Persistence and Transcription of Paternal mtDNA Dependent on the Delivery Strategy Rather than Mitochondria Source in Fish Embryos.

BACKGROUND/AIMS: Mitochondria (MT) and mitochondrial DNA (mtDNA) show maternal inheritance in most eukaryotic organisms; the sperm mtDNA is usually delivered to the egg during fertilization and then rapidly eliminated to avoid heteroplasmy, which can affect embryogenesis. In our previous study, fertilization-delivered sperm mtDNA exhibited late elimination and transcriptional quiescence in cyprinid fish embryos. However, the mechanisms underlying elimination and transcriptional quiescence of paternal mtDNA are unclear. METHODS: Goldfish and zebrafish were used to investigate the fate of mtDNAs with different parental origins delivered by fertilization or microinjection in embryos. Goldfish MT from heart, liver and spermatozoa were microinjected into zebrafish zygotes, respectively. Specific PCR primers were designed so that the amplicons have different sizes to characterize goldfish and zebrafish cytb genes or their cDNAs. RESULTS: The MT injection-delivered paternal mtDNA from sperm, as well as those from the heart and liver, was capable of persistence and transcription until birth, in contrast to the disappearance and transcriptional quiescence at the heartbeat stage of fertilization-delivered sperm mtDNA. In addition, the exogenous MT-injected zebrafish embryos have normal morphology during embryonic development. CONCLUSIONS: The fate of paternal mtDNA in fishes is dependent on the delivery strategy rather than the MT source, suggesting that the presence of sperm factor(s) is responsible for elimination and transcriptional quiescence of fertilization-delivered sperm mtDNA. These findings provide insights into the mechanisms underlying paternal mtDNA fate and heteroplasmy in cyprinid fishes.

Identification and Expression of Conserved and Novel RNA Variants of Medaka pax6b Gene.

Gene duplication is a major driving force of evolution. How gene duplicates have evolved remains a mystery. A highly conserved gene such as Pax6 is an ideal model to study functional conservation and divergence via comparisons among diverse organisms. One pax6 gene has been characterized in the Japanese medaka (Oryzias latipes), which is annotated as pax6b on chromosome 3. Here, we report that Medaka pax6b is homolog to Pax6 of mammals in sequence, chromosomal synteny, and genomic organization. Cloning and sequencing led to the identification of up to 43 pax6b RNA variants predicting six protein isoforms, 22 of which are similar to those reported in other organisms and 21 represent novel RNA variants. By RT-PCR, the pax6b transcripts were found to be most abundant in the adult eye and easily detectable in the adult brain and pancreas but not detectable in developing embryos until gastrulation. Interestingly, apparently differential expression in adult organs was observed for several major variants. In situ hybridization revealed that pax6b exhibited highly conserved RNA expression in pancreas, brain, and eye of adult animals and developing embryos. Therefore, by sequence, chromosomal synteny, gene structure, conserved alternative transcription and splicing, and most importantly, conserved expression patterns in adulthood and embryogenesis, medaka pax6b represents a ortholog of Pax6 gene in mammals and is capable of generating differentially expressed RNA variants.

Insights of sex determination and differentiation from medaka as a teleost model.

The mechanisms of sex determination and differentiation in fish are highly divergent with a broad range of gonadal differentiation types from hermaphroditism to gonochorism. Multiple triggers regulate the process of sexual differentiation including genetic or environmental factors (temperature, light, hormones and/or pH value, etc.). In recent years, with the advances of molecular technologies and genetic engineering approaches, there are significant breakthroughs in identifying the master genes of vertebrate sex determination and differentiation. In this review, we explore the fundamental and molecular mechanisms underlying the sexual differentiation in teleost fish, using medaka (Oryzias latipes) as a model. We focus on the male pathways and factors, particularly on dmrt1, gsdf and amh genes involved in testicular differentiation, sexual reversal and plasticity. It is anticipated that new techniques will likely be developed in the field of sex manipulations and monosex breeding for fish aquaculture in the future.

Correlative light and electron microscopic analyses of mitochondrial distribution in blastomeres of early fish embryos.

