E(nos)/CG4699 required for nanos function in the female germ line of Drosophila

The translational repressor Nanos is required in the germ line stem cells of the Drosophila ovary to maintain their capacity for self‐renewal. Following division of the stem cells, Nanos is inhibited in the daughters that differentiate into cysts and ultimately become mature oocytes. The control of Nanos activity is thus an important aspect of the switch from self‐renewal to differentiation. In this report, we describe a genetic interaction between nanos and Enhancer of nos, an allele of the previously uncharacterized locus CG4699. We find that E(nos) protein is required for normal accumulation of Nanos in the ovary and thus for maintenance of the germ line. The mechanism by which E(nos)/CG4699 protein acts is not clear, although it has been found in a complex with Mof acetylase. Consistent with the finding that E(nos) interacts with Mof, we observe that nanos and mof also interact genetically to maintain normal oogenesis. genesis 48:161–170, 2010. © 2010 Wiley‐Liss, Inc.     

Dead end1 is an essential partner of NANOS2 for selective binding of target RNAs in male germ cell development

RNA‐binding proteins (RBPs) play important roles for generating various cell types in many developmental processes, including eggs and sperms. Nanos is widely known as an evolutionarily conserved RNA‐binding protein implicated in germ cell development. Mouse NANOS2 interacts directly with the CCR4‐NOT (CNOT) deadenylase complex, resulting in the suppression of specific RNAs. However, the mechanisms involved in target specificity remain elusive. We show that another RBP, Dead end1 (DND1), directly interacts with NANOS2 to load unique RNAs into the CNOT complex. This interaction is mediated by the zinc finger domain of NANOS2, which is essential for its association with target RNAs. In addition, the conditional deletion of DND1 causes the disruption of male germ cell differentiation similar to that observed in Nanos2‐KO mice. Thus, DND1 is an essential partner for NANOS2 that leads to the degradation of specific RNAs. We also present the first evidence that the zinc finger domain of Nanos acts as a protein‐interacting domain for another RBP, providing a novel insight into Nanos‐mediated germ cell development.     

Nanos3 of the frog Rana rugosa: Molecular cloning and characterization

Nanos is expressed in the primordial germ cells (PGCs) and also the germ cells of a variety of organisms as diverse as Drosophila, medaka fish, Xenopus and mouse. In Nanos3‐deficient mice, PGCs fail to incorporate into the gonad and the size of the testis and ovary is thereby dramatically reduced. To elucidate the role of Nanos in an amphibian species, we cloned Nanos3 cDNA from the testis of the R. rugosa frog. RT‐PCR analysis showed strong expression of Nanos3 mRNA in the testis of adult R. rugosa frogs, but expression was not sexually dimorphic during gonadal differentiation. In Nanos3‐knockdown tadpoles produced by the CRISPR/Cas9 system, the number of germ cells decreased dramatically in the gonads of both male and female tadpoles before sex determination and thereafter. This was confirmed by three dimensional imaging of wild‐type and Nanos3 knockdown gonads using serial sections immunostained for Vasa, a marker specific to germ cells. Taken together, these results suggest that Nanos3 protein function is conserved between R. rugosa and mouse.     

Functional redundancy among Nanos proteins and a distinct role of Nanos2 during male germ cell development

The mouse Nanos proteins, Nanos2 and Nanos3, are required for germ cell development and share a highly conserved zinc-finger domain. The expression patterns of these factors during development, however, differ from each other. Nanos3 expression in the mouse embryo commences in the primordial germ cells (PGCs) just after their formation, and a loss of this protein results in the germ cell-less phenotype in both sexes. By contrast, Nanos2 expression begins only in male PGCs after their entry into the genital ridge and a loss of this protein results in a male germ cell deficiency, irrespective of the co-expression of Nanos3 in these cells. These results indicate that these two Nanos proteins have distinct functions, which depend on the time and place of their expression. To further elucidate this, we have generated transgenic mouse lines that express Nanos2 under the control of the Oct4DeltaPE promoter and examined Nanos2 function in a Nanos3-null genetic background. We find that ectopically produced Nanos2 protein rescues the Nanos3-null defects, because the germ cells fully develop in both sexes in the transgenic mice. This result indicates that Nanos2 can substitute for Nanos3 during early PGC development. By contrast, our current data show that Nanos3 does not rescue the defects in Nanos2-null mice. Our present findings thus indicate that there are redundant functions of the Nanos proteins in early PGC development, but that Nanos2 has a distinct function during male germ cell development in the mouse.     

