Zoological Science

Sea urchin embryos are though to possess a body plan characteristic of early deuterostomes. Sea urchins contain homologs of Otx, Lim, T-brain and Hox gene cluster, which are involved in head and segment formation in vertebrate development, although the sea urchin has not evolved a head or segments. We described here that sea urchin Otx is involved in various aspects of early development and that the Hox genes do not obey spatial colinearity in sea urchin embryo. The Otx and Hox genes seems to be used subsequently for head formation and determining the anteroposterior axis respectively during chordate evolution. We propose that the Precambrian was a period where these regulatory genes were utilized in many different combinations during animal development, leading to the evolution of a wide range of body plans, many of which were successful. We also discuss the role of chromatin boundaries and the mechanism of cell specification along animal vegetal axis, especially differentiation of the large micromere progeny, which are the prospective primary mesenchyme cells and play a role as an organizer in sea urchin embryos.

Treatment by fenoxycarb (> 0.1 μg/animal) during a short developmental period in the pharate pupal stage of the silkworm, Bombyx mori, induced alterations in the normal pupal-adult development. All fenoxycarb-treated animals showed accelerated development and precocious adult cuticle deposition very early in the pupal stage. Various internal and external organs of the developing adults underwent abnormal differentiation and were extensively malformed. Consequently, pupal-adult development and adult eclosion behaviour were disturbed. Similar effects were induced by injections of high doses (≥ 4 μg/animal) of 20-hydroxyecdysone (20E) only around the time of pupal ecdysis. Treatment with 1 μg of fenoxycarb in the pharate pupal stage did not affect haemolymph ecdysteroid titers early in the pupal stage. Our results, therefore, suggest that fenoxycarb, a potent juvenile hormone mimic, can imitate the developmental effects of excess 20E and behave as an ecdysteroid mimic after its application during the short developmental period of the pharate pupal stage of B. mori. These results shed light on the timely and coordinated increase of endogenous juvenile hormone titer before pupal ecdysis.

Isolated individual foragers of Apis cerana japonica could be entrained under a light-dark cycle, and the predominant activity was concentrated to the later part of the photophase. Foragers showed circadian rhythm under conditions of constant light and constant dark with free-running periods of more and less than 24 hr, respectively. These observations indicated that A. cerana possesses a circadian clock controlling locomotor activity. To investigate the molecular mechanism underlying the circadian system we cloned cDNA for a homolog of the clock gene period (per) from the honeybee by a PCR-strategy. The cloned percDNAs consisted of two types, α and β, encoding a putative protein of 1124 amino acids and 1116 amino acids, respectively. The sequences of types α and β were identical except that the former possessed an additional 24 bp stretch corresponding to 8 amino acids in the conserved C2 block. These two types were assumed to be differentially spliced variants and found also in per cDNA of A. mellifera. In support of this idea, Southern blotting experiments showed that per of A. cerana is a single copy gene. RT-PCR analysis and subcloning of the products revealed that the both types α and β are expressed in the brain of the forager. A quantitative RT-PCR assay by which the level of per mRNA in one single brain can be detected was established. Per mRNA level showed daily oscillation under a light-dark cycle with a change of the ratio of type α to β.

In the air-puff-evoked escape behavior of the cricket Gryllus bimaculatus, the effect of a uni-lateral cercal ablation and the process of behavioral recovery were investigated during postembryonic development. The response rate (relative occurrence of the escape behavior in response to an air puff stimulus) and the escape direction (relative to the stimulus direction) in first-, third-, sixth- and last-instar nymphs were almost identical with those of adults. A unilateral cercal ablation in the nymphs caused a decrease in the response rate and an increase in the number of misoriented escapes as have been observed in adults. However, the effect of ablation on the response rate was less in younger insects, i.e., the escape-eliciting potential of one cercus decreased during postembryonic development. Instead, facilitation of sensory inputs from both cerci essentially occurs, thus explaining the constant response rate throughout the developmental period. The response rate of the ablated insects measured after the final molt showed a compensational increase even when the cercal regenerates were removed at each molt. Although the final response rate was higher in crickets ablated from earlier stages, the recovery ratio was larger in crickets ablated from later stages. Regarding escape direction, a compensational change was observed in insects ablated from the first and the third instars. However, crickets ablated from the sixth and the last instar did not show any recovery in escape direction. The time course of the recovery in escape direction appears different between adults and nymphs.

