Green fluorescent protein labeling of primordial germ cells using a nontransgenic method and its application for germ cell transplantation in salmonidae

Transplanting primordial germ cells (PGCs) has a number of potential applications in fish bioengineering. Previously, we established a system to visualize live PGCs in the rainbow trout by introducing the green fluorescent protein (Gfp) gene driven by rainbow trout vasa gene regulatory regions. However, for PGC transplantation to be practically useful in aquaculture, visualization of PGCs using a nontransgenic technique is required. In this study, we demonstrate a method for labeling PGCs from various fish species by introducing chimeric RNAs composed of the Gfp coding region and vasa gene 3'-untranslated regions (UTRs); these sequences play a critical role in stabilizing mRNA in zebrafish PGCs. The GFP chimeric RNAs, including vasa 3'-UTR RNAs from rainbow trout, Nibe croaker, and zebrafish, were microinjected into the cytoplasm of fertilized eggs of several Salmonidae species. All the resulting embryos showed specific labeling in PGCs after the somatogenesis stage, which continued to be visible for at least 50 days. To apply this technique to PGC transplantation, PGCs labeled with chimeric RNA were microinjected into the peritoneal cavity of newly hatched salmonid embryos. The GFP labeling was sufficiently long-lived for the initial stage of donor PGC behavior to be followed in the recipient embryos. Importantly, donor PGCs from brown trout and masu salmon were incorporated into xenogeneic genital ridges in recipient rainbow trout. This nontransgenic method for labeling fish PGCs should be extremely useful for applications of PGC transplantation where the resulting progeny are to be released into the environment, such as PGC cryopreservation for fish stocks and surrogate brood stock technology.     

Isolation of teleost primordial germ cells using flow cytometry

Primordial germ cells (PGCs) generate gametes, the only cells that can transmit genetic information to the next generation. A previous report demonstrated that a fusion construct of green fluorescent protein (gfp) and zebrafish nos 1 3UTR mRNA could be used to label PGCs in a number of fish species. Here, we sought to exploit this labeling strategy to isolate teleost PGCs by flow cytometry (FCM), and to use these isolated PGCs to examine germ cell migration to the gonadal region. In zebrafish, medaka and goldfish, the PGCs were labeled by injecting the gfp-nos1 3UTR mRNA into 1- 4 cell embryos. When the embryos had developed to the somitogenesis or later stages, they were enzymatically disaggregated and GFP positive cells isolated using FCM. PGCs in the different species clustered in the same segments of the FCM scatter diagrams for total embryonic cells produced by plotting the forward scatter intensity against GFP intensity. In situ hybridization showed that the sorted zebrafish cells expressed vasa RNA in their cytoplasm, suggesting that they were PGCs. When the migration ability of the sorted cells from zebrafish was examined in an in vivo transplantation experiment, approximately 30% moved to the gonadal region of host embryos. These observations demonstrate that PGCs can be isolated without use of transgenic fishes and that the isolated PGCs retain the ability to migrate. Our data indicate that this technique will be of value for isolating PGCs from a range of fish species.     

Visualization of primordial germ cells in vivo using GFP-nos1 3'UTR mRNA

In some teleost fish, primordial germ cells (PGCs) inherit specific maternal cytoplasmic factors such as vasa and nanos 1 (nos1) mRNA. It has been shown that the 3'untranslated regions (UTRs) of vasa and nos1 have critical roles for stabilization of these RNAs in zebrafish PGCs. In this study, to determine whether this role of the nos 1 3'UTR is conserved between teleost species, we injected artificially synthesized mRNA, combining green fluorescent protein (GFP) and the zebrafish nos 1 3'UTR (GFP-nos 1 3'UTR mRNA), into the fertilized eggs of various fish species. The 3'UTR of the Oryzias latipes vasa homologue (olvas ) mRNA was assayed in the same manner. We demonstrate that the PGCs of seven teleost species could be visualized using GFP-nos 1 3'UTR mRNA. GFP-olvas 3'UTR mRNA did not identify PGCs in herring or loach embryos, but did enable visualization of the PGCs in medaka embryos. Our results indicate that the 3'UTR of the zebrafish nos1 mRNA can promote maintenance of RNAs in the PGCs of different fish species. Finally, we describe and compare the migration routes of PGCs in seven teleost species.     

