Ocular lesions in leptin receptor-deficient medaka (Oryzias latipes).

Ocular lesions in leptin receptor-deficient medaka were examined histopathologically at 10, 28, and 37 weeks post hatching. Leptin receptor-deficient medaka at 28 and 37 weeks old showed hyperglycemia and hypoinsulinemia. Histopathologically, vacuolation, swelling, fragmentation, and liquefaction of the lens fibers and dilatation of the retinal central veins, retinal capillaries, iridal veins and capillaries, and choroidal veins were observed in leptin receptor-deficient medaka at 28 and 37 weeks old. Thinning of the total retina, pigment epithelial layer, layer of rods and cones, outer granular layer, outer plexiform layer, inner granular layer, and inner plexiform layer was observed in leptin receptor-deficient medaka at 28 and 37 weeks compared with in control medaka. These histopathological characteristics in leptin receptor-deficient medaka are similar to characteristics in ocular lesions of rodent models for type II diabetes mellitus, making leptin receptor-deficient medaka a useful model of diabetic cataract and retinopathy.

Leptin receptor-deficient (knockout) medaka, Oryzias latipes, show chronical up-regulated levels of orexigenic neuropeptides, elevated food intake and stage specific effects on growth and fat allocation.

The first studies that identified leptin and its receptor (LepR) in mammals were based on mutant animals that displayed dramatic changes in body-weight and regulation of energy homeostasis. Subsequent studies have shown that a deficiency of leptin or LepR in homoeothermic mammals results in hyperphagia, obesity, infertility and a number of other abnormalities. The physiological roles of leptin-mediated signaling in ectothermic teleosts are still being explored. Here, we produced medaka with homozygous LepR gene mutation using the targeting induced local lesions in a genome method. This knockout mutant had a point mutation of cysteine for stop codon at the 357th amino acid just before the leptin-binding domain. The evidence for loss of function of leptin-mediated signaling in the mutant is based on a lack of response to feeding in the expression of key appetite-related neuropeptides in the diencephalon. The mutant lepr−/− medaka expressed constant up-regulated levels of mRNA for the orexigenic neuropeptide Ya and agouti-related protein and a suppressed level of anorexigenic proopiomelanocortin 1 in the diencephalon independent of feeding, which suggests that the mutant did not possess functional LepR. Phenotypes of the LepR-mutant medaka were analyzed in order to understand the effects on food intake, growth, and fat accumulation in the tissues. The food intake of the mutant medaka was higher in post-juveniles and adult stages than that of wild-type (WT) fish. The hyperphagia led to a high growth rate at the post-juvenile stage, but did not to significant alterations in final adult body size. There was no additional deposition of fat in the liver and muscle in the post-juvenile and adult mutants, or in the blood plasma in the adult mutant. However, adult LepR mutants possessed large deposits of visceral fat, unlike in the WT fish, in which there were none. Our analysis confirms that LepR in medaka exert a powerful influence on the control on food intake. Further analyses using the mutant will contribute to a better understanding of the role of leptin in fish. This is the first study to produce fish with leptin receptor deficiency.

Homologue gene of bile acid transporters ntcp, asbt, and ost-alpha in rainbow trout Oncorhynchus mykiss: tissue expression, effect of fasting, and response to bile acid administration.

Bile acid transporters belonging to the SLC10A protein family, Na+ taurocholate cotransporting polypeptide (NTCP or SLC10A1), apical sodium-dependent bile salt transporter (ASBT or SLC10A2), and organic solute transporter alpha (Ost-alpha) have been known to play critical roles in the enterohepatic circulation of bile acids in mammals. In this study, ntcp, asbt, and ost-alpha-1/-2 cDNA were cloned, their tissue distributions were characterized, and the effects of fasting and bile acid administration on their expression were examined in rainbow trout Oncorhynchus mykiss. The structural characteristics of Ntcp, Asbt, and Ost-alpha were well conserved in trout, and three-dimensional structure analysis showed that Ntcp and Asbt were similar to each other. Tissue distribution analysis revealed that trout asbt was primarily expressed in the hindgut, while ntcp expression occurred in the brain, and ost-alpha-1/-2 was mainly expressed in the liver or ovary. Although asbt and ost-alpha-1 mRNA levels in the gut increased in response to fasting for 4 days, ost-alpha-1 expression in the liver decreased. Similarly, bile acid administration increased asbt and ost-alpha-1 expression levels in the gut, while those of ntcp and ost-alpha-2 in the liver decreased. These results suggested that the genes asbt, ntcp, and ost-alpha are involved in bile acid transport in rainbow trout.