Early embryos of vertebrates undergo remarkable dynamic molecular events, such as embryonic gradient, cellular polarity, and asymmetry necessary for cell fate decisions. Correlative light and electron microscopy (CLEM) is a powerful tool to investigate rare or dynamic molecular events and has been developed for relatively small cells in culture and tissues but is not yet available for large cells of early development stage embryos. Here we report the capability of CLEM in blastomeres of medaka fish by using the mitochondria detection system. A short N-terminal signal peptide of the mitochondrial protein Tom20 was linked to green fluorescent protein (GFP), resulting in a fusion protein termed Tom20:GFP. The subcellular location of Tom20:GFP in medaka blastomeres reveals the lack of mitochondrial distribution in pseudopodia as well as inconspicuous redistribution during divisions. Blastomeres, after sample preparation procedures including high-pressure freezing and freeze substitution, are able to preserve fluorescence, antigenicity, and fine structures, which allows for precise correlation between the Tom20:GFP fluorescence and mitochondria on merged light and electron micrographs. Furthermore, nanogold immunostaining for Tom20:GFP and endogenous Tom20 revealed their specific localization on the mitochondrial outer membrane. Our results extend the CLEM approach to early development stage embryos of a vertebrate.

Proton gradient-induced water transport mediated by water wires inside narrow aquapores of aquafoldamer molecules.

Hollow tubular aquapores inside aquafoldamers can be created via the "sticky" end-mediated formation of 1D chiral helical stacks involving same-handed helices, and are capable of aligning H-bonded water molecules in a chain-like fashion. These aquapores uniquely feature a small cavity of ∼2.8 Šin diameter, a size identical to that of the water molecule and also comparable to the narrowest opening in naturally occurring aquaporins measuring ∼3 Šacross, and hence allow not only proton transport but also unique proton-gradient-induced water transport across the lipid membranes in the presence of proton gradient.

Derivation of stable zebrafish ES-like cells in feeder-free culture.

Zebrafish offers an excellent opportunity to combine embryological, genetic and molecular analyses of vertebrate development in vivo. Embryonic stem (ES) cells have enormous potential to study developmental potency and differentiation in vitro and thus to complement in vivo approaches. Zebrafish ES-like cells have been produced on a feeder cell layer. Here, we report the derivation of Z428, a zebrafish ES-like cell line, from blastula embryos in feeder-free culture. Fetal bovine serum, fish serum, fish embryo extract, basic fibroblast growth factor, non-essential amino acids and 2-mercaptoethanol were found to be important for Z428 growth. After more than 120 passages and many freezing/thawing cycles over a period of 20 years, Z428 exhibits stable growth and manifests many ES cell features including an ES cell phenotype, high alkaline phosphatase activity and spontaneous differentiation in culture. Most importantly, Z428 was transplantable to blastula hosts and capable of contributing to embryonic tissues and organ systems of the three germ layers. Therefore, Z428 is a stable cell line and contains ES-like cells with pluripotency in vitro and in vivo, and a feeder layer is dispensable for ES-like cell derivation in zebrafish. The derivation and easy maintenance of zebrafish ES-like cells under feeder-free conditions provide a useful extension of the present toolbox for studying development and differentiation in the zebrafish model.

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.

Medaka haploid embryonic stem cells are susceptible to Singapore grouper iridovirus as well as to other viruses of aquaculture fish species.

Viral infection is a challenge in high-density aquaculture, as it leads to various diseases and causes massive or even complete loss. The identification and disruption of host factors that viruses utilize for infection offer a novel approach to generate viral-resistant seed stocks for cost-efficient and sustainable aquaculture. Genetic screening in haploid cell cultures represents an ideal tool for host factor identification. We have recently generated haploid embryonic stem (ES) cells in the laboratory fish medaka. Here, we report that HX1, one of the three established medaka haploid ES cell lines, was susceptible to the viruses tested and is thus suitable for genetic screening to identify host factors. HX1 cells displayed a cytopathic effect and massive death upon inoculation with three highly infectious and notifiable fish viruses, namely Singapore grouper iridovirus (SGIV), spring viremia of carp virus (SVCV) and red-spotted grouper nervous necrosis virus (RGNNV). Reverse transcription-PCR and Western blot analyses revealed the expression of virus genes. SGIV infection in HX1 cells elicited a host immune response and apoptosis. Viral replication kinetics were determined from a virus growth curve, and electron microscopy revealed propagation, assembly and release of infectious SGIV particles in HX1 cells. Our results demonstrate that medaka haploid ES cells are susceptible to SGIV, as well as to SVCV and RGNNV, offering a unique opportunity for the identification of host factors by genetic screening.

