Based on recent advances in experimental embryology and molecular genetics, the morphogenetic program for the vertebrate cranium is summarized and several unanswered classical problems are reviewed. In particular, the presence of mesodermal segmentation in the head, the homology of the trabecular cartilage, and the origin of the dermal skull roof are discussed. The discovery of the neural-crest-derived ectomesenchyme and the roles of the homeobox genes have allowed the classical concept of head segmentation unchanged since Goethe to be re-interpreted in terms of developmental mechanisms at the molecular and cellular levels. In the context of evolutionary developmental biology, the importance of generative constraints is stressed as the developmental factor that generates the homologous morphological patterns apparent in various groups of vertebrates. Furthermore, a modern version of the germ-layer theory is defined in terms of the conserved differentiation of cell lineages, which is again questioned from the vantage of evolutionary developmental biology.

Butterfly wing color-patterns are determined in the prospective wing tissues during the late larval and early pupal stages. To study the cellular differentiation process of wings, morphological knowledge on pupal wings is prerequisite. Here we systematically examined morphological patterns of the pupal wing cuticular surface in a wide variety of nymphalid butterflies in relation to adult color-patterns. Several kinds of pupal wing patterns corresponding to particular adult color-pattern elements were widely observed in many species. Especially noteworthy were the pupal “focal” spots corresponding to the adult border ocelli system, which were detected in many species of Nymphalinae, Apaturinae, Argynninae, Satyrinae, and Danainae. Striped patterns on the pupal wing cuticle seen in some species of Limenitinae, Ariadnae, and Marpesiinae directly corresponded to those of the adult wings. In Vanessa cardui, eyespot-like pattern elements were tentatively produced during development in the wing tissue underneath the pupal spots and subsequently erased, suggesting a mechanism for producing novel color-patterns in the course of development and evolution. The pupal focal spots reasonably correlated with the adult eyespots in size in Precis orithya and Ypthima argus. We physically damaged the pupal focal spots and their corresponding cells underneath in these species, which abolished or inhibited the formation of the adult eyespots. Taken together, our results clarified that pupal cuticle patterns were often indicative of the adult color-patterns and apparently reflect molecular activity of organizing centers for the adult color-pattern formation at least in nymphalid butterflies.

Two ecologically distinct forms, fresh- and brackish-water types, of ninespine stickleback coexist in several freshwater systems on the coast of eastern Hokkaido. Recent genetic analyses of 13 allozyme loci revealed genetic separation between the two types even though their spawning grounds were in close proximity. On the other hand, there is only a small difference in mitochondrial DNA (mtDNA) sequence between the two types suggesting that they diverged quite recently or that mtDNA introgression occurred between them. To test for postzygotic reproductive isolating mechanisms and hybrid mediated gene flow, we examined the viability and reproductive performance of reciprocal F₁ hybrids. The hybrids grew to the adult size normally and both sexes expressed secondary sexual characters in the reciprocal crosses. The female hybrids were reciprocally fertile, while the male hybrids were reciprocally sterile. Histological and flow-cytometric analyses of the hybrid testis revealed that the sterility pattern was classified as ‘gametic sterility,’ with gonads of normal size but abnormal spermatogenesis. To our knowledge, the present finding is a novel example of one sex hybrid sterility in the stickleback family (Gasterosteidae).

In the presence of 30% glycerol, the cilia of a permeabilized cell model from Paramecium exhibit dynamic orientation changes while displaying only a restricted cyclic beating with a very small amplitude. The direction of cilia under these conditions corresponds to the direction of the effective power stroke of cilia beating in the absence of glycerol, i.e., pointing posteriorly in the absence of Ca² and anteriorly at > 10⁻⁶ M Ca² . Ciliary reorientation toward the posterior in response to the removal of Ca² is particularly conspicuous; all the cilia become predominantly pointing to the posterior end all through their beating phases. Previous studies suggested that the effect of glycerol is caused through modification of cAMP-dependent protein phosphorylation. To determine whether glycerol in fact affects ciliary reorientation through changes in protein phosphorylation, here we examined protein phosphorylation in the axonemes. Glycerol stimulated cAMP-induced phosphorylation of 29-kDa and 65-kDa proteins. The stimulation of phosphorylation was found to be partly due to the inhibition of endogenous phosphodiesterase (PDE), and partly due to the inhibition of the dephosphorylation of the 29-kDa and 65-kDa phosphoproteins within the axoneme. Thus glycerol appears to cause predominant posterior orienation of cilia by stimulating cAMP-dependent phosphorylation on those proteins. In addition, glycerol appears to inhibit ciliary beating through inhibition of dynein ATPase.

