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Vertebrate pigmentation is a fundamentally important, multifaceted phenotype. Zebrafish, Danio rerio, has been a valuable model for understanding genetics and development of pigment pattern formation due to its genetic and experimental tractability, advantages that are shared across several Danio species having a striking array of pigment patterns. Here, we use the sister species D. quagga and D. kyathit, with stripes and spots, respectively, to understand how natural genetic variation impacts phenotypes at cellular and organismal levels. We first show that D. quagga and D. kyathit phenotypes resemble those of wild-type D. rerio and several single locus mutants of D. rerio, respectively, in a morphospace defined by pattern variation along dorsoventral and anteroposterior axes. We then identify differences in patterning at the cellular level between D. quagga and D. kyathit by repeated daily imaging during pattern development and quantitative comparisons of adult phenotypes, revealing that patterns are similar initially but diverge ontogenetically. To assess the genetic architecture of these differences, we employ reduced-representation sequencing of second-generation hybrids. Despite the similarity of D. quagga to D. rerio, and D. kyathit to some D. rerio mutants, our analyses reveal a complex genetic basis for differences between D. quagga and D. kyathit, with several quantitative trait loci contributing to variation in overall pattern and cellular phenotypes, epistatic interactions between loci, and abundant segregating variation within species. Our findings provide a window into the evolutionary genetics of pattern-forming mechanisms in Danio and highlight the complexity of differences that can arise even between sister species. Further studies of natural genetic diversity underlying pattern variation in D. quagga and D. kyathit should provide insights complementary to those from zebrafish mutant phenotypes and more distant species comparisons., Author summary Pigment patterns of fishes are diverse and function in a wide range of behaviors. Common pattern themes include stripes and spots, exemplified by the closely related minnows Danio quagga and D. kyathit, respectively. We show that these patterns arise late in development owing to alterations in the development and arrangements of pigment cells. In the closely related model organism zebrafish (D. rerio) single genes can switch the pattern from stripes to spots. Yet, we show that pattern differences between D. quagga and D. kyathit have a more complex genetic basis, depending on multiple genes and interactions between these genes. Our findings illustrate the importance of characterizing naturally occurring genetic variants, in addition to laboratory induced mutations, for a more complete understanding of pigment pattern development and evolution.

Vertebrate pigmentation is a fundamentally important, multifaceted phenotype. Zebrafish,Danio rerio, has been a valuable model for understanding genetics and development of pigment pattern formation due to its genetic and experimental tractability, advantages that are shared across severalDaniospecies having a striking array of pigment patterns. Here, we use the sister speciesD. quaggaandD. kyathit, with stripes and spots, respectively, to understand how natural genetic variation impacts phenotypes at cellular and organismal levels. We first show thatD. quaggaandD. kyathitphenotypes resemble those of wild-typeD. rerioand several single locus mutants ofD. rerio, respectively, in a morphospace defined by pattern variation along dorsoventral and anteroposterior axes. We then identify differences in patterning at the cellular level betweenD. quaggaandD. kyathitby repeated daily imaging during pattern development and quantitative comparisons of adult phenotypes, revealing that patterns are similar initially but diverge ontogenetically. To assess the genetic architecture of these differences, we employ reduced-representation sequencing of second-generation hybrids. Despite the similarity ofD. quaggatoD. rerio, andD. kyathitto someD. reriomutants, our analyses reveal a complex genetic basis for differences betweenD. quaggaandD. kyathit, with several quantitative trait loci contributing to variation in overall pattern and cellular phenotypes, epistatic interactions between loci, and abundant segregating variation within species. Our findings provide a window into the evolutionary genetics of pattern-forming mechanisms inDanioand highlight the complexity of differences that can arise even between sister species. Further studies of natural genetic diversity underlying pattern variation inD. quaggaandD. kyathitshould provide insights complementary to those from zebrafish mutant phenotypes and more distant species comparisons.Author SummaryPigment patterns of fishes are diverse and function in a wide range of behaviors. Common pattern themes include stripes and spots, exemplified by the closely related minnowsDanio quaggaandD. kyathit, respectively. We show that these patterns arise late in development owing to alterations in the development and arrangements of pigment cells. In the closely related model organism zebrafish (D. rerio) single genes can switch the pattern from stripes to spots. Yet, we show that pattern differences betweenD. quaggaandD. kyathithave a more complex genetic basis, depending on multiple genes and interactions between these genes. Our findings illustrate the importance of characterizing naturally occuring genetic variants, in addition to laboratory induced mutations, for a more complete understanding of pigment pattern development and evolution.

Using a recombinant chimeric single-chain follicle stimulating hormone (FSH), we established a radioimmunoassay (RIA) for red seabream (Pagrus major) FSH (pmFSH) which became a powerful tool for studying reproductive physiology. We studied the profiles in plasma and pituitary concentrations of FSH and luteinizing hormone (LH) during sexual maturation. A pre-established RIA for red seabream LH was used for the LH measurements. The regulation of FSH and LH secretion from the pituitary was investigated using a gonadotropin-releasing hormone analog (GnRHa) in vivo and in vitro. Marked differences in plasma and pituitary FSH levels were observed between males and females; pituitary FSH content in males was much higher than that in females during all seasons, and plasma FSH levels in males were high during the spawning season, whereas those in females were unchanged. In contrast, plasma and pituitary levels of LH were elevated before and during the spawning season in males and females. Injecting or implanting (cholesterol pellet) a GnRHa into adult and juvenile red seabream resulted in significant increases in plasma LH concentrations; however, no significant change was observed in plasma FSH. Moreover, GnRHa stimulated only LH secretion in an in vitro experiment using dispersed pituitary cells. The discrete FSH and LH secretion profiles revealed suggest differential roles for the two gonadotropins during red seabream gametogenesis. In addition, the marked difference in pituitary FSH levels in males and females suggests the relative significance of FSH in male reproduction.