Vascular cell adhesion molecule-1 (VCAM-1), which has several alternatively splicing variants, plays a role in myotube formation. To investigate which form functions in myogenesis, we analyzed VCAM-1 mRNA expression in bovine skeletal muscle cells. We detected the expression of two VCAM-1 splice forms in the muscle tissue and in the primary satellite cell culture. The longer form was predominantly expressed at the muscle and during myotube formation of the cells. The nucleotide sequences of the two forms were determined by cDNA direct sequencing. The sequence data showed that the predominant form in skeletal muscle was a full-length VCAM-1 (VCAM-7D) that consists of seven immunoglobulin-like (Ig) domains, and the minor form was a novel six-domain form that lacks the seventh Ig domain. Compared to this, bovine pulmonary artery endothelial cells also express a variant which lacks domain 7, but VCAM-7D was not detected by RT-PCR in the culture. No VCAM-1 expression was detected in bovine kidney epithelial cell, lymph node epithelial cell, or leukemic B-lymphocyte culture even under stimulation by tumor necrosis factor-α. These data suggest that the splicing of the VCAM-1 gene alternatively varies depending on the cell type where it is expressed, and that VCAM-7D plays a predominant role in myotube formation.

Strong fluorescence emission occurred in sea urchin larvae when irradiated with blue light under a fluorescence microscope. The blue light irradiation first broke red granules in the pigment cells, releasing green fluorescent substance(s) into the cytoplasm of the pigment cells. The released and dispersed fluorescent substance(s) then made the entire pigment cell emit strong green fluorescence. With prolonged blue light irradiation, the pigment cell itself bursted, dispersing the fluorescent substance(s) into the body cavity or seawater. This resulted in “explosive emission” of the fluorescence. Cells that can emit such fluorescence first appeared at the late-blastula stage, proliferated with development, and changed into the red-pigmented cells. Similar fluorescence emission was observed in the larvae of Clypeaster japonicus, Hemicentrotus pulcherrimus, Anthocidaris crassispina and Pseudocentrotus depressus, of which C. japonicus larvae displayed the strongest fluorescence.

We obtained the full-length cDNA and genomic DNA clones of HpGCS-β₁ encoding the β subunit of soluble guanylyl cyclase (soluble GC), which is expressed in the testis and ovary of the sea urchin Hemicentrotus pulcherrimus. Reverse transcription-polymerase chain reaction analysis demonstrated that the HpGCS-β₁ transcript was detected in unfertilized eggs as well as in the testis and ovary. The open reading frame predicted a protein of 604 amino acids with a putative cyclase catalytic domain conserved in membrane GCs and adenylyl cyclases. ¹⁰⁵His, ⁷⁸Cys, and ¹²²Cys, important amino acids for heme-binding in the β₁ subunit of soluble GCs in vertebrates, were conserved in the corresponding positions of HpGCS-β₁. The HpGCS-β₁ gene consisted of 8 exons and had a span of 26 kbp. A comparison of genomic structure of the HpGCS-β₁ gene with that of the soluble GC β₁ subunit gene (OlGCS-β₁) of the medaka fish indicated that exon 4 in the HpGCS-β₁ gene corresponds to exons 4 to 9 in the OlGCS-β₁ gene.

Exposure of neonatal mice to an estrogenic endocrine disruptor, bisphenol A, resulted in a malfunction of the testes when the animals became adults. The effect of bisphenol A was cancelled out by concurrent administration of retinol acetate, a naturally occurring metabolite of vitamin A. In contrast, the effect of endocrine disruption became more severe in mice neonatally exposed to bisphenol A and nursed by mothers fed a vitamin A-deficient diet only a few days before and after parturition. These results clearly show that maternal vitamin A is important for relieving in a baby the effect of endocrine disruption caused by environmental xenoestrogens, and suggest that the changes in the content of vitamin A and similar physiological factors in the habitat may be worth considering in studies on environmental disruptors.

