Primordial germ cells (PGCs) are the precursors of gametes responsible for genetic transmission to the next generation. They provide an ideal system for cryopreservation and restoration of ...biodiversity. Recently, considerable attention has been raised to visualize, isolate and transplant PGCs within and between species. In fish, stable PGC visualization in live embryo and individual has been limited to laboratory fish models such as medaka and zebrafish. One exception is the rainbow trout, which represents the only species with aquaculture importance and has GFP-labeled germ cells throughout development. PGCs can be transiently labeled by embryonic injection of mRNA containing green fluorescence protein gene (GFP) and 3'-untranslated region (3'-UTR) of a maternal germ gene such as vasa, nos1, etc. Stable PGC labeling can be achieved through production of transgenic animals by some transcriptional regulatory sequences from germ genes, such as the vasa promoter and 3'-UTR. In this study, we reported the functional analyses of the red seabream vasa (Pmvas) regulatory sequences, using medaka as a model system. It was showed that injection of GFP-Pmvas3'UTR mRNA was able to label medaka PGCs during embryogenesis. Besides, we have constructed pPmvasGFP transgenic vector, and established a stable transgenic medaka line exhibiting GFP expression in germ cells including PGCs, mitotic and meiotic germ cells of both sexes, under control of the Pmvas transcriptional regulatory sequences. It is concluded that the Pmvas regulatory sequences examined in this study are sufficient for germ cell expression and labeling.
Fish, like many other animals, have two major cell lineages, namely the germline and soma. The germ-soma separation is one of the earliest events of embryonic development. Germ cells can be ...specifically labeled and isolated for culture and transplantation, providing tools for reproduction of endangered species in close relatives, such as surrogate production of trout in salmon. Haploid cell cultures, such as medaka haploid embryonic stem cells have recently been obtained, which are capable of mimicking sperm to produce fertile offspring, upon nuclear being directly transferred into normal eggs. Such fish originated from a mosaic oocyte that had a haploid meiotic nucleus and a transplanted haploid mitotic cell culture nucleus. The first semi-cloned fish is Holly. Here we review the current status and future directions of understanding and manipulating fish germ cells in basic research and reproductive technology.
Gene targeting (GT) provides a powerful tool for the generation of precise genetic alterations in embryonic stem (ES) cells to elucidate gene function and create animal models for human diseases. ...This technology has, however, been limited to mouse and rat. We have previously established ES cell lines and procedures for gene transfer and selection for homologous recombination (HR) events in the fish medaka (Oryzias latipes).
Here we report HR-mediated GT in this organism. We designed a GT vector to disrupt the tumor suppressor gene p53 (also known as tp53). We show that all the three medaka ES cell lines, MES1∼MES3, are highly proficient for HR, as they produced detectable HR without drug selection. Furthermore, the positive-negative selection (PNS) procedure enhanced HR by ∼12 folds. Out of 39 PNS-resistant colonies analyzed, 19 (48.7%) were positive for GT by PCR genotyping. When 11 of the PCR-positive colonies were further analyzed, 6 (54.5%) were found to be bona fide homologous recombinants by Southern blot analysis, sequencing and fluorescent in situ hybridization. This produces a high efficiency of up to 26.6% for p53 GT under PNS conditions. We show that p53 disruption and long-term propagation under drug selection conditions do not compromise the pluripotency, as p53-targeted ES cells retained stable growth, undifferentiated phenotype, pluripotency gene expression profile and differentiation potential in vitro and in vivo.
Our results demonstrate that medaka ES cells are proficient for HR-mediated GT, offering a first model organism of lower vertebrates towards the development of full ES cell-based GT technology.
Balbiani body （BB） is a large distinctive organelle aggregate uniquely present in developing oocytes of diverse animal species. BB is thought as a stage-specific structure that resembles germ plasm, ...the cytoplasmic organelle of germ cells. The role and function of BB have remained speculative because of a highly dynamic structure and a lack of genetic and molecular data. BB has been found to contain proteins and RNAs, none of them -- except the zebrafish foxH1 RNA, is or encodes a transcription factor. Here we report in the fish medaka （Oryzias latipes） that RNAs encoding microphthalmia-associated transcription factor （Mitf） are prominent components of the BB. By fluorescence in situ hybridization on ovarian section, we revealed that the transcripts of both mitfl and mitf2 genes concentrated in the BB, in which they co-localized with the dazl RNA, a definitive BB marker highly conserved in vertebrates. Therefore, the mitfproduct may play dual roles in germ gene transcription and BB formation and/or function in this organism. Our data provide the second evidence that the RNA of a transcription factor can be a prominent component of the BB in a vertebrate.
