Search

Cartilaginous fishes have various unique physiological features such as a cartilaginous skeleton and a urea-based osmoregulation strategy for adaptation to their marine environment. Also, because they are a sister group of bony vertebrates, understanding their unique features is important from an evolutionary perspective. However, genetic engineering based on gene functions as well as cellular behavior has not been effectively utilized in cartilaginous fishes. This is partly because their reproductive strategy involves internal fertilization, which results in difficulty in microinjection into fertilized eggs at the early developmental stage. Here, to identify efficient gene transfer methods in cartilaginous fishes, we examined the effects of various methods both in vitro and in vivo using the cloudy catshark, a candidate model cartilaginous fish species. In all methods, green fluorescent protein (GFP) expression was used to evaluate exogenous gene transfer. First, we examined gene transfer into primary cultured cells from cloudy catshark embryos by lipofection, polyethylenimine (PEI) transfection, adenovirus infection, baculovirus infection, and electroporation. Among the methods tested, lipofection, electroporation, and baculovirus infection enabled the successful transfer of exogenous genes into primary cultured cells. We then attempted in vivo transfection into cloudy catshark embryos by electroporation and baculovirus infection. Although baculovirus-injected groups did not show GFP fluorescence, electroporation successfully introduced GFP into muscle cells. Furthermore, we succeeded in GFP transfer into adult tissues by electroporation. The in vitro and in vivo gene transfer methods that worked in this study may open ways for genetic manipulation including knockout experiments and cellular lineage analysis in cartilaginous fishes.

Expression patterns of paralogous genes in the functionally homologous cells sometimes show differences across species. However, no reasonable explanation for the mechanism underlying such phenomena has been discovered. To understand this mechanism, the present study focused on the hypophysiotropic GnRH neurons in vertebrates as a model. These neurons express eithergnrh1orgnrh3paralogs depending on species, and apparent switching of the expressed paralogs in them occurred at least four times in vertebrate evolution. First, we found redundant expressions ofgnrh1andgnrh3in a single neuron in piranha and hypothesized that this situation may indicate an ancestral condition. We tested this hypothesis by examining the activity of piranhagnrh1/gnrh3enhancers in zebrafish and medaka, in which the twognrhparalogs are not co-expressed. Here, thegnrh1/gnrh3enhancer of piranha induced reporter RFP/GFP co-expressions in a single hypophysiotropic GnRH neuron in both zebrafish and medaka. From these results, we propose that long-lasting (∼550 My) redundancy aftergnrh1/3duplication in 1R/2R WGD may be the key to apparent switching of the paralog usage among the present-day species. Moreover, interspecies analyses of enhancers indicated that the loss of enhancers rather than changes in trans-regulatory elements drove the role-division of these paralogs.

We have previously shown that the prostaglandin E

The medaka is a small freshwater fish found in ponds and rivers in Japan and has been a familiar pet since the 17th century. It has also been used as a model of basic biology for more than 100 years. Recently, because of the development of molecular genetic tools, the medaka has drawn attention because of its amenability to genetic modification. In this chapter, we will introduce a brief history, biological features, and application techniques for genetic modification, including disruption and foreign gene introduction. In addition to such basic approaches involving the generation of transgenic and knockout medaka, we will introduce several techniques for increasing the efficiency and range of possible applications: bacterial artificial chromosome/Fosmid-based vector construction, the dual promoter method, transposase, and phiC31 integrase. Lastly, we will introduce several gene knock-in techniques that involve Cas9/clustered regularly interspaced short palindromic repeats-mediated genome cleavage and the following of various DNA repair pathways.

Germ cells give rise to eggs or sperm. However, recent analyses in medaka (Oryzias latipes) showed that germ cells are also important for feminization of gonads, although this novel role of germ cells has not been characterized in detail. Here, we show that the feminizing effect is inherent to germ cells and is not affected by gametogenic stages or the sexual fate of germ cells. Three medaka mutants were generated to demonstrate this effect: figlα mutants, in which follicle formation is disrupted; meioC mutants, in which germ cells are unable to commit to gametogenesis and meiosis; and dazl mutants, in which germ cells do not develop into gonocytes. All these different stages of germ cells in XX mutants have an ability to feminize the gonads, resulting in the formation of gonads with ovarian structures. In addition to normal ovarian development, we also suggest that the increased number of gonocytes is sufficient for male to female sex reversal in XY medaka. These results may genetically demonstrate that the mechanism underlying the feminizing effect of germ cells is activated before the sexual fate decision of germ cells and meiosis, probably by the time of gonocyte formation in medaka. Author summary Germ cells are the only cells that can transfer genetic materials to the next generation via the sperm or egg. However, recent analyses in teleosts revealed another essential role of germ cells: feminizing the gonads. In our study, medaka mutants in which gametogenesis was blocked at specific stages provides the novel view that the feminizing effect of germ cells occurs in parallel with other reproductive elements, such as meiosis, the sexual fate decision of germ cells, and gametogenesis. Germ cells in medaka may have a potential to feminize gonads at the moment they have developed.

