pmar1/phb homeobox genes and the evolution of the double-negative gate for endomesoderm specification in echinoderms.

In several model animals, the earliest phases of embryogenesis are regulated by lineage-specific genes, such as Drosophila bicoid Sea urchin (echinoid) embryogenesis is initiated by zygotic expression of pmar1, a paired-class homeobox gene that has been considered to be present only in the lineage of modern urchins (euechinoids). In euechinoids, Pmar1 promotes endomesoderm specification by repressing the hairy and enhancer of split C (hesC) gene. Here, we have identified the basal echinoid (cidaroid) pmar1 gene, which also promotes endomesoderm specification but not by repressing hesC A further search for related genes demonstrated that other echinoderms have pmar1-related genes named phb Functional analyses of starfish Phb proteins indicated that, similar to cidaroid Pmar1, they promote activation of endomesoderm regulatory gene orthologs via an unknown repressor that is not HesC. Based on these results, we propose that Pmar1 may have recapitulated the regulatory function of Phb during the early diversification of echinoids and that the additional repressor HesC was placed under the control of Pmar1 in the euechinoid lineage. This case provides an exceptional model for understanding how early developmental processes diverge.

Anteroposterior molecular registries in ectoderm of the echinus rudiment.

BACKGROUND: Echinoderms and hemichordates are sister taxa that both have larvae with tripartite coeloms. Hemichordates inherit the coelom plan and ectoderm from larvae, whereas echinoderms form the adult rudiment comprising rearranged coeloms and a vestibule that then develops into adult oral ectoderm. Molecular networks that control patterns of the ectoderm and the central nervous system along the anteroposterior (AP) axis are highly conserved between hemichordates and chordates, respectively. In echinoderms, however, little is known about the AP registry in the ectoderm. RESULTS: We isolated ectodermal AP map genes from the sand dollar Peronella japonica and examined their expression. Comparative expression analyses showed that (1) P. japonica orthologs of hemichordate anterior markers are expressed in the larval apical plate, which degenerates during metamorphosis; (2) P. japonica orthologs of the medial markers are expressed in the ambulacral ectoderm of the rudiment; and (3) few P. japonica orthologs of the posterior markers are expressed in ectoderm. CONCLUSIONS: We suggest that echinoids only inherit the ambulacral ectoderm from a common ambulacrarian ancestor, which largely corresponds to the collar ectoderm in hemichordates. The ectodermal AP registry provides insights into the AP axis and evolutionary processes of echinoderms from a common ambulacrarian ancestor. Developmental Dynamics 247:1297-1307, 2018. © 2018 Wiley Periodicals, Inc.

α-Melanocyte-stimulating hormone promotes bone resorption resulting from increased osteoblastic and osteoclastic activities in goldfish.

We examined the effects of α-melanocyte-stimulating hormone (α-MSH) on bone metabolism using regenerating goldfish scales. Normally developed scales on the bodies of goldfish were removed to allow the regeneration of scales under anesthesia. Thereafter, the influence of α-MSH on the regeneration of goldfish scales was investigated in vivo. In brief, α-MSH was injected at a low dose (0.1 µg/g body weight) or a high dose (1 µg/g body weight) into goldfish every other day. Ten days after removing the scales, we collected regenerating scales and analyzed osteoblastic and osteoclastic activities as respective marker enzyme (alkaline phosphatase for osteoblasts, tartrate-resistant acid phosphatase for osteoclasts) activity in the regenerating scales as well as plasma calcium levels. At both doses, osteoblastic and osteoclastic activities in the regenerating scales increased significantly. Plasma calcium concentrations in the α-MSH-treated group (high doses) were significantly higher than those in the control group. Next, in vitro experiments were performed to confirm the results of in vivo experiments. In the cultured regenerating scales, osteoblastic and osteoclastic activities significantly increased with α-MSH (10-7 and 10-6 M) treatment. In addition, real-time PCR analysis indicated that osteoclastogenesis in α-MSH-treated scales was induced by the receptor activator of the NF-κB/receptor activator of the NF-κB ligand/osteoprotegerin pathway. Furthermore, we found that α-MSH receptors (melanocortin receptors 4 and 5) were detected in the regenerating scales. Thus, in teleosts, we are the first to demonstrate that α-MSH functions in bone metabolism and promotes bone resorption via melatonin receptors 4 and/or 5.

Calcitonin-typical suppression of osteoclastic activity by amphioxus calcitonin superfamily peptides and insights into the evolutionary conservation and diversity of their structures.

