Mingyou Li

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.

Boule Is Present in Fish and Bisexually Expressed in Adult and Embryonic Germ Cells of Medaka

Background The DAZ family genes boule, daz and dazl encode RNA binding proteins essential for fertility of diverse animals including human. dazl has bisexual expression in both mitotic and meiotic germ cells, whereas daz has male premeiotic expression, and boule is largely a unisexual meiotic regulator. Although boule has been proposed as the ancestor for dazl/daz by gene duplication, it has been identified only in invertebrates and mammals. It has, however, remained unclear when and how the DAZ family has evolved in vertebrates. Methodology and Principal Findings This study was aimed at identifying and characterizing the DAZ family genes in fish as the basal vertebrate. We show that boule and dazl coexist in medaka and stickleback. Similar to the medaka dazl (Odazl), the medaka boule (Obol) is maternally supplied and segregates with primordial germ cells. Surprisingly, Obol is expressed in adult germ cells at pre-meiotic and meiotic stages of spermatogenesis and oogenesis. However, the maximal meiotic Obol expression in spermatocytes contrasts with the predominant pre-meiotic Odazl expression in spermatogonia, and the diffuse cytoplasmic Obol distribution in early oocytes contrasts with the Odazl concentration in the Balbinanis body. Conclusions The identification of fish boule and dazl genes provides direct evidence for the early gene duplication during vertebrate evolution. Our finding that Obol exhibits bisexual expression in both embryonic and adult germ cells considerably extends the diversity of boule expression patterns and offers a new insight into the evolutions of DAZ family members, expression patterns and functions in animal fertility.

Fish germ cells

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.

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.

Differential conservation and divergence of fertility genes boule and dazl in the rainbow trout.

Background The genes boule and dazl are members of the DAZ (Deleted in Azoospermia) family encoding RNA binding proteins essential for germ cell development. Although dazl exhibits bisexual expression in mitotic and meiotic germ cells in diverse animals, boule shows unisexual meiotic expression in invertebrates and mammals but a bisexual mitotic and meiotic expression in medaka. How boule and dazl have evolved different expression patterns in diverse organisms has remained unknown. Methodology and Principal Findings Here we chose the fish rainbow trout (Oncorhynchus mykiss) as a second lower vertebrate model to investigate the expression of boule and dazl. By molecular cloning and sequence comparison, we identified cDNAs encoding the trout Boule and Dazl proteins, which have a conserved RNA-recognition motif and a maximal similarity to their homologs. By RT-PCR analysis, adult RNA expression of trout boule and dazl is restricted to the gonads of both sexes. By chromogenic and two-color fluorescence in situ hybridization, we revealed bisexual and germline-specific expression of boule and dazl. We found that dazl displays conserved expression throughout gametogenesis and concentrates in the Balbinanis body of early oocytes and the chromatoid body of sperm. Surprisingly, boule exhibits mitotic and meiotic expression in the male but meiosis-specific expression in the female. Conclusions Our data underscores differential conservation and divergence of DAZ family genes during vertebrate evolution. We propose a model in which the diversity of boule expression in sex and stage specificity might have resulted from selective loss or gain of its expression in one sex and mitotic germ cells.

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.

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 Oct4 is Essential for Pluripotency in Blastula Formation and ES Cell Derivation

