Ascidians, or sea squirts, are lower chordates, and share basic gene repertoires and many characteristics, both developmental and physiological, with vertebrates. Therefore, decoding cis-regulatory systems in ascidians will contribute toward elucidating the genetic regulatory systems underlying the developmental and physiological processes of vertebrates. cis-Regulatory DNAs can also be used for tissue-specific genetic manipulation, a powerful tool for studying ascidian development and physiology. Because the ascidian genome is compact compared with vertebrate genomes, both intergenic regions and introns are relatively small in ascidians. Short upstream intergenic regions contain a complete set of cis-regulatory elements for spatially regulated expression of a majority of ascidian genes. These features of the ascidian genome are a great advantage in identifying cis-regulatory sequences and in analyzing their functions. Function of cis-regulatory DNAs has been analyzed for a number of tissue-specific and developmentally regulated genes of ascidians by introducing promoter-reporter fusion constructs into ascidian embryos. The availability of the whole genome sequences of the two Ciona species, Ciona intestinalis and Ciona savignyi, facilitates comparative genomics approaches to identify cis-regulatory DNAs. Recent studies demonstrate that computational methods can help identify cis-regulatory elements in the ascidian genome. This review presents a comprehensive list of ascidian genes whose cis-regulatory regions have been subjected to functional analysis, and highlights the recent advances in bioinformatics and comparative genomics approaches to cis-regulatory systems in ascidians.

Phylogenetic relationships among 36 species of major coleoid cephalopods from Japanese waters were studied using partial sequences of three mitochondrial genes, 16S rDNA, 12S rDNA, and cytochrome c oxidase subunit I gene. Octopoda and Decapoda were monophylic groups. Within Sepioidea, Sepiadariidae and Sepiolidae were not closely related to Sepiidae, but rather related to Teuthoidea. Sepiidae with a distinct calcareous shell formed a single cluster. Myopsida was closely related to Oegopsida. Within Octopoda, Opisthoteuthis depressa and Argonauta argo diverged earlier than Octopodiidae. The common octopuses in Japanese waters were separated into three clusters. The first cluster occupied a basal position, and includes large-sized octopuses, such as Enteroctopus dofleini and Octopus (Paroctopus) conispadiceus from the continental shelf and upper slope. The second cluster consisted of long-armed octopuses, such as O. ornatus, O. minor, and O. sasakii. The third cluster contained small- to medium-sized octopus, such as Amphioctopus fangsiao, A. areolatus, O. cyaneus, and O. vulgaris, in which several species possess ocelli on the web. The second cluster formed the sister group to the third cluster.

In order to elucidate phylogenetic relationships and intraspecific variations and to infer the evolutionary process of loaches of the genus Lefua, we analyzed nucleotide sequences of the mitochondrial D-loop region of 100 specimens obtained from 97 localities in Japan and Korea. The genus Lefua includes three described species, L. nikkonis, L. echigonia, and L. costata and an undescribed species, Lefua sp. Our results showed that each species of Lefua formed a monophyletic group, indicating clearly that Lefua species can be genetically distinguished from one another. Lefua nikkonis was the most closely related to L. costata, while L. sp. was the most closely related to L. echigonia. Specimens of L. sp. were grouped into two intraspecific populations and specimens of L. echigonia were grouped into six populations. These populations were well separated geographically from one another by mountain ranges and highlands. We estimated the evolutionary time for splitting of the species and intraspecific populations, and speculated on the evolutionary process of the genus Lefua. Species of Lefua are severely threatened. Fundamental genetic information is indispensable for conservation. We presented genetic background in order to protect these threatened loaches.

Impaired insulin receptor (IR) signaling leads to insulin resistance and type 2 diabetes mellitus. Several inhibitors of the IR tyrosine kinase activity have recently been described and associated with human insulin resistance. Among these negative regulators, protein tyrosine phosphatases (PTPs) are likely to play a pivotal role in IR signaling. Transgenic studies revealed that PTP1B and TCPTP are primary candidates but IR of these animals can be finally dephosphorylated, suggesting that other PTPs are also involved in the dephosphorylation of IR. In this study, we showed that receptor-type PTPϵ (PTPϵM) dephosphorylated IR in rat primary hepatocytes and tyrosines 972, 1158, 1162 and 1163 were primary targets of PTPϵM. Wild type as well as substrate-trapping DA forms of PTPϵM suppressed phosphorylation of IR downstream enzymes such as Akt, extracellular regulated kinase (ERK) and glycogen synthase kinase 3 (GSK3). It was also demonstrated that PTPϵM suppressed insulin-induced glycogen synthesis and inhibited insulin-induced suppression of phosphoenol pyruvate carboxykinase (PEPCK) expression in primary hepatocytes. Furthermore, adenovirally introduced PTPϵM also exhibited inhibitory activity against suppression of PEPCK expression in mouse liver. These results suggest that PTPϵM is a negative regulator of IR signaling and involved in insulin-induced glucose metabolism mainly through direct dephospho-rylation and inactivation of IR in hepatocytes and liver.

