The sex-determining locus in the tiger pufferfish, Takifugu rubripes

The tiger pufferfish (fugu), Takifugu rubripes, is a model fish that has had its genome entirely sequenced. By performing genomewide linkage analyses, we show that the sex of fugu is determined by a single chromosomal region on linkage group 19 in an XX-XY system.     

A genetic linkage map for the tiger pufferfish, Takifugu rubripes

The compact genome of the tiger pufferfish, Takifugu rubripes (fugu), has been sequenced to the "draft" level and annotated to identify all the genes. However, the assembly of the draft genome sequence is highly fragmented due to the lack of a genetic or a physical map. To determine the long-range linkage relationship of the sequences, we have constructed the first genetic linkage map for fugu. The maps for the male and female spanning 697.1 and 1213.5 cM, respectively, were arranged into 22 linkage groups by markers heterozygous in both parents. The resulting map consists of 200 microsatellite loci physically linked to genome sequences spanning approximately 39 Mb in total. Comparisons of the genome maps of fugu, other teleosts, and mammals suggest that syntenic relationship is more conserved in the teleost lineage than in the mammalian lineage. Map comparisons also show a pufferfish lineage-specific rearrangement of the genome resulting in colocalization of two Hox gene clusters in one linkage group. This map provides a foundation for development of a complete physical map, a basis for comparison of long-range linkage of genes with other vertebrates, and a resource for mapping loci responsible for phenotypic differences among Takifugu species.     

Involvement of gelatinases (MMP-2 and MMP-9) in the development of airway inflammation and pulmonary fibrosis

Pulmonary fibrosis has an aggressive course and is usually fatal an average of 3 to 6 years after the onset of symptoms. Pulmonary fibrosis is associated with deposition of extracellular matrix (ECM) components in the lung interstitium. Matrix metalloproteinases (MMPs) are a major group of proteinases known to regulate the ECM remodeling and so they are hypothesized to be important in the process of lung fibrosis. These led to the concept that modulation of airway remodeling including excessive proteolytic damage of the tissue may be of interest for future treatment. The excessive airway remodeling as a result of an imbalance in the equilibrium of the normal processes of synthesis and degradation of extracellular matrix components could argue in favor of antiprotease treatments. Moreover, these observations emphasize that effective therapies for these disorders must be given early in the natural history of the disease, prior to the development of expensive lung destruction and fibrosis. This revised version was published online in August 2006 with corrections to the Cover Date.     

Characterization and tissue distribution of multiple agouti-family genes in pufferfish, Takifugu rubripes

Four types of agouti-family genes (AGRP1, AGRP2, ASIP1 and ASIP2) were obtained from torafugu, Takifugu rubripes. Their characterization and structure were analyzed to elucidate the relationship among the torafugu agouti-family genes. Both AGRP1 and AGRP2 showed genomic synteny with the human AGRP gene. Phylogenetic tree analysis showed that AGRP1 formed a cluster with human AGRP. We inferred that torafugu AGRP1 and AGRP2 are orthologs of human AGRP and that they are paralogous genes derived from genome duplication occurred in the teleost phylogeny. Torafugu ASIP1 showed genomic synteny with the human ASIP, but ASIP2 did not. The ASIP1 expression level was about five times higher in the white ventral skin than in the black dorsal skin. Therefore, we concluded that torafugu ASIP1 is an ortholog of human ASIP, nevertheless, we are unable to determine if torafugu ASIP2 is a paralog of ASIP1 or not.     

