Full-Length Transcriptomes and Sex-Based Differentially Expressed Genes in the Brain and Ganglia of Giant River Prawn Macrobrachium rosenbergii.

Macrobrachium rosenbergii is an important aquaculture prawn that exhibits sexual dimorphism in growth, with males growing much faster than females. However, the mechanisms controlling these complex traits are not well understood. The nervous system plays an important role in regulating life functions. In the present work, we applied PacBio RNA-seq to obtain and characterize the full-length transcriptomes of the brains and thoracic ganglia of female and male prawns, and we performed comparative transcriptome analysis of female and male prawns. A total of 159.1-Gb of subreads were obtained with an average length of 2175 bp and 93.2% completeness. A total of 84,627 high-quality unigenes were generated and annotated with functional databases. A total of 6367 transcript factors and 6287 LncRNAs were predicted. In total, 5287 and 6211 significantly differentially expressed genes (DEGs) were found in the brain and thoracic ganglion, respectively, and confirmed by qRT-PCR. Of the 435 genes associated with protein processing pathways in the endoplasmic reticula, 42 DEGs were detected, and 21/26 DEGs with upregulated expression in the male brain/thoracic ganglion. The DEGs in this pathway are regulated by multiple LncRNAs in polypeptide folding and misfolded protein degradation in the different organs and sexes of the prawn. Our results provide novel theories and insights for studying the nervous system, sexual control, and growth dimorphism.

Generation of a Normal Long-Term-Cultured Chinese Hook Snout Carp Spermatogonial Stem Cell Line Capable of Sperm Production In Vitro.

Opsariichthys bidens belongs to the family Cyprinidae and is a small freshwater economic fish widely distributed in China. In recent years, the natural resources of O. bidens have been drastically reduced due to overfishing and the destruction of the water environment. The in vitro culture and long-term preservation of germ stem cells are the key technologies to keep genetic resources from degeneration. However, except for the establishment of the first long-term cultured medaka spermatogonia cell line (SSC) capable of producing sperm in vitro in 2004, no other long-term cultured SSC line has been found in other fish species. In this study, we successfully established another long-term-cultured spermatogonial stem cell line from Opsariichthys bidens (ObSSC). After more than 2 years of culture, ObSSC had a diploid karyotype and stable growth, with the typical gene expression patterns of SSC. Under in vitro culture, ObSSC could be induced to differentiate into sperm and other different types of somatic cells. In vivo, ObSSC could differentiate into different cells of three germ layers upon being transplanted into zebrafish embryos. Our research helps to explore the potential and regulation mechanism of fish SSC differentiation and spermatogenesis in vitro, provides a new way for solving the problem of fish genetic resource degradation and lays a foundation for further research on fish germ cell transplantation.

Quick detection of Carassius auratus herpesvirus (CaHV) by recombinase-aid amplification lateral flow dipstick (RAA-LFD) method.

Crucian carp (Carassius auratus) is one of the major freshwater species and is also a common food fish in China. Recently, Carassius auratus herpesvirus (CaHV) could induce fatal viral disease with high mortality of crucian carp, which had caused huge economic losses. In this study, we described a rapid and simple recombinase-aid amplification (RAA) assay coupled with lateral flow dipstick (LFD), which could achieve sensitive diagnosis of tumor necrosis factor receptor (TNFR) of CaHV within 35 min at 40°C. Our RAA-LFD method had a satisfactory detection limit of 100 gene copies per reaction, which was 100-fold more sensitive than traditional PCR. In addition, no cross-reaction was observed with other viral pathogens, including koi herpesvirus (KHV), cyprinid herpesvirus 2 (CyHV-2), infectious hematopoietic necrosis virus (IHNV), spring viremia of carp virus (SVCV) and grass carp reovirus (GCRV). Furthermore, the overall cost of the method was cut in half compared to previous studies. In conclusion, RAA-LFD assay is therefore, a promising alternative for point-of-care testing (POCT) of CaHV, which is feasible and of certain value in application of aquatic disease control.

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.

Alternative transcription generates multiple Mitf isoforms with different expression patterns and activities in medaka.

Microphthalmia-associated transcription factor (Mitf) is best known for distinct functions in multiple cell lineages including melanocytes, mast cells, and osteoclasts. In mammals, mitf produces multiple Mitf isoforms by alternative transcription and splicing. The fish medaka has two mitf genes, mitf1 and mitf2. Here, we report differential expression and activities of medaka Mitf isoforms. Molecular cloning identified four mitf1 variants encoding isoforms Mitf1A, MitfB, MitfH, and MitfM, and only one mitf2RNA encoding Mitf2M, which exhibited differential expression. Mitf1 isoforms and Mitf2M differed dramatically in activating the dazl and tyrosinase promoters DAZ and TYR. Interestingly, Mitf1ΔN, an N-terminus-less Mitf1 mutant form, retained activity to activate TYR but not DAZ. Importantly, Mitf1B was also sufficient for inducing melanocyte differentiation and endogenous tyrosinase RNA expression in medaka embryonic stem cells. Intriguingly, Mitf1 isoforms possessed considerable differences in inducing the expression of multiple cell lineage marker genes. Therefore, alternative mitf transcription is a conserved mechanism from fish to mammals, and medaka Mitf1 isoforms show differences in expression and activity. We conclude that differential expression of isoforms contributes to multiple distinct functions of Mitf in vertebrates.

p53 gene targeting by homologous recombination in fish ES cells.

BACKGROUND: 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). METHODOLOGY AND PRINCIPAL FINDINGS: 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. CONCLUSIONS: 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.

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.

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.