Meisheng Yi

Generation of Medaka Fish Haploid Embryonic Stem Cells

Haploid Medaka Stem Cells Although diploid embryonic stem cells have been generated by various means, there would also be value in deriving haploid stem cells. In these cells, recessive mutations in essential genes would show phenotypes that would not be apparent in heterozygous animals. Yi et al. (p. 430) used the medaka fish model system to generate haploid stem cells that show stable growth and pluripotency. In addition, a fertile female medaka fish was produced by haploid embryonic stem cell nuclear transfer into a normal egg. This system has potential for analyzing recessive genes, for example, in disease phenotypes or in various cell lineages in culture. Stem cells that are haploid can sustain stable growth, pluripotency, and genetic integrity in fish cell cultures. Haploid embryonic stem (ES) cells combine haploidy and pluripotency, enabling direct genetic analyses of recessive phenotypes in vertebrate cells. Haploid cells have been elusive for culture, due to their inferior growth and genomic instability. Here, we generated gynogenetic medaka embryos and obtained three haploid ES cell lines that retained pluripotency and competitive growth. Upon nuclear transfer into unfertilized oocytes, the haploid ES cells, even after genetic engineering, generated viable offspring capable of germline transmission. Hence, haploid medaka ES cells stably maintain normal growth, pluripotency, and genomic integrity. Mosaic oocytes created by combining a mitotic nucleus and a meiotic nucleus can generate fertile fish offspring. Haploid ES cells may offer a yeast-like system for analyzing recessive phenotypes in numerous cell lineages of vertebrates in vitro.

Medaka vasa is required for migration but not survival of primordial germ cells

Vasa is essential for germline development. However, the precise processes in which vasa involves vary considerably in diverse animal phyla. Here we show that vasa is required for primordial germ cell (PGC) migration in the medakafish. vasa knockdown by two morpholinos led to the PGC migration defect that was rescued by coinjection of vasa RNA. Interestingly, vasa knockdown did not alter the PGC number, identity, proliferation and motility even at ectopic locations. We established a cell culture system for tracing PGCs at the single cell level in vitro. In this culture system, control and morpholino-injected gastrulae produced the same PGC number and the same time course of PGC survival. Importantly, vasa-depleted PGCs in culture had similar motility and locomotion to normal PGCs. Expression patterns of wt1a, sdf1b and cxcr4b in migratory tissues remained unchanged by vasa knockdown. By chimera formation we show that PGCs from vasa-depleted blastulae failed to migrate properly in the normal environment, whereas control PGCs migrated normally in vasa-disrupted embryos. Furthermore, ectopic PGCs in vasa-depleted embryos also retained all the PGC properties examined. Taken together, medaka vasa is cell-autonomously required for PGC migration, but dispensable to PGC proliferation, motility, identity and survival.

Medaka dead end encodes a cytoplasmic protein and identifies embryonic and adult germ cells.

dead end (dnd) was identified in zebrafish as a gene encoding an RNA-binding protein essential for primordial germ cell (PGC) development and gametogenesis in vertebrates. The adult dnd RNA expression has been restricted to the ovary in Xenopus or to the testis in mouse. Its protein product is nuclear in chicken germ cells but both cytosolic and nuclear in mouse cell cultures. Here we report the cloning and expression pattern of Odnd, the medakafish (Oryzias latipes) dnd gene. Sequence comparison, gene structure, linkage analysis and expression demonstrate that Odnd encodes the medaka Dnd orthologue. A systematic comparison of Dnd proteins from five fishes and tetrapod representatives led to the identification of five previously unidentified conserved regions besides the RNA recognition motif. The Odnd RNA is maternally supplied and preferentially segregated with PGCs. Its adult expression occurs in both sexes and is restricted to germ cells. In the testis, Odnd is abundant in spermatogonia and meiotic cells but absent in sperm. In the ovary, Odnd RNA persists throughout oogenesis. Furthermore, we developed a dual color fluorescent in situ hybridization procedure allowing for precise comparisons of expression and distribution patterns between two genes in medaka embryos and adult tissues. Importantly, this procedure co-localized Odnd and Ovasa in testicular germ cells and PGCs. Surprisingly, by cell transfection and embryo RNA injection we show that ODnd is cytoplasmic in cell cultures, cleavage embryos and PGCs. Therefore, medaka dnd encodes a cytoplasmic protein and identifies embryonic and adult germ cells of both sexes.

