Left-right pigmentation pattern of Japanese flounder corresponds to expression levels of melanocortin receptors (MC1R and MC5R), but not to agouti signaling protein 1 (ASIP1) expression.

Body coloration in flatfish is one of the most distinctive asymmetries in the animal kingdom, although the fundamental molecular mechanism of the pigmentation is unclear. In the dorso-ventral coloration (countershading) of other teleost fishes, ventral-specific expression of agouti signaling protein 1 (ASIP1), an endogenous antagonist of melanocortin 1 receptor (MC1R), has been reported to play a pivotal role. Contribution of ASIP1 is also suggested in the asymmetrical pigmentation of flatfish. In order to confirm the contribution of ASIP1 and further examine receptor function in the body coloration of Japanese flounder, expression levels of asip1, mc1r, melanocortin 5 receptor (mc5r), and melanin-concentrating hormone receptor 2 (mchr2) were measured in the normally pigmented area of the left side, the normally non-pigmented area of the right side, and the abnormally pigmented (exhibiting hypermelanosis) area of the right side. Measurement was also carried out under conditions of hypermelanosis stimulated by cortisol and during the transition from non-pigmentation to pigmentation in areas of hypermelanosis. Contrary to our expectations, no difference was detected in asip1 expression between pigmented and non-pigmented areas. There was also no difference between normal and hormonally stimulated pigmented conditions in areas of hypermelanosis or during the transition process. Instead, the expression levels of mc1r, mc5r, and mchr2 were consistently higher in pigmented areas, and were especially increased under hormonally stimulated conditions. In addition, expressions of these receptor genes increased prior to pigmentation in areas of future hypermelanosis. Our results suggest that MC1Rand MC5R, but not necessarily ASIP1, contribute to pigmentation and hypermelanosis in Japanese flounder. We propose a yet unknown molecular mechanism for asymmetrical pigmentation in flatfish that is distinct from that of countershading in other vertebrates.

Ontogenetic transition from aquatic to amphibious life in the mudskipper, Periophthalmus modestus.

Vertebrates first emerged from water to land in the Paleozoic. Our understanding about the process has been steadily refined through paleontological studies, although the soft-body traits and behavior of these early animals remain poorly known. Mudskippers, extant amphibious gobies, could give insight into this question. This study reports on the ontogenetic transition from water to land of the mudskipper Periophthalmus modestus under laboratory conditions. After about 30 days after hatching (dah), the fish gradually changed their preference from water to an artificial shore and then to land. After about five days of periodic volitional emersion, the fish became able to propel themselves on land using the pectoral fins and after a further 13 days they began feeding on land. During the transition, the head morphology altered to suit for terrestrial existence. Tissue contents of triiodothyronine (T3) and thyroxine (T4) sharply increased at 30 dah. Forced underwater confinement of larvae at the last pelagic stage (27-29 dah) for 40-42 days resulted in no statistically significant difference in survival or gross morphology of the body and the gills. Growth was slightly stimulated. Our results show that mudskippers emerge on land with little morphological alteration during ontogenesis, much less than the changes observed for amphibians, and that emersion was not indispensable for survival or growth under our laboratory conditions. Further analysis of how and why mudskippers make their way across the water's edge will shed valuable light on what morphological, behavioral and physiological traits were needed for, and what environmental conditions may have driven the earliest steps of the water-to-land transition in ancient fishes.

Developmental changes in melanophores and their asymmetrical responsiveness to melanin-concentrating hormone during metamorphosis in barfin flounder (Verasper moseri).

Barfin flounder larvae exhibit unique black coloration, as well as left-right asymmetry in juvenile stage as in other flatfish. In this study, we first assessed the changes in melanophores with development and then investigated their responsiveness to melanin-concentrating hormone (MCH) during metamorphosis. Larval-type melanophores appeared on both sides of the body before metamorphosis, whereas adult-type melanophores appeared only on the ocular side after metamorphosis. Even in the individuals of this species displaying black coloration, the density of larval-type melanophores was similar to that in transparent larvae of other species. However, unlike in transparent larvae, larval-type melanophores completely dispersed in the black larvae of this species. Therefore, the black coloration during larval stages was mainly due to dispersion, and not the density, of larval-type melanophores. In vitro MCH treatment revealed, for the first time, the responsiveness of melanophores in larval stages. On the ocular side, larval-type melanophores aggregated against MCH during larval stages, while, in the larvae at later metamorphic stages and in juveniles, larval-type melanophores did not aggregate, although aggregation of adult-type melanophores was noted. In contrast, on the blind side, the responsiveness of larval-type melanophores to MCH was consistently present from larval to juvenile stages. The metamorphic transition of MCH responsiveness from larval- to adult-type melanophores only on the ocular side suggests the larval (therefore, immature) nature of the blind side skin. We propose that the inhibited development, and thus the retention of the larval-type skin leads to the formation of the blind side characteristics and is the central mechanism for the flatfish asymmetry.

