagation. The nanos ( nos) gene plays an evolutionarily conserved role in germline development and is also essential for abdominal patterning in Drosophila. A small fraction of nos mRNA is localized to the germ plasm at the posterior pole of the Drosophila embryo, where it becomes incorporated into the germ cells. Germ plasm associated nos mRNA is translated to produce a gradient of Nos protein that patterns the abdomen, whereas the remaining unlocalized RNA is translationally repressed to allow anterior development. Using transgenes that compromise nos mRNA localization and translational regulation, we show that wild-type body patterning can ensue without nos mRNA localization provided that nos translation is properly modulated. In contrast, localization of nos to the germ plasm, but not translational regulation, is essential for nos function in the developing germ cells. We propose that an imperative for nos localization in producing a functional germline has preserved an inefficient localization mechanism.

Nanos gene control DNA mediates developmentally regulated transposition in the yellow fever mosquito Aedes aegypti.

Transposable elements (TEs) are proposed as a basis for developing drive systems to spread pathogen resistance genes through vector mosquito populations. The use of transcriptional and translational control DNA elements from genes expressed specifically in the insect germ line to mediate transposition offers possibilities for mitigating some of the concerns about transgene behavior in the target vector species and eliminating effects on nontarget organisms. Here, we describe the successful use of the promoter and untranslated regions from the nanos (nos) orthologous gene of the yellow fever mosquito, Aedes aegypti, to control sex- and tissue-specific expression of exogenously derived mariner MosI transposase-encoding DNA. Transgenic mosquitoes expressed transposase mRNA in abundance near or equal to the endogenous nos transcript and exclusively in the female germ cells. In addition, MosI mRNA was deposited in developing oocytes and localized and maintained at the posterior pole during early embryonic development. Importantly, four of five transgenic lines examined were capable of mobilizing a second MosI transgene into the mosquito genome, indicating that functional transposase was being produced. Thus, the nos control sequences show promise as part of a TE-based gene drive system.

Translational repression restricts expression of the C. elegans Nanos homolog NOS-2 to the embryonic germline

Members of the nanos gene family are evolutionarily conserved regulators of germ cell development. In several organisms, Nanos protein expression is restricted to the primordial germ cells (PGCs) during early embryogenesis. Here, we investigate the regulation of the Caenorhabditis elegans nanos homolog nos-2. We find that the nos-2 RNA is translationally repressed. In the adult germline, translation of the nos-2 RNA is inhibited in growing oocytes, and this inhibition depends on a short stem loop in the nos-2 3′UTR. In embryos, nos-2 translation is repressed in early blastomeres, and this inhibition depends on a second region in the nos-2 3′UTR. nos-2 RNA is also degraded in somatic blastomeres by a process that is independent of translational repression and requires the CCCH finger proteins MEX-5 and MEX-6. Finally, the germ plasm component POS-1 activates nos-2 translation in the PGCs. A combination of translational repression, RNA degradation, and activation by germ plasm has also been implicated in the regulation of nanos homologs in Drosophila and zebrafish, suggesting the existence of conserved mechanisms to restrict Nanos expression to the germline.

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.

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.

Nanos1: a mouse nanos gene expressed in the central nervous system is dispensable for normal development

A mouse nanos ( nanos1) gene was cloned and its function was examined by generating a gene-knockout mouse. The nanos1 gene encodes an RNA-binding protein, which contains a putative zinc-finger motif that exhibits similarity with other nanos-class genes in vertebrates and invertebrates. Although nanos1 is not detected in primordial germ cells, it is observed in seminiferous tubules of mature testis. Interestingly, maternally expressed nanos1 is observed in substantial amounts in oocytes, but the amount of maternal RNA is rapidly reduced after fertilization, and the transient zygotic nanos1 expression is observed in eight-cell embryos. At 12.5 days postcoitum, nanos1 is re-expressed in the central nervous system and the expression continues in the adult brain, in which the hippocampal formation is the predominant region. The nanos1 -deficient mice develop to term without any detectable abnormality and they are fertile. No significant neural defect is observed in terms of their behavior to date.