Despite the common structure of vertebrates, the development of the vertebral column differs widely between teleosts and tetrapods in several respects, including the ossification of the centrum and the function of the notochord. In contrast to tetrapods, vertebral development in teleosts is not fully understood, particularly for large fish with highly ossified bones. We therefore examined the histology and gene expression profile of vertebral development in fugu, Takifugu rubripes, a model organism for genomic research. Ossification of the fugu centrum is carried out by outer osteoblasts expressing col1a1, col2a1, and sparc, and the growing centra completely divide the notochord into double cone-shaped segments that function as intercentral joints. In this process, the notochord basal cells produce a thick notochord sheath exhibiting Alcian-blue-reactive cartilaginous properties and composing the intercentral ligament in cooperation with the external ligament connective tissue. Synthesis of the matrix by the basal cells was ascertained by an in vitro test. Expression of twist2 indicates that this connective tissue is descended from the embryonic sclerotome. Notochord basal cells express sox9, ihhb, shh, and col2a1a, suggesting that the signaling system involved in chondrocyte proliferation and matrix production also functions in notochord cells for notochord sheath formation. We further found that the notochord expression of both ntla and shh is maintained in the fugu vertebral column, whereas it is turned off after embryogenesis in zebrafish. Thus, our results demonstrate that, in contrast to zebrafish, a dynamic morphogenesis and molecular network continues to function in fugu until the establishment of the adult vertebral column.

Pleuronectiform fish develop marked external asymmetry in eye location and skin color at metamorphosis. The bamboo sole, Heteromycteris japonica, also exhibits loss of the pectoral fins at metamorphosis. Because of its small body size, short generation time, and long spawning season, we focused on the bamboo sole as an experimental model to investigate metamorphic asymmetry and pectoral fin loss during development. In the present study, we utilized a small-scale culture system to evaluate bamboo sole larvae and larval development, and a microinjection system for fertilized eggs. The culture system described here uses an 18 L culture tank for rotifers (the first diet for larvae) and 5 L plastic beakers for larval culture. Under this system, most larvae completed metamorphosis, including one-eye migration and pigmentation of the ocular side, by 23 days postfertilization (dpf) at 25°C. Larvae at density of 120–150 per liter were grown from hatching to 23 dpf with a survival ratio of 60–75% per beaker. Pectoral fins, including coracoid and disk cartilage, formed but were completely lost in late metamorphosis without formation of proximal radials and fin rays. The microinjection system designed here is adequate for the bamboo sole and allows injection of 100 one-cell-stage embryos per day. We expect that the culture and microinjection systems described here will facilitate the use of the bamboo sole as an experimental model organism in developmental biology.

The effects of the removal of the oily surface film on initial swim bladder inflation and survival in cultured seven-band grouper Hyporthodus septemfasciatus were investigated during the larval stage. Additionally, the relationship between swim bladder inflation failure and malformations in later stages was examined. To explore promotion conditions for inflation, duplicate experiments were conducted under three different water surface conditions: removal of the oily surface film (ROF group), covering the water surface with an oil film (COF group), and sealing the water surface with liquid paraffin (SLP group). Larval swim bladder inflation was first observed 14 days after hatching (DAH) in the ROF group; thereafter, swim bladder inflation rates increased to 11.1% and 38.7% until the end of the experiments. This indicates that initial swim bladder inflation occurs at the pre-flexion stage in larval development. Conversely, bladder inflation was rarely observed in the COF or SLP groups, indicating the promotional effect of surface film removal on larval swim bladder inflation and suggesting that gulping atmospheric air is necessary for initial inflation. Although larval water surface death occurred frequently in the ROF group, no significant difference existed in any group between the survival rates at 10 DAH and at the end of the experiment. The incidence rate of spinal curvature and deformation of the centra was significantly higher in individuals without initial swim bladder inflation, and these malformations mainly occurred in the caudal vertebra. Therefore, the removal of the oily surface film promotes initial swim bladder inflation and reduces larval malformations.

The cause of abnormal musculature on both sides of the same myotome in artificially cultured seven-band grouper Epinephelus septemfasciatus (Thunberg) larvae remains unclear. We evaluated the effect of three factors: changes in temperature (between 22 and 24°C), disinfection with ozonated seawater (0.3 mg TROs L−1, 1 min), and hypoxia (22 or 14% saturation) during the early (20 h post-fertilization, hpf) and mid- (24 hpf) somite stages on trunk muscular development in larval seven-band grouper. The thermal regimes and disinfection with ozonated seawater had no effect on muscle development (P > 0.05, G-test). In contrast, exposure to hypoxia during both the early and mid-somite stages increased the incidence of abnormal musculature in the trunk (P

The kelp grouper (Epinephelus bruneus), belonging to one of the largest genera among the subfamily Epinephelinae, is a commercially important fish in Japan. There are limited data about the genomics of this species. To provide tools for addressing both population genetics studies and gene mapping, dito pentanucleotide simple sequence repeat (SSR) markers were developed using 454 pyrosequencing. Among the 1466 SSR markers developed, 1244 primer sets produced strong PCR products, of which 905 (72.7%) were polymorphic in kelp grouper. Cross-species utility of the 905 polymorphic SSR markers was tested in four additional Epinephelinae species of Hyporthodus septemfasciatus, Plectropomus leopardus, Epinephelus lanceolatus and Epinephelus coioides. Results revealed that, respectively, 401 (44.3%), 136 (15.0%), 434 (49.0%) and 538 (59.4%) SSRs showed specific polymorphic products. Of these, 40 SSR markers (33 di-, 1 tri- and 6 tetra-nucleotides) showed polymorphism in all species tested. Additionally, three AGAT SSR motifs which accounted for 42.9% of the nondi-nucleotide markers were found in the 40 SSR markers. This indicates that the AGAT SSR motif has a high potential as a highly versatile SSR marker in grouper Epinephelinae. The SSR markers developed in this study can be employed to obtain reliable genetic variability estimates for groupers (Epinephelinae).

