Katsuhisa Uchida

Excellent salinity tolerance of Mozambique tilapia (Oreochromis mossambicus): elevated chloride cell activity in the branchial and opercular epithelia of the fish adapted to concentrated seawater.

Abstract Changes in morphology and cellular activity of the chloride cells in branchial and opercular epithelia were examined in tilapia, Oreochromis mossambicus, adapted to fresh water (FW), seawater (SW) and concentrated SW (180% SW). The tilapia are adaptable to a wide range of salinity, maintaining the plasma osmolality within physiological levels. Gill Na+, K+-ATPase activity was remarkably increased in response to elevated environmental salinity. Using immunocytochemical staining with an antiserum specific for α-subunit of Na+, K+-ATPase, chloride cells were detected on the afferent half of the filament epithelia. The size of immunoreactive chloride cells was twice larger in SW and four times larger in 180% SW than in FW. Confocal laser scanning microscopic observations revealed the frequent occurrence of chloride cell complexes under hypersaline conditions. By electron microscopy, a deeply invaginated apical crypt and well-developed tubular network were observed in chloride cells of SW- and 180% SW-adapted fish, indicating enhanced cellular activity. Chloride cells present in the opercular membrane were also developed in response to increased salinity. These findings suggest that highly activated chloride cells in branchial and opercular epithelia may be responsible for salt secretion in hyperosmotic environments. The excellent salinity tolerance of tilapia appears to be attributed to their ability to develop chloride cells in response to increased environmental salinity.

On the carapacial ridge in turtle embryos: its developmental origin, function and the chelonian body plan

The chelonian carapace is composed of dorsolaterally expanded ribs; an evolutionary change in the rib-patterning program is assumed to be related to this novelty. Turtle embryos exhibit a longitudinal ridge called the carapacial ridge (CR) on the flank, and its histological resemblance to the apical ectodermal ridge of the limb bud implies its inductive activity in the unique patterning of the ribs. We studied the Chinese soft-shelled turtle, Pelodiscus sinensis, and confirmed by labeling with a lipophilic dye, DiI, that the CR contains the somite-derived dermis and that it is a unique structure among amniotes. Using electroporation of a dominant-negative form of LEF-1, the CR-specific gene, we showed that CR-specific genes function in the growth and maintenance of the CR. Microcauterization or implantation of the CR did not change the dorsoventral pattern of the ribs, and only their fan-shaped pattern was arrested by CR removal. We conclude that the CR is a true embryonic novelty among amniotes and, because of the specific expression of regulatory genes, it functions in the marginal growth of the carapacial primordium, thereby inducing the fan-shaped arrangement of the ribs.

Evolutionary origin of a functional gonadotropin in the pituitary of the most primitive vertebrate, hagfish

Hagfish, which lack both jaws and vertebrae, are considered the most primitive vertebrate known, living or extinct. Hagfish have long been the enigma of vertebrate evolution not only because of their evolutionary position, but also because of our lack of knowledge on fundamental processes. Key elements of the reproductive endocrine system in hagfish have yet to be elucidated. Here, the presence and identity of a functional glycoprotein hormone (GPH) have been elucidated from the brown hagfish Paramyxine atami. The hagfish GPH consists of two subunits, α and β, which are synthesized and colocalized in the same cells of the adenohypophysis. The cellular and transcriptional activities of hagfish GPHα and -β were significantly correlated with the developmental stages of the gonad. The purified native GPH induced the release of gonadal sex steroids in vitro. From our phylogenetic analysis, we propose that ancestral glycoprotein α-subunit 2 (GPA2) and β-subunit 5 (GPB5) gave rise to GPHα and GPHβ of the vertebrate glycoprotein hormone family, respectively. The identified hagfish GPHα and -β subunits appear to be the typical gnathostome GPHα and -β subunits based on the sequence and phylogenetic analyses. We hypothesize that the identity of a single functional GPH of the hagfish, hagfish GTH, provides critical evidence for the existence of a pituitary-gonadal system in the earliest divergent vertebrate that likely evolved from an ancestral, prevertebrate exclusively neuroendocrine mechanism by gradual emergence of a previously undescribed control level, the pituitary, which is not found in the Protochordates.

Turtle–chicken chimera: An experimental approach to understanding evolutionary innovation in the turtle

Turtles have a body plan unique among vertebrates in that their ribs have shifted topographically to a superficial layer of the body and the trunk muscles are greatly reduced. Identifying the developmental factors that cause this pattern would further our understanding of the evolutionary origin of the turtles. As the first step in addressing this question, we replaced newly developed epithelial somites of the chicken at the thoracic level with those of the Chinese soft‐shelled turtle Pelodiscus sinensis (P. sinensis somites into a chicken host) and observed the developmental patterning of the grafted somites in the chimera. The P. sinensis somites differentiated normally in the chicken embryonic environment into sclerotomes and dermomyotomes, and the myotomes differentiated further into the epaxial and hypaxial muscles with histological morphology similar to that of normal P. sinensis embryos and not to that of the chicken. Epaxial dermis also arose from the graft. Skeletal components, however, did not differentiate from the P. sinensis sclerotome, except for small fragments of cartilage associated with the host centrum and neural arches. We conclude that chicken and P. sinensis share the developmental programs necessary for the early differentiation of somites and that turtle‐specific traits in muscle patterning arise mainly through a cell‐autonomous developmental process in the somites per se. However, the mechanism for turtle‐specific cartilage patterning, including that of the ribs, is not supported by the chicken embryonic environment. Developmental Dynamics 232:149–161, 2005.