Primordial germ cells: the blueprint for a piscine life

Germ cells prior to sex differentiation are called primordial germ cells (PGCs). They are the progenitor cells of the germ cell lineage and possess the ability to differentiate into either spermatogonia or oogonia. In all animals examined to date, PGCs are set aside from somatic cell lineages during early embryonic development. Recent studies of fish germ cells have revealed that (1) germ plasm-like structures are found in fertilized eggs of zebrafish, and structurally and morphologically similar structures called `nuage' are found in the gonadal PGCs of various fish species; (2) RNA transcripts of the vasa and nanos genes, which are known to be components of the germ plasm of Xenopus laevis, are also present as maternally inherited molecules in fishes; and (3) PGCs emerge only from the lower part of the blastoderm and this specification occurs prior to zygotic gene expression. Based on these findings, asymmetrical distribution of maternally inherited germ plasm appears to be a major system for germ cell specification in fishes. This mechanism is different from that in mammals, in which PGCs are specified epigenetically. PGCs separate from somatic cell lineages in extragonadal areas and then migrate to coalesce with somatic gonadal support cells. These migrations are performed at least partly by active movement of PGCs themselves in zebrafish and rainbow trout. On the other hand, in medaka, the PGC migration may be the result of passive movement caused by surrounding somatic cell migration. During these migration periods, fish PGCs are mitotically silent and resume proliferation after they reach the gonadal anlagen. PGCs, in addition to being biologically important as founders of the germ cell lineage, may have many applications for bioengineering of fish because of their ability to be converted into individual fishes. Some of these potential applications are discussed.

Nos-1 and nos-2, two genes related to Drosophila nanos, regulate primordial germ cell development and survival in Caenorhabditis elegans

In Drosophila, the posterior determinant nanos is required for embryonic patterning and for primordial germ cell (PGC) development. We have identified three genes in Caenorhabditis elegans that contain a putative zinc-binding domain similar to the one found in nanos, and show that two of these genes function during PGC development. Like Drosophila nanos, C. elegans nos-1 and nos-2 are not generally required for PGC fate specification, but instead regulate specific aspects of PGC development. nos-2 is expressed in PGCs around the time of gastrulation from a maternal RNA associated with P granules, and is required for the efficient incorporation of PGCs into the somatic gonad. nos-1 is expressed in PGCs after gastrulation, and is required redundantly with nos-2 to prevent PGCs from dividing in starved animals and to maintain germ cell viability during larval development. In the absence of nos-1 and nos-2, germ cells cease proliferation at the end of the second larval stage, and die in a manner that is partially dependent on the apoptosis gene ced-4. Our results also indicate that putative RNA-binding proteins related to Drosophila Pumilio are required for the same PGC processes as nos-1 and nos-2. These studies demonstrate that evolutionarily distant organisms utilize conserved factors to regulate early germ cell development and survival, and that these factors include members of the nanos and pumilio gene families.

NANOS-3 and FBF proteins physically interact to control the sperm–oocyte switch in Caenorhabditis elegans

Background: The Caenorhabditis elegans FBF protein and its Drosophila relative, Pumilio, define a large family of eukaryotic RNA-binding proteins. By binding regulatory elements in the 3′ untranslated regions (UTRs) of their cognate RNAs, FBF and Pumilio have key post-transcriptional roles in early developmental decisions. In C. elegans, FBF is required for repression of fem-3 mRNA to achieve the hermaphrodite switch from spermatogenesis to oogenesis.Results: We report here that FBF and NANOS-3 (NOS-3), one of three C. elegans Nanos homologs, interact with each other in both yeast two-hybrid and in vitro assays. We have delineated the portions of each protein required for this interaction. Worms lacking nanos function were derived either by RNA-mediated interference (nos-1 and nos-2) or by use of a deletion mutant (nos-3). The roles of the three nos genes overlap during germ-line development. In certain nos-deficient animals, the hermaphrodite sperm–oocyte switch was defective, leading to the production of excess sperm and no oocytes. In other nos-deficient animals, the entire germ line died during larval development. This germ-line death did not require CED-3, a protease required for apoptosis.Conclusions: The data suggest that NOS-3 participates in the sperm–oocyte switch through its physical interaction with FBF, forming a regulatory complex that controls fem-3 mRNA. NOS-1 and NOS-2 also function in the switch, but do not interact directly with FBF. The three C. elegans nanos genes, like Drosophila nanos, are also critical for germ-line survival. We propose that this may have been the primitive function of nanos genes.

Novel genetic screen for genes involved in posterior body patterning in Drosophila.

Much of our current understanding of how cytoplasmic determinants are localized and activated stems from genetic analyses in Drosophila. Characterization of mutants defective in establishing the initial embryonic body pattern has revealed the importance of mRNA localization and translational control and identified several factors required for these processes. Nevertheless, many additional genes are likely to be involved. Here, we describe a novel genetic screen designed to identify genes that participate in posterior body patterning, an elaborate process involving the sequential use of two localized cytoplasmic determinants, the products of the oskar and nanos genes. From the screen, we recovered new alleles of genes known to be required for posterior body patterning, demonstrating the validity of the approach. In addition, we isolated numerous other mutants. Further characterization of one mutant, P58, revealed that it is a novel allele of bullwinkle. We find that in bullwinkle mutants, oskar mRNA localization is not maintained in the embryo and oskar protein accumulates ectopically and to abnormally high levels. These defects are distinct from previously described perturbations in oskar activity and provide new insights into the regulation of oskar.