The effects of rearing temperature (23–29 °C) during the larval and juvenile stage on survival, growth and skeletal malformations in the seven-band grouper Epinephelus septemfasciatus were investigated. The survival rate of juveniles 30–40 mm in total length emerging from eggs was higher at 25 and 26 °C (0.1–1.3 %) than at 23 °C or 27–29 °C (0.004–1.5 %). Growth (increase in total length) was accelerated at higher temperatures. The frequency of malformed individuals was lower at 25–27 °C (36.0–61.5 %) than at 23, 28 or 29 °C (65.3–76.9 %). Specific incidences of spinal curvature and centrum fusion or defects in juveniles were not related to rearing temperature. However, incidences of twisted or compressed vertebrae (6.5–64.0 %) were higher at higher temperatures, while the incidence of bifurcated neural spine was significantly higher at 23 °C (43.6–54.4 %) than at other temperatures (3.3–22.7 %). The incidence rate of spinal curvature (23.3 %) was significantly higher in juveniles with a deflated swim bladder, regardless of rearing temperature. The results of this study suggest that the optimum culture temperature for seven-band grouper is 25–26 °C, collectively considering the survival, growth and incidences of abnormalities. Our results also demonstrate the significance of identifying the conditions for swim bladder inflation to prevent spinal curvature in seven-band grouper.

Muscle development in the bamboo sole Heteromycteris japonicus was investigated, focusing primarily on the cranial muscles, using an improved whole mount immunohistochemical staining method with potassium hydroxide, hydrogen peroxide and trypsin. Larvae of H. japonicus had branchial levators, but not all of them were retained in adults, a condition also seen in the Japanese flounder Paralichthys olivaceus. In particular, larval branchial levators II and III disappeared during development, while I and IV remained to become the levator internus I and levator posterior, which were well-defined muscles in adults. In place of the atrophied muscles, levatores externi and levator internus II developed and regulated the branchial arches. The results showed that the muscle composition in the dorsal branchial arches changed to the adult form before metamorphosis in H. japonicus, as seen in P. olivaceus, and this transformation may be common to all members of that group.

There are a number of reports documenting skeletal development in groupers, but surprisingly little is known about muscle development. We have documented muscle development in the seven-band grouper Epinephelus septemfasciatus (Thunberg), focusing primarily on the cranial muscles from 0 days post-hatching (dph) to 28 dph, using a modified whole mount immunohistochemical staining method. The eye muscles developed first followed by the cheek muscles, which completed development prior to first feeding. The muscles associated with the opercle and the levator internus 1 developed gradually and were not linked to first feeding. Later, between 12 and 16 dph, the muscle composition in the dorsal branchial arches changed to the adult form. We observed a number of cultured larvae that exhibited abnormal musculature in the body and jaw. Our results provide the first documentation of muscle development in larval seven-band grouper. This information can be used to detect abnormalities during development and provide a marker to assess the effect of changes in larval rearing techniques.

Muscles play important roles in feeding, respiration and swimming during not only adult stage but also larval stage in fish. However, there is no information of the muscle development in larval greater amberjack, Seriola dumerili (Risso). Here, we investigated muscle development in the greater amberjack, focusing primarily on the cranial muscles from 0 days post hatch (dph) to 12 dph using a modified whole-mount immunohistochemical staining method. We found that the muscles required for feeding develop by 3 dph, when the larvae begin to feed. Subsequently, muscle composition in the dorsal branchial arches changes to the adult form between 5 and 8 dph. At 8 dph, all the muscles required for feeding and respiration appear, whereas the dorsal, pelvic and caudal fin muscles required for swimming develop later. This report provides fundamental information on larval greater amberjack muscle development, which will enable the detection of abnormal larval muscles and improve larval rearing techniques by modifying environmental conditions.

In mammals, the role of the suprachiasmatic nucleus (SCN) as the primary circadian clock that coordinates the biological rhythms of peripheral oscillators is well known. However, in teleosts, it remains unclear whether the SCN also functions as a circadian pacemaker. We used in situ hybridization (ISH) techniques to demonstrate that the molecular clock gene, per2, is expressed in the SCN of flounder (Paralichthys olivaceus) larvae during the day and down-regulated at night, demonstrating that a circadian pacemaker exists in the SCN of this teleost. The finding that per2 expression in the SCN was also observed in the amberjack (Seriola dumerili), but not in medaka (Oryzias latipes), implies that interspecific variation exists in the extent to which the SCN controls the circadian rhythms of fish species, presumably reflecting their lifestyle. Rhythmic per2 expression was also detected in the pineal gland and pituitary, and aperiodic per2 expression was observed in the habenula, which is known to exhibit circadian rhythms in rodents. Since the ontogeny of per2 expression in the brain of early flounder larvae can be monitored by whole mount ISH, it is possible to investigate the effects of drugs and environmental conditions on the functional development of circadian clocks in the brain of fish larvae. In addition, flounder would be a good model for understanding the rhythmicity of marine fish. Our findings open a new frontier for investigating the role of the SCN in teleost circadian rhythms.

Fugu (Takifugu rubripes) has contributed as an ideal model organism for understanding the structure and evolution of vertebrate genomes, but also has potential as a good model organism for developmental biology because of the availability of the genome information. However, there is no comprehensive report describing the developmental stages, which is fundamental data for developmental biology. Here we describe a series of stages of the embryonic development of fugu during the first 8 days after fertilization, i.e. from fertilization to hatching. We define seven periods of embryogenesis – the zygote, cleavage, blastula, gastrula, segmentation, pharyngula, and hatching periods. Stages subdividing these periods are defined based on morphological characteristics. In addition, as a model experiment of gene expression analysis using this staging series, we performed in situ hybridization of aldh1a2, aldh1a3 and cyp26a1 that play regulatory roles in retinoic acid (RA) metabolism essential for embryogenesis. This report provides fundamental information on fugu embryogenesis, which is anticipated to facilitate the use of fugu as a model organism for developmental studies.