Characterization of novel RFamide peptides in the central nervous system of the brown hagfish: Isolation, localization, and functional analysis

RFamide (RFa) peptides play various important roles in the central nervous system in both invertebrates and vertebrates. However, there is no evidence of the existence of any RFamide peptide in the brain of hagfish, one of the oldest lineages of vertebrates. In this study, we sought to identify novel RFamide peptides from the brains of hagfish (Paramyxine atami). We identified four novel RFamide peptides, which had the C-terminal Pro-Gln-Arg-Phe-NH2 structure. cDNA cloning revealed that the identified RFamide peptides are encoded in two types of cDNA. Molecular phylogenetic analysis of the two precursors indicated that the hagfish RFamide peptides belong to the PQRFamide peptide group that includes mammalian neuropeptide FF and AF. Based on immunohistochemistry and in situ hybridization, hagfish PQRFamide peptide precursor mRNA and its translated peptides were localized in the infundibular nucleus of the hypothalamus. Immunoreactive fibers were terminated on blood vessels in the infundibular nucleus. Dense immunoreactive fibers were also observed in other brain regions. We further showed that one of the hagfish PQRFamide peptides significantly stimulated the expression of gonadotropin-β mRNA in the cultured hagfish pituitary. These results indicate that the control mechanism of gonadotropin expression by a hypothalamic neuropeptide evolved in the agnathan brain. This is the first evidence describing the identification of RFamide peptides in the hagfish brain. This is also the first report showing the regulation of gonadotropin expression by a homolog of neuropeptide FF that belongs to the PQRFamide peptide group in any vertebrate.

cDNA cloning and isolation of somatolactin in Mozambique tilapia and effects of seawater acclimation, confinement stress, and fasting on its pituitary expression.

Somatolactin (SL) is a member of the growth hormone (GH)/prolactin (PRL) family of pituitary hormones, and is found in a variety of teleost species. Somatolactin is thought to be involved in a wide range of physiological actions, including reproduction, stress response, the regulation of Ca(2+) and acid-base balance, growth, metabolism, and immune response. We report here on the cDNA structure of SL from the pituitary of Mozambique tilapia, Oreochromis mossambicus, and its gene expression in response to seawater acclimation, stress, and fasting. Tilapia SL cDNA (1573bp long) encoded a prehormone of 230 amino acids. Sequence analysis of purified SL revealed that the prehormone is composed of a signal peptide of 23 amino acids and a mature protein of 207 amino acids, which has a possible N-glycosylation site at position 121 and seven Cys residues. Tilapia SL shows over 80% amino acid identity with SLalpha of advanced teleosts such as medaka and flounder, and around 50% identity with SLbeta of carp and goldfish. Acclimation to seawater had no effect on pituitary expression of SL or on hepatic expression of the putative tilapia SL receptor (GHR1). By contrast, seawater acclimation resulted in significant increases in pituitary GH expression and in hepatic expression of tilapia GH receptor (GHR2). Confinement stress had no effect on pituitary expression of either SL or GH, or on hepatic expression of GHR1, whereas a significant increase was seen in GHR2 expression in the liver. Fasting for 4 weeks resulted in significant reductions in SL transcripts both in fresh water and seawater. It is highly likely that SL is involved in metabolic processes in tilapia along with the GH/IGF-I axis.

Effects of environmental salinity on somatic growth and growth hormone/insulin-like growth factor-I axis in juvenile tilapia Oreochromis mossambicus

Hormonal control of growth was examined in juvenile Mozambique tilapia Oreochromis mossambicus under different environmental salinities. When juve nile tilapia were acclimated to fresh water (FW) or to full-strength sea water (SW) for 3 weeks, growth rate of the SW-acclimated fish doubled compared to FW fish. Pituitary mRNA levels of growth hormone (GH) were significantly higher in SW-adapted tilapia compared with FW fish, while the gene expression of prolactin (PRL188 and PRL177) in SW fish was lower than in the FW group. The release and synthesis of insulin-like growth factor-1 (IGF-1) were significantly elevated in the SW fish. This in dicates that the GH/IGF-1 axis is up-regulated in juvenile tilapia in SW and likely leads to the observed elevation in growth. Transfer of the juveniles from FW to 70% SW, while PRL188 mRNA levels were significantly elevated after transfer of the 70% SW fish to FW. Our findings indicate that the GH/IGF-I axis plays important roles both in growth and in osmoregulation in juvenile tilapia in SW.

