Retrovirus (RV) is efficient for gene transfer and integration in dividing cells of diverse organisms. RV provides a powerful tool for insertional mutagenesis (IM) to identify and functionally ...analyze genes essential for normal and pathological processes. Here we report RV-mediated gene transfer and genome-wide IM in fish stem cells from medaka and zebrafish. Three RVs were produced for fish cell transduction: rvLegfp and rvLcherry produce green fluorescent protein (GFP) and mCherry fluorescent protein respectively under control of human cytomegalovirus immediate early promoter upon any chromosomal integration, whereas rvGTgfp contains a splicing acceptor and expresses GFP only upon gene trapping (GT) via intronic in-frame integration and spliced to endogenous active genes. We show that rvLegfp and rvLcherry produce a transduction efficiency of 11~23% in medaka and zebrafish stem cell lines, which is as 30~67% efficient as the positive control in NIH/3T3. Upon co-infection with rvGTgfp and rvLcherry, GFP-positive cells were much fewer than Cherry-positive cells, consistent with rareness of productive gene trapping events versus random integration. Importantly, rvGTgfp infection in the medaka haploid embryonic stem (ES) cell line HX1 generated GTgfp insertion on all 24 chromosomes of the haploid genome. Similar to the mammalian haploid cells, these insertion events were presented predominantly in intergenic regions and introns but rarely in exons. RV-transduced HX1 retained the ES cell properties such as stable growth, embryoid body formation and pluripotency gene expression. Therefore, RV is proficient for gene transfer and IM in fish stem cells. Our results open new avenue for genome-wide IM in medaka haploid ES cells in culture.
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.
Stem cell cultures can be derived directly from early developing embryos and indirectly from differentiated cells by forced expression of pluripotency transcription factors. Pluripotency genes are ...routinely used to characterize mammalian stem cell cultures at the molecular level. However, such genes have remained unknown in lower vertebrates. In this regard, the laboratory fish medaka is uniquely suited because it has embryonic stem (ES) cells and genome sequence data. We identified seven medaka pluripotency genes by homology search and expression in vivo and in vitro. By RT-PCR analysis, the seven genes fall into three groups of expression pattern. Group I includes nanog and oct4 showing gonad-specific expression; Group II contains sall4 and zfp281 displaying gonad-preferential expression; Group III has klf4, ronin and tcf3 exhibiting expression also in several somatic tissues apart from the gonads. The transcripts of the seven genes are maternally supplied and persist at a high level during early embryogenesis. We made use of early embryos and adult gonads to examine expression in stem cells and differentiated derivatives by in situ hybridization. Strikingly, nanog and oct4 are highly expressed in pluripotent blastomeres of 16-cell embryos. In the adult testis, nanog expression was specific to spermatogonia, the germ stem cells, whereas tcf3 expression occurred in spermatogonia and differentiated cells. Most importantly, all the seven genes are pluripotency markers in vitro, because they have high expression in undifferentiated ES cells but dramatic down-regulation upon differentiation. Therefore, these genes have conserved their pluripotency-specific expression in vitro from mammals to lower vertebrates.
Gene targeting (GT) can introduce subtle alterations into a particular locus and represents a powerful tool for genome editing. Engineered zinc finger nucleases (ZFNs) are effective for generating ...minor allelic alterations. Efficient detection of such minor alterations remains one of the challenges in ZFN-mediated GT experiments. Here, we report the establishment of procedures allowing for efficient detection, quantification and enrichment of such subtle alterations. In a biallelic model, polyacrylamide gel electrophoresis (PAGE) is capable of detecting rare allelic variations in the form of DNA heteroduplexes at a high efficiency of ~0.4% compared with ~6.3% by the traditional T7 endonuclease I-digestion and agarose gel electrophoresis. In a multiple allelic model, PAGE could discriminate different alleles bearing addition or deletion of 1-18 bp as distinct bands that were easily quantifiable by densitometry. Furthermore, PAGE enables enrichment for rare alleles. We show for the first time that direct endogenous GT is possible in medaka by ZFN RNA injection, whereas PAGE allows for detection and cloning of ZFN-targeted alleles in adults arising from ZFN-injected medaka embryos. Therefore, PAGE is effective for detection, quantification and enrichment of multiple fine allelic differences and thus offers a versatile tool for screening targeted subtle gene alterations.
Medaka sex is determined by dmy/drnrtlbY, the doublesex and mab-3 domain gene on the Y chromosome 1,2. Most recent stud- ies have revealed a germ cell-intrinsic cue for the sperm-egg fate decision ...present in medaka, leading to spermatogenesis occurrence in a female gonadal environment without the presence of dmy 3. Targeted disruption of dmy, or sex related genes, such as DM domain related transcription factor dmrtl and anti-Mullerian hormone receptor 2 （arnhr2） were partially or fully resulted in XY feminization 4-6.