Isolation of seven IL-17 family genes from the Japanese pufferfish Takifugu rubripes

In humans, the IL-17 family is composed of six members (A–F). The A, E and F forms have been extensively studied in numerous mammalian species. However, there are few reports regarding IL-17 expression in teleost. In this study, IL-17 family genes were isolated from the Japanese pufferfish (Fugu) and their structure and expression profile were analyzed. Screening of the Fugu genome database revealed the existence of five scaffolds containing IL-17 family homologous genes. Scaffold_1 contained three IL-17 family homologues including IL-17A/F1, 2 and C2, and IL-17A/F1 and two located in tandem. This was similar to the IL-17A/F1 and two genes in zebrafish and to human IL-17A and F. Other scaffolds 38, 143 and 430, contained IL-17 family homologous genes that were identical to IL-17D, A/F3 and C1 in Fugu, respectively. Moreover, IL-17 family homologues on scaffold_264 included a novel type of IL-17 family genes in teleost. These isolates contained four cysteine residues that were involved in the formation of a typical cysteine knot consisting of two disulphide linkages. However, IL-17A/F2 did not demonstrate any conservation at the second and fourth cysteine residues. The tissue distribution of the Fugu IL-17 family genes was also found to differ. In particular, IL-17 family genes were highly expressed in the head kidney and gill. Moreover, expression of IL-17 family genes was significantly up-regulated in the lipopolysaccharide-stimulated head kidney. These results suggested that Fugu IL-17 family members were involved in inflammatory responses.     

Identification of three isoforms for mitochondrial adenine nucleotide translocator in the pufferfish Takifugu rubripes

Three adenine nucleotide translocator (ANT) genes were identified through in silico data mining of the Fugu genome database along with isolation of their corresponding cDNAs in vivo from the pufferfish (Takifugu rubripes). As a result of phylogenetic analysis, the ANT gene on scaffold_254 corresponded to mammalian ANT1, whereas both of those on scaffold_6 and scaffold_598 to mammalian ANT3. The ANT gene encoded by scaffold_6 was expressed ubiquitously in various tissues, whereas the ANT genes encoded by scaffold_254 and scaffold_598 were predominantly expressed in skeletal muscle and heart, respectively.     

A trans-species missense SNP in Amhr2 is associated with sex determination in the tiger pufferfish, Takifugu rubripes (fugu)

Heterogametic sex chromosomes have evolved independently in various lineages of vertebrates. Such sex chromosome pairs often contain nonrecombining regions, with one of the chromosomes harboring a master sex-determining (SD) gene. It is hypothesized that these sex chromosomes evolved from a pair of autosomes that diverged after acquiring the SD gene. By linkage and association mapping of the SD locus in fugu (Takifugu rubripes), we show that a SNP (C/G) in the anti-Müllerian hormone receptor type II (Amhr2) gene is the only polymorphism associated with phenotypic sex. This SNP changes an amino acid (His/Asp384) in the kinase domain. While females are homozygous (His/His384), males are heterozygous. Sex in fugu is most likely determined by a combination of the two alleles of Amhr2. Consistent with this model, the medaka hotei mutant carrying a substitution in the kinase domain of Amhr2 causes a female phenotype. The association of the Amhr2 SNP with phenotypic sex is conserved in two other species of Takifugu but not in Tetraodon. The fugu SD locus shows no sign of recombination suppression between X and Y chromosomes. Thus, fugu sex chromosomes represent an unusual example of proto-sex chromosomes. Such undifferentiated X-Y chromosomes may be more common in vertebrates than previously thought.     

Developmental genetic basis for the evolution of pelvic fin loss in the pufferfish Takifugu rubripes

Paired appendages were a key developmental innovation among vertebrates and they eventually evolved into limbs. Ancient developmental control systems for paired fins and limbs are broadly conserved among gnathostome vertebrates. Some lineages including whales, some salamanders, snakes, and many ray-fin fish, independently lost the pectoral, pelvic, or both appendages over evolutionary time. When different taxa independently evolve similar developmental morphologies, do they use the same molecular genetic mechanisms? To determine the developmental genetic basis for the evolution of pelvis loss in the pufferfish Takifugu rubripes (fugu), we isolated fugu orthologs of genes thought to be essential for limb development in tetrapods, including limb positioning (Hoxc6, Hoxd9), limb bud initiation (Pitx1, Tbx4, Tbx5), and limb bud outgrowth (Shh, Fgf10), and studied their expression patterns during fugu development. Results showed that bud outgrowth and initiation fail to occur in fugu, and that pelvis loss is associated with altered expression of Hoxd9a, which we show to be a marker for pelvic fin position in three-spine stickleback Gasterosteus aculeatus. These results rule out changes in appendage outgrowth and initiation genes as the earliest developmental defect in pufferfish pelvic fin loss and suggest that altered Hoxd9a expression in the lateral mesoderm may account for pelvis loss in fugu. This mechanism appears to be different from the mechanism for pelvic loss in stickleback, showing that different taxa can evolve similar phenotypes by different mechanisms.     

