Kiyoshi Kikuchi

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.

Novel sex‐determining genes in fish and sex chromosome evolution

Although the molecular mechanisms underlying many developmental events are conserved across vertebrate taxa, the lability at the top of the sex‐determining (SD) cascade has been evident from the fact that four master SD genes have been identified: mammalian Sry; chicken DMRT1; medaka Dmy; and Xenopus laevis DM‐W. This diversity is thought to be associated with the turnover of sex chromosomes, which is likely to be more frequent in fishes and other poikilotherms than in therian mammals and birds. Recently, four novel candidates for vertebrate SD genes were reported, all of them in fishes. These include amhy in the Patagonian pejerrey, Gsdf in Oryzias luzonensis, Amhr2 in fugu and sdY in rainbow trout. These studies provide a good opportunity to infer patterns from the seemingly chaotic picture of sex determination systems. Here, we review recent advances in our understanding of the master SD genes in fishes. Developmental Dynamics 242:339–353, 2013.

Integration of the Genetic Map and Genome Assembly of Fugu Facilitates Insights into Distinct Features of Genome Evolution in Teleosts and Mammals

Abstract The compact genome of fugu (Takifugu rubripes) has been used widely as a reference genome for understanding the evolution of vertebrate genomes. However, the fragmented nature of the fugu genome assembly has restricted its use for comparisons of genome architecture in vertebrates. To extend the contiguity of the assembly to the chromosomal level, we have generated a comprehensive genetic map of fugu and anchored the scaffolds of the assembly to the 22 chromosomes of fugu. The map consists of 1,220 microsatellite markers that provide anchor points to 697 scaffolds covering 86% of the genome assembly (http://www.fugu-sg.org/). The integrated genome map revealed a higher recombination rate in fugu compared with other vertebrates and a wide variation in the recombination rate between sexes and across chromosomes of fugu. We used the extended assembly to explore recent rearrangement events in the lineages of fugu, Tetraodon, and medaka and compared them with rearrangements in three mammalian (human, mouse, and opossum) lineages. Between the two pufferfishes, fugu has experienced fewer chromosomal rearrangements than Tetraodon. The gene order is more highly conserved in the three teleosts than in mammals largely due to a lower rate of interchromosomal rearrangements in the teleosts. These results provide new insights into the distinct patterns of genome evolution between teleosts and mammals. The consolidated genome map and the genetic map of fugu are valuable resources for comparative genomics of vertebrates and for elucidating the genetic basis of the phenotypic diversity of ∼25 species of Takifugu that evolved within the last 5 My.

Fugu immunoglobulin D: a highly unusual gene with unprecedented duplications in its constant region

We have isolated and characterized the cDNA that encodes IgD of fugu (Takifugu rubripes). Though the splicing of μ1 with the δ1 domain was similar to those reported for teleost IgDs, highly unusual and unprecedented domain duplications were found in the constant region of the fugu IgD. The structure of the fugu IgD is like VDJ-μ1-(δ1-δ2-δ3-δ4-δ5-δ6)2-δ7-δm1-δm2. Genomic sequence analysis of the fugu IgD gene supported the results of cDNA sequencing that the first six δ domains in the constant region are duplicated. Such a novel duplication pattern has not been reported in any other vertebrates. However, IgD secretory domains could not be identified in this study. The deduced amino acid sequence of the fugu IgD constant region showed high identity (35–55%) to the sequences of previously reported teleost IgDs. Gene expression analyses based on RT-PCR demonstrated that the IgD gene is preferentially expressed in presumptive lymphoid tissues; moreover, in situ hybridization showed that IgD-positive cells are distributed throughout the spleen and head kidney. The expression pattern is similar to that of IgM, corroborating the hypothesis that IgD plays an important role in the humoral immune system of this species.