Body plan of turtles: an anatomical, developmental and evolutionary perspective

The evolution of the turtle shell has long been one of the central debates in comparative anatomy. The turtle shell consists of dorsal and ventral parts: the carapace and plastron, respectively. The basic structure of the carapace comprises vertebrae and ribs. The pectoral girdle of turtles sits inside the carapace or the rib cage, in striking contrast to the body plan of other tetrapods. Due to this topological change in the arrangement of skeletal elements, the carapace has been regarded as an example of evolutionary novelty that violates the ancestral body plan of tetrapods. Comparing the spatial relationships of anatomical structures in the embryos of turtles and other amniotes, we have shown that the topology of the musculoskeletal system is largely conserved even in turtles. The positional changes seen in the ribs and pectoral girdle can be ascribed to turtle-specific folding of the lateral body wall in the late developmental stages. Whereas the ribs of other amniotes grow from the axial domain to the lateral body wall, turtle ribs remain arrested axially. Marginal growth of the axial domain in turtle embryos brings the morphologically short ribs in to cover the scapula dorsocaudally. This concentric growth appears to be induced by the margin of the carapace, which involves an ancestral gene expression cascade in a new location. These comparative developmental data allow us to hypothesize the gradual evolution of turtles, which is consistent with the recent finding of a transitional fossil animal, Odontochelys, which did not have the carapace but already possessed the plastron.

Regulation of growth hormone (GH) receptor (GHR1 and GHR2) mRNA level by GH and metabolic hormones in primary cultured tilapia hepatocytes

Growth hormone (GH) regulates essential physiological functions in teleost fishes, including growth, metabolism, and osmoregulation. Recent studies have identified two clades of putative receptors for GH (GHR1 clade and GHR2 clade) in fishes, both of which are highly expressed in the liver. Moreover, the liver is an important target for the anabolic effects of GH via endocrine IGFs, and liver sensitivity to GH is modulated by metabolic hormones. We investigated the effects of GH, insulin, glucagon, cortisol and triiodothyronine on GHR1 and GHR2 mRNA levels in primary cultured tilapia hepatocytes. Physiological concentrations of GH strongly stimulated GHR2 mRNA level (0.5-50×10(-9) M), but did not affect GHR1 mRNA level. Insulin suppressed stimulation of GHR2 mRNA level by GH (10(-8)-10(-6) M). Insulin increased basal GHR1 mRNA level (10(-8)-10(-6) M). Cortisol increased basal GHR2 mRNA level (10(-7)-10(-6) M), but did not consistently affect GH-stimulated GHR2 mRNA level. Cortisol increased basal GHR1 mRNA level (10(-9)-10(-6) M). Glucagon suppressed GH-stimulated GHR2 mRNA level and increased basal GHR1 mRNA level at a supraphysiological concentration (10(-6) M). A single injection of GH (5 μg/g) increased liver GHR2 mRNA level, and insulin injection (5 μg/g) decreased both basal and GH-stimulated GHR2 mRNA levels after 6 h. In contrast, insulin and GH injection had little effect on liver GHR1 mRNA level. This study shows that GHR1 and GHR2 gene expression are differentially regulated by physiological levels of GH and insulin in tilapia primary hepatocytes.

Gonadotropin-like and adrenocorticotropin-like cells in the pituitary gland of hagfish, Paramyxine atami : Immunohistochemistry in combination with lectin histochemistry

The pituitary system of the hagfish remains an enigma. The present study has aimed to detect possible adenohypophysial hormones in the pituitary gland of the brown hagfish, Paramyxine atami, by means of immunohistochemistry in combination with lectin histochemistry. Rabbit antisera raised against ovine luteinizing hormone (LH)β, proopiomelanocortin (POMC)-related peptides, and the growth hormone/prolactin family of tetrapod and fish species were used, and 25 kinds of lectins were tested. Three different types of adenohypophysial cells were revealed in the pituitary of brown hagfish. The first was stained with both anti-ovine LHβ and several D-mannose-binding lectins, such as Lens culinaris agglutinin and Pisum sativum agglutinin. This cell type predominated in the adenohypophysis in adults with developing gonads and thus appeared to be involved in the regulation of gonadal functions. The second was negative for anti-ovine LHβ but was stained with several N-acetylglucosamine-binding lectins, such as wheat germ agglutinin and Lycopersicon esculentum lectin. This cell type exhibited a weak positive reaction with anti-lamprey adrenocorticotropin (ACTH) and thus appeared to be related to POMC-like cells. The second cell type was found in the adenohypophysis regardless of the developmental state of the gonads. The third cell type was negative for both antisera and lectins. Since this cell type was numerous in juveniles and adults without developing gonads, most cells of this type were probably undifferentiated. These findings suggest that GTH and ACTH are major adenohypophysial hormones in the hagfish.