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.     

Nanos3 maintains the germ cell lineage in the mouse by suppressing both Bax-dependent and -independent apoptotic pathways

Cell death in the germ line is controlled by both positive and negative mechanisms that maintain the appropriate number of germ cells and that prevent the possible formation of germ cell tumors. In the mouse embryo, Steel/c-Kit signaling is required to prevent migrating primordial germ cells (PGCs) from undergoing Bax-dependent apoptosis. In our current study, we show that migrating PGCs also undergo apoptosis in Nanos3-null embryos. We assessed whether the Bax-dependent apoptotic pathway is responsible for this cell death by knocking out the Bax gene together with the Nanos3 gene. Differing from Steel-null embryos, however, the Bax elimination did not completely rescue PGC apoptosis in Nanos3-null embryos, and only a portion of the PGCs survived in the double knockout embryo. We further established a mouse line, Nanos3-Cre-pA, to undertake lineage analysis and our results indicate that most of the Nanos3-null PGCs die rather than differentiate into somatic cells, irrespective of the presence or absence of Bax. In addition, a small number of surviving PGCs in Nanos3/Bax-null mice are maintained and differentiate as male and female germ cells in the adult gonads. Our findings thus suggest that heterogeneity exists in the PGC populations and that Nanos3 maintains the germ cell lineage by suppressing both Bax-dependent and Bax-independent apoptotic pathways.     

A selective screen reveals discrete functional domains in Drosophila Nanos

The Drosophila protein Nanos encodes an evolutionarily conserved protein with two zinc finger motifs. In the embryo, Nanos protein function is required for establishment of the anterior-posterior body pattern and for the migration of primordial germ cells. During oogenesis, Nanos protein is involved in the establishment and maintenance of germ-line stem cells and the differentiation of oocyte precursor cells. To establish proper embryonic patterning, Nanos acts as a translational regulator of hunchback RNA. Nanos' targets for germ cell migration and development are not known. Here, we describe a selective genetic screen aimed at isolating new nanos alleles. The molecular and genetic analysis of 68 new alleles has allowed us to identify amino acids critical for nanos function. This analysis shows that the CCHC motifs, which coordinate two metal ions, are essential for all known functions of Nanos protein. Furthermore, a region C-terminal to the zinc fingers seems to constitute a novel functional domain within the Nanos protein. This "tail region" of Nanos is required for abdomen formation and germ cell migration, but not for oogenesis.     

Crystal structure of zinc-finger domain of Nanos and its functional implications

Nanos is an RNA-binding protein that is involved in the development and maintenance of germ cells. In combination with Pumilio, Nanos binds to the 3' untranslated region of a messenger RNA and represses its translation. Nanos has two conserved Cys-Cys-His-Cys zinc-finger motifs that are indispensable for its function. In this study, we have determined the crystal structure of the zinc-finger domain of zebrafish Nanos, for the first time revealing that Nanos adopts a novel zinc-finger structure. In addition, Nanos has a conserved basic surface that is directly involved in RNA binding. Our results provide the structural basis for further studies to clarify Nanos function.     