The gastric glands of marine aquaculture fish species are known to start secretion of pepsinogens (Pep) during larval development, far after first feeding. Although characterization of gastric gland development is considered useful for managing larval feeding, the timing of initial Pep synthesis by the gastric gland has only been reported for a few species. Consequently, this study employed in situ hybridization (ISH) to characterize the onset of expression of genes encoding the gastric enzymes Pep and Chitinase (Chi), as well as the complete development of gastric glands in the larval stomach of Japanese flounder ( Paralichthys olivaceus ), spotted halibut ( Verasper variegatus ), seven-band grouper ( Epinephelus septemfasciatus ) and greater amberjack ( Seriola dumerili ). The expression of both Chi and Pep was observed to begin in the cardiac region of the larval stomach at 25 days post-hatching (dph) in Japanese flounder, 36 dph in spotted halibut, 38 dph in seven-band grouper and 13 dph in greater amberjack. The gastric glands of these species were fully developed in the stomach approximately 5–12 days after the initiation of Pep expression. Development of gastric glands was observed to coincide with external manifestations of metamorphosis, such as the development of adult coloration in Japanese flounder and spotted halibut of the Pleuronectiformes, while it finished prior to adult-type coloration in seven-band grouper and greater amberjack of the Perciformes.

The bilateral symmetry of flounder larvae changes through the process of morphogenesis to produce external asymmetry at metamorphosis. The process is characterized by the lateral migration of one eye and pigmentation at the ocular side. Migration of the left or right eye to produce either dextral or sinistral forms, respectively, is usually fixed within a species. Here we propose a mechanism for the mediation of lateralization by the nodal-lefty-pitx2 (NLP) pathway in flounders, in which pitx2, the final left-right determinant of the NLP pathway, is re-expressed in the left habenula at pre-metamorphosis. After the initiation of left-sided pitx2 re-expression, the eye commences migration, when the habenulae shift their position on the ventral diencephalon rightwards in sinistral flounder (Paralichthys olivaceus) and leftwards in dextral flounder (Verasper variegatus). In addition, the right habenula increases in size relative to the left habenula in both species. Loss of pitx2 re-expression induces randomization of eye-sidedness, manifesting as normal, reversed or bilateral symmetry, with laterality of the structural asymmetry of habenulae being entirely inverted in reversed flounders compared with normal ones. Thus, flounder pitx2 appears to be re-expressed in the left habenula at metamorphosis to direct eye-sidedness by lateralizing the morphological asymmetry of the habenulae.

Studies on the cellulose utilization by animals have been conducted in keeping with the recent developments in molecular biology. In mollusks, endogenous cellulases have been reported from blue mussel, abalone, and freshwater snail. We previously reported the possibility of cellulose assimilation by Corbicula japonica , a representative bivalve dominant in brackish water environments in Japan, and the cloning of its endogenous cellulase (β-1,4-glucanase) gene (Sakamoto, K., Touhata, K., Yamashita, M., Kasai, A. and Toyohara, H., 2007. Cellulose digestion by common Japanese freshwater clam Corbicula japonica . Fish. Sci. 73, 675–683). However, the gene of β-glucosidase, another enzyme essential for the complete cellulose decomposition to glucose units, has not yet been isolated from the mollusk. Therefore, we attempted the molecular cloning of endogenous β-glucosidase from C. japonica and succeeded in the isolation of a cDNA with a 2832-bp open reading frame (ORF) encoding 943 amino acid residues ( CjCel1A ). CjCEL1A has 2 repeated GHF-1(Glycosyl Hydrolase family 1)-like domains and showed high similarity with known insect β-glucosidases and mammalian lactase-phlorizin-hydrolases. Reverse transcription (RT)-PCR analysis and in situ hybridization revealed that CjCEL1A is likely to be produced in the secretory cells in the digestive gland, suggesting that CjCEL1A is a digestive β-glucosidase of C. japonica and is not derived from symbionts.

The occurrence of morphological deformities under different rearing water temperatures (18, 20, 22, 24, 26, 28, and 30 C) was examined in Japanese eel larvae. The rates of hatching and survival until yolk resorption at 22–26 C were higher than those at other water temperatures. Fertilized eggs never hatched at 18 and 30 C. The rates of occurrence of abnormal larvae reared at the water temperatures 24–28 C were lower than those at 20 or 22 C. Pericardial edema and lower jaw deformities occurred most frequently at lower temperatures (20 and 22 C). In contrast, the incubation temperature did not significantly affect the relative frequency of some neurocranial deformities and of spinal curvature. These results imply that the optimal temperatures for rearing Japanese eel eggs and embryos are 24–26 C from the viewpoints of survival and deformity.

Flounders form left-right asymmetry in body coloration during metamorphosis through differentiation of adult-type melanophores and xanthophores on the ocular side. As the first step in investigating the formation of flounder body coloration asymmetry, in this study, we aimed to determine where the precursors of adult-type chromatophores distribute in larvae before metamorphosis. In Paralichthys olivaceus and Verasper variegatus, GTP cyclohydrolase 2 (gch2), a common marker of melanoblasts and xanthoblasts, was found to be transiently expressed in cells located along the bilateral skeletal muscles at the basal parts of the dorsal and anal fins of premetamorphic larvae. When V. variegatus larvae were fed with a strain of Artemia collected in Brazil, this gch2 expression was abolished and the differentiation of adult-type melanophores was completely inhibited, while the density of larval melanophores was not affected. In a cell trace test in which the cells at the basal part of the dorsal fin were labeled with DiI at the premetamorphic stage, adult-type melanophores labeled with DiI were found in the skin on the ocular side after metamorphosis. These data suggest that, in flounder larvae, adult-type melanophores are distributed at the basal parts of the dorsal and anal fins as unpigmented precursor cells.