An investigation on gastropod fauna was carried out on a tidal flat in the Nagura Estuary on Ishigaki Island, the Ryukyu Islands in 1989 and 1998 using similar methods. 470–480 quadrats covering ca. 1900 m² were surveyed during low tides from February to April in each year. Of the total 19 species recorded, the ranges of eight species had varied significantly between the two surveys, with six species expanding their range and two species contracting their range. Percentage in abundance of muddy-bottom species and tropical (<29°N) species increased significantly between the two years. Topography of the flat also changed: the mouth of the river was narrowed and the elevated sections of the tidal flat expanded. During the period from 1984 to 1998, the farmland development around the study site caused influxes of soil into the estuary and the sea-water temperature was rising. These results suggest that the topographical changes due to soil influx and the rising temperature affected the gastropod assemblage at the study site, by increasing the abundance of muddy-bottom species and tropical species. The methodology used in this study, i.e. surface observation at low tides, includes more than 95% of the gastropod fauna, demonstrating the usefulness of surface counts for the study of soft-bottom fauna.

Carassius RFamide (C-RFa) is a peptide, isolated originally from the brain of Japanese crucian carp and sharing homologies with mammalian prolactin-releasing peptides. From the physiological aspect, it is known that C-RFa has contraction-promoting action on fish intestines, but its localization in peripheral tissues is unknown. We observed the localization of C-RFa in teleost guts using an immunohistochemical technique. C-RFa-like immunoreactive (irC-RFa) sites were observed in not only the smooth muscle cells in the longitudinal muscle layer, but also in both Auerbach's and Meissner's nerve plexus in the stomach, pyloric ceca and intestine. In epithelial mucous cells, irC-RFa sites were observed in the surface mucous cells in the stomach in freshwater fish (FW), and in the goblet cells of the apical sites in the villi of the pyloric ceca and intestine in all fish. In the stomach, irC-RFa sites were found in the fundic glands of the body regions in seawater (SW) and brackish water (BW) fish, but not in FW fish. This study confirmed that one of the functions of C-RFa is the smooth muscle contraction of the longitudinal muscle layer in digestive organs. We suggest that C-RFa may have functional roles in both central and peripheral neurotransmission. In addition, it appears that the difference in C-RFa localization of SW, BW, and FW fish reflects the adaptation of the stomach function to different salinity habitats.

Daily and circadian variations of melatonin contents in the diencephalic region containing the pineal organ, the lateral eyes, and plasma were studied in a urodele amphibian, the Japanese newt (Cynops pyrrhogaster), to investigate the possible roles of melatonin in the circadian system. Melatonin levels in the pineal region and the lateral eyes exhibited daily variations with higher levels during the dark phase than during the light phase under a light-dark cycle of 12 h light and 12 h darkness (LD12:12). These rhythms persisted even under constant darkness but the phase of the rhythm was different from each other. Melatonin levels in the plasma also exhibited significant day-night changes with higher values at mid-dark than at mid-light under LD 12:12. The day-night changes in plasma melatonin levels were abolished in the pinealectomized (Px), ophthalmectomized (Ex), and Px Ex newts but not in the sham-operated newts. These results indicate that in the Japanese newts, melatonin production in the pineal organ and the lateral eyes were regulated by both environmental light-dark cycles and endogenous circadian clocks, probably located in the pineal organ and the retina, respectively, and that both the pineal organ and the lateral eyes are required to maintain the daily variations of circulating melatonin levels.