Calcitonin (CT) is a hormone that decreases serum calcium level by suppressing osteoclastic activity in the vertebrate bone. In vertebrates, the structure-function relationship of CTs has been studied extensively. We recently identified three CT superfamily peptides, Bf-CTFP1 to 3, and clarified the molecular and functional characteristics of their receptor and receptor activity-modifying protein in amphioxus, Branchiostoma floridae. However, the CT activity of Bf-CTFPs has yet to be investigated. In the present study, a functional analysis of Bf-CTFPs was performed using goldfish scales having both osteoclasts and osteoblasts. All Bf-CTFPs suppressed osteoclastic activity via a goldfish CT receptor. Although the primary amino acid sequences of the Bf-CTFPs showed low sequence similarity to vertebrate CTs, Bf-CTFP1 to 3 share three amino acids, Thr25, Thr27, and Pro32-NH2, that are required for receptor binding, with salmon CT. Moreover, homology model analysis revealed that the Bf-CTFPs form alpha-helical structures. The alpha-helical position and length of Bf-CTFP1 and 2 were conserved with those of a highly potent ligand, teleost CT. Interestingly, the composition of the alpha-helix of Bf-CTFP3 differed from those of teleost CT, despite that the action of Bf-CTFP3 on goldfish scales was the same as that of Bf-CTFP1 and 2. Collectively, the present study provides new insights into the structure-function relationship of CT and its functional evolution in chordates.

The evolutionally-conserved function of group B1 Sox family members confers the unique role of Sox2 in mouse ES cells.

BACKGROUND: In mouse ES cells, the function of Sox2 is essential for the maintenance of pluripotency. Since the Sox-family of transcription factors are well conserved in the animal kingdom, addressing the evolutionary origin of Sox2 function in pluripotent stem cells is intriguing from the perspective of understanding the origin of pluripotency. RESULTS: Here we approach this question using a functional complementation assay in inducible Sox2-null ES cells. Assaying mouse Sox proteins from different Groups, we found that only Group B1 and Group G proteins were able to support pluripotency. Interestingly, invertebrate homologs of mammalian Group B1 Sox proteins were able to replace the pluripotency-associated function of mouse Sox2. Moreover, the mouse ES cells rescued by the Drosophila SoxNeuro protein are able to contribute to chimeric embryos. CONCLUSIONS: These data indicate that the function of mouse Sox2 supporting pluripotency is based on an evolutionally conserved activity of the Group B1 Sox family. Since pluripotent stem cell population in developmental process could be regarded as the evolutional novelty in vertebrates, it could be regarded as a co-optional use of their evolutionally conserved function.

Seawater Polluted with Highly Concentrated Polycyclic Aromatic Hydrocarbons Suppresses Osteoblastic Activity in the Scales of Goldfish, Carassius auratus.

We have developed an original in vitro bioassay using teleost scale, that has osteoclasts, osteoblasts, and bone matrix as each marker: alkaline phosphatase (ALP) for osteoblasts and tartrate-resistant acid phosphatase (TRAP) for osteoclasts. Using this scale in vitro bioassay, we examined the effects of seawater polluted with highly concentrated polycyclic aromatic hydrocarbons (PAHs) and nitro-polycyclic aromatic hydrocarbons (NPAHs) on osteoblastic and osteoclastic activities in the present study. Polluted seawater was collected from two sites (the Alexandria site on the Mediterranean Sea and the Suez Canal site on the Red Sea). Total levels of PAHs in the seawater from the Alexandria and Suez Canal sites were 1364.59 and 992.56 ng/l, respectively. We were able to detect NPAHs in both seawater samples. Total levels of NPAHs were detected in the seawater of the Alexandria site (12.749 ng/l) and the Suez Canal site (3.914 ng/l). Each sample of polluted seawater was added to culture medium at dilution rates of 50, 100, and 500, and incubated with the goldfish scales for 6 hrs. Thereafter, ALP and TRAP activities were measured. ALP activity was significantly suppressed by both polluted seawater samples diluted at least 500 times, but TRAP activity did not change. In addition, mRNA expressions of osteoblastic markers (ALP, osteocalcin, and the receptor activator of the NF-κB ligand) decreased significantly, as did the ALP enzyme activity. In fact, ALP activity decreased on treatment with PAHs and NPAHs. We conclude that seawater polluted with highly concentrated PAHs and NPAHs influences bone metabolism in teleosts.

Low-intensity pulsed ultrasound induces apoptosis in osteoclasts: Fish scales are a suitable model for the analysis of bone metabolism by ultrasound.