Preparation of high quality eggs is essential in aquaculture. While egg quality is widely regarded as being largely dependent on the nutrients that are stored in the yolk, the identity of the factors responsible for affecting this egg quality is not yet known. Thus, in order to ensure the quality of better quality eggs, it is essential to elucidate how the nutrients in yolk influence embryogenesis. Using a microinjection system, we performed two tests with zebrafish embryos to evaluate the effects of nutrients on embryogenesis. To determine whether retinoic acid, excess retinoids and carotenoids in yolk cause malformations, we injected retinoic acid, retinal, retinol and β-carotene into the yolk of 1–4 cell cell-stage embryos, and compared their effects on embryogenesis. The injection of retinal into yolk causes similar anomalies to those induced by retinoic acid in the brain, jaw, heart, as well as suppressing hatching rate. Retinol had a lower tetragenic effect than retinal and toxicity was not detected for β-carotene. These findings suggest that excess retinal and retinol in yolk can impair egg quality by causing malformations and decreasing hatching rate. We then compared the protective effects of astaxanthin, β-carotene and α-tocopherol against oxidative stress in embryos. After injecting one of these compounds into the yolk of a 1–4 cell-stage embryo, embryos were exposed to H 2 O 2 for 2 h from 24 h post-fertilization (hpf) and mortality and apoptosis were observed at 48 hpf. Astaxanthin conferred embryos with a marked tolerance against mortality and apoptosis due to H 2 O 2 exposure, suggesting its potential application to the improvement of egg quality by increasing the tolerance of embryos toward oxidative stress. The methodology presented herein describes a quick method for evaluating the effects of nutrients on egg quality.

Leptin is a key factor for the regulation of food intake and energy homeostasis in mammals, but information regarding its role in teleosts is still limited. There are large differences between mammalian and teleost leptin at both gene and protein levels, and in order to characterize the function of leptin in fish, preparation of species-specific leptin is therefore a key step. In this study, full-length cDNA coding for rainbow trout leptin was identified. In spite of low amino acid sequence similarity with other animals, leptin is highly conserved between trout and salmon (98.7%). Based on the cDNA, we produced pure recombinant trout leptin (rt-leptin) in E. coli , with a final yield of 20 mg/L culture medium. We then examined the effects of intraperitoneal (IP) injection of rt-leptin on feeding behavior and gene expression of hypothalamic NPY and POMCs (POMC A1, A2 and B) in a short-term (8 h) experiment. The rt-leptin suppressed food intake and led to transient reduction of NPY mRNA levels, while the expression of POMCs A1 and A2, was elevated compared with vehicle-injected controls. These results for rainbow trout are the first that describe a physiological role of leptin using a species-specific orthologue in teleosts, and they suggest that leptin suppresses food intake mediated by hypothalamic regulation. This anorexic effect is similar to that observed in mammals and frogs and supports that the neuroendocrine pathways that control feeding by leptin are ancient and have been conserved through evolution.

The processes underlying vertebral development in teleosts and tetrapods differ markedly in a variety of ways. At present, the molecular basis of teleost vertebral development and growth is poorly understood. Understanding vertebral development at the molecular level is important for aquaculture to prevent vertebral anomalies that can arise from a variety of factors, including excess vitamin A (all-trans retinol, VA) in the diet. To facilitate studies on teloest vertebral development, we performed transcriptome analysis of four month old flounder, Paralichthys olivaceus, vertebrae using next-generation sequencing. Expression profile obtained demonstrates that some members of the hh, bmp, fgf, wnt gene families, and their receptors, hox, pax, sox, dlx and tbx gene families and ntl, which are known to function in notochord and somite development in embryos, are expressed in the vertebrae. It was also showed that in addition to the retinoic acid receptor (Rar), the vertebrae express alcohol dehydrogenase 1 and retinal dehydrogenase 2 which convert VA to all-trans-retinoic acid (RA). The assembled contigs also included cytochrome p450 family members, which inactivate RA, as well as phosphatidylcholine-retinol O-acetyltransferase, which converts VA to all-trans-retinyl ester, a stock form of VA. These data suggest that in teleost vertebrae, expression of various signals and transcription factors which function in the notochord and somite development is maintained until adult stage, and RA metabolism and signaling are active to regulate transcription of RA-responsible genes, such as hedgehog and hox genes. This is the first transcriptome analysis of teleost fish vertebrae.

We identified cDNA coding for a homologue to mammalian leptin in puffer, Takifugu rubripes, using genomic synteny around the human leptin gene. In addition to significant sequence homologies, the puffer leptin (pLEP) displays characteristic structural features in common with mammalian leptin. The pLEP mRNA was expressed mostly in the liver that contained abundant lipids. In addition, homologues to pLEP were found in the databanks for three fish species (salmon, medaka, and Tetraodon) and two amphibians (salamander and Xenopus). The phylogenetic analysis shows rapid rates of molecular divergence among leptins from different vertebrate classes, but not between mammals and avians.