The forkhead box genes (Fox genes) are expressed in many tissues during embryogenesis and their products play a key role in organogenesis. We cloned two Fox genes from an embryonic cDNA library of medaka (Oryzias latipes). One was MeHNF3β, a homologue of HNF3β (FoxA2). The other was Mefkh1 (medaka-forkhead1), related to HNF3γ (FoxA3). We found that the expression pattern of FoxA3 differs in some aspects between medaka and zebrafish. Mefkh1 is expressed in the dorsal marginal zone, embryonic shield, polster, gut and slightly axial mesoderm, whereas zebrafish FoxA3 is expressed in the yolk syncytial layer (YSL), axial mesoderm, polster, gut and posterior neural crest cells (Odenthal and Nüsslein-Volhard, 1998, Dev Genes Evol 208, 245–258). In zebrafish, the level of expression of FoxA3 in the axial mesoderm is similar to that of FoxA2 (axial); in medaka, FoxA3 (Mefkh1) expression is mainly detected in the polster, and FoxA2 (MeHNF3β) is expressed in the axial mesoderm. This indicates that the combined functioning of FoxA2 and FoxA3 in the organogenesis of the polster and axial mesoderm differs between medaka and zebrafish.

The ascidian embryo has been long thought to show a mosaic mode of development. However, recent studies revealed significance of cell-cell communication during cleavage stages of embryogenesis. FGF and BMP signalings play critical roles in determination of cell types. Little is, however, known about regulation of competence of cells to the signals. Here we report the isolation of ascidian smad genes; Hrsmad4 which encodes a homolog of smad4 of vertebrates, Hrsmad6/7 which encodes a homologous gene of smad 6 and smad7 of vertebrates, and Hrsmad2/3 which encodes a homolog of smad2 and smad3 of vertebrates. The mRNAs of the isolated smad family genes were maternally inherited in egg and early embryos. While Hrsmad4 and Hrsmad6/7 RNAs distributed broadly in the early embryos, Hrsmad2/3 RNA was preferentially accumulated in the animal hemisphere.

Photoperiodic sensitivity of gonad in relation to pineal gland activity was noted during two important reproductive phases i.e. reproductively active (February- March) and inactive (July- August) phases of nocturnal flying mammal, Cynopterus sphinx of Indian tropical origin. They were exposed to experimental long (Light: Dark in hours; 16L: 8D) and short (8L: 16D) photoperiods for 30 days during both the reproductive phases. Exposure to the long photoperiod (LP) during reproductively active phase had no significant effect on pineal gland, gonad and its hormonal concentration. However, exposure to short photoperiod (SP) induced a decrease in gonadal activity and increase in pineal activity (as judged by the gland weight, histology, low level of estradiol / testosterone and high level of melatonin in plasma). Increased melatonin and decreased estrogen levels following the short photoperiod exposure caused abortion in females, which were undergoing delayed embryonic development during reproductive inactive phase. Exposure to LP during reproductively inactive phase reactivated the gonadal axis and inhibited the pineal activity (as judged by the gland weight, histology, high level of estradiol / testosterone and low level of melatonin in plasma), while exposure to SP had no significant effect on pineal and gonad during this phase.

From these observations, it is suggested that depending upon the reproductive status, short and long photo-period affected differently the gonadal and pineal gland activity of this bat. SP induced gonadal regression during reproductive active phase and LP reactivated the gonadal axis during reproductive inactive phase. Under experimental condition the inverse relationship between pineal and gonadal activity was maintained as observed in nature.