Sperm nuclear transfer or intracytoplasmic sperm injection (ICSI) is a powerful assisted reproductive technology (ART) for treating human male infertility. Controversial reports of increased birth ...defects have raised concerns about the ART's safety. The cause for birth defects, however, has remained elusive for analysis in human because of the sample size, male infertility genetics, physiological heterogeneity and associated procedures such as embryo manipulations. Animal models are required to evaluate factors leading to the increased birth defects. Here we report the establishment of medakafish model for ICSI and transgenic production. This small laboratory fish has high fecundity and easy embryology. We show that ICSI produced a 5% high percentage of fertile animals that exhibited both paternal and maternal contribution as evidenced by the pigmentation marker. Furthermore, when sperm were pre-incubated with a plasmid ubiquitously expressing RFP and subjected to ICSI, 50% of sperm nuclear transplants showed germline transmission. We conclude that medaka is an excellent model for ICSI to evaluate birth defects and that sperm nuclear transfer can mediate stable gene transfer at high efficiency. Although more demanding for experimentation, sperm-mediated transgenesis should be particularly applicable for aquaculture species with a lengthy generation time and/or a large adult body size.
The fish medaka is the first vertebrate capable of full spermatogenesis in vitro from self-renewing spermatogonial stem cells to motile test-tube sperm. Precise staging and molecular dissection of ...this process has been hampered by the lack of suitable molecular markers.
We have generated a normalized medaka testis cDNA library and obtained 7040 high quality sequences representing 3641 unique gene clusters. Among these, 1197 unique clusters are homologous to known genes, and 2444 appear to be novel genes. Ontology analysis shows that the 1197 gene products are implicated in diverse molecular and cellular processes. These genes include markers for all major types of testicular somatic and germ cells. Furthermore, markers were identified for major spermatogenic stages ranging from spermatogonial stem cell self-renewal to meiosis entry, progression and completion. Intriguingly, the medaka testis expresses at least 13 homologs of the 33 mouse X-chromosomal genes that are enriched in the testis. More importantly, we show that key components of several signaling pathways known to be important for testicular function in mammals are well represented in the medaka testicular EST collection.
Medaka exhibits a considerable similarity in testicular gene expression to mammals. The medaka testicular EST collection we obtained has wide range coverage and will not only consolidate our knowledge on the comparative analysis of known genes' functions in the testis but also provide a rich resource to dissect molecular events and mechanism of spermatogenesis in vivo and in vitro in medaka as an excellent vertebrate model.
Existing zebrafish embryonic stem (ES) cell lines are derived and maintained using feeder layers. We describe here the derivation and long-term culture of an ES cell-like line derived from zebrafish ...blastomeres without the use of feeder cells. This line, designated as ZES1, has been maintained for more than 800 days in defined Dulbecco's modified Eagle's medium supplemented with fetal bovine serum, zebrafish embryo extract, trout serum, and human basic fibroblast growth factor. ZES1 cells possessed a morphology typical of ES cells, being round or polygonal in shape with a large nucleus and sparse cytoplasm and were mostly diploid. The cells formed individual colonies consisting of tightly packed cells that stained positively for alkaline phosphatase. ZES1 cells also formed embryoid bodies when transferred onto uncoated wells. The pluripotent nature of ZES1 cells was confirmed when they could be induced to differentiate in vitro into several cell types, through low- or high-density culture conditions. Treatment with retinoic acid also induced the differentiation of ZES1 cells into primarily neuronal cells. Using immunostaining and real-time polymerase chain reaction, we showed that Sox2, a known pluripotent marker in mammalian ES cells, was also present in ZES1 cells. Chimera experiments revealed that fluorescent-labeled ZES1 cells microinjected into zebrafish blastulas participated in the formation of all three germ layers. Using GFP-labeled ZES1 cells, chimera germline transmission was also demonstrated at the F1 generation. In conclusion, ZES1 cells possess both in vitro and in vivo pluripotency characteristics, indicating that nonmammalian ES cells can be readily derived and maintained for a long term under feeder-free culture conditions.