Molecular cloning and expression of pyruvate kinase from globefish (Fugu rubripes) skeletal muscle

A cDNA clone encoding pyruvate kinase (PK) was isolated from a skeletal muscle cDNA library of globefish (Fugu rubripes), which is a kind of lower vertebrate. The full-length cDNA of globefish skeletal muscle pyruvate kinase (FM-PK) is approximately 2 kb and encodes a protein comprising 530 amino acids. The FM-PK gene is spanning approximately 4.8 kb and consists of 11 exons. FM-PK mRNA was detected in muscle and heart using Northern blots. The recombinant FM-PK (rFM-PK) was expressed in a baculovirus-insect cell system and purified using ion-exchange chromatography. The purified rFM-PK was shown to exist a 230 kDa homotetramer composed of 57 kDa subunits. Gel filtration showed 230000 as the tetramer of the subunit. The apparent K(m) (or S(0.5)) and the Hill coefficient for phosphoenolpyruvate (PEP) and ADP are 0.14 mM, 1.3 and 0.30 mM 0.98 at pH 7.4, respectively, when the enzyme is saturated with the second substrate. The rFM-PK is strongly activated by fructose-1,6-bisphosphate, the apparent K(m) for PEP changes to 0.059 mM and the Hill coefficient to 1.1. ATP, which is the product of the enzyme reaction, inhibits activity. This is the first report to show the full-length cDNA and amino acid sequence of PK for a species of fish.     

Serum gelatinases (MMP-2 and MMP-9) and VCAM-1 as a guideline in a therapeutic approach in Graves' ophthalmopathy

INTRODUCTION: The aim of this study was to estimate the influence of corticosteroids on soluble MMP-2, MMP-9 and VCAM-1 in patients with Graves ophthalmopathy (GO) in order to assess their usefulness as a guideline in a therapeutic approach. MATERIAL AND METHODS: Serum gelatinases and VCAM-1 were detected in three groups of subjects: 20 patients with GO (CAS > or = 3, anamnesis of GO > or = 1 yr), 12 patients with no clinical symptoms of ophthalmopathy (Gd) and 10 healthy volunteers. Corticosteroid therapy consisted of intravenous infusions (2 series, 3 grams each time) of methylprednisolone (MP) and subsequent treatment with oral prednisone (60 mg per day) in a tapering schedule. The serum samples were collected 24 hours before MP, 24 hours after MP, after 14 days of treatment with prednisone and at the end of corticosteroid therapy. The levels of soluble MMP-2, MMP-9 and VCAM-1 were determined by the ELISA method. RESULTS: We have found no differences in serum MMP-2 between the groups studied and a significant reduction after MP only in corticosteroid-resistant GO patients. Soluble MMP-9 was highest in the GO group compared with both the Gd and control individuals. Moreover serum MMP-9 decreased in corticosteroid-responsive GO patients after MP and remained at the lower level at the end of the study. Positive correlations between MMP-2 and MMP-9 before and after MP administration were observed. Serum VCAM-1 was significantly elevated both in GO and Gd subjects and pre-treatment VCAM-1 levels were elevated in corticosteroid-responders compared with non-responders. CONCLUSIONS: Our results suggest that serum VCAM-1 may serve as a marker predicting the efficacy of corticosteroids and that soluble MMP-9 may be helpful in monitoring corticosteroid administration and in decision-making with regard to further GO treatment.