We developed a quantitative PCR assay to investigate transcript levels of gill cortisol receptor (CR) in masu salmon (Oncorhynchus masou). Using this system, we examined changes in transcript levels of gill CR during smoltification of wild masu salmon while tracking serum cortisol and growth hormone (GH) concentrations patterns. The masu salmon were parr in January and February, and thereafter smoltified, migrating from the river to the sea in May. Gill CR transcript levels were very low in January and February, but thereafter increased and reached a maximum in April (5 fold increase over levels observed in January). In May, when smolt enter the sea, the gill CR transcript levels decreased. Serum cortisol concentrations were also low from January to March and increased to the peak in April. These changes correlated well with changes in CR transcript levels from March to April. In contrast, serum GH concentration began to increase in January, peaked in March and decreased from March to May. These results elucidated patterns in transcript levels of gill CR during smoltification of wild masu salmon and suggested that gill CR transcription was positively regulated by cortisol until serum cortisol level reached a peak, and negatively done after the peak in masu salmon.

The natriuretic peptide (NP) family consists of endocrine ANP/BNP/VNP in the heart and paracrine CNP in the brain in vertebrates ranging from teleosts to mammals. In elasmobranchs, however, only CNP has been identified thus far in the heart and brain. To delineate the molecular evolution of this hormone family, it is essential to determine whether CNP is the only NP in this primitive fish group. In the present study, PCR cloning of all types of piscine NP was performed from the heart and brain of a dogfish, Triakis scyllia, using degenerate primers that amplified eel ANP, VNP and CNP. However, only CNP cDNA with an identical sequence was cloned from the heart and brain. Southern blot analysis showed that the CNP gene is a single copy gene, showing that endocrine CNP from the heart and paracrine CNP in the brain originate from the same gene. Since expression of the CNP gene was so high as demonstrated by Northern blot analysis, the abundantly expressed CNP mRNA could have interfered the amplification of other NP mRNAs expressed in small amounts. Therefore, a method was developed to cleave the cloned CNP mRNA specifically at the 3′-untranslated region with RNase H. After removal of the cloned CNP mRNA by this technique, no other NP cDNAs could be cloned, but small amounts of CNP cDNAs with shorter 3′ sequence were amplified. These results strongly suggest that only CNP is present in elasmobranchs. Thus, it is likely that CNP is an ancestral form of the NP family and endocrine ANP/BNP/VNP have appeared later in the vertebrate evolution.

The morphologies of epithelial cells throughout the alimentary canal of the wood-feeding termite Nasutitermes takasagoensis (Shiraki) were examined. The digestive tract consists of four principal portions, which are the foregut, the midgut, the mixed segment and the hindgut. The midgut epithelium is primarily composed of columnar cells and degenerative cells. Most columnar cells have one or more autophagic vacuoles at cell apexes, suggesting a rapid turnover of the midgut cells. In the mixed segment, the mesenteric epithelium occupies half of the gut wall and the proctodeal epithelium covers the remaining wall. Extensive invaginations of the basal membrane are characteristic of the mesenteric columnar cells, suggesting active transport of an ionic fluid. The hindgut can be divided into five segments, the first of which is a simple tube lined with a thick cuticle, termed the first proctodeal segment. The epithelium of the third segment, the paunch, consists of cuboidal cells, which are covered by multiple cuticular layers. The apical membrane of these epithelial cells forms regular invaginations, suggesting that they have an absorptive function. In the anterior paunch, numerous spirochetes are found adhered to the gut wall. Our observations indicate that termites such as N. takasagoensis appear to have developed structures that enable more efficient interactions with intestinal microorganisms, particularly by the elongation and differentiation of the hindgut and the creation of the mixed segment.

Three new species of solitary entoprocts of the genus Loxosomella, L. monocera sp. nov., L. lappa sp. nov., and L. aloxiata sp. nov., are described from coral reefs in Okinawa Island and Sesoko Island, Ryukyu Archipelago, Japan. This is the first report on entoprocts from this archipelago. Whereas most of Loxosomella species are known to be epizoic, the three species were found on stones and shell remains in the shallow reef flat or on the slide glasses settled there. It is thus probable that the present species do not depend on host animals unlike most other congeneric species.