The Deleted in Azoospermia (DAZ) family of RNA binding proteins consists of highly conserved genes boule, daz and daz-like (dazl) essential for germ cell development. boule is known for its unisexual ...meiotic expression in invertebrates and mammals, but meiotic-specific female expression plus meiosis-preferential male expression in trout, and meiosis-preferential bisexual expression in medaka. dazl shows highly conserved bisexual expression throughout gametogenesis in diverse species. Here we report the cloning and expression of boule and dazl in the Nile tilapia (Oreochromis niloticus), an important aquaculture fish. Molecular cloning and sequence analysis led to the identification of tilapia boule and dazl cDNAs. The predicted partial Boule contains a conserved RRM motif and Dazl has the C-terminal sequence. On a phylogenetic tree, tilapia Boule and Dazl are in separate clades of Boule and Dazl homologs from other species, indicating their divergence during early vertebrate evolution. By RT-PCR analysis, boule and dazl showed bisexual gonad-specific expression. By in situ hybridization analysis, both boule and dazl RNAs were restricted to female and male germ cells of adult gonads but absent in gonadal soma. In the ovary, boule and dazl RNAs were abundant in oocytes. In the testis, boule and dazl RNAs were prominent in meiotic spermatocytes but barely detectable in meiotic products. These data show that boule and dazl are expressed bisexually in germ cells and provide useful markers to study gametogenesis in the adult tilapia.
•Cloning and expression pattern analysis of boule and dazl RNA in the Nile tilapia.•Both the genes have gonad specific expression in both the sexes.•The boule and dazl have stage specific expression during gametogenesis.•Both genes can be markers for stages of gametogenesis in the Nile tilapia.
Embryonic stem (ES) cell lines have provided very useful models to analyse differentiation processes. We present here the development of a differentiation system using ES-like cell lines from medaka. ...These cells were transfected with the melanocyte specific isoform of the microphtalmia-related transcription factor (Mitf). Mitf is a basic helix-loop-helix-leucine zipper transcription factor whose M isoform is restricted to neural crest derived melanocytes and is essential for the development of these cells in vertebrates from mammals to fish. What is not clear yet is whether Mitf is a downstream factor or a master regulator of melanocyte commitment and differentiation. Expression of Mitf in the ES-like cells from medaka led to the induction of cells that, by morphology, physiology and gene expression pattern, were confirmed to be fully differentiated pigment cells. Mitf expression is therefore sufficient for the proper differentiation of medaka pluripotent stem cells into melanocytes.
Targeted gene disruption (GD) is powerful for generating genetic alterations in animal genomes. Engineered endonucleases such as zinc finger nucleases and transcription activator-like effector ...nucleases (TALENs) allow for GD directly in animal embryos to achieve germline transmission. Here we report procedures and parameters of TALEN-mediated GD in the fish medaka by using a germ cell-specific gene dnd as a model. Embryos at the 1-cell stage were microinjected with synthetic TALEN mRNAs and examined for the survival rate and GD efficiency. Medaka embryos can tolerate a high dosage of TALEN-mRNA injection and exhibit a steadily increasing GD efficiency with increasing mRNA dosages before peaking at 100ng/μl. This dosage produced ~24% efficiency for somatic GD. Some of the animals from manipulated embryos developed into fertile female and male. Most importantly, four fish (3 males and 1 female) examined by progeny-test were able to produce GD-bearing male and female gametes for germline transmission to F1 generation at ~10% efficiency. Therefore, TALEN is proficient for somatic and germline GD in medaka embryos, and disruption of one dnd copy does not compromise somatic development and gamete production.
•Procedures and key parameters of TALEN-mediated gene disruption were established.•Gene dnd was efficiently disrupted by TALEN in medaka.•Direct gene disruption by TALEN is germline transmittable and highly mosaic.•Genomic dnd is dispensable during the post-meiotic haploid phase.
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