The origin and evolution of molecular mechanisms underlying cellular pluripotency is a fundamental question in stem cell biology. The transcription factor Oct4 or Pou5f1 identified in mouse features pluripotency expression and activity in the inner cell mass and embryonic stem (ES) cells. Pou2 identified in zebrafish is the non-mammalian homolog prototype of mouse Oct4. The genes oct4 and pou2 have reportedly evolved by pou5 gene duplication in the common ancestor of vertebrates. Unlike mouse oct4, however, zebrafish pou2 lacks pluripotency expression and activity. Whether the presence of pluripotency expression and activity is specific for mammalian Oct4 or common to the ancestor of vertebrate Oct4 and Pou2 proteins has remained to be determined. Here we report that Oloct4, the medaka oct4/pou2, is essential for early embryogenesis and pluripotency maintenance. Oloct4 exists as a single copy gene and is orthologous to pou2 by sequence and chromosome synteny. Oloct4 expression occurs in early embryos, germ stem cells and ES cells like mouse oct4 but also in the brain and tail bud like zebrafish pou2. Importantly, OlOct4 depletion caused blastula lethality or blockage. We show that Oloct4 depletion abolishes ES cell derivation from midblastula embryos. Thus, Oloct4 has pluripotency expression and is essential for early embryogenesis and pluripotency maintenance. Our results demonstrate the conservation of pluripotency expression and activity in vertebrate Oct4 and Pou2 proteins. The finding that Oloct4 combines the features of mouse oct4 and zebrafish pou2 in expression and function suggests that Oloct4 might represent the ancestral prototype of vertebrate oct4 and pou2 genes.

Mitf is a transcriptional activator of medaka germ genes in culture.

Germ cells express a unique subset of genes called germ genes mostly encoding RNA-binding proteins such as Dazl, Dnd and Vasa. How germ gene expression is controlled remains illusive, because in no organism has a transcription factor been identified that regulate expression of these genes. Microphthalmia-associated transcription factor (Mitf) has been reported to show expression in male mouse germ cells of the adult testis. Here we report in the fish medaka (Oryzias latipes) that Mitf is a transcription activator of germ gene expression. Mitf is a master regulator of melanocyte development, which activates melanogenic genes through binding to the E-box containing consensus CANNTG. The E-box was found to be present in 23-26 copies in the promoters of medaka germ genes dazl, dnd and vasa. Importantly, forced Mitf expression enhanced the transcriptional activity of the three gene promoters by up to more than 10 fold and remarkably increased the level of endogenous dazl, dnd and vasa transcripts in cell culture. Transfection of Mitf expression vectors was sufficient to induce directed differentiation of medaka embryonic stem cells into melanocytes. Fluorescence in situ hybridization revealed the expression of both medaka mitf genes in adult germ cells of male and female gonads. Mitf is well-known as the melanocyte master regulator. Our results offer first evidence that Mitf may act as a transcriptional activator of germ gene expression in medaka.

Light and electron microscopic analyses of Vasa expression in adult germ cells of the fish medaka.

Germ cells of diverse animal species have a unique membrane-less organelle called germ plasm (GP). GP is usually associated with mitochondria and contains RNA binding proteins and mRNAs of germ genes such as vasa. GP has been described as the mitochondrial cloud (MC), intermitochondrial cement (IC) and chromatoid body (CB). The mechanism underlying varying GP structures has remained incompletely understood. Here we report the analysis of GP through light and electron microscopy by using Vasa as a marker in adult male germ cells of the fish medaka (Oryzias latipes). Immunofluorescence light microscopy revealed germ cell-specific Vasa expression. Vasa is the most abundant in mitotic germ cells (oogonia and spermatogonia) and reduced in meiotic germ cells. Vasa in round spermatids exist as a spherical structure reminiscent of CB. Nanogold immunoelectron microscopy revealed subcellular Vasa redistribution in male germ cells. Vasa in spermatogonia concentrates in small areas of the cytoplasm and is surrounded by mitochondria, which is reminiscent of MC. Vasa is intermixed with mitochondria to form IC in primary spermatocytes, appears as the free cement (FC) via separation from mitochondria in secondary spermatocyte and becomes condensed in CB at the caudal pole of round spermatids. During spermatid morphogenesis, Vasa redistributes and forms a second CB that is a ring-like structure surrounding the dense fiber of the flagellum in the midpiece. These structures resemble those described for GP in various species. Thus, Vasa identifies GP and adopts varying structures via dynamic reorganization at different stages of germ cell development.