Evidence for an ontogenetic change from pre-programmed to meal-responsive cck production in Atlantic herring, Clupea harengus L.

The effects of up to three days of food deprivation on the cholecystokinin (CCK)-producing cells in the Atlantic herring gut were assessed by quantifying the number of cells detected by in situ hybridization at three ontogenetic stages. In feeding larvae that still possessed yolk-sacs (2 and 8days after hatch, DAH), intestinal cck expression appeared to be maintained regardless of external nutritional conditions. In 30 DAH-old herring larvae with well-established exogenous feeding only, very few CCK-producing cells could be identified, indicating that cck production in the gut had shut down after three days of starvation. This suggests that cck transcription is pre-programmed by a local timer in the midgut during the yolk-sac stage, regardless of the nutritional status and presence of nutrients in the gut lumen; however, it becomes strongly influenced by the external nutritional conditions after the yolk has been completely absorbed. Our results suggest that CCK-producing cells in the gut develop "meal-responsiveness" later in post-hatch development.

Further evidence on acetylation-induced inhibition of the pigment-dispersing activity of α-melanocyte-stimulating hormone.

Our previous studies showed that in barfin flounder, α-melanocyte-stimulating hormone (α-MSH) stimulates pigment dispersion in xanthophores, while it shows negligible effects in melanophores. The present study was undertaken to evaluate whether these results are limited to barfin flounder by using Japanese flounder. Three subtypes of proopiomelanocortin gene encoding melanocortins (MCs) were expressed in the Japanese flounder pituitary, one of which was also expressed in the skin. Expression of melanocortin 5 receptor gene (Mc5r) was observed in isolated xanthophores, while that of Mc1r and Mc5r was found in melanophores. In the xanthophores of Japanese flounder skin, α-MSH as well as desacetyl (Des-Ac)-α-MSH and diacetyl (Di-Ac)-α-MSH exhibited dose-dependent pigment-dispersing activities, indicating that the signals of α-MSH-related peptides were mediated by MC5R. On the other hand, α-MSH did not stimulate pigment dispersion in melanophores, while Des-Ac-α-MSH and Di-Ac-α-MSH did, thus indicating that the expression of two different types of Mcr is related to the decrease in α-MSH activity. Thus, the molecular repertoire in MC system observed in Japanese flounder is similar to that in barfin flounder. Moreover, the relationship between the pigment-dispersing activities of α-MSH-related peptides and the expression of Mcr subtypes in xanthophores and melanophores were also similar between Japanese flounder and barfin flounder. Consequently, we hypothesize that inhibition of α-MSH activity could be due to the formation of heterodimers comprising MC1R and MC5R, often observed in G-protein-coupled receptors.

Defective fin regeneration in medaka fish (Oryzias latipes) with hypothyroidism.

Wild-type medaka are known to have remarkable capabilities of fin, or epimorphic, regeneration. However, a hypothyroid mutant, kamaitachi (kmi), frequently suffers from injury in fins, suggesting an important role of thyroid hormone in fin regeneration. This led us to examine the relationship between thyroid hormone and fin regeneration using medaka as a model. For this, we first set up a medaka experimental system in which the rate of regeneration was statistically analyzed after caudal fin amputation under normal and hypothyroid conditions. As expected, the regeneration of amputated caudal fins was delayed in hypothyroid kmi -/- mutants. We then examined wild-type medaka with thiourea-induced hypothyroidism to evaluate the requirement of thyroid hormone during epimorphic fin regeneration. The results demonstrate that the growth rate of regenerates was much reduced in severely hypothyroid medaka throughout the regeneration period. This reduction in regenerative rate was recovered by exogenous administration of L-thyroxine. The present study is thus the first to report the direct involvement of thyroid hormone in teleost fin regeneration, and provides a basic framework for future molecular and genetic analyses.

Isolation and characterization of a Japanese flounder clonal line, reversed, which exhibits reversal of metamorphic left-right asymmetry.