PGC-Enriched miRNAs Control Germ Cell Development

Non-coding microRNAs (miRNAs) regulate the translation of target messenger RNAs (mRNAs) involved in the growth and development of a variety of cells, including primordial germ cells (PGCs) which play an essential role in germ cell development. However, the target mRNAs and the regulatory networks influenced by miRNAs in PGCs remain unclear. Here, we demonstrate a novel miRNAs control PGC development through targeting mRNAs involved in various cellular pathways. We reveal the PGC-enriched expression patterns of nine miRNAs, including miR-10b, -18a, -93, -106b, -126-3p, -127, -181a, -181b, and -301, using miRNA expression analysis along with mRNA microarray analysis in PGCs, embryonic gonads, and postnatal testes. These miRNAs are highly expressed in PGCs, as demonstrated by Northern blotting, miRNA in situ hybridization assay, and miRNA qPCR analysis. This integrative study utilizing mRNA microarray analysis and miRNA target prediction demonstrates the regulatory networks through which these miRNAs regulate their potential target genes during PGC development. The elucidated networks of miRNAs disclose a coordinated molecular mechanism by which these miRNAs regulate distinct cellular pathways in PGCs that determine germ cell development.     

Formation and cultivation of medaka primordial germ cells

Primordial germ cell (PGC) formation is pivotal for fertility. Mammalian PGCs are epigenetically induced without the need for maternal factors and can also be derived in culture from pluripotent stem cells. In egg-laying animals such as Drosophila and zebrafish, PGCs are specified by maternal germ plasm factors without the need for inducing factors. In these organisms, PGC formation and cultivation in vitro from indeterminate embryonic cells have not been possible. Here, we report PGC formation and cultivation in vitro from blastomeres dissociated from midblastula embryos (MBEs) of the fish medaka (Oryzias latipes). PGCs were identified by using germ-cell-specific green fluorescent protein (GFP) expression from a transgene under the control of the vasa promoter. Embryo perturbation was exploited to study PGC formation in vivo, and dissociated MBE cells were cultivated under various conditions to study PGC formation in vitro. Perturbation of somatic development did not prevent PGC formation in live embryos. Dissociated MBE blastomeres formed PGCs in the absence of normal somatic structures and of known inducing factors. Most importantly, under culture conditions conducive to stem cell derivation, some dissociated MBE blastomeres produced GFP-positive PGC-like cells. These GFP-positive cells contained genuine PGCs, as they expressed PGC markers and migrated into the embryonic gonad to generate germline chimeras. Our data thus provide evidence for PGC preformation in medaka and demonstrate, for the first time, that PGC formation and derivation can be obtained in culture from early embryos of medaka as a lower vertebrate model.     

Isolation of highly pure and viable primordial germ cells from rainbow trout by GFP-dependent flow cytometry

A highly pure and viable primordial germ cell (PGC) population appears to be an essential tool for establishing a cell line that can differentiate into a germ cell lineage and for studying the molecular biology and biochemistry of fish PGCs. Therefore, the aim of the present study was to establish a flow cytometric method for isolating highly pure and viable PGCs. As the material for PGC isolation, we used transgenic rainbow trout possessing the green fluorescent protein (GFP) gene driven by trout vasa-gene regulatory sequences (pvasa-GFP). Four independent transgenic strains were subjected to fluorescence microscopy and GFP-dependent flow cytometric analyses. We found that some of the pvasa-GFP transgenic strains exhibited ectopic background green fluorescence in the somatic cells aside from strong fluorescence in PGCs. Although flow cytometric analysis of genital ridge somatic cells in the four pvasa-GFP transgenic strains revealed a wide range of GFP intensities, we proved that somatic cell contamination of the GFP-positive cell population was markedly reduced if transgenic strains without the ectopic background green fluorescence were used. In addition, the forward light-scattering (FS) property, which is an indication of relative cell size, and the side light-scattering (SS) property, which is determined by cell shape and granularity, were employed to remove non-PGC contaminants from the GFP-positive cell population. By isolating GFP-positive cells with high FS/SS values, we were able to effectively remove cell blebs and the apoptotic fraction. Consequently, the purities and survival rates of isolated PGCs were greatly improved compared with those using GFP intensity as a single indicator. Thus, our flow cytometric method, in combination with the selection of suitable transgenic strains without the ectopic background green fluorescence, is capable of isolating highly pure and viable PGCs from rainbow trout. By using this method in combination with cell-cryopreservation and cell transplantation techniques, the isolated PGCs may also be used for preserving the genetic resources of endangered fish species and domesticated fish strains carrying commercially valuable traits. Mol. Reprod. Dev. 67: 91-100, 2004.     