A neuropeptide, pituitary adenylate cyclase–activating polypeptide (PACAP) has possible potency as a hypothalamic factor mediating the release of pituitary hormones, especially growth hormone (GH), in the fish pituitary. We used double-immunostaining to examine the relationship between PACAP nerve fibers and adenohypophysial hormone-producing cells in the pituitary of a teleost, the stargazer Uranoscopus japonicus, and enzyme immunoassay to determine the quantity of PACAP in the stargazer brain, in conjunction with the body mass and gonad somatic index (GSI) of fish. In adult stargazer, PACAP-like immunoreactive (PACAP-LI) nerve fibers and endings were identified in both the neurohypophysis and adenohypophysis in close proximity to pituitary cells containing immunoreactive hormones such as prolactin, somatolactin, the N-terminal peptide of proopiomelanocortin, and N-acetyl endorphin. PACAP-LI nerve fibers were also identified close to immunoreactive GH cells in the pituitary of young fish. The concentration of immunoreactive PACAP in whole brain ranged from 100 to 800 pmol/g wet weight, in fish with weighing 70–480 g. A negative correlation was found between the concentration of immunoreactive PACAP in the whole brain and body weight, but there was no relation between the former and GSI. These results suggest that PACAP may act as a hypophysiotropic factor in the stargazer pituitary.

Atrial natriuretic peptide (ANP) decreases plasma Na concentration and promtes seawater (SW) adaptation in eels. The hyponatremia may most probably be caused by increased branchial extrusion of Na , but the mechanism has not been determined yet. The present study examined initially the effects of ANP on branchial Na efflux in vivo using isotopic ²²Na. However, the efflux rate was not altered by infusion of a hyponatremic dose of ANP (5 pmol·kg⁻¹·min⁻¹). Therefore, we sought to examine whether the ANP-mediated hyponatremia is caused by a decrease in the uptake of Na from the environment. Since a decrease in drinking was highly correlated with a degree of hyponatremia, conscious SW eels were infused with dilute SW into the stomach at a normal drinking rate to offset the antidipsogenic effect of ANP. Under this regimen, the hyponatremic effect of ANP was abolished. Then, we examined the site of Na absorption in the alimentary tract by measuring the changes in ion composition of intraluminal fluid along the tract. Since Na was absorbed at the esophagus and anterior/middle intestine, a sac was prepared at each site and the effects of ANP were examined in situ in conscious SW eels. ANP infusion did not alter Na absorption at the esophagus, but it profoundly reduced the absorption at the intestine. Together with our previous finding that ANP does not alter renal Na excretion, we propose that ANP reduces plasma Na concentration in SW eels by inhibiting drinking and subsequent absorption of Na by the intestine.

The phylogenetic relationships between gobies of the genus Gymnogobius were analyzed using mitochondrial cytochrome b gene sequences, focusing on the species currently classified as G. taranetzi and G. castaneus that occur in Japan, South Korea, and Russia. Gobies of the two species collected at 12 localities in Japan, South Korea, and Russia formed a monophyletic clade (called the “castaneus species complex” here) with G. breunigii as the sister clade. Within the species complex, six lineages were recognized: (L1) G. castaneus from the Akigawa River, Tokyo, Japan; (L2) G. castaneus from Yuza, Yamagata, Japan; (L3) G. taranetzi from Russia and South Korea; (L4) G. castaneus from the Tonegawa River, Chiba, Japan; (L5a) G. taranetzi from Shimane, Japan; and (L5b) G. castaneus G. taranetzi from the Japan Sea coast of northern Japan. The two local lineages of G. castaneus (L1 and L2) are highly divergent from the others. The Japanese populations of G. taranetzi have diverged from the continental G. taranetzi populations, while one mitochondrial lineage (L5b) is shared with G. castaneus of northeast Japan. Therefore, the current species G. taranetzi and G. castaneus as defined morphologically are polyphyletic, necessitating a taxonomic revision. The genetic differentiation of isolated local lineages and the evolution of taranetzi- and castaneus-type gobies have likely occurred repeatedly in brackish/freshwater habitats around the Sea of Japan. We discussed the time of divergence for these gobies based on a tree with the molecular clock assumption.