We have isolated a clonal line reversed (rev) of homozygous Japanese flounder through gynogenesis. The homozygous offspring gynogenetically produced from rev exhibited reversal of organization of the metamorphic L/R asymmetry such as the direction of eye-migration at a high frequency (20-30%). The molecular analysis using a left-specific marker pitx2 revealed that the embryonic L/R axis was ambiguously established: in more than half of rev embryos, pitx2 was expressed bilaterally in the lateral plate mesoderm (LPM). Previous studies in other animals demonstrated that ectopic pitx2 expression in the LPM could cause laterality defects of the visceral organs. Likewise, our results using rev imply that bilateral pitx2 expression could lead to randomization of the visceral organs. Coincidence of ectopic pitx2 expression and reversal of the direction of eye-migration in the population of rev offspring suggests that the rev locus is critical in specification of both the metamorphic and the visceral L/R asymmetries. However, reversal of the sidedness of the orientation of the visceral organs was not always accompanied by reversal of the direction of metamorphic eye-migration, suggesting that different mechanisms should be involved downstream of the rev locus in directing these two phases of asymmetric morphogenesis in the Japanese flounder.

Cholecystokinin mRNA in Atlantic herring, Clupea harengus--molecular cloning, characterization, and distribution in the digestive tract during the early life stages.

The mRNA of the peptide hormone cholecystokinin (CCK) was isolated from juvenile Atlantic herring, Clupea harengus, by RT-PCR. The open reading frame encodes a 137 amino acid-long precursor protein. The peptide sequence of herring CCK-8, DYMGWMDF, is identical to that of higher vertebrates and elasmobranchs, and contains methionine in the sixth position from the C-terminus, which has not been reported previously in teleosts. Expression analysis by in situ hybridization shows that positive endocrine-like cells were mainly located in the pyloric caeca and to a less extent in the rectum of the juvenile. A few positive cells were also found in the pyloric portion of the stomach and the intestine. CCK cells were present in all the larvae examined from the day of hatching onwards. Although the CCK cells were scattered throughout the whole midgut, no signals were detected in either the foregut or the hindgut. Since herring larvae have a straight gut, the distribution pattern of CCK cells seems to be reflected in the anatomy of the gut.

Fine structure of soft and hard tissues involved in eye migration in metamorphosing Japanese flounder (Paralichthys olivaceus).

The body of a Japanese flounder (Paralichthys olivaceus) changes from a symmetrical to an asymmetrical form during metamorphosis. To obtain detailed information on the mechanisms of the migration of the right eye to the left side, soft and hard tissues in the head of larval flounders were examined using transmission electron microscopy (TEM). Retrorbital vesicles (Rvs) are pairs of sac-like structures under the eyes. It has been suggested that the asymmetrical development of Rvs, with the right (blind) one being bigger than the left, is the driving force behind eye migration. The present study revealed that the ultrastructure of the Rv sheath is quite similar to that of a lymphatic capillary. Thus, it is possible that the Rv is a part of the lymph system, and is probably related to the secondary vascular system in teleosts. If we assume that the Rv sheath has a high permeability to liquid, similar to lymphatic capillaries, it is not plausible that the active expansion of the Rv pushes the eyeball. On the other hand, the pseudomesial bar (Pb) is a bone that is unique to flounders and is present only on the right (blind) side. At the beginning of eye migration, an aggregation of fibroblast-like cells is observed in the dermis under the right eye, where the Pb will subsequently be formed. These cells have a well-developed rough endoplasmic reticulum (rER) and mitochondria, and are probably responsible for formation of the thick layers of collagen fibrils around them. Since it is unlikely that the active expansion of the Rv causes eye migration, the role played by the Pb and its rudiment becomes more significant in right eye migration in the Japanese flounder becomes more significant.

Thyroid gland development in a neotenic goby (ice goby, Leucopsarion petersii) and a common goby (ukigori, Gymnogobius urotaenia) during early life stages.

In order to study the characteristics of neoteny in teleosts, development of the thyroid system and digestive tract of a neotenic goby (ice goby, Leucopsarion petersii) and a non-neotenic goby (ukigori, Gymnogobius urotaenia) were compared. In juvenile ukigori, the intestine was found to be convoluted once in the antero-midpart, and gastric glands were present. In the ice goby, the alimentary canal was straight, and no gastric gland was observed even in adult, suggesting that the ice goby retains larval features, not only in appearance but also in internal organs. A marked difference was also found in the thyroid system. In ukigori, activity of the thyroid gland and thyroid stimulating hormone (TSH) cells increased between flexion and postflexion larval phases. However, in the ice goby, thyroid glands remained inactive, and no TSH cells were observed. A delayed development of the thyroid system was suggested as a major factor contributing to neoteny in the ice goby.

Response of a neotenic goby, ice goby (Leucopsarion petersii), to thyroid hormone and thiourea treatments.