New approach for fish breeding by chemical mutagenesis: establishment of TILLING method in fugu (Takifugu rubripes) with ENU mutagenesis.

BACKGROUND: In fish breeding, it is essential to discover and generate fish exhibiting an effective phenotype for the aquaculture industry, but screening for natural mutants by only depending on natural spontaneous mutations is limited. Presently, reverse genetics has become an important tool to generate mutants, which exhibit the phenotype caused by inactivation of a gene. TILLING (Targeting Induced Local Lesions IN Genomes) is a reverse genetics strategy that combines random chemical mutagenesis with high-throughput discovery technologies for screening the induced mutations in target genes. Although the chemical mutagenesis has been used widely in a variety of model species and also genetic breeding of microorganisms and crops, the application of the mutagenesis in fish breeding has been only rarely reported. RESULTS: In this study, we developed the TILLING method in fugu with ENU mutagenesis and high-resolution melting (HRM) analysis to detect base pair changes in target sequences. Fugu males were treated 3 times at weekly intervals with various ENU concentrations, and then the collected sperm after the treatment was used to fertilize normal female for generating the mutagenized population (F1). The fertilization and the hatching ratios were similar to those of the control and did not reveal a dose dependency of ENU. Genomic DNA from the harvested F1 offspring was used for the HRM analysis. To obtain a fish exhibiting a useful phenotype (e.g. high meat production and rapid growth), fugu myostatin (Mstn) gene was examined as a target gene, because it has been clarified that the mstn deficient medaka exhibited double-muscle phenotype in common with MSTN knockout mice and bovine MSTN mutant. As a result, ten types of ENU-induced mutations were identified including a nonsense mutation in the investigated region with HRM analysis. In addition, the average mutation frequency in fugu Mstn gene was 1 mutant per 297 kb, which is similar to values calculated for zebrafish and medaka TILLING libraries. CONCLUSIONS: These results demonstrate that the TILLING method in fugu was established. We anticipate that this TILLING approach can be used to generate a wide range of mutant alleles, and be applicable to many farmed fish that can be chemically mutagenized.

Postprandial response and tissue distribution of the bile acid synthesis-related genes, cyp7a1, cyp8b1 and shp, in rainbow trout Oncorhynchus mykiss.

In mammals, cholesterol 7α-hydroxylase (CYP7A1) and sterol 12α-hydroxylase (CYP8B1) are rate-limiting enzymes in bile acid synthesis. In addition, a small heterodimer partner (SHP) is also known to inhibit bile acid synthesis via the suppression of CYP7A1 and CYP8B1 expression. However, little information is currently available regarding primary structure of the genes involved in bile acid synthesis in fish. We therefore cloned cyp7a1, cyp8b1 and shp genes from rainbow trout and obtained cDNAs encoding two isoforms each of Cyp7a1 (-1 and -2), Cyp8b1 (-1 and -2) and Shp (-1 and -2). Both cyp7a1-1 and -2 encoded proteins of 512 amino acids. Trout cyp7a1-1 was expressed not only primarily in the kidney, pyloric caecum and mid-gut, but also weakly in the liver, eye, gill and ovary. cyp7a1-2 was highly expressed in the liver, pyloric caecum and mid-gut. cyp8b1-1 and -2, which encoded proteins of 512 and 509 amino acids, respectively, were principally expressed in the liver. Both shp-1 and -2, which encoded proteins of 288 and 290 amino acids, respectively, were strongly expressed in the liver, but shp-2 was also highly expressed in the gallbladder and digestive tract. The temporal changes in the expression of cyp7a1-1/-2, cyp8b1-1/-2 and shp-1/-2 in the liver were assessed after consumption of a single meal. Expression of cyp7a1-1/-2 and cyp8b1-1/-2 increased within 3h post feeding (hpf) when the stomach was still approximately 84% full and the gallbladder was almost completely empty. Although the expression of shp-1 did not change after feeding, the expression pattern of shp-2 was inversely related to the expression patterns of cyp7a1-1/-2 and cyp8b1-1/-2. Specifically, shp-2 expression decreased until 3 hpf before returning to initial levels at 24 hpf. These findings suggest that Cyp7a1s/8b1s and Shp-2 function antagonistically in bile acid synthesis in rainbow trout.