In teleosts, the embryonic fin fold consists of a peridermis, an underlying epidermis and a small number of mesenchymal cells. Beginning from such a simple structure, the fin skeletons, including the proximal and distal radials and lepidotrichia (finrays), develop in the dorsal fin fold at the larval stage. Their process of skeletogenesis and embryonic origin are unclear. Using flounder larvae, we report the differentiation process for chondrocytes and scleroblasts prior to fin skeletogenesis and the effects of retinoic acid (RA) on it. In early larvae, the mesenchymal cells grow between the epidermis and spinal cord to form a line of periodical condensations, which are proximal radial primordia, to produce chondrocytes. The prescleroblasts, which ossify the proximal radial cartilages, differentiate in the mesenchymal cells remaining between the cartilages. Then, mesenchymal condensations occur between the distal ends of the proximal radials, forming distal radial primordia, to produce chondrocytes. Simultaneously, condensations occur between the distal radial primordia and peridermis, which are lepidotrichia primordia, to produce prescleroblasts. Exogenous RA specifically inhibits the mesenchymal condensation prior to the proximal radial formation together with the down-regulation of sonic hedgehog (shh) and patched (pta) expression, resulting in the loss of proximal radials. Thus, it was indicated that differentiation of the precursor cells of radials and lepidotrichia begins in the proximal part of the fin fold and that the initial mesenchymal condensation prior to the proximal radial formation is highly susceptible to the effects of RA. Lepidotrichia formation does not occur where proximal radials are absent, indicating that lepidotrichia differentiation requires interaction with the radial cartilages. To examine the suggestion that neural crest cells contribute to the medial fin skeletons, we localized the HNK-1 positive cells in flounder embryos and slug and msxb-positive cells in pufferfish, Fugu rubripes, embryos. That the positive cells commonly arrive at the proximal part of the fin fold does not contradict the suggestion, but their final destiny as radial chondrocytes or lepidotrichia scleroblasts, should be further investigated.

We have cloned for the first time two cDNAs encoding distinct types of tissue inhibitor of metalloproteinases-2 (TIMP-2) from teleost fish, Japanese flounder, and designated these types as jfTIMP-2a and jfTIMP-2b. The open reading frames of the jfTIMP-2a and jfTIMP-2b cDNAs are composed of 663 and 657 nucleotides and 221 and 219 amino acids, respectively. Both jfTIMP-2s contain 12 cysteine residues, which might form six disulfide bonds as in other animals' TIMP-2s. The predicted full-length amino acid sequence of jfTIMP-2a has lower identity to jfTIMP-2b (63%) than to those of human (74%) and chicken (73%) TIMP-2s, but higher than to those of other human TIMPs (TIMP-1: 39%, TIMP-3: 43%, TIMP-4: 45%), indicating that jfTIMP-2a is a common TIMP-2, while jfTIMP-2b is unique to Japanese flounder. However, the C-terminal region including the last three disulfide bonds of jfTIMP-2b has higher amino acid identity to those of other animal TIMP-2s than to that of jfTIMP-2a. Reverse-transcribed polymerase chain reaction (RT-PCR) analysis showed the mRNAs of jfTIMP-2a and jfTIMP-2b to be ubiquitously expressed in all tissues examined, but with different expression patterns. These findings suggest that the two distinct jfTIMP-2s might perform different functions in teleost tissues.

Circadian rhythms enable organisms to coordinate multiple physiological processes and behaviors with the earth's rotation. In mammals, the suprachiasmatic nuclei (SCN), the sole master circadian pacemaker, has entrainment mechanisms that set the circadian rhythm to a 24-h cycle with photic signals from retina. In contrast, the zebrafish SCN is not a circadian pacemaker, instead the pineal gland (PG) houses the major circadian oscillator. The SCN of flounder larvae, unlike that of zebrafish, however, expresses per2 with a rhythmicity of daytime/ON and nighttime/OFF. Here, we examined whether the rhythm of per2 expression in the flounder SCN represents the molecular clock. We also examined early development of the circadian rhythmicity in the SCN and PG. Our three major findings were as follows. First, rhythmic per2 expression in the SCN was maintained under 24 h dark (DD) conditions, indicating that a molecular clock exists in the flounder SCN. Second, onset of circadian rhythmicity in the SCN preceded that in the PG. Third, both 24 h light (LL) and DD conditions deeply affected the development of circadian rhythmicity in the SCN and PG. This is the first report dealing with the early development of circadian rhythmicity in the SCN in fish.

In order to better understand the endocrine aberrations related to abnormal metamorphic pigmentation that appear in flounder larvae reared in tanks, this study examined the effects of continuous 24-h illumination (LL) through larval development on the expression of tyrosine hydroxylase-1 (th1), proopiomelanocortin (pomc), α-melanophore-stimulating hormone (α-MSH) and melanin concentrating hormone (MCH), which are known to participate in the control of background adaptation of body color. We observed two conspicuous deviations in the endocrine system under LL when compared with natural light conditions (LD). First, LL severely suppressed th1 expression in the dopaminergic neurons in the anterior diencephalon, including the suprachiasmatic nucleus (SCN). Second, pomc and α-MSH expression in the pars intermedia melanotrophs was enhanced by LL. Skin color was paler under LL than LD before metamorphic pigmentation, and abnormal metamorphic pigmentation occurred at a higher ratio in LL. We therefore hypothesize that continuous LL inhibited dopamine synthesis in the SCN, which resulted in up-regulation of pomc mRNA expression in the melanotrophs. In spite of the up-regulation of pomc in the melanotrophs, larval skin was adjusted to be pale by MCH which was not affected by LL. Accumulation of α-MSH in the melanotrophs is caused by uncoupling of α-MSH synthesis and secretion due to inhibitory role of MCH on α-MSH secretion, which results in abnormal metamorphic pigmentation by affecting differentiation of adult-type melanophores. Our data demonstrate that continuous illumination at the post-embryonic stage has negative effects on the neuroendocrine system and pituitary in flounder.

Fugu (Takifugu rubripes) has contributed as an ideal model organism for understanding the structure and evolution of vertebrate genomes, but also has potential as a good model organism for developmental biology because of the availability of the genome information. However, there is no comprehensive report describing the developmental stages, which is fundamental data for developmental biology. Here we describe a series of stages of the embryonic development of fugu during the first 8 days after fertilization, i.e. from fertilization to hatching. We define seven periods of embryogenesis - the zygote, cleavage, blastula, gastrula, segmentation, pharyngula, and hatching periods. Stages subdividing these periods are defined based on morphological characteristics. In addition, as a model experiment of gene expression analysis using this staging series, we performed in situ hybridization of aldh1a2, aldh1a3 and cyp26a1 that play regulatory roles in retinoic acid (RA) metabolism essential for embryogenesis. This report provides fundamental information on fugu embryogenesis, which is anticipated to facilitate the use of fugu as a model organism for developmental studies.