Using fish scales in which osteoclasts and osteoblasts coexist on the calcified bone matrix, we examined the effects of low-intensity pulsed ultrasound (LIPUS) on both osteoclasts and osteoblasts. At 3h of incubation after LIPUS treatment, osteoclastic markers such as tartrate-resistant acid phosphatase (TRAP) and cathepsin K mRNA expressions decreased significantly while mRNA expressions of osteoblastic markers, osteocalcin, distal-less homeobox 5, runt-related transcription factor 2a, and runt-related transcription factor 2b, increased significantly. At 6 and 18h of incubation, however, both osteoclastic and osteoblastic marker mRNA expression did not change at least present conditions. Using GeneChip analysis of zebrafish scales treated with LIPUS, we found that cell death-related genes were upregulated with LIPUS treatment. Real-time PCR analysis indicated that the expression of apoptosis-related genes also increased significantly. To confirm the involvement of apoptosis in osteoclasts with LIPUS, osteoclasts were induced by autotransplanting scales in goldfish. Thereafter, the DNA fragmentation associated with apoptosis was detected in osteoclasts using the TUNEL (TdT-mediated dUTP nick end labeling) method. The multi-nuclei of TRAP-stained osteoclasts in the scales were labeled with TUNEL. TUNEL staining showed that the number of apoptotic osteoclasts in goldfish scales was significantly elevated by treatment with LIPUS at 3h of incubation. Thus, we are the first to demonstrate that LIPUS directly functions to osteoclasts and to conclude that LIPUS directly causes apoptosis in osteoclasts shortly after exposure.

Molecular Identification of Ptychodera flava (Hemichordata: Enteropneusta): Reconsideration in Light of Nucleotide Polymorphism in the 18S Ribosomal RNA Gene.

Seven nuclear and mitochondrial DNA markers were examined in 12 specimens of Ptychodera flava, a model acorn worm used in molecular biology, collected in Japan from three local populations with different modes of living. A comparison of intraspecific results did not show genetically isolated populations despite the species' enclave habitats and asexual reproduction. Moreover, both the nuclear 18S ribosomal RNA gene and mitochondrial 16S ribosomal RNA gene sequences were identical to those from Moorea in French Polynesia, nearly 10,000 kilometers away from Japan. I also provide the first definitive information regarding polymorphisms in 18S ribosomal RNA gene, the external transcribed spacer (ETS), internal transcribed spacers (ITS), and mitochondrial cytochrome c oxidase subunit 1 (mtCO1) sequence in hemichordates using newly designed primer sets, and I show both high larval vagility and certain criteria for the molecular identification of this species.

Neurogenesis in directly and indirectly developing enteropneusts: of nets and cords.

Concerning the evolution of deuterostomes, enteropneusts (acorn worms) occupy a pivotal role as they share some characteristics with chordates (e.g., tunicates and vertebrates) but are also closely related to echinoderms (e.g., sea urchin). The nervous system in particular can be a highly informative organ system for evolutionary inferences, and advances in fluorescent microscopy have revealed overwhelming data sets on neurogenesis in various clades. However, immunocytochemical descriptions of neurogenesis of juvenile enteropneusts are particularly scarce, impeding the reconstruction of nervous system evolution in this group. We followed morphogenesis of the nervous system in two enteropneust species, one with direct (Saccoglossus kowalevskii) and the other with indirect development (Balanoglossus misakiensis), using an antibody against serotonin and electron microscopy. We found that all serotonin-like immunoreactive (LIR) neurons in both species are bipolar ciliary neurons that are intercalated between other epidermal cells. Unlike the tornaria larva of B. misakiensis, the embryonic nervous system of S. kowalevskii lacks serotonin-LIR neurons in the apical region as well as an opisthotroch neurite ring. Comparative analysis of both species shows that the projections of the serotonin-LIR somata initially form a basiepidermal plexus throughout the body that disappears within the trunk region soon after settlement before the concentrated dorsal and ventral neurite bundles emerge. Our data reveal a highly conserved mode of neurogenesis in enteropneusts that is independent of the developing mode and is inferred to be a common feature for Enteropneusta. Moreover, all detected serotonin-LIR neurons are presumably receptor cells, and the absence of serotonin-LIR interneurons from the enteropneust nervous system, which are otherwise common in various bilaterian central nervous systems, is interpreted as a loss that might have occurred already in the last common ancestor of Ambulacraria.

Development of the swimming acorn worm Glandiceps hacksi: similarity to holothuroids.