Carp expresses fast skeletal myosin isoforms with altered motor functions and structural stabilities to compensate for changes in environmental temperature

1. 1. Myosin and its subfragment-1 (Sl) from carp acclimated to 10°C showed higher actin-activated Mg2+-ATPase activity and lower thermostability than their counterparts from carp acclimated to 30°C. Accordingly, filament velocity for the 10°C-acclimated carp myosin was higher at any measuring temperatures from 3 to 23°C than that for the 30°C-acclimated carp myosin. 2. 2. Three types of cDNA clones encoding myosin heavy chains were isolated from thermally acclimated carp. The 10 and 30°C types were predominating in carp acclimated to 10 and 30°C, respectively, whereas the intermediate type was found as a minor component in the 10°C-acclimated carp with an intermediate feature in both DNA nucleotide and deduced amino acid sequences between those of the 10 and 30°C types. 3. 3. The three types of myosin rod all showed a typical coiled-coil structure of α-helices. DSC scans demonstrated that myosin rod prepared from carp acclimated to 10°C had a lower thermostability than that from carp acclimated to 30°C, showing that low thermostability in cold-acclimated carp myosin prevails over the entire molecule. 4. 4. cDNA clones encoding myosin alkali light chains were isolated from thermally acclimated carp. Northern blot analysis showed that the ratios of LC3LC1 mRNAs were significantly higher (3.92) in the 30°C- than 10°C-acclimated (3.10) carp.

Characterization of CD8+ leukocytes in fugu (Takifugu rubripes) with antiserum against fugu CD8α

We have investigated the characteristics of CD8+ leukocytes by using an anti-CD8alpha antiserum raised in mouse by DNA-immunization. The magnetically sorted CD8alpha+ peripheral blood leukocyte (PBL) population comprised lymphocytes/thrombocytes and monocytes, whereas CD8alpha- PBLs consisted of lymphocytes/thrombocytes, monocytes, and neutrophils. Expression analysis demonstrated that both groups of cells expressed the CD3epsilon and TCRalpha genes. The CD8alpha and CD8beta genes were detected only in CD8alpha+ cells, whereas expression of CD4 and immunoglobulin light chain (IgL) was observed only in CD8alpha- cells. These results suggest that fugu CD8alpha+ leukocytes contain CD8+ T cells, but not CD4+ T cells or B cells. Furthermore, mitogenesis of the CD8+ lymphocyte/thrombocyte population was induced by phytohemaglutinin stimulation, suggesting that fish CD8+ lymphocytes/thrombocytes (probably CD8+ T cells) have characteristics similar to mammalian CD8+ T cells. Neutrophils and monocytes/macrophages infiltrating a subcutaneous inflammatory site expressed only CD8alpha, but not CD8beta, CD4, TCRalpha, or IgL. This result suggests that similar to mammalian dendritic cells, fugu monocytes/macrophages express CD8alpha.

Characterization and expression analysis of CD3ɛ and CD3γ/δ in fugu, Takifugu rubripes

CD3 is an essential component of the CD3-TCR complex. In this report, we describe the cloning, characterization, and expression analysis of the CD3ɛ and CD3γ/δ chain genes from fugu, Takifugu rubripes. Two distinct CD3ɛ homologue cDNAs, designated as CD3ɛ-1 and CD3ɛ-2, and a CD3γ/δ homologue cDNA were isolated from the fugu thymus. The deduced amino acid sequences of these cDNAs exhibit conserved essential CD3 chain motifs and overall structures. RT-PCR analysis demonstrated that the CD3ɛ and CD3γ/δ genes were expressed in lymphoid organs (e.g. thymus, head kidney, trunk kidney and spleen), mucosal tissues (gill, skin, and intestine), and peripheral blood leucocytes (PBL). The CD3 and TCRα genes were expressed only in the surface IgM− population, which were separated from PBL using an anti-fugu IgM monoclonal antibody. In addition, in situ hybridization confirmed that CD3-expressing cells were distributed randomly in the head kidney, trunk kidney, and spleen, but in the thymus were restricted to the lymphoid outer zone and epithelioid inner zone only. Collectively, these results suggest that CD3 molecules are useful markers for the identification of T cells in teleost fish. The present study thus provides a critical step in identifying T cells in this model organism.

