The mouse dead-end gene isoform alpha is necessary for germ cell and embryonic viability

Inactivation of the dead-end (Dnd1) gene in the Ter mouse strain results in depletion of primordial germ cells (PGCs) so that mice become sterile. However, on the 129 mouse strain background, loss of Dnd1 also increases testicular germ cell tumor incidence in parallel to PGC depletion. We report that inactivation of Dnd1 also affects embryonic viability in the 129 strain. Mouse Dnd1 encodes two protein isoforms, DND1-isoform alpha (DND1-alpha) and DND1-isoform beta (DND1-beta). Using isoform-specific antibodies, we determined DND1-alpha is expressed in embryos and embryonic gonads whereas DND1-beta expression is restricted to germ cells of the adult testis. Our data implicate DND1-alpha isoform to be necessary for germ cell viability and therefore its loss in Ter mice results in PGC depletion, germ cell tumor development and partial embryonic lethality in the 129 strain.     

BAX-mediated cell death affects early germ cell loss and incidence of testicular teratomas in Dnd1 mice

A homozygous nonsense mutation (Ter) in murine Dnd1 (Dnd1^Ter/Ter) results in a significant early loss of primordial germ cells (PGCs) prior to colonization of the gonad in both sexes and all genetic backgrounds tested. The same mutation also leads to testicular teratomas only on the 129Sv/J background. Male mutants on other genetic backgrounds ultimately lose all PGCs with no incidence of teratoma formation. It is not clear how these PGCs are lost or what factors directly control the strain-specific phenotype variation. To determine the mechanism underlying early PGC loss we crossed Dnd1^Ter/Ter embryos to a Bax-null background and found that germ cells were partially rescued. Surprisingly, on a mixed genetic background, rescued male germ cells also generated fully developed teratomas at a high rate. Double-mutant females on a mixed background did not develop teratomas, but were fertile and produced viable off-spring. However, when Dnd1^Ter/Ter XX germ cells developed in a testicular environment they gave rise to the same neoplastic clusters as mutant XY germ cells in a testis. We conclude that BAX-mediated apoptosis plays a role in early germ cell loss and protects from testicular teratoma formation on a mixed genetic background.     

Identification and migration of primordial germ cells in Atlantic salmon,Salmo salar: Characterization of Vasa,Dead End,and Lymphocyte antigen 75 genes

No information exists on the identification of primordial germ cells (PGCs) in the super‐order Protacanthopterygii, which includes the Salmonidae family and Atlantic salmon (Salmo salar L.), one of the most commercially important aquatic animals worldwide. In order to identify salmon PGCs, we cloned the full‐length cDNA of vasa, dead end (dnd), and lymphocyte antigen 75 (ly75/CD205) genes as germ cell marker candidates, and analyzed their expression patterns in both adult and embryonic stages of Atlantic salmon. Semi‐quantitative RT‐PCR results showed that salmon vasa and dnd were specifically expressed in testis and ovary, and vasa, dnd, and ly75 mRNA were maternally deposited in the egg. vasa mRNA was consistently detected throughout embryogenesis while dnd and ly75 mRNA were gradually degraded during cleavages. In situ analysis revealed the localization of vasa and dnd mRNA and Ly75 protein in PGCs of hatched larvae. Whole‐mount in situ hybridization detected vasa mRNA during embryogenesis, showing a distribution pattern somewhat different to that of zebrafish; specifically, at mid‐blastula stage, vasa‐expressing cells were randomly distributed at the central part of blastodisc, and then they migrated to the presumptive region of embryonic shield. Therefore, the typical vasa localization pattern of four clusters during blastulation, as found in zebrafish, was not present in Atlantic salmon. In addition, salmon PGCs could be specifically labeled with a green fluorescence protein (GFP) using gfp‐rt‐vasa 3′‐UTR RNA microinjection for further applications. These findings may assist in understanding PGC development not only in Atlantic salmon but also in other salmonids. Mol. Reprod. Dev. © 2013 Wiley Periodicals, Inc.     

Expression of EGFL7 in primordial germ cells and in adult ovaries and testes

We have previously reported the isolation and characterization of a novel endothelial-restricted gene, Egfl7, that encodes a secreted protein of about 30-kDa. We and others demonstrated that Egfl7 is highly expressed by endothelial cells during embryonic development and becomes down-regulated in the adult vasculature. In the present paper, we show that during mouse embryonic development, Egfl7 is also expressed by primordial germ cells (PGC). Expression is down-regulated when PGCs differentiate into pro-spermatogonia and oogonia, and by 15.5 dpc Egfl7 can no longer be detected in the germ line of both sexes. Notably, Egfl7 is again transiently up-regulated in germ cells of the adult testis. In contrast, expression in the ovary remains limited to the vascular endothelium. Our results provide the first evidence of a non-endothelial expression of EGFL7 and suggest distinctive roles for Egfl7 in vascular development and germ cell differentiation.     

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.     