Fusion gene vectors allowing for simultaneous drug selection, cell labeling, and reporter assay in vitro and in vivo.

Vector systems allowing simultaneously for rapid drug selection, cell labeling, and reporter assay are highly desirable in biomedical research including stem cell biology. Here, we present such a vector system including pCVpf or pCVpr, plasmids that express pf or pr, a fusion protein between puromycin acetyltransferase and green or red fluorescent protein from CV, the human cytomegalovirus enhancer/promoter. Transfection with pCVpf or pCVpr produced a ∼10% efficiency of gene transfer. A 2-day pulse puromycin selection resulted in ∼13-fold enrichment for transgenic cells, and continuous puromycin selection produced stable transgenic stem cell clones with retained pluripotency. Furthermore, we developed a PAC assay protocol for quantification of transgene expression. To test the usefulness for cell labeling and PAC assay in vivo, we constructed pVASpf containing pf linked to the regulatory sequence of medaka germ gene vasa and generated transgenic fish with visible GFP expression in germ cells. PAC assay revealed the highest expression in the testis. Interestingly, PAC activity was also detectable in somatic organs including the eye, which was validated by fluorescence in situ hybridization. Therefore, the pf and pr vectors provide a useful system for simultaneous drug selection, live labeling, and reporter assay 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 (i (1) , i (3) and af), only strain i (1) generated pigmented chimeras. Strikingly, this accessibility is completely lost in i (1) but acquired in i (3) after host gamma-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 gastrulation, central nervous system development and angiogenesis.

Gene oct4 (also called oct3/4 or pou5f1) encodes an octamer-binding transcription factor and is best known for its pluripotency-specific expression and pluripotency-maintaining role in early embryos and embryonic stem cells of mouse and human. Its fish paralog oct4 (also called pou2 or pou5f3) plays divergent roles in embryos and stem cells development. Here the expression and function of the medaka oct4 (Oloct4) during gastrulation and organogenesis were analysed. Oloct4 RNA was abundant in pluripotent cells and differentiated extraembryonic cells of blastula embryos. It was also detectable in primordial germ cells, brain, eye and tail bud at advanced stages. Importantly, oct4 depletion at high dosages severely affected gastrulation and axis formation. Surprisingly, Oloct4 depletion at low dosages also led to embryos that either had defective brain, eye and/or blood vessels or completely lacked them. Oloct4 depletion in transgenic embryos caused the loss of rx2-positive retinal stem cells in the developing eye. Therefore, Oloct4 is essential for gastrulation, central nervous system development as well as angiogenesis in medaka besides its role in pluripotency maintenance. These results together with previous studies suggest that Oloct4 play pleiotropic roles and represent the ancestral prototype of vertebrate oct4 and pou2 genes.

Evolutionary conservation of Dazl genomic organization and its continuous and dynamic distribution throughout germline development in gynogenetic gibel carp.

To investigate germline development and germ cell specification, we identified a Dazl homolog (CagDazl) from gynogenetic gibel carp (Carassius auratus gibelio). Its cDNA sequence and BAC clone sequence analyses revealed the genomic organization conservation and conserved synteny of the Dazl family members and their neighborhood genes among vertebrates, especially in fish. Moreover, a polyclonal antibody specific to CagDazl was produced and used to examine its expression and distribution throughout germline development at protein level. Firstly, ovary-specific expression pattern of CagDazl was confirmed in adult tissues by RT-PCR and Western blot. In addition, in situ hybridization and immunofluorescence localization demonstrated its specific expression in germ cells, and both its transcript and protein were localized to germ plasm. Then, co-localization of CagDazl and mitochondrial cloud was found, confirming that CagDazl transcript and its protein are germ plasm component and move via METRO pathway during oogenesis. Furthermore, the CagDazl is abundant and continuous throughout germline development and germ cell specification including primordial germ cell (PGC) formation, oogonium differentiation, oocyte development, and embryogenesis, and the dynamic distribution occurs at different development stages. The data suggest that maternal CagDazl might play an important role in gibel carp PGC formation. Therefore, CagDazl is a useful and specific marker for tracing germ plasm and germ cell development in the gynogenetic gibel carp. In addition, in comparison with previous studies in sexual reproduction species, the continuous and dynamic distribution of CagDazl protein in the germ plasm throughout the life cycle seems to have significant implication in sex evolution of vertebrates.