Zinc finger nucleases (ZFNs) can generate targeted gene disruption (GD) directly in developing embryos of zebrafish, mouse and human. In the fish medaka, ZFNs have been attempted on a transgene. ...Here, we developed procedures and parameters for ZFN-mediated direct GD on the gonad-specifically expressed
gsdf
locus in medaka. A pair of ZFNs was designed to target the first exon of
gsdf
and their synthetic mRNAs were microinjected into 1-cell stage embryos. We reveal dose-dependent survival rate and GD efficiency. In fry, ZFN mRNA injection at 10 ng/μl led to a GD efficiency of 30 %. This value increased up to nearly 100 % when the dose was enhanced to 40 ng/μl. In a typical series of experiments of ZFN mRNA injection at 10 ng/μl, 420 injected embryos developed into 94 adults, 4 of which had altered
gsdf
alleles. This leads to a GD efficacy of ∼4 % in the adulthood. Sequencing revealed a wide variety of subtle allelic alterations including additions and deletions of 1∼18 bp in length in ZFN-injected samples. Most importantly, one of the 4 adults examined was capable of germline transmission to 15.2 % of its F1 progeny. Interestingly, ontogenic analyses of the allelic profile revealed that GD commenced early in development, continued during subsequent stages of development and in primordia for different adult organs of the three germ layers. These results demonstrate the feasibility and—for the first time to our knowledge—the efficacy of ZFN-mediated direct GD on a chromosomal gene in medaka embryos.
Paedocypris is a newly established genus of fish in Southeast Asia. Paedocypris is characterized by several unique features, including a tiny adult size (thus named miniature fish or minifish), ...fragmentary habitats of acidic peat blackwater swamps, an unusual reproduction mode and truncated development. These peculiarities lend themselves excellent for studying chromosome evolution and rapid speciation in vertebrates but also make them highly controversial for the phylogenetic position.
We have established an organ procedure to prepare chromosome spreads from tiny organs of minifish and performed a cytogenetic study on two species of the genus Paedocypris, namely P. carbunculus (Pc) and P. sp. "Singkep" (Ps). We found 30 and 34 chromosomes in diploid cells of Pc and Ps, respectively, which are unusual in teleost fishes. The diploid metaphase has 5 pairs of metacentrics and 7 pairs of subtelocentrics in Pc compared to 3 pairs of metacentrics and 11 pairs of subtelocentrics in Ps, whereas the haploid metaphase contains 5 metacentrics and 7 subtelocentrics in Pc compared to 3 metacentrics and 11 subtelocentrics Ps. Chromosome behavior in first meiosis revealed the presence of a chromosomal ring consisting of 2 metacentrics in Pc, suggesting that centric fusion rather than fission was responsible for the karyotypic evolution from Ps to Pc. Flow cytometry revealed that Pc had a 45% nuclear staining intensity relative to medaka whose genome is 700 Mb in size and contains 0.81 pg DNA. The Pc genome should have 315 Mb in length and 0.36 pg of DNA, which represent one of the smallest values in vertebrates, suggesting genome miniaturization in this organism.
Our data demonstrate that gross chromosome rearrangements and genome miniaturization have accompanied the evolution of Paedocypris fishes. Our data also place Paedocypris outside currently described taxa of the Cypriniformes.
Spermatogonia are the male germ stem cells that continuously produce sperm for the next generation. Spermatogenesis is a complicated process that proceeds through mitotic phase of stem cell renewal ...and differentiation, meiotic phase, and postmeiotic phase of spermiogenesis. Full recapitulation of spermatogenesis in vitro has been impossible, as generation of normal spermatogonial stem cell lines without immortalization and production of motile sperm from these cells after long-term culture have not been achieved. Here we report the derivation of a normal spermatogonial cell line from a mature medakafish testis without immortalization. After 140 passages during 2 years of culture, this cell line retains stable but growth factor-dependent proliferation, a diploid karyotype, and the phenotype and gene expression pattern of spermatogonial stem cells. Furthermore, we show that this cell line can undergo meiosis and spermiogenesis to generate motile sperm. Therefore, the ability of continuous proliferation and sperm production in culture is an intrinsic property of medaka spermatogonial stem cells, and immortalization apparently is not necessary to derive male germ cell cultures. Our findings and cell line will offer a unique opportunity to study and recapitulate spermatogenesis in vitro and to develop approaches for germ-line transmission.
Augmenter of Liver Regeneration (ALR) is a sulfhydryl oxidase carrying out fundamental functions facilitating protein disulfide bond formation. In mammals, it also functions as a hepatotrophic growth ...factor that specifically stimulates hepatocyte proliferation and promotes liver regeneration after liver damage or partial hepatectomy. Whether ALR also plays a role during vertebrate hepatogenesis is unknown. In this work, we investigated the function of alr in liver organogenesis in zebrafish model. We showed that alr is expressed in liver throughout hepatogenesis. Knockdown of alr through morpholino antisense oligonucleotide (MO) leads to suppression of liver outgrowth while overexpression of alr promotes liver growth. The small-liver phenotype in alr morphants results from a reduction of hepatocyte proliferation without affecting apoptosis. When expressed in cultured cells, zebrafish Alr exists as dimer and is localized in mitochondria as well as cytosol but not in nucleus or secreted outside of the cell. Similar to mammalian ALR, zebrafish Alr is a flavin-linked sulfhydryl oxidase and mutation of the conserved cysteine in the CxxC motif abolishes its enzymatic activity. Interestingly, overexpression of either wild type Alr or enzyme-inactive Alr(C131S) mutant promoted liver growth and rescued the liver growth defect of alr morphants. Nevertheless, alr(C131S) is less efficacious in both functions. Meantime, high doses of alr MOs lead to widespread developmental defects and early embryonic death in an alr sequence-dependent manner. These results suggest that alr promotes zebrafish liver outgrowth using mechanisms that are dependent as well as independent of its sulfhydryl oxidase activity. This is the first demonstration of a developmental role of alr in vertebrate. It exemplifies that a low-level sulfhydryl oxidase activity of Alr is essential for embryonic development and cellular survival. The dose-dependent and partial suppression of alr expression through MO-mediated knockdown allows the identification of its late developmental role in vertebrate liver organogenesis.
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 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.
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