We cloned an Achaearanea tepidariorum (Chelicerata, Arachnida) gene related to Drosophila twist (twi), which encodes a basic helix-loop-helix transcription factor required to specify mesoderm fate in the Drosophila embryo. The cloned spider gene was designated At.twist (At.twi). We examined its expression by whole-mount in situ hybridization. At.twi transcripts were first detected in cells located at the polar and equatorial areas of the spherical embryo when the cumulus reached the equator. As the extra-embryonic area expanded, more cells expressed At.twi transcripts. The At.twi-expressing cells became distributed nearly uniformly in the embryonic area. At these stages, some At.twi-expressing cells were found in the surface epithelial cell layer, but other At.twi-expressing cells were at slightly deeper positions from the surface. When the embryo was transformed into a germ band, all At.twi-expressing cells were situated just beneath the surface ectoderm, where they became metamerically arranged. Although little expression was observed in the caudal lobe of the elongating germ band, new stripes of At.twi expression appeared beneath the ectoderm in accordance with the posterior growth. These observations suggested that the cells expressing At.twi were most likely mesoderm. We propose that At.twi can be used as a molecular marker for analyzing mesoderm development in the spider embryo. Moreover, comparison of the expression patterns of twi and At.twi revealed divergent aspects of mesoderm development in the fly and spider. In addition, we cloned an Achaearanea gene related to snail, which is another mesoderm-determining gene in Drosophila, and showed that its expression was restricted to the ectoderm with no indication for a role in mesoderm development.

Newly laid eggs of many insect species store maternal ecdysteroids as physiologically inactive phosphoric esters. In the silkworm Bombyx mori, we previously reported the presence of a specific enzyme, called ecdysteroid-phosphate phosphatase (EPPase), which catalyzes the dephosphorylation of ecdysteroid-phosphates to increase the amount of free ecdysteroids during early embryonic development. In this study, we demonstrated that (1) EPPase is found in the cytosol of yolk cells, (2) ecdysteroid-phosphates are localized in yolk granules, being bound to the yolk protein vitellin (Vn), and (3) Vn-bound ecdysteroid-phosphates are scarcely hydrolyzed by EPPase, although free ecdysteroid-phosphates are completely hydrolyzed by EPPase. Thus, we investigated the mechanism by which ecdysteroid-phosphates dissociate from the Vn-ecdysteroid-phosphate complex, and indicated that the acidification of yolk granules causes the dissociation of ecdysteroid-phosphates from the Vn-ecdysteroid-phosphate complex and thereby ecdysteroid-phosphates are released from yolk granules into the cytosol. Indeed, the presence of vacuolar-type proton-translocating ATPase in the membrane fraction of yolk granules was also verified by Western blot analysis. Our experiments revealed that Vn functions as a reservoir of maternal ovarian ecdysteroid-phosphates as well as a nutritional source during embryonic development. This is the first report showing the biochemical mechanism by which maternal Vn-bound ecdysteroid-phosphates function during early embryonic development.

As a first step towards understanding the functional role of neuroactive substances in the first olfactory center of the male silkworm moth Bombyx mori, we carried out an immunocytochemical identification of antennal lobe neurons. Antibodies against γ-aminobutyric acid (GABA), FMRFamide, serotonin, tyramine and histamine were applied to detect their existence in the antennal lobe. In the present immunocytochemical study, we clarified four antenno-cerebral tracts from their origin and projection pathways to the protocerebrum, and revealed the following immunoreactive cellular organization in the antennal lobe. 1) Local interneurons with cell bodies in the lateral cell cluster showed GABA, FMRFamide and tyramine immunoreactivity. 2) Projection neurons passing through the middle antenno-cerebral tract with cell bodies in the lateral cell cluster showed GABA and FMRFamide immunoreactivity. Projection neurons passing through the outer antenno-cerebral tract with cell bodies in the lateral cell cluster showed FMRFamide immunoreactivity. 3) Centrifugal neurons passing through the inner antenno-cerebral tract b with cell bodies located outside the antennal lobe showed serotonin and tyramine immunoreactivity. Our results revealed basic distribution patterns of neuroactive substances in the antennal lobe and indicated that each projection pathway from the antennal lobe to the protocerebrum contains specific combination of neuro-active substances.

In the silkworm Bombyx mori, diapause hormone (DH) is produced in the female subesophageal ganglion (SG) and induces embryonic diapause by targeting developing ovaries. DH is processed from a precursor protein consisting of DH, pheromone biosynthesis activating neuropeptide (PBAN) and three other neuropeptides (SGNPs). Because these five neuropeptides share a common sequence, FXPRLamide, at the C-terminus, a direct and specific assay for DH itself is required in order to understand the profile of concentration changes. In this study, we produced a mouse monoclonal antibody (anti-DH[N] mAb) against the N-terminal region of DH and developed a sandwich enzyme-linked immunosorbent assay using the anti-DH[N] mAb and a rabbit polyclonal antibody against the C-terminus of DH. This procedure enabled us to specifically quantify the DH molecule at femtomolar levels (equivalent to 1/10 of SG). We then plotted DH levels in eggs and SGs during embryonic and post-embryonic development. DH was present in late-stage embryos that had been destined for the production of both diapause and nondiapause eggs. DH levels in SG gradually increased in both types during larval development and peaked at the early pupal stage. At the middle pupal stage, DH levels in SG and SG-brain complex decreased markedly in the diapause-egg producing type, thus indicating active release of DH into the hemolymph. From 5th instar larva to adult, no sexual differences in DH levels were observed in SGs or SG-brain complexes from diapause and nondiapause egg-producing types.