The novel sequences of major plasma apolipoproteins in the eel Anguilla japonica

cDNAs encoding major plasma apolipoproteins (apo) were cloned from the eel Anguilla japonica liver and their nucleotide sequences determined. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that eel lipoproteins contain apolipoproteins of 28 kDa and 14 kDa as major components. Each of the two apolipoproteins showed two isoforms having different isoelectric points as demonstrated by two-dimensional electrophoresis. The two 28 kDa components had different N-terminal amino acid sequences, whereas the two 14 kDa components had an identical one. Then cDNA clones encoding these apolipoproteins were isolated from a cDNA library constructed from the eel liver. An acidic 28 kDa component (28 kDa-1) consisted of 259 amino acids including a putative signal peptide of 27 residues, whereas a basic 28 kDa component (28 kDa-2) was composed of 260 amino acids containing a putative signal peptide of 23 residues. The tandem repeating units, which are characteristic of apolipoproteins, for 28 kDa-1 showed 27.8% identity to that of porcine apoA-IV, although mammalian apoA-IV is about 40 kDa and much larger than 28 kDa-1. However, the repeating units of 28 kDa-2 showed 52.5% identity to that of Atlantic salmon apoA-I. The 14 kDa apolipoprotein consisted of 142 amino acids containing a putative signal peptide of 20 residues. It has a novel sequence differing from apolipoproteins of other vertebrates. The transcriptional expressions of 28 kDa-1, 28 kDa-2, and 14 kDa components were all restricted to the liver, except for the transcripts of 28 kDa-2 which were also slightly expressed in the intestine.

Teleost B7 Expressed on Monocytes Regulates T Cell Responses

In mammals, professional APCs induce adaptive immunity via the activation of T cells. During this process, B7 family molecules present upon APCs are known to play crucial roles in optimal T cell stimulation. In contrast, the confirmation of APCs in a nonmammalian vertebrate has yet to be achieved. To obtain further insights into the evolutionary origin of APCs, we have identified three members of the B7 family in the teleost Takifugu rubripes (fugu): B7-H1/DC, B7-H3, and B7-H4. The three fugu B7s were expressed on the surface of blood monocytes. The B7+ monocytes, which are composed of at least two distinct populations, expressed the MHC class II component gene. The fugu B7 molecules bound to activated T cells, indicating that putative B7 receptors were expressed upon T cells. Fugu B7-H1/DC inhibited T cell proliferation concomitant with increasing levels of both IL-10 and IFN-γ expression, whereas both B7-H3 and B7-H4 promoted T cell growth following IL-2 induction and the suppression of IL-10. These observations indicate that fugu B7s regulate T cell responses via receptors upon T cells. We suggest that fish B7+ monocytes are APCs and that a costimulatory system has already developed in fish via the evolutionary process.

The 65-kDa cytosolic protein associated with warm temperature acclimation in goldfish, Carassius auratus

Goldfish Carassius auratus were acclimated to either 10 or 30°C for a minimum of 5 weeks. A 65-kDa protein specific to warm-temperature-acclimated fish was extracted from the gel with 70% formic acid after two-dimensional electrophoresis of the muscle cytoplasmic protein fraction. The 65-kDa protein thus prepared to homogeneity was used to raise specific antibodies in rabbit by conventional methods. The antibody produced exhibited specific reaction with a protein having the same molecular weight from brain and liver tissue, suggesting that the 65-kDa protein is a ubiquitous cytosolic component in warm-acclimated goldfish. When water temperature was increased from 20 to 30°C over a 20-h period, a prominent amount of the 65-kDa protein was observed in muscle tissue extracts within 5 days of additional rearing; this was demonstrated by immunoblotting with the specific antibody. The N-terminal amino acid sequence of the 65-kDa protein was determined as Asp-Glu-Pro-Gln-Gly-His-Gln-His (or Asp)-Glu-Leu, differing from that of a family of known heat-shock proteins having about 70 kDa in molecular mass (hsp 70). No interaction between ATP and the 65-kDa protein revealed by ATP-agarose affinity chromatography further confirmed the different properties of the 65-kDa protein from those of hsp 70.