The Xdsg protein in presumptive primordial germ cells (pPGCs) is essential to their differentiation into PGCs in Xenopus

In order to know the role of the Xdsg gene in presumptive PGCs (pPGCs) of Xenopus, we attempted to inhibit the translation of Xdsg mRNA in pPGCs by injecting antisense morpholino oligo (asMO), together with Fluorescein Dextran-Lysine (FDL), into single germ plasm-bearing cells of 32-cell embryos. Among three types of asMOs complementary to different parts of the 5'-untranslated region of Xdsg mRNA tested, only one asMO, designated as Xdsg-3, inhibited the translation of the mRNA in FDL-labeled pPGCs, resulting in the absence of labeled PGCs in experimental tadpoles. On the other hand, two other asMOs, Xdsg-1 and -2, did not inhibit the translation, so that a similar number of labeled PGCs found in FDL-injected but asMO-uninjected control tadpoles were observed in experimental tadpoles derived from asMO-injected embryos. Surprisingly, use of Xdsg-3 asMO resulted in the disappearance of the protein of Xenopus vasa homolog (Xenopus vasa-like gene 1, XVLG1) from FDL-labeled pPGCs by inhibiting the translation of XVLG1 mRNA. However, the effect of Xdsg-3 asMO on the translation of Xdsg and XVLG1 mRNAs and PGC formation could be canceled by the coinjection with Xdsg mRNA. Consequently, the Xdsg protein in pPGCs may play an important role in the formation of PGCs by regulating the production of XVLG1 protein.     

Distinct contributions of CXCR4b and CXCR7/RDC1 receptor systems in regulation of PGC migration revealed by medaka mutants kazura and yanagi

Migratory pathways of PGCs to the gonad vary depending on the vertebrate species, yet the underlying regulatory mechanisms guiding PGCs are believed to be largely common. In teleost medaka embryo, PGC migration follows two major steps before colonizing in gonadal areas: (1) bilateral lineup in the trunk and (2) posterior drift of PGCs. kazura (kaz) and yanagi (yan) mutants of medaka isolated in mutagenesis screening were defective in the first and second steps, respectively. kaz^j2-15D was identified as a missense mutation in chemokine receptor gene cxcr4b expressed in PGCs. Embryonic injection of cxcr4b mRNA with vasa 3′ UTR rescued the PGC phenotype of kaz mutant, indicating a cell-autonomous function of cxcr4b in PGCs. yan^j6-29C was identified as a nonsense mutation in the cxcr7/rdc1 gene encoding another chemokine receptor. cxcr7 transgene with genomic flanking sequences rescued the yan mutant phenotype efficiently at the G0 generation. cxcr7 was expressed in somites rather than PGCs. cxcr7-expressing somitic domain expanded posteriorly with its margin immediately anterior of posteriorly drifting PGCs, as if PGCs were thrusted toward the gonadal area. kaz and yan mutants are also defective in lateral line positioning, suggesting combined employment of these receptor systems in various cell migratory processes.     

Mass isolation of primordial germ cells from transgenic rainbow trout carrying the green fluorescent protein gene driven by the vasa gene promoter

Mass isolation of live primordial germ cells (PGCs) was demonstrated for the first time in ectothermal vertebrates. To establish a stem cell-mediated gene transfer system in fish, a stem cell line that retains the ability to develop into gametes is necessary. PGCs are well suited for use as the initial material for such a stem cell line. We established transgenic rainbow trout (Oncorhynchus mykiss) strains carrying the green fluorescent protein (GFP) gene driven by a rainbow trout vasa-like gene (RtVLG) promoter/enhancer. Because GFP expression was specific to the PGCs, PGCs were successfully visualized in all developmental stages examined. Isolated genital ridges containing GFP-labeled PGCs were enzymatically dissociated. To isolate PGCs from the complex pools of dissociated genital ridges, GFP-labeled cells were sorted by flow cytometry. The sorted GFP-positive cells were large and round with a large nucleus, typical characters of PGC morphology. The expression of RtVLG was detected only in the GFP-positive cell population, confirming that these cells were PGCs. This simple and efficient technique to purify a large number of viable PGCs opens the way for establishing a stem cell line, which can differentiate into the germline. The purified PGCs would also be a novel tool for cellular and molecular study of vertebrate germline stem cells.     