Derivation and characterization of haploid embryonic stem cell cultures in medaka fish

Embryonic stem (ES) cells are pluripotent cells capable of differentiation into various cell types. Haploid ES cells elegantly combine the advantages of haploidy and pluripotency and offer a unique in vitro system for genetic analyses of molecular, cellular and developmental events in various cell lineages. Our recent success in generating haploid ES cell lines from gynogenetic embryos of the medaka fish suggests that haploidy can support ES cell derivation and maintenance in a vertebrate. In this study, we present a step-by-step protocol for derivation and characterization of medaka haploid ES cells. We have used this procedure to produce three haploid ES cell lines from five primary cultures. It takes about 15 weeks to generate stable cultures, 5–8 weeks to obtain pure haploid cells and 5–6 weeks to characterize ES cells in vitro and in vivo.

Accessibility of host cell lineages to medaka stem cells depends on genetic background and irradiation of recipient embryos

Chimera formation is a powerful tool for analyzing pluripotency in vivo. It has been widely accepted that host cell lineages are generally accessible to embryonic stem (ES) cells with the actual contribution depending solely on the intrinsic pluripotency of transplanted donor cells. Here, we show in the fish medaka (Oryzias latipes) that the host accessibility to ES cell contribution exhibits dramatic differences. Specifically, of three albino host strains tested (i1, i3 and af), only strain i1 generated pigmented chimeras. Strikingly, this accessibility is completely lost in i1 but acquired in i3 after host γ-irradiation. Host irradiation also differentially affected ES cell contribution to somatic organs and gonad. Therefore, the accessibility of various host cell lineages can vary considerably depending on host strains and cell lineages as well as on irradiation. Our findings underscore the importance of host genotypes for interpreting donor cell pluripotency and for improving ES-derived chimera production.

Interordinal chimera formation between medaka and zebrafish for analyzing stem cell differentiation.

Chimera formation is a standard test for pluripotency of stem cells in vivo. Interspecific chimera formation between distantly related organisms offers also an attractive approach for propagating endangered species. Parameters influencing interspecies chimera formation have remained poorly elucidated. Here, we report interordinal chimera formation between medaka and zebrafish, which separated ∼320 million years ago and exhibit a more than 2-fold difference in developmental speed. We show that, on transplantation into zebrafish blastulae, both noncultivated blastomeres and long-term cultivated embryonic stem (ES) cells of medaka adopted the zebrafish developmental program and differentiated into physiologically functional cell types including pigment cells, blood cells, and cardiomyocytes. We also show that medaka ES cells express differentiation gene markers during chimeric embryogenesis. Therefore, the evolutionary distance and different embryogenesis speeds do not produce donor-host incompatibility to compromise chimera formation between medaka and zebrafish, and molecular markers are valuable for analyzing lineage commitment and cell differentiation in interspecific chimeric embryos.

Establishment of medakafish as a model for stem cell-based gene therapy: efficient gene delivery and potential chromosomal integration by baculoviral vectors.