Nanos is an evolutionarily conserved organizer of anterior-posterior polarity.

In Drosophila melanogaster, nanos functions as a localized determinant of posterior pattern. Nanos RNA is localized to the posterior pole of the maturing egg cell and encodes a protein that emanates from this localized source. Nanos acts as a translational repressor and thereby establishes a gradient of the morphogen Hunchback. Here we show that the mechanism by which nanos acts in Drosophila is a common developmental strategy in Dipteran insects. We used cytoplasmic transplantation assays to demonstrate that nanos activity is found in posterior poleplasm of five diverse Dipteran species. Genes homologous to nanos were identified from Drosophila virilis, the housefly Musca domestica, and the midge Chironomus samoensis. These genes encode RNAs that are each localized, like nanos, to the embryonic posterior pole. Most importantly, we demonstrate that these homologues can functionally substitute for nanos in D. melanogaster. These results suggest that nanos acts in a similar pathway for axis determination in other insects. Comparison of the Nanos sequences reveals only 19% overall protein sequence similarity; high conservation of a novel zinc finger near the carboxy terminus of the protein defines a region critical for nanos gene function.

Genetics of nanos localization in Drosophila.

The Drosophila gene nanos is required for two processes. During oogenesis, nanos function is required for the continued production of egg chambers, and nanos is expressed in the early germarium. During embryogenesis, nanos is required maternally to specify abdominal segmentation. Nanos shares this latter function with nine other genes, collectively known as the posterior group. Of this group, nanos encodes a determinant, and is localized as an RNA to the posterior pole of early embryos. This RNA is translated to form a gradient of nanos protein with highest concentrations at the posterior. Analysis of the distribution of nanos gene products in embryos mutant for posterior group genes shows that eight of these genes are required for localization, but not stability, of the nanos RNA. Embryos mutant for posterior group alleles which produce weak abdominal phenotypes show reduced amounts of localized nanos RNA. This correlation between nanos RNA localization and abdominal phenotype suggests that nanos acts as a localization-dependent posterior determinant. Localization of nanos is not affected by mutations in bicoid or torso, confirming that the three maternal systems of anterior-posterior determination initially act independently.

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.

Nanos is the localized posterior determinant in Drosophila

Segmental pattern in the Drosophila embryo is established by two maternal factors localized to the anterior and posterior poles of the egg cell. Here we provide molecular evidence that the locallzed posterior factor is the RNA of the nanos (nos) gene. nos RNA is localized to the posterior pole of early embryos, and nos protein acts at a distance to direct abdomen formation. Synthetic nos RNA has biological activity identical to that of the posterior pole plasm. Injection of nos RNA rescues the segmentation defect of embryos derived from females mutant for all nine known posterior group genes. Injection of nos RNA Into the anterior is able to direct formation of ectopic posterior structures. Our results demonstrate that a localized source of nos RNA is sufficient to specify abdominal segmentation and imply that other posterior group genes are required for localization, stabilization, or distribution of the nos gene product.

Interactions of the Drosophila gap gene giant with maternal and zygotic pattern-forming genes

The Drosophila gene giant (gt) is a segmentation gene that affects anterior head structures and abdominal segments A5-A7. Immunolocalization of the gt product shows that it is a nuclear protein whose expression is initially activated in an anterior and a posterior domain. Activation of the anterior domain is dependent on the maternal bicoid gradient while activation of the posterior domain requires maternal nanos gene product. Initial expression is not abolished by mutations in any of the zygotic gap genes. By cellular blastoderm, the initial pattern of expression has evolved into one posterior and three anterior stripes of expression. The evolution, position and width of these stripes are dependent on interactions between gt and the other gap genes. In turn, gt activity in these domains affects the expression of the other gap genes. These interactions, typical of the cross-regulation previously observed among gap genes, confirm that gt is a member of the gap gene class whose function is necessary to establish the overall pattern of gap gene expression. After cellular blastoderm, gt protein continues to be expressed in the head region in parts of the maxillary and mandibular segments as well as in the labrum. Expression is never detected in the labial or thoracic segment primordia but persists in certain head structures, including the ring gland, until the end of embryonic development.