Pepsinogen is the precursor form of the gastric-specific digestive enzyme, pepsin. Ghrelin is a representative gastric hormone with multiple functions in vertebrates, including the regulation of growth hormone release, stimulation of food intake and gastrointestinal motility function. We investigated chronological changes in the distribution of pepsinogen-expressing cells by in situ hybridization and ghrelin-immunoreactive cells by immunohistochemistry in the Japanese eel (Anguilla japonica) during metamorphosis from the leptocephalus sage to the elver stage. The ghrelin-producing cells first appeared in the gastric cecum and pyloric portion of the stomach in the late phase of metamorphosing leptocephali, whereas the pepsinogen-producing cells were first detected in the early phase of the glass-eel stage. These suggest that endocrine cells differentiated earlier than exocrine cells in the eel stomach. Accompanying eel development, the distribution of ghrelin-producing cells spread to the esophagus and other regions of the stomach, but not to the intestine. These results may be related to the changes in dietary habits during metamorphosis in the Japanese eel.

We investigated muscle development in the Japanese flounder Paralichthys olivaceus, focusing primarily on the cranial muscles, using a whole mount immunohistochemical staining method. It is well established that during the very early stages of morphogenesis, until 4 days post hatching (dph), muscles required for feeding develop. Later, between 8 and 16 dph, the muscle composition in the dorsal branchial arches changes to the adult form. We discovered the presence of larval-specific muscles in this ontogenetic period, termed the larval branchial levators 2 and 3, located in the dorsal branchial arches. The larval branchial levators 2 and 3 disappear during the course of development, whereas the others remain as levator internus 1 and levator posterior, which have also been described in adult fish. In place of these regressed muscles, the levatores externi and levator internus 2 develop and regulate the branchial arches. In addition, we found that the levator posterior, which is thought to represent the fifth levator externus, and the levatores externi exhibit different origins. We also found that at least a part of the caudal fin musculature develops from the trunk myotome.

The bilateral symmetry of flounder larvae changes through the process of morphogenesis to produce external asymmetry at metamorphosis. The process is characterized by the lateral migration of one eye and pigmentation at the ocular side. Migration of the left or right eye to produce either dextral or sinistral forms, respectively, is usually fixed within a species. Here we propose a mechanism for the mediation of lateralization by the nodal-lefty-pitx2 (NLP) pathway in flounders, in which pitx2, the final left-right determinant of the NLP pathway, is re-expressed in the left habenula at pre-metamorphosis. After the initiation of left-sided pitx2 re-expression, the eye commences migration, when the habenulae shift their position on the ventral diencephalon rightwards in sinistral flounder (Paralichthys olivaceus) and leftwards in dextral flounder (Verasper variegatus). In addition, the right habenula increases in size relative to the left habenula in both species. Loss of pitx2 re-expression induces randomization of eye-sidedness, manifesting as normal, reversed or bilateral symmetry, with laterality of the structural asymmetry of habenulae being entirely inverted in reversed flounders compared with normal ones. Thus, flounder pitx2 appears to be re-expressed in the left habenula at metamorphosis to direct eye-sidedness by lateralizing the morphological asymmetry of the habenulae.

In addition to altering the phenotypes of gene-modified animals, transgenesis also has the potential to facilitate access to the various mechanisms underlying the development and functioning of specific phenotypes and genes, respectively. Myostatin (MSTN) is implicated in double-muscling when mutated in mammals, indicating that MSTN is a negative regulator of skeletal muscle formation. In order to elucidate the role of an MSTN equivalent in fish muscle formation, we created a transgenic medaka strain that expresses dominant-negative MSTN exclusively in skeletal muscle, d-rR-Tg(OlMA1-C315Y-MSTN-hrGFPII-FLAG). The transgenic fish exhibited increased production of skeletal muscle fibers at the adult stage (hyperplasia), although gross muscle mass was not altered. During embryogenesis, ectopic accumulation and misalignment of muscle fibers, possibly due to muscle-fiber hypertrophy, were observed in the transgenic medaka. Our findings suggest that MSTN function is required for regulating the appropriate growth of skeletal muscle in medaka. Unlike in mammals, MSTN loss-of-function failed to induce double-muscling in medaka, despite the highly conserved nature of MSTN function among taxa.

Flounders form left-right asymmetry in body coloration during metamorphosis through differentiation of adult-type melanophores and xanthophores on the ocular side. As the first step in investigating the formation of flounder body coloration asymmetry, in this study, we aimed to determine where the precursors of adult-type chromatophores distribute in larvae before metamorphosis. In Paralichthys olivaceus and Verasper variegatus, GTP cyclohydrolase 2 (gch2), a common marker of melanoblasts and xanthoblasts, was found to be transiently expressed in cells located along the bilateral skeletal muscles at the basal parts of the dorsal and anal fins of premetamorphic larvae. When V. variegatus larvae were fed with a strain of Artemia collected in Brazil, this gch2 expression was abolished and the differentiation of adult-type melanophores was completely inhibited, while the density of larval melanophores was not affected. In a cell trace test in which the cells at the basal part of the dorsal fin were labeled with DiI at the premetamorphic stage, adult-type melanophores labeled with DiI were found in the skin on the ocular side after metamorphosis. These data suggest that, in flounder larvae, adult-type melanophores are distributed at the basal parts of the dorsal and anal fins as unpigmented precursor cells.