Zebrafish Staufen1 and Staufen2 are required for the survival and migration of primordial germ cells

In sexually reproducing organisms, primordial germ cells (PGCs) give rise to the cells of the germ line, the gametes. In many animals, PGCs are set apart from somatic cells early during embryogenesis. Work in Drosophila, C. elegans, Xenopus, and zebrafish has shown that maternally provided localized cytoplasmic determinants specify the germ line in these organisms (Raz, E., 2003. Primordial germ-cell development: the zebrafish perspective. Nat. Rev., Genet. 4, 690--700; Santos, A.C., Lehmann, R., 2004. Germ cell specification and migration in Drosophila and beyond. Curr. Biol. 14, R578-R589). The Drosophila RNA-binding protein, Staufen is required for germ cell formation, and mutations in stau result in a maternal effect grandchild-less phenotype (Schupbach,T., Weischaus, E., 1989. Female sterile mutations on the second chromosome of Drosophila melanogaster:1. Maternal effect mutations. Genetics 121, 101-17). Here we describe the functions of two zebrafish Staufen-related proteins, Stau1 and Stau2. When Stau1 or Stau2 functions are compromised in embryos by injecting antisense morpholino modified oligonucleotides or dominant-negative Stau peptides, germ layer patterning is not affected. However, expression of the PGC marker vasa is not maintained. Furthermore, expression of a green fluorescent protein (GFP):nanos 3'UTR fusion protein in germ cells shows that PGC migration is aberrant, and the mis-migrating PGCs do not survive in Stau-compromised embryos. Stau2 is also required for survival of neurons in the central nervous system (CNS). These phenotypes are rescued by co-injection of Drosophila stau mRNA. Thus, staufen has an evolutionarily conserved function in germ cells. In addition, we have identified a function for Stau proteins in PGC migration.     

Analysis of the molecular dynamics of medaka nuage proteins by fluorescence correlation spectroscopy and fluorescence recovery after photobleaching

The nuage is a unique organelle in animal germ cells that is known as an electron-dense amorphous structure in the perinuclear region. Although the nuage is essential for primordial germ cell (PGC) determination and development, its roles and functions are poorly understood. Herein, we report an analysis of the diffusion properties of the olvas gene product of the medaka fish (Oryzias lapites) in PGCs prepared from embryos, using fluorescence correlation spectroscopy and fluorescence recovery after photobleaching. Olvas-green fluorescent protein (GFP) localized in granules thought to be nuages, and exhibited a constraint movement with two-component diffusion constants of 0.15 and 0.01 microm(2).s(-1). On the other hand, cytosolic Olvas-GFP was also observed to have a diffusion movement of 7.0 microm(2).s(-1). Interestingly, Olvas-GFP could be expressed in HeLa cells, and formed granules that were similar to nuages in medaka PGCs. Olvas-GFP also exhibited a constraint movement in the granules and diffused in the cytosol of HeLa cells, just as in the medaka embryo. The other two gene products, Nanos and Tudor of the medaka, which are known as constituents of the nuage, could also be expressed in HeLa cells and formed granules that colocalized with Olvas-GFP. Nanos-GFP and Tudor-GFP exhibited constraint movement in the granules and diffused in the cytosol of HeLa cells. These results suggest that these granules in the HeLa cell are not simple aggregations or rigid complexes, but dynamic structures consisting of several proteins that shuttle back and forth between the cytosol and the granules.     

Mutations affecting early distribution of primordial germ cells in Medaka (Oryzias latipes) embryo

The development of germ cells has been intensively studied in Medaka (Oryzias latipes). We have undertaken a large-scale screen to identify mutations affecting the development of primordial germ cells (PGCs) in Medaka. Embryos derived from mutagenized founder fish were screened for an abnormal distribution or number of PGCs at embryonic stage 27 by RNA in situ hybridization for the Medaka vasa homologue (olvas). At this stage, PGCs coalesce into two bilateral vasa-expressing foci in the ventrolateral regions of the trunk after their migration and group organization. Nineteen mutations were identified from a screen corresponding to 450 mutagenized haploid genomes. Eleven of the mutations caused altered PGC distribution. Most of these alterations were associated with morphological abnormalities and could be grouped into four phenotypic classes: Class 1, PGCs dispersed into bilateral lines; Class 2, PGCs dispersed in a region more medial than that in Class 1; Class 3, PGCs scattered laterally and over the yolk sac area; and Class 4, PGCs clustered in a single median focus. Eight mutations caused a decrease in the number of PGCs. This decrease was observed in the offspring of heterozygous mothers, indicating the contribution of a maternal factor in determining PGC abundance. Taken together, these mutations should prove useful in identifying molecular mechanisms underlying the early PGC development and migration.