Vibrios adhere to epithelial cells in the intestinal tract of red sea bream, Pagrus major, utilizing GM4 as an attachment site.

Vibrios, distributed in marine and brackish environments, can cause vibriosis in fish and shellfish under appropriate conditions. Previously, we clarified by thin-layer chromatography (TLC) overlay assay that (35)S-labeled Vibrio trachuri adhered to GM4 isolated from red sea bream intestine. However, whether GM4 actually functions on epithelial cells as an attachment site for vibrios still remains to be uncovered. We found that six isolates, classified as V. harveyi, V. campbellii, and V. splendidus, from intestinal microflora of red sea bream adhered to GM4 but not galactosylceramide (GalCer) by TLC-overlay assay. Tissue-overlay assays revealed that V. harveyi labeled with green fluorescent protein (GFP) adhered to epithelial cells of red sea bream intestine where GM4 and GalCer were found to be distributed on the top layer of actin filaments by immunohistochemical analysis using corresponding antibodies. The number of adhering vibrios was diminished by pretreatment with anti-GM4 antibody, but not anti-GalCer antibody. These results clearly indicate that vibrios adhere to epithelial cells of red sea bream intestine utilizing GM4 as an attachment site.

Myostatin-deficient medaka exhibit a double-muscling phenotype with hyperplasia and hypertrophy, which occur sequentially during post-hatch development.

Myostatin (MSTN) functions as a negative regulator of skeletal muscle mass. In mammals, MSTN-deficient animals result in an increase of skeletal muscle mass with both hyperplasia and hypertrophy. A MSTN gene is highly conserved within the fish species, allowing speculation that MSTN-deficient fish could exhibit a double-muscled phenotype. Some strategies for blocking or knocking down MSTN in adult fish have been already performed; however, these fish show either only hyperplastic or hypertrophic growth in muscle fiber. Therefore, the role of MSTN in fish myogenesis during post-hatch growth remains unclear. To address this question, we have made MSTN-deficient medaka (mstnC315Y) by using the targeting induced local lesions in a genome method. mstnC315Y can reproduce and have the same survival period as WT medaka. Growth rates of WT and mstnC315Y were measured at juvenile (1-2wk post-hatching), post-juvenile (3-7wk post-hatching) and adult (8-16wk post-hatching) stages. In addition, effects of MSTN on skeletal muscle differentiation were investigated at histological and molecular levels at each developmental stage. As a result, mstnC315Y show a significant increase in body weight from the post-juvenile to adult stage. Hyper-morphogenesis of skeletal muscle in mstnC315Y was accomplished due to hyperplastic growth from post-juvenile to early adult stage, followed by hypertrophic growth in the adult stage. Myf-5 and MyoD were up-regulated in mstnC315Y at the hyperplastic growth phase, while myogenin was highly expressed in mstnC315Y at the hypertrophic growth phase. These indicated that MSTN in medaka plays a dual role for muscle fiber development. In conclusion, MSTN in medaka regulates the number and size of muscle fiber in a temporally-controlled manner during posthatch growth.

Zebrafish and mouse alpha2,3-sialyltransferases responsible for synthesizing GM4 ganglioside.