Distinct modes of recruitment of the CCR4–NOT complex by Drosophila and vertebrate Nanos

Nanos proteins repress the expression of target mRNAs by recruiting effector complexes through non‐conserved N‐terminal regions. In vertebrates, Nanos proteins interact with the NOT1 subunit of the CCR4–NOT effector complex through a NOT1 interacting motif (NIM), which is absent in Nanos orthologs from several invertebrate species. Therefore, it has remained unclear whether the Nanos repressive mechanism is conserved and whether it also involves direct interactions with the CCR4–NOT deadenylase complex in invertebrates. Here, we identify an effector domain (NED) that is necessary for the Drosophila melanogaster (Dm) Nanos to repress mRNA targets. The NED recruits the CCR4–NOT complex through multiple and redundant binding sites, including a central region that interacts with the NOT module, which comprises the C‐terminal domains of NOT1–3. The crystal structure of the NED central region bound to the NOT module reveals an unanticipated bipartite binding interface that contacts NOT1 and NOT3 and is distinct from the NIM of vertebrate Nanos. Thus, despite the absence of sequence conservation, the N‐terminal regions of Nanos proteins recruit CCR4–NOT to assemble analogous repressive complexes.     

Evolution of the Malagasy endemic genus Nanos Westwood, 1842 (Coleoptera,Scarabaeidae, Epilissini)

The traditionally defined ‘Nanos group’, composed of the genera Nanos Westwood, 1842, Cambefortantus Paulian, 1986 and Apotolamprus Olsoufieff, 1947, represents the most recent Malagasy dung beetle radiation. Species in this group have been ecologically very successful with many being numerically dominant in local dung beetle communities in Madagascar. In this study the phylogenetic relationships of species in this group are inferred using molecular data from mitochondrial (cytochrome c oxidase I) and nuclear (rudimentary, topoisomerase I and 28S) genes.The monophyly of Apotolamprus is supported both by molecular and morphological characters, but that of Nanos, supported by only one morphological character, is questioned. Congruent species groups can be defined within Nanos on the base of morphology and molecular results. In addition to the phylogenetic study, the revision of the genus Nanos Westwood, 1842, s.l., is presented. Nanos antsihanakensis (Lebis, 1953) stat.n . is re‐established. Thirteen new species – Nanos pseudofusconitens sp.n ., Nanos magnus sp.n ., Nanos marojejyensis sp.n ., Nanos bemarahaensis sp.n ., Nanos andreiae sp.n ., Nanos mirjae sp.n ., Nanos pseudorubromaculatus sp.n ., Nanos pseudominutus sp.n ., Nanos mixtus sp.n ., Nanos ranomafanaensis sp.n ., Nanos manongorivoensis sp.n ., N. pseudoviettei sp.n . and N. constricticollis sp.n . – are described and compared with their most closely related taxa. Sphaerocanthon fallaciosus Lebis, 1953, is synonymised with Nanos fusconitens (Fairmaire, 1899) syn.n . and Nanos neoelectrinus Montreuil & Viljanen, 2007, with Nanos humeralis Paulian, 1975 syn.n . Lectotypes are designated for Epilissus fusconitens var. agaboides Boucomont, 1937, Epilissus punctatus Boucomont, 1937, Epilissus sinuatipes Boucomont, 1937, Epilissus semiscribrosus Fairmaire, 1898, Epilissus fusconitens Fairmaire, 1899, and Sphaerocanthon vadoni Lebis, 1953. Aedeagus and male pro‐ and metatibiae are illustrated for each species. This published work has been registered in Zoobank, http://zoobank.org/urn:lsid:zoobank.org:pub:C1F29A37‐E380‐4D87‐871F‐039227547156 .     

Mouse germ cell development during embryogenesis

The discrimination and differentiation of germ cells from somatic cells is a fundamental issue during development. The early specification of mouse primordial germ cells (PGCs) is achieved by the induction of Blimp1, a key regulator of germ cells. Nanos3 is one of the genes activated in early PGCs and prevents apoptosis during their migration stage. Once PGCs enter the embryonic gonads, they differentiate according to the somatic sex of the organism. During this process, Nanos2 plays an important role as it promotes male germ cell pathway by suppressing the female fate. In this review, the process of germ cell development in the mouse is discussed with a particular focus on the functions of the key proteins, Blimp1, Nanos, and Dead end1.     