Spawnings of Glandiceps hacksi (Hemichordata: Enteropneusta) were stimulated in the laboratory by a brief increase in temperature, and the development from fertilization through metamorphosis is described for the first time for a member of the family Spengelidae. When fertilized, the spawned female gametes, which are primary oocytes, rapidly raise a fertilization membrane and undergo two maturation divisions. Holoblastic, radial cleavage produces a blastula; a gastrula then forms by invagination from the vegetal pole, and the blastopore closes soon thereafter. In previously described enteropneust embryos, the archenteron buds off the protocoel before the latter connects to the exterior via the proboscis pore. By contrast, in G. hacksi the archenteron precociously connects with the exterior before the protocoel forms. Soon thereafter, the embryo becomes uniformly ciliated and then hatches from the fertilization envelope at approximately 32 h (15°C culture temperature). At day 3 of development, the protocoel separates from the gut, which establishes a mouth opening to the exterior; by this time, the gut has differentiated into an esophagus, a stomach, and an intestine that opens posteriorly as an anus. The larva grows to form a tornaria with distinctive pigment patches along its ciliary bands.

Deep ocean water alters the cholesterol and mineral metabolism of squid Todarodes pacificus and suppresses its weight loss.

This study is the first to demonstrate that deep ocean water (DOW) has physiological significant effects on squid. After 36 h of rearing squids, those reared with DOW had significantly higher total and free cholesterol levels and lower alanine transaminase activity in hemolymph as compared with those reared with surface sea water (SSW). SSW rearing also resulted in 6.95% weight loss, while DOW rearing caused only 2.5% weight loss, which might be due to liver metabolism suppression. Furthermore, both monovalent (sodium, chloride, and potassium ions) and divalent (calcium, inorganic phosphorus, and magnesium ions) ions in hemolymph were elevated when reared with DOW compared to those when reared with SSW. A study of genes expressed in the brain revealed that five genes were specifically remarked in DOW rearing. Most altered genes were neuropeptides, including those from vasopressin superfamily. These neuropeptides are involved in cholesterol and/or mineral metabolisms and physiological significant effects on squid. This study is the first report the effects of DOW on cholesterol and mineral metabolism of squid and will contribute to squid aquaculture using DOW.

Diversification of acorn worms (Hemichordata, Enteropneusta) revealed in the deep sea.

Enteropneusts (phylum Hemichordata), although studied extensively because of their close relationship to chordates, have long been considered shallow-water, burrowing animals. The present paper more than doubles the number of enteropneust species recorded in the deep sea based on high-resolution imaging and sampling with remotely operated vehicles. We provide direct evidence that some enteropneusts are highly mobile-using changes in posture and currents to drift between feeding sites-and are prominent members of deep, epibenthic communities. In addition, we provide ecological information for each species. We also show that despite their great morphological diversity, most deep-living enteropneusts form a single clade (the rediagnosed family Torquaratoridae) on the basis of rDNA sequences and morphology of the proboscis skeleton and stomochord. The phylogenetic position of the torquaratorids indicates that the group, after evolving from near-shore ancestors, radiated extensively in the deep sea.

Biology of the swimming acorn worm Glandiceps hacksi from the Seto Inland Sea of Japan.

The enteropneust hemichordate Glandiceps hacksi inhabits the muddy bottoms of the intertidal to subtidal zones of Koguno-shima Island, located in the central part of the Seto Inland Sea of Japan. Monthly collections from October 2005 to September 2007 revealed that their spawning occurs once a year, in the latter half of May. Parameters such as density and sex ratio, as well as the type of sediment, were also examined. Worm behavior and type of burrows revealed that G. hacksi are infaunal burrowers. Autotomy and regeneration of their posterior regions, and swimming behavior were also observed in an aquarium environment. This is the first comprehensive study on the biology of G. hacksi, the swimming acorn worm.

Ameliorative effects of jamun seed and orange peel extracts on microcystin LR induced alterations in calcitonin cells and parathyroid gland of rats.

This study investigated changes in calcitonin cells (C-cells) and parathyroid glands (PTG) induced by microcystin LR (MCLR) exposure to rats and evaluated ameliorative effects of jamun (Syzygium cumini) seed (JSE) and orange (Citrus sinensis) peel (OPE) extracts. Wistar rats were treated as-Group A (control), Group B (MCLR), Group C (MCLR + JSE), Group D (MCLT + OPE), Group E (OPE) and Group F (JSE). Microcystin dose was (10 µg/kg body wt/day whereas OPE and JSE dose was 200 mg/kg body wt/day. Thyroid and PTG were fixed on 15 and 30 days following the treatment. C-cells of treated rats for 15 days with MCLR; MCLR + JSE and MCLR + OPE exhibit degranulation, mitochondrial swelling and prominent RER. In MCLR treated rats few cells completely lack secretory granules. After 30 days MCLR treatment accumulation of secretory granules and degeneration were noticed in C-cells. C-cell nuclear volume (NV) of MCLR, MCLR + JSE and MCLT + OPE treated rats show an increase. In MCLR, MCLR + JSE and MCLR + OPE treated rats PTG exhibit hyperchromatic nuclei, nuclear elongation and increased NV after 15 days. After 30 days MCLR treatment nuclei of PTG become more hyperchromatic, more elongated, show degeneration of nuclei and increase in NV. NV is increased in Group C and Group D. PTG remain unaltered 30 days following treatment with OPE and JSE. Microcystin LR provoke physiological effects on the blood calcium and alterations in C cells and PTG, which cause serious threat to organism. These changes can be protected by JSE and OPE.