Direct electrochemistry and electrocatalytic mechanism of evolved Escherichia coli cells in microbial fuel cells.

E. coli cells evolved under electrochemical tension in a microbial fuel cell possess direct electrochemical behavior due to the excretion of hydroquinone derivatives through a highly permeable outer membrane, and their catalyzed fuel cell demonstrates excellent performance.

Developmental expression of CagMdkb during gibel carp embryogenesis.

Midkine (Mdk) genes have been revealed to have different expression patterns in vertebrates and therefore, additional studies on Mdk expression patterns are required in more species. In this study, CagMdkb has been cloned and characterized from a SMART cDNA library of 10-somite stage embryos of Carassius auratus gibelio. Its full length cDNA is 1091 bp and encodes a sequence of 147 amino acids, which shows 97.3% identity to zebrafish Mdkb on the amino acid level. RT-PCR analysis reveals that CagMdkb is first transcribed in gastrula embryos and maintains a relatively stable expression level during subsequent embryogenesis. Western blot analysis reveals a 19 kDa maternal CagMdkb protein band and the zygotic CagMdkb protein is expressed from gastrula stage. At around 10 somite stage, the 19 kDa CagMdkb is processed to another protein band of about 17 kDa, which might be the secreted form with the 21-residue signal peptide removed. With immunofluorescence analysis, maternal CagMdkb protein was found to be localized in each blastamere cell of early embryos. The zygotic CagMdkb positive fluorescence signal was detected from a pair of large neurons at 18-somite stage. At the later stages, CagMdkb protein was also extended to numerous small neurons in the forebrain, midbrain and hindbrain, as well as to nerve fibers in the spinal cord. Co-localization with 3A10 antibody revealed CagMdkb immunoreactivity on developing Mauthner neurons, a member of reticulospinal neurons. In addition, ectopic expression of CagMdkb in early embryos of gibel carp and zebrafish suppressed head formation and CagMdkb function was found to depend on secretory activity. All these findings indicate that CagMdkb plays an important role in neural development during gibel carp embryogenesis and there is functional conservation of Mdkb in fish head formation.

Efficient genome editing using CRISPR/Cas9 ribonucleoprotein approach in cultured Medaka fish cells.

Gene editing with CRISPR/Cas9 is a powerful tool to study the function of target genes. Although this technology has demonstrated wide efficiency in many species, including fertilized zebrafish and medaka fish embryos when microinjected, its application to achieve efficient gene editing in cultured fish cells have met some difficulty. Here, we report an efficient and reliable approach to edit genes in cultured medaka (Oryzias latipes) fish cells using pre-formed gRNA-Cas9 ribonucleoprotein (RNP) complex. Both medaka fish haploid and diploid cells were transfected with the RNP complex by electroporation. Efficient gene editing was demonstrated by polymerase chain reaction (PCR) amplification of the target gene from genomic DNA and heteroduplex mobility assay carried out with polyacrylamide gel electrophoresis (PAGE). The heteroduplex bands caused by RNP cleavage and non-homologous end joining could be readily detected by PAGE. DNA sequencing confirmed that these heteroduplex bands contains the mutated target gene sequence. The average gene editing efficiency in haploid cells reached 50%, enabling us to generate a clonal cell line with ntrk3b gene mutation for further study. This RNP transfection method also works efficiently in diploid medaka cells, with the highest mutation efficiency of 61.5%. The specificity of this synthetic RNP CRISPR/Cas9 approach was verified by candidate off-target gene sequencing. Our result indicated that transfection of pre-formed gRNA-Cas9 RNP into fish cells is efficient and reliable to edit target genes in cultured medaka fish cells. This method will be very useful for gene function studies using cultured fish cells.

Cloning and expression of medaka dazl during embryogenesis and gametogenesis.