Conditional expression of Lodavin, an avidin-tagged LDL receptor, for biotin-mediated applications in vivo

Lodavin represents an engineered fusion protein that consists of a cytoplasmic and a transmembrane domain of the human low‐density lipoprotein receptor coupled to an extracellular avidin monomer. Biotinylated compounds have been successfully targeted to Lodavin‐expressing cells that have been transduced by a Lodavin‐containing virus, and the targeting is based on the high affinity between biotin and avidin. We engineered a Rosa26 (R26R) knock‐in Lodavin mouse to develop biotin‐based applications such as targeted drug delivery, cell purification, and tissue imaging in vivo. A cDNA encoding Lodavin was inserted downstream of a floxed βgeo resistance gene in the R26R locus in embryonic stem cells, and a germ line‐derived R26RLodavin mouse line was generated. Efficient removal of the floxed βgeo cassette and conditional activation of Lodavin expression was achieved as a result of crossing the R26RLodavin mice with HoxB7‐Cre, Wnt4‐Cre, or Tie1‐Cre mice. In summary, the R26RLodavin mouse line may provide a useful tool for testing and developing applications with the aid of avidin and biotin interaction. genesis 50:693–699, 2012. © 2012 Wiley Periodicals, Inc.     

Estrogen rescues masculinization of genetically female medaka by exposure to cortisol or high temperature

Medaka (Oryzias latipes) is a teleost fish with an XX/XY sex determination system. Recently, it was reported that XX medaka can be sex‐reversed into phenotypic males by exposure to high water temperature (HT) during gonadal sex differentiation, possibly by elevation of cortisol, the major glucocorticoid produced by the interrenal cells in teleosts. Yet, it remains unclear how the elevation of cortisol levels by HT causes female‐to‐male sex reversal. This paper reports that exposure to cortisol or HT after hatching inhibited both the proliferation of female‐type germ cells and the expression of ovarian‐type aromatase (cyp19a1), which encodes a steroidogenic enzyme responsible for the conversion of androgens to estrogens, and induced the expression of gonadal soma‐derived growth factor (gsdf) in XX gonads during gonadal sex differentiation. In contrast, exposure to either cortisol or HT in combination with 17β‐estradiol (E2) did not produce these effects. Moreover, E2 completely rescued cortisol‐ and HT‐induced masculinization of XX medaka. These results strongly suggest that cortisol and HT cause female‐to‐male sex reversal in medaka by suppression of cyp19a1 expression, with a resultant inhibition of estrogen biosynthesis. This mechanism may be common among animals with temperature‐dependent sex determination. Mol. Reprod. Dev. 79: 719–726, 2012. © 2012 Wiley Periodicals, Inc.     

Induction of primordial germ cells from mouse induced pluripotent stem cells derived from adult hepatocytes

Pluripotent stem cells can be established by various methods, but they share several cytological properties, including germ cell differentiation in vitro, independently of their origin. Although mouse induced pluripotent stem (iPS) cells can produce functional gametes in vivo, it is still unclear whether or not they have the ability to produce presumptive germ cells in vitro. Here, we show that mouse iPS cells derived from adult hepatocytes were able to differentiate into presumptive germ cells marked by mouse vasa homolog (Mvh) expression in feeder‐free or suspension cultures. Embryoid body (EB) formation from iPS cells also induced the formation of round‐shaped cells resembling immature oocytes. Mvh⁺ cells formed clumps by co‐aggregation with differentiation‐supporting cells, and increased expression of germ cell markers was detected in these cell aggregates. Differentiation culture of presumptive germ cells from iPS cells could provide a conventional system for facilitating our understanding of the mechanisms underlying direct reprogramming and germline competency. Mol. Reprod. Dev. 77: 802–811, 2010. © 2010 Wiley‐Liss, Inc.