Ultrastructural study of embryonic and early adult germ cells,and their support cells,in both sexes of Xiphophorus (Teleostei:Poeciliidae)

The ultrastructure of the early spermatogonia in mature testes of the platyfish, Xiphophorus maculatus, was compared to that of oogonia in mature ovaries of X. maculatus and the related X. nigrensis. Both cell types were very similar and, characterized as being large, oval to round cells containing large, central nuclei with prominent nucleoli. Abundant mitochondria with sparse transverse cristae were located at one pole or around the nucleus. Annulate lamellae and electron-dense granular material (nuage) were present. Other organelles were not prominent. A female that had received a testis graft had testicular tissue containing mature spermatozoa within the ovary, indicating that cells were present that could develop along the male line. Special crosses were carried out to obtain all-male embryos of X. maculatus and all-female embryos of X. nigrensis. The ultrastructure of the germ cells in all embryonic gonads was similar to that of the adult cells. These results suggest the presence of sexually undifferentiated germ cells in the adult gonads of both sexes. The support cells investing all of these germ cells were also similar structurally and appeared to be undifferentiated.     

Molecular characterization,sexually dimorphic expression,and functional analysis of 3′-untranslated region of vasa gene in half-smooth tongue sole (Cynoglossus semilaevis)

Vasa is a highly conserved ATP-dependent RNA helicase expressed mainly in germ cells. The vasa gene plays a crucial role in the development of germ cell lineage and has become an excellent molecular marker in identifying germ cells in teleosts. However, little is known about the structure and function of the vasa gene in flatfish. In this study, the vasa gene (Csvasa) was isolated and characterized in half-smooth tongue sole (Cynoglossus semilaevis), an economically important flatfish in China. In the obtained 6425-bp genomic sequence, 23 exons and 22 introns were identified. The Csvasa gene encodes a 663-amino acid protein, including highly conserved domains of the DEAD-box protein family. The amino acid sequence also shared a high homology with other teleosts. Csvasa expression was mainly restricted to the gonads, with little or no expression in other tissues. Real-time quantitative polymerase chain reaction analysis revealed that Csvasa expression levels decreased during embryonic and early developmental stages and increased with the primordial germ cell proliferation. A typical sexually dimorphic expression pattern of Csvasa was observed during early development and sex differentiation, suggesting that the Csvasa gene might play a differential role in the proliferation and differentiation of male and female primordial germ cells (PGCs). Csvasa mRNA expression levels in neomales were significantly lower than those in normal males and females, indicating that the Csvasa gene might be implicated in germ cell development after sex reversal by temperature treatment. In addition, medaka (Oryzias latipes) PGCs could be transiently labeled by microinjection of synthesized mRNA containing the green fluorescence protein gene and 3′-untranslated region of Csvasa, which confirmed that the Csvasa gene has the potential to be used as a visual molecular marker of germ cells and laid a foundation for manipulation of PGCs in tongue sole reproduction.     

Control of Dead end localization and activity – Implications for the function of the protein in antagonizing miRNA function

Dead end (dnd) is a vertebrate-specific component of the germ plasm and germ-cell granules that is crucial for germ-cell development in zebrafish and mouse. Dnd counteracts the inhibitory function of miRNAs, thereby facilitating the expression of proteins such as Nanos and Tdrd7 in the germ cells. Here, we show that cis-acting elements within dnd mRNA and the RNA recognition motive (RRM) of the protein are essential for targeting protein expression to the germ cells and to the perinuclear granules, respectively. We demonstrate that as it executes its function, Dnd translocates between the germ-cell nucleus and germ-cell granules. This phenomenon is not observed in proteins mutated in the RRM motif, correlating with loss of function of Dnd. Based on molecular modeling, we identify the putative RNA binding domain of Dnd as a canonical RRM and propose that this domain is important for protein subcellular localization and function.     

A novel function for KIF13B in germ cell migration

Primordial germ cell (PGC) development in Xenopus embryos relies on localised maternal determinants. We report on the identification and functional characterisation of such one novel activity, a germ plasm associated mRNA encoding for the Xenopus version of a kinesin termed KIF13B. Modulations of xKIF13B function result in germ cell mismigration and in reduced numbers of such cells. PGCs explanted from Xenopus embryos form bleb-like protrusions enriched in PIP3. Knockdown of xKIF13B results in inhibition of blebbing and PIP3 accumulation. Interference with PIP3 synthesis leads to PGC mismigration in vivo and in vitro. We propose that xKIF13B function is linked to polarized accumulation of PIP3 and directional migration of the PGCs in Xenopus embryos.     

Loss of Dnd1 facilitates the cultivation of genital ridge-derived rat embryonic germ cells

Pluripotent cells referred to as embryonic germ cells (EGCs) can be derived from the embryonic precursors of the mature gametes: the primordial germ cells (PGCs). A homozygous mutation (ter) of the dead-end homolog 1 gene (Dnd1) in the rat causes gonadal teratocarcinogenesis and sterility due to neoplastic transformation and loss of germ cells. We mated heterozygous ter/+ WKY-Dnd1^ter/Ztm rats and were able to cultivate the first genital ridge-derived EGCs of the rat embryo at day 14.5 post coitum (pc). Genotyping revealed that ten EGC lines were Dnd1 deficient, while only one wild type cell line had survived in culture. This suggests that the inactivation of the putative tumor suppressor gene Dnd1 facilitates the immortalization of late EGCs in vitro. Injection of the wild type EGCs into blastocysts resulted in the first germ-line competent chimeras. These new pluripotent rat EGCs offer an innovative approach for studies on germ cell tumor development as well as a new tool for genetic manipulations in rats.