In order to clarify the mechanisms of neoteny in the ice goby (Leucopsarion petersii), we examined effects of thyroid hormone and thiourea (TU) treatments on their neotenic characteristics and the pituitary-thyroid axis. Adult ice goby were exposed to 3, 5, 3'-triiodothyronine (T3, 0.1 ppm), TU (inhibitor of thyroid hormone synthesis, 30 ppm), or the combination of the two for 2 weeks. Observations of whole body T3 levels, thyroid follicles and TSH immunoreactive cells in the pituitary suggests the presence of a functioning thyroidal system. However, all of the neotenic features did not disappear in T3-treated fish, suggesting the absence of T3 responsiveness in peripheral tissues. These results indicate the similarity between neoteny of the ice goby and obligatory-type neoteny of urodeles.

Induction of ambicoloration by exogenous cortisol during metamorphosis of spotted halibut Verasper variegatus.

Cortisol, the main glucocorticoid in fish, increases during flatfish metamorphosis and peaks before the surge of thyroxine. A large body of evidence indicates the essential role of thyroxine in flatfish metamorphosis, whereas information on cortisol is limited. We administered cortisol to spotted halibut Verasper variegatus larvae in order to examine the effect on pigmentation during metamorphosis. Administration of 10 µg cortisol per mL of water from before the onset of metamorphosis (stage E) to metamorphic climax (stage G) induced the development of adult type pigment cells on the blind side of the metamorphosed juveniles and increased the occurrence of ambicolored juveniles. When 10 µg/mL cortisol was administered during stage D, stages E-F, stage G or stage H, only the administration during stages E-F induced the development of adult type pigment cells on the blind side. In addition, the expression of the gene dopachrome tautomerase (dct), a marker of melanoblasts, was enhanced at Stage E by cortisol administration. These results clearly indicated, for the first time, the enhancement of pigmentation by exogenous high-dose cortisol. Since endogenous cortisol is secreted in response to various kinds of stress in rearing conditions, these results indicate a possible influence of stress conditions in the occurrence of ambicoloration in flatfish.

Production of symmetrical flatfish by controlling the timing of thyroid hormone treatment in spotted halibut Verasper variegatus.

The thyroid hormone plays an essential role in the metamorphosis in flatfish, during which external asymmetry (for example, eye relocation and pigmentation) is established. However, no information is available on the expression mechanisms of metamorphic asymmetry. Since the presence of malformed juveniles (having either ocular or blind side characteristics on both sides) was known in spotted halibut (Verasper variegatus), the effect of the timing of thyroid hormone treatment was investigated. When thyroxine (T4, 10 or 30 ng/ml) was administered to the hypothyroid larvae (continuously receiving 30 microg/ml thiourea) from 25 days after hatching (DAH), the occurrence of one type of symmetrical juvenile (symmetrical pseudoalbino having blind side characteristics on both sides) increased to more than three times, and the occurrence was significantly greater than that observed in the control group. In the fish in which T4 treatment was initiated prior to 15 DAH or after 60 DAH, the occurrence of another type of symmetrical juvenile (symmetrical ambicolorate having ocular side characteristics on both sides) became more than two times, and its occurrence was significantly greater than that observed in the control group. These results suggest that both sides of the larval body independently possess the potential to become either the ocular or the blind side of the juvenile body and that the timing of thyroid hormone increase is the determining factor of "which side to become." Based on previous studies, since thyroid hormone is expected to induce blind side characteristics, the differential responsiveness to thyroid hormone-longer in the left side (blind side in normal juvenile) than the right-is strongly suggested as the central mechanism for metamorphic asymmetry in spotted halibut.

Distribution of cholecystokinin-immunoreactive cells in the digestive tract of the larval teleost, ayu, Plecoglossus altivelis.

The ontogenetic development of cholecystokinin-immunoreactive (CCK-IR) cells was studied in larval ayu, Plecoglossus altivelis. This species has a straight digestive tract during the larval phase. CCK-IR cells were present in all the larvae from the day of hatching (0 days after hatching, DAH). An immunoreaction to anti-trypsinogen antibody was also detected in the pancreas at this stage. The distribution pattern of the CCK-IR cells was quantified by recording the location of CCK-IR cells at 1, 16, and 76 DAH. Although the number of CCK-IR cells increased during development, the distribution pattern of CCK-IR cells did not change until 76 DAH. The CCK-IR cells were scattered throughout the midgut, with the exception of the regions adjacent to the pyloric and rectal sphincters. No CCK-IR cells were detected in the foregut or the hindgut. This distribution pattern differs from species with rotated digestive tracts, whose CCK-IR cells are only found in the anterior part of the midgut. CCK-IR cells seem to be located in regions where the ingested food is retained and thus can easily receive chemical signals from the food and the digestive process in order to control the release of the hormone.