A cDNA clone coding for the collagen type IV alpha1 chain was obtained from the ovary of the medaka, Oryzias latipes. The clone encoded a protein of 1639 amino acids including a putative 21-residue signal peptide, and the deduced amino acid sequence of the alpha1 chain was homologous to those of the proteins from other species. The mRNA of the collagen type IV alpha1 chain was expressed in various tissues of the adult fish. In situ hybridization analysis revealed that the alpha1 chain mRNA was localized in the follicle layer of all growing follicles. In the post-ovulatory follicle that had released its oocyte during ovulation, the alpha1 chain transcript was detected in a winding line surrounding the tissue. This localization pattern was different from that of gelatinase B, a marker gene for granulosa cells. A specific antibody was prepared for the medaka collagen type IV alpha1 chain. Immunohistochemical analysis with this antibody yielded results consistent with those obtained by in situ hybridization. These data indicate that, in the medaka ovary, collagen type IV is synthesized by theca cells and is localized in the basement membrane.

Ovulation is the process of liberating oocytes from the preovulatory follicles, and is observed in the ovaries of virtually all female vertebrate animals. Compared with mammalian species, there have been far fewer studies that address the ovulatory mechanisms of non-mammalian species. We have examined the molecular mechanism of follicle rupture during ovulation using the teleost model, medaka, or Oryzias latipes. Follicle rupture in medaka ovulation involves the cooperation of the tissue inhibitor of metalloproteinase-2b protein with at least three matrix metalloproteinases (MMP): membrane type-1 MMP (MT1-MMP), MT2-MMP, and gelatinase A. Our studies also indicate that the serine protease, i.e., plasmin, participates in the rupture for only a few hours prior to the activation of MMP-mediated hydrolysis at ovulation. The involvement of prostaglandin E2 (PGE2) in medaka ovulation was also demonstrated. Cyclooxygenase-2 and PGE2 receptor subtype EP4b were respectively shown to be an enzyme responsible for PGE2 synthesis and a receptor for the generated ligand in the preovulatory follicles. Based on the results obtained from our studies of fish, we discuss the similarities and differences in vertebrate ovulation compared with mammalian species.

A cDNA for a prostaglandin E(2) (PGE(2)) receptor subtype 4, EP4b (Ptger4b), was cloned from the medaka ovary. The effect of PGE(2) was examined using COS-7 cells expressing the recombinant Ptger4b protein. An increase in intracellular cAMP levels was observed when the cells were incubated with PGE(2), but the increase in cAMP levels was nullified by the addition of the EP4 antagonist GW627368X. The expression of ptger4b mRNA was drastically induced by the addition of pregnant mare serum gonadotropin to the in vitro culture of large preovulatory follicles. In in vitro ovulation studies of the effect of GW627368X addition on follicle ovulation, the critical timing of the PGE(2)/Ptger4b interaction was suggested to be between -1 and 0 h of ovulation. These results further substantiate that PGE(2)/Ptger4b signaling is involved in follicle rupture during ovulation in the medaka ovary.

Follicle rupture during ovulation is a well-regulated biological process of extracellular matrix degradation in the vertebrate ovary. Although proteolytic enzymes responsible for the rupture have recently been identified in the medaka, Oryzias latipes , the lack of knowledge about the ovarian expression and distribution of extracellular matrix components in lower vertebrates prevents the understanding of this process's molecular mechanism. To approach the problem, we cloned a cDNA coding for the medaka collagen type-I alpha1 chain and examined its mRNA expression in the fish ovary. The deduced amino acid sequence of the collagen type-I alpha1 chain was homologous to those of the proteins from other vertebrate species. The alpha1 chain mRNA was expressed in various tissues of the adult fish. In the ovary sections of mature female fish, this mRNA was detected in a line surrounding ovarian follicles of all sizes. A comparison with the distribution of gelatinase B mRNA in follicles that had just ovulated indicated that the collagen type-I alpha1 gene is expressed in the theca cells. The current results strongly suggest that collagen type I is synthesized by theca cells and is localized in the same cell layer of the follicles.