Adult amphibian skin actively transports Na from its apical to basolateral side while in turn, K is recycled through Na , K -ATPase and K channels located in the basolateral membrane. We previously found that PRL stimulates Na transport in the skin of the adult tree frog (Hyla arborea japonica) via an increase in the open-channel density of the epithelial Na channel (ENaC). If PRL also activates basolateral K channels, this activation would help to stimulate Na transport, too. Whether PRL does indeed stimulate basolateral K channels in the adult tree frog was examined by measuring the short-circuit current across nystatin-treated skin. Both tolbutamide, a K_ATP channel blocker, and tetrapentylammonium (TPA), a K_Ca channel blocker, blocked the current, the effect of TPA being more powerful than that of tolbutamide. Contrary to expectation, PRL inhibited the basolateral K channels in this skin. In the presence of basolateral amiloride, PRL still inhibited the basolateral K current, suggesting that the Na -H exchanger located in the basolateral membrane does not mediate the inhibitory effect of PRL on the basolateral K channels in Hyla.

We incubated different radiolabeled steroid precursors with intact chub mackerel ovarian follicles to clarify the synthetic pathways of steroid hormones during vitellogenesis and following final oocyte maturation (FOM). During vitellogenesis, estradiol-17β (E2) was synthesized from pregnenolone via 17-hydroxypregnenolone, 17-hydroxyprogesterone, androstenedione, and testosterone. The physiological significance of the intermediate metabolites of E2 in the ovarian follicles was examined by comparing follicular steroidogenesis between gonochoric and hermaphroditic fish species. After vitellogenesis, the steroidogenic pathway shifted from E2 to maturation-inducing hormone (MIH) production owing to the inactivation of 17,20-lyase and the activation of 20β-hydroxysteroid dehydrogenase. Of the new steroids produced during FOM, 17,20β-dihydroxy-4-pregnen-3-one (17,20β-P) was most effective at inducing germinal vesicle breakdown in vitro. Circulating levels of 17,20β-P increased specifically around the time of germinal vesicle migration, while another FOM-specific 20β-hydroxylated progestin, 17,20β,21-trihydroxy-4-pregnen-3-one, was present at consistently low levels during FOM. These results indicate that 17,20β-P is the MIH of chub mackerel.

We compared the reproductive characteristics of two populations of Bufo bankorensis in central Taiwan, one inhabiting a temperate climate (Meifong, 2100 m), the other inhabiting a subtropical climate (Wushe, 1100 m). We determined ovary status, spermatogenetic activity, fat body and liver mass cycles, and plasma 17-βestradiol and androgen levels over a 14 month period. B. bankorensis from both populations are prolonged breeders but the temperate population exhibits breeding activity throughout the year, while the subtropical population only breeds from September to March. Their spermatogenic cycles are continuous, and their spermatogenetic activities are invariably at stage 6, in which spermatozoa are predominant in the seminiferous tubule. Both populations show monthly variations in plasma androgen and 17-βestradiol levels, but follow different patterns. Ovary mass is larger in the temperate than in the subtropical population. The reproduction differences of two elevation toads could be reflected by adaptations to the local environmental regimes of its habitat. The reproductive patterns of these populations of Bufo bankorensis are also compared to those of sympatric and allopatric populations of five anurans studied from sites throughout Taiwan.

Fertilization and development in the ovarian cavity of oviparous fish, Oryzias latipes, were examined using the S-rR strain in which the sex genotype can be easily distinguished by the body color of the fish. Mature eggs were fertilized within the ovarian cavity after a sperm suspension was artificially introduced with a small bore-pipette through the urinogenital opening. Three batches of eggs ovulated within 48 hrs were fertilized and began to develop in the ovarian cavity, while eggs ovulated 72 hrs post-insemination (PI) were no longer fertilized. These observations indicate that ovulation occurs irrespective of the existence of developing embryos within the ovarian cavity. All embryos developing in the ovarian cavity were, however, retarded and ceased development before the stage of initiation of blood circulation at room temperature. These embryos developed normally and hatched after they were transferred from the ovarian cavity into regular saline 48 or 72 hrs PI. When these individuals matured sexually, their sex differentiation was found to be normal, and sex reversal was not observed.