Additivity in the circadian phototransduction system of the mouse has not been tested directly. Because of this, accurate prediction of circadian phase shifts elicited by polychromatic light stimuli cannot be derived from the results of studies using monochromatic light stimuli. This limitation also makes it impossible to deduce the relative contributions of the photoreceptive mechanisms (rods, cones and melanopsin-containing retinal ganglion cells) underlying circadian phototransduction in the mouse. Using nearly monochromatic light stimuli of different spectral composition, and combinations thereof, we demonstrated that murine circadian phototransduction exhibits additivity. Based on the locomotor activity phase shifts elicited by these stimuli, we developed the first quantitative assessment of the relative contributions of conventional and novel photoreceptive mechanisms for circadian functioning in the mouse.

Skeletal muscle wasting is a common symptom in the adrenal insufficiency such as Addison's disease. Although it has been suspected that several cytokines and/or growth factors are responsible for the manifestation of the symptom, the precise mechanisms underlying the phenomenon have so far been poorly understood. Myostatin is predominantly expressed in skeletal muscles and involved in the regulation of skeletal muscle mass. Recently, several reports indicated that myostatin is secreted into the circulation and the increased levels of circulating myostatin is associated with the induction of skeletal muscle wasting in adult animals. We, therefore, hypothesized that the increased levels of circulating myostatin may account for the development of skeletal muscle wasting in adrenal insufficiency. To test the validity of this hypothesis, we compared the serum levels of myostatin in normal with those in bilaterally adrenalectomized (ADX) rats, a model of Addison's disease, by Western blot analysis. The active form of myostatin (13 kDa) was barely detectable in the sera collected either 1 month or 2 month after adrenalectomy, but present at conspicuously detectable levels in those obtained 3 month after the operation, while the total amounts of myostatin proteins (sum of the precursor and the active forms) remained constant at all the time points examined post-operatively. These results are consistent with the hypothesis that the increased serum levels of active form of myostatin protein, induced yet unknown post-translational control mechanisms may be responsible, at least in part, for the muscle wasting associated with the adrenal insufficiency syndromes.

Tenascin and fibronectin are components of the extracellular matrices that oppose and promote adhesion, respectively. Using immunohistochemical techniques, we studied the distribution of tenascin and fibronectin in the mouse ovary, in which dynamic reconstruction and degeneration occur during folliculogenesis, atresia, ovulation, corpus luteum formation and luteolysis. In growing follicles, tenascin was only detected in the theca externa layer, while fibronectin was detected in the theca externa layer, theca interna layer and basement membrane. During follicular atresia, granulosa cells, which are surrounded by the basement membrane, began to die through apoptosis. In atretic follicles, tenascin was detected in the basement membrane and theca externa layer. Distribution of fibronectin in atretic follicles was similar to that in healthy growing follicles, except that granulosa cells were slightly immunopositive for fibronectin. In young corpus luteum, luteal cells exhibit high 3 β -hydroxysteroid dehydrogenase (3 β -HSD) activity, an enzyme indispensable for progesterone production. Tenascin was barely detected in young luteal cells. 3 β -HSD activity in luteal cells declines with corpus luteum age, and in older corpus luteum there is an increase in apoptotic death of luteal cells. Tenascin was intensely immunopositive in old luteal cells. In contrast, fibronectin immunostaining in luteal cells was relatively constant during corpus luteum formation and luteolysis. Our observations suggest that tenascin is critical in controlling the degenerative changes of tissues in mouse ovaries. Moreover, in all circumstances observed in this study, tenascin always co-localized with fibronectin, suggesting fibronectin is indispensable for the function of tenascin.

In order to obtain data on the reproductive pattern of the clonal cnidarian Zoanthus sansibaricus, polyps were sampled by scuba gear at Taisho Lava Field, Sakurajima, for 24 months between April 2000 and March 2002 (polyps collected weekly for breeding season). According to cross-sections, Zoanthus polyps were divided into three sexual types; male, female and asexual, and were found in the same colony. At Sakurajima, Zoanthus sansibaricus spawned in the middle of July, releasing oocytes and sperm. These spawning events occur synchronously with moon phase. In gametogenesis of Zoanthus sansibaricus, oocytes became recognizable in February and grew rapidly from the end of June onward. Spermatocytes became recognizable in June and matured rapidly in the middle of July. After spawning events, oocytes still remaining in the endoderm were absorbed into Zoanthus tissue quickly.

The present paper deals with 19 species in four subgenera of the attelabid genus Euops from China (including Taiwan), of which six species and a subgenus are new to science. The subgenus Synaptops is newly divided into three species-groups. A key to species, with photographs and line illustrations of important features of new species is provided.