We have previously reported that fish pathogens causing vibriosis specifically adhere to GM4 on the epithelial cells of fish intestinal tracts (Chisada, S., Horibata, Y., Hama, Y., Inagaki, M., Furuya, N., Okino, N., and Ito, M. (2005) Biochem. Biophys. Res. Commun. 333, 367-373). To identify the gene encoding the enzyme for GM4 synthesis in the fish intestinal tract, a phylogenetic tree of vertebrate ST3GalVs, including Danio rerio and Oryzias latipes, was generated in which two putative subfamilies of fish ST3GalVs were found. Two putative ST3GalVs of zebrafish (zST3GalV-1 and -2), each belonging to different subfamilies, were cloned from the zebrafish cDNA library. Interestingly, zST3GalV-1 synthesized GM3 (NeuAcalpha2-3Galbeta1-4Glcbeta1-1'Cer) but not GM4, whereas zSTGalV-2 synthesized both gangliosides in vitro when expressed in CHO-K1 and RPMI1846 cells. Flow cytometric analysis using anti-GM4 antibody revealed that the transformation of RPMI1846 cells with zST3GalV-2 but not zST3GalV-1 cDNA increased the cell-surface expression of GM4. Whole mount in situ hybridization showed that the zST3GalV-2 transcript was strongly expressed in the gastrointestinal tract, whereas zST3GalV-1 was expressed in the brain and esophagus but not gastrointestinal tract in 3-day post-fertilization embryos. It has long been a matter of controversy which enzyme is responsible for the synthesis of GM4 in mammals. We found that three isoforms of mouse ST3GalV (mST3GalV) having different N-terminal sequences can synthesize GM4 as well as GM3 when expressed in RPMI1846 and CHO-K1 cells. Furthermore, mST3GalV knock-out mice were found to lack GM4 synthase activity and GM4 in contrast to wild-type mice. These results clearly indicate that zST3GalV-2 and mST3GalV are the enzymes responsible for the synthesis of GM4 in zebrafish and mice, respectively.

Ganglioside from eel serum high density lipoprotein (HDL) and its role as a ligand for HDL binding protein.

First, we attempted to isolate glycosphingolipids from eel serum HDL. A single ganglioside containing N-acetylneuraminic acid (NeuAc), which is positive with resorcinol and orcinol reactions, was purified. The mobilities of the purified ganglioside and its lyso-form on high performance TLC were similar as those of authentic GM4 and its lyso-form, respectively. The mass of the purified ganglioside was determined by TOF mass spectrometer, and the mass of its oligosaccharide was the same as that of authentic GM4 from human brain consisting of disaccharide of NeuAc and galactose. The ganglioside from eel HDL was not hydrolyzed by recombinant endoglycoceramidase II, which cannot hydrolyze between galactose and ceramide of gangliosides, but hydrolyzes between glucose and ceramide. We concluded from these results that the ganglioside purified from eel serum HDL is GM4. Second, we investigated the effects of the ganglioside on binding of HDL labeled with fluorescein isothiocyanate (FITC-HDL) to cultured eel hepatocytes and on FITC-HDL ligand blotting by using plasma membrane proteins of the hepatocytes. Stimulatory effect of GM4 on FITC-HDL binding to the hepatocytes and FITC-HDL ligand blotting suggests strongly that GM4 is a ligand for HDL binding protein of eel hepatocytes.

The glycosphingolipid receptor for Vibrio trachuri in the red sea bream intestine is a GM4 ganglioside which contains 2-hydroxy fatty acids.

Three major glycosphingolipids (tentatively designated IGL-1, 2, and 3) were isolated from the intestine of red sea bream (Pagrus major) and were subjected to a TLC-overlay assay with (35)S-labeled Vibrio trachuri which causes vibriosis of fish. The bacteria adhered to IGL-2, which was determined to be a GM4 ganglioside (NeuAcalpha2-3Galbeta1-ceramide). The fatty acid portion of IGL-2 was composed of 2-hydroxy C22:0, C24:0, and C24:1, in addition to the non-hydroxy C16:0 and C18:0, while the sphingoid base was composed exclusively of sphingenine (d18:1). Among glycosphingolipids tested, V. trachuri adhered to GM4 the most strongly followed by adherence to GM3 and GalCer, but the bacteria did not adhere to GM1a, GM2, LacCer, or GlcCer. V. trachuri was found to aggregate with the erythrocytes coated with GM4, but not with those coated with GM1a or GM2, thus indicating that specific adhesion occurs on intact cells. Interestingly, the dynamics for adhesion of V. trachuri to glycosphingolipids was defined by the structure of not only the sugar moiety but also the ceramide moiety, since the bacteria adhered to GM4 which contained 2-hydroxy fatty acids much more strongly than to that which contained non-hydroxy fatty acids.