The Deleted in azoospermia family consists of RNA-binding proteins Boule, Daz, and Daz-like (Dazl) that are expressed in the germline. Here, we report the cloning and expression of the medakafish (Oryzias latipes) dazl gene (odazl). Interestingly, although the predicted medaka Dazl protein (oDazl) contains a RRM motif and a DAZ repeat characteristic of its mammalian homologs, it lacks 80 aa at the C-terminus. By RT-PCR, RNA in situ hybridization, Western blotting and fluorescent immunohistochemistry using a rabbit anti-Dazl antibody (alphaDazl), we analyzed the expression patterns of odazl and its protein. The odazl transcript persists throughout embryogenesis and delineates with primordial germ cells. In adults, the expression of odazl RNA and its protein is restricted to germ cells of both the testis and ovary. We observed differential expression of RNA and protein at critical stages of gametogenesis. In the testis, the odazl RNA is low at premeiotic stages, abundant at meiotic stages, but absent in postmeiotic stages; whereas the oDazl protein is rich in premeiotic stages, reduced at meiotic stages, becomes barely detectable or absent in postmeiotic round spermatids or sperm, respectively. This is in sharp contrast to the human situation where the Dazl transcript and protein are present in mature spermatozoa. In the ovary, the odazl RNA and protein persist throughout oogenesis and also show differential expression at premeiotic, meiotic and postmeiotic stages. Thus, the odazl or its protein is a marker for germ cells during embryogenesis and at critical stages of gametogenesis in both sexes of medaka.

Fish ES cells and applications to biotechnology.

ES cells provide a promising tool for the generation of transgenic animals with site-directed mutations. When ES cells colonize germ cells in chimeras, transgenic animals with modified phenotypes are generated and used either for functional genomics studies or for improving productivity in commercial settings. Although the ES cell approach has been limited to mice, there is strong interest for developing the technology in fish. We describe the step-by-step procedure for developing ES cells in fish. Key aspects include avoiding cell differentiation, specific in vitro traits of pluripotency, and, most importantly, testing for production of chimeric animals as the main evidence of pluripotency. The entire process focuses on two model species, zebrafish and medaka, in which most work has been done. The achievements attained in these species, as well as their applicability to other commercial fish, are discussed. Because of the difficulties relating to germ line competence, mostly of long-term fish ES cells, alternative cell-based approaches such as primordial germ cells and nuclear transfer need to be considered. Although progress to date has been slow, there are promising achievements in homologous recombination and alternative avenues yet to be explored that can bring ES technology in fish to fruition.

Pluripotency and chimera competence of an embryonic stem cell line from the sea perch (Lateolabrax japonicus).

A stable GFP-expressing (GFP(+)LJES1) cell strain was developed from the LJES1 cells obtained from sea perch (Lateolabrax japonicus,) embryos. GFP(+)LJES1 cells were induced in vitro by RA to differentiate into a variety of cell types and also had the ability to form embryoid body-like structures in suspension culture. To determine the differentiation potential of LJES1 cells in vivo, GFP(+)LJES1 cells were transplanted into sea perch and zebrafish embryos at mid-blastula stage. Twenty out of 478 transplanted sea perch embryos contained GFP-expressing LJES1 cells 24 h after microinjection. Fifteen chimera embryos developed into fry. In these chimeras, the GFP(+)LJES1 cells contributed to a variety of tissues including the head and trunk. In zebrafish, 221 embryos were microinjected with GFP(+)LJES1 cells and 22 chimera embryos and fries expressing GFP were obtained. Donor GFP(+)LJES1 cells contributed to various tissues in head and trunk of zebrafish embryos and hatched fry.

Medaka insulin-like growth factor-2 supports self-renewal of the embryonic stem cell line and blastomeres in vitro.

Insulin-like growth factors (IGFs) regulate diverse processes including energy metabolism, cell proliferation and embryonic development. They activate the IGF signaling pathway via binding to cell surface receptors. Here we report an essential role of IGF2 in maintaining the pluripotency of embryonic stem (ES) cell from medaka (Oryzias latipes). The medaka igf2 gene was cloned for prokaryotically expression of IGF2 ligand and green fluorescent protein-tagged IGF2 namely IGF2:GFP. With flow cytometry analysis, we demonstrated that the IGF2:GFP can bind to the cultured ES cells from medaka and zebrafish respectively. We also verified that IGF2 is able to activate the phosphorylation of Erk1/2 and Akt, and sustain the viability and pluripotency of medaka ES cells in culture. Furthermore, we characterized the binding of IGF2:GFP to freshly isolated blastomeres by fluorescence microscopy and electron microscopy. Most importantly, we revealed the important role of IGF2 in supporting the derivation of blastomeres in short-term culture. Therefore, Medaka IGF2 is essential for the self-renewal of cultured ES cells and blastomeres from fish embryos. This finding underscores a conserved role of the IGF signaling pathway in stem cells from fish to mammals.