Viral vectors hold promise and challenges in gene therapy. Specifically, we have previously shown that baculoviral (BV) vectors have a high efficiency of gene delivery in human embryonic stem (ES) cells. Here we report the development of a complementary system to further our evaluation by utilizing the laboratory fish medaka that has ES cell lines and tools for experimental analyses in vitro and in vivo. We show that BV vectors can give rise to almost 100% of transient gene delivery in the medaka ES cell line MES1. BV-transduced MES1 cells reproducibly (at approximately 10(-5)) produce GFP-expressing colonies that, upon manual isolation, develop into stable clones during 300 days of culture. Surprisingly, BV transduction can also mediate efficient gene integration in the medaka genome, as fluorescent in situ hybridization revealed the presence of the BV-delivered gfp transgene in multiple locations in nuclei and on various chromosomes of metaphase spreads. We show that BV transduction does not compromise the genome stability and pluripotency of MES1 cells. We conclude that BV can efficiently mediate gene delivery and chromosomal integration in medaka ES cells. Therefore, medaka provides a powerful system for analyzing the potential of BV-mediated gene delivery in stem cells and gene therapy.

Medaka fish stem cells and their applications

Stem cells are present in developing embryos and adult tissues of multicellular organisms. Owing to their unique features, stem cells provide excellent opportunities for experimental analyses of basic developmental processes such as pluripotency control and cell fate decision and for regenerative medicine by stem cell-based therapy. Stem cell cultures have been best studied in 3 vertebrate organisms. These are the mouse, human and a small laboratory fish called medaka. Specifically, medaka has given rise to the first embryonic stem (ES) cells besides the mouse, the first adult testis-derived male stem cells spermatogonia capable of test-tube sperm production, and most recently, even haploid ES cells capable of producing Holly, a semi-cloned fertile female medaka from a mosaic oocyte created by microinjecting a haploid ES cell nucleus directly into a normal oocyte. These breakthroughs make medaka a favoring vertebrate model for stem cell research, the topic of this review.

Nanos3 Gene Targeting in Medaka ES Cells

Gene targeting (GT) by homologous recombination offers the best precision for genome editing in mice. nanos3 is a highly conserved gene and encodes a zinc-finger RNA binding protein essential for germ stem cell maintenance in Drosophila, zebrafish and mouse. Here we report nanos3 GT in embryonic stem (ES) cells of the fish medaka as a lower vertebrate model organism. A vector was designed for GT via homologous recombination on the basis of positive-negative selection (PNS). The ES cell line MES1 after gene transfer and PNS produced 56 colonies that were expanded into ES cell sublines. Nine sublines were GT-positive by PCR genotyping, 4 of which were homologous recombinants as revealed by Southern blot. We show that one of the 4, A15, contains a precisely targeted nanos3 allele without any random events, demonstrating the GT feasibility in medaka ES cells. Importantly, A15 retained all features of undifferentiated ES cells, including stable self-renewal, an undifferentiated phenotype, pluripotency gene expression and differentiation during chimeric embryogenesis. These results provide first evidence that the GT procedure and genuine GT on a chromosomal locus such as nanos3 do not compromise pluripotency in ES cells of a lower vertebrate.

A Syntenic Region Conserved from Fish to Mammalian X Chromosome

Sex chromosomes bearing the sex-determining gene initiate development along the male or female pathway, no matter which sex is determined by XY male or ZW female heterogamety. Sex chromosomes originate from ancient autosomes but evolved rapidly after the acquisition of sex-determining factors which are highly divergent between species. In the heterogametic male system (XY system), the X chromosome is relatively evolutionary silent and maintains most of its ancestral genes, in contrast to its Y counterpart that has evolved rapidly and degenerated. Sex in a teleost fish, the Nile tilapia (Oreochromis niloticus), is determined genetically via an XY system, in which an unpaired region is present in the largest chromosome pair. We defined the differences in DNA contents present in this chromosome with a two-color comparative genomic hybridization (CGH) and the random amplified polymorphic DNA (RAPD) approach in XY males. We further identified a syntenic segment within this region that is well conserved in several teleosts. Through comparative genome analysis, this syntenic segment was also shown to be present in mammalian X chromosomes, suggesting a common ancestral origin of vertebrate sex chromosomes.