Full-length cDNAs encoding the leptin receptor (tfLEPR), leptin receptor overlapping transcript (tfLEPROT) and leptin receptor overlapping transcript-like 1 (tfLEPROTL1) were cloned and sequenced from the pufferfish, Takifugurubripes. The tfLEPR gene encoded an 1116-amino acid protein that includes almost all functionally important domains conserved among vertebrate LEPR such as three fibronectin type III domains, the immunoglobulin (Ig) C2-like domain and a pair of repeated tryptophan/serine motifs. The tfLEPR mRNA was abundantly expressed in the pituitary and ovary and moderately expressed in brain, eye, heart, kidney, liver and testis. Both tfLEPROT and tfLEPROTL1 genes encoded a 130-amino acid protein. Human LEPR gene shares the first and second exons with the LEPROT gene, and they are continuously located on chromosome 1p31. In contrast, TakifuguLEPR and LEPROT were located at different regions of the chromosome. However, both Takifugu regions showed genomic synteny with the human genome around LEPR gene on chromosome 1p31. This result could mean that the Takifugu chromosomes around LEPR and LEPROT genes are paralogous genomic regions derived from genome duplication early in the teleost lineage and the overlapping LEPR and LEPROT genes were subsequently lost.

We previously reported on the endogenous cellulase gene of Corbicula japonica, CjCel9A. In this study, the tissue localization of the mRNA and translated products of CjCel9A was investigated in order to understand how this gene is physiologically involved in cellulose decomposition by C. japonica. Antiserum against recombinant CjCel9A protein was prepared. Multiple bands were observed mainly on western blot analysis of the crystalline style, and the band sizes partially corresponded to the active bands detected using zymographic analysis. In situ hybridization and immunohistochemical analyses clarified the exclusive production and secretion of this cellulase by the secretory cells localized in the epithelium of the digestive tubules in the digestive gland. These data strongly support our previous assumption that the endogenous cellulase of C. japonica is produced in the digestive gland and transported to the crystalline style to act as a component of its cellulolytic activity.

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.

The lefty gene encodes a member of the TGF-β superfamily that regulates L–R axis formation during embryogenesis via antagonistic activity against Nodal, another TGF-β superfamily member. Both mouse and zebrafish have two lefty genes, lefty1 and lefty2. Interestingly, the expression domains of mouse and zebrafish lefty are different from one another. At present, the orthology and functional diversity of the mouse and zebrafish lefty genes are not clear. Here, we report that flounder and two fugu species, Takifugu and Tetraodon, have a single lefty gene in their genomes. In addition, we provide evidence that the mouse lefty genes were duplicated on a single chromosome but the zebrafish lefty genes arose from a whole-genome duplication that occurred early in the divergence of ray-finned fishes. These independent origins likely explain the difference in the expression domains of the mouse and zebrafish lefty gene pairs. Furthermore, we found that the duplication corresponding to the zebrafish lefty2 gene was lost from the fugu genome, suggesting that loss of lefty2 in the fugu/flounder lineage occurred after its divergence from the zebrafish lineage. During L–R patterning, the single lefty gene of flounder covers two expression domains, the left side of the dorsal diencephalon and the left LPM, which are regulated separately by lefty1 and lefty2 in zebrafish. We infer that the lefty genes of the ray-finned fishes and mammals underwent independent gene duplication events that resulted in independent regulation of lefty expression.

Retinaldehyde dehydrogenases (raldhs) synthesize retinoic acid (RA), which is required for pattern formation and organogenesis during embryogenesis. To elucidate the common role of RA on vertebrate embryos, we first sought to clone a homologous gene to human raldh2 from fugu, Takifugu rubripes. We cloned a 1837 bp cDNA that encodes fugu raldh. The deduced amino acid sequence of the fugu raldh comprises 502 amino acids. The fugu Raldh showed highest sequence identity to zebrafish, Danio rerio, Raldh2 (79.9%). The fugu Raldh also showed high sequence identity to other vertebrate Raldh2: Xenopus laevis (77.2%), human (77.4%), mouse (74.3%) and chick (73.9%). Comparative genomic analysis showed that the gene arrangement around fugu raldh agreed with that of human raldh2. Fugu raldh mRNA was expressed through embryogenesis similarly to raldh2 in other vertebrates. These results and phylogenetic analyses suggest that pufferfish raldh is a fugu orthologue of other species' raldh2.

Pepsinogen is a precursor of pepsin, a gastric specific protease belonging to the aspartic proteinase family. In teleosts, several species, such as zebrafish and puffer, have independently lost gastric glands. So whether puffer have pepsinogen gene or not is an interesting issue. A search of GSS database for pufferfish, Takifugu rubripes, revealed five different aspartic proteinase genes in its genome. One of them (pPep) has typical pepsinogen structure and belongs to the fish pepsinogen cluster by phylogenic analysis. The pPep antisense probe hybridized to the gastric glands of flounder stomach. Therefore, we concluded that pPep is a pufferfish pepsinogen. The pufferfish pepsinogen mRNA was not expressed in the digestive organs but specifically in the skin. We speculated that while Tetraodontiformes evolutionarily lost gastric glands, pufferfish pepsinogen acquired an alternative function to food digestion.

Three different chitinase genes (fChi1, fChi2 and fChi3) were identified from Japanese flounder, Paralichthys olivaceus. The deduced amino-acid sequences of flounder chitinases revealed a typical chitinase structure containing a catalytic glyco-18 domain, a hinge region and a chitin binding domain type 2. The fChi1 and fChi2 mRNAs were predominantly expressed in the gastric glands of stomach. In contrast, expression of fChi3 was found in spleen, pancreas, stomach, intestine, liver, kidney and gonads of adult flounder by RT-PCR. The expression level of fChi3 in the adult tissues was below the detection limit of in situ hybridization (ISH) analysis; however, ISH signals were detected in the liver of flounder larvae. These results suggest that fChi1 and fChi2 are acidic chitinases that digest dietary chitin and that fChi3 probably is a macrophage specific chitinase (chitotriosidase) for biodefense and has an important unknown role in the liver during larval stages.