Influence of Benz[a]anthracene on Bone Metabolism and on Liver Metabolism in Nibbler Fish, Girella punctata.

It has been reported that spinal deformity was induced in developing fish by the addition of polycyclic aromatic hydrocarbons (PAHs). To examine the mechanism of the disruption of fish bone metabolism, the effect of benz[a]anthracene (BaA), a kind of PAH, on plasma calcium, inorganic phosphorus, osteoblasts, and osteoclasts was investigated in this study. We also measured several plasma components to analyze the toxicity of BaA on other metabolisms. BaA (1 or 10 ng/g body weight) was intraperitoneally injected (four times) into nibbler fish during breeding, for 10 days, and it was indicated, for the first time, that injecting high doses of BaA to nibbler fish induced both hypocalcemia and hypophosphatemia. Furthermore, in the scales of nibbler fish treated with high doses of BaA, both osteoclastic and osteoblastic marker messengerRNA (mRNA) expressions decreased. These results are a cause of disruption of bone metabolism and, perhaps, the induction of spinal deformities. In addition, we found that total protein, metabolic enzymes in the liver, total cholesterol, free cholesterol, and high-density lipoprotein cholesterol levels significantly decreased in BaA-injected fish. These results indicate that BaA may affect liver diseases and emphasize the importance of prevention of aquatic PAH pollution.

The Hox8 of the hemichordate Balanoglossus misakiensis.

Deuterostomes comprise a monophyletic group of animals that include chordates, xenoturbellids, and the Ambulacraria, which consists of echinoderms and hemichordates. The ancestral chordate probably had 14 Hox genes aligned linearly along the chromosome, with the posterior six genes showing an independent duplication compared to protostomes. In contrast, ambulacrarians are characterized by a duplication of the posterior Hox genes, resulting in three genes known as Hox11/13a, Hox11/13b, and Hox11/13c. Here, we isolated 12 Hox genes from the hemichordate Balanoglossus misakiensis and found an extra Hox gene that has not been reported in hemichordates. The extra B. misakiensis gene was suggested to be Hox8 from paralog-characteristic residues in its hexapepetide motif and homeodomain and a comparison with Strongylocentrotus purpuratus Hox genes. Our data suggest that the ancestor of echinoderms and hemichordates may have had a full complement of 12 Hox genes.

Larval development of the oriental lancelet, Branchiostoma belcheri, in laboratory mass culture.

We are successfully maintaining a laboratory colony of the lancelet Branchiostoma belcheri bred in the laboratory. Based on living individuals in this mass culture, morphological characteristics from the seven-day larval to benthic juvenile stages have been studied. Most striking was that later larval development of B. belcheri showed great individual variation even in a rather stable culture environment. Metamorphosis first occurred on 60 days post fertilization (dpf) and was continuously observed throughout the present study up to 100 dpf. Morphological traits such as the number of primary gill slits and body size at the start of metamorphosis are apparently affected by culture condition. Body size measured in the largest individuals showed nearly linear growth at 0.087 mm/day. The variability found in larval development calls for caution when developmental stages and chronological ages are compared between populations. However, the developmental flexibility of this animal also raises the possibility that growth and sexual maturation could be controlled artificially in captivity.

Laboratory culture of the Oriental lancelet Branchiostoma belcheri.

To overcome difficulties in getting research materials of cephalochordate lancelets, which has severely hampered experimental studies of this animal, we have attempted to establish a culture system in the laboratory. Adult animals collected from the wild were maintained in 2.5-L plastic containers filled with natural seawater without sand substratum. They were fed daily with unicellular algae. About 25% of the animals collected in 2003, 2004, and 2005 developed gonads in our culture system. Some of the sexually mature animals collected in the breeding seasons in 2005 and 2006 spawned spontaneously in the plastic containers of this system. Broods obtained in 2005 were maintained longer than a year in a glass tank without sand substratum. The progeny born in the laboratory showed great individual variation in growth but metamorphosed normally, and some of them started to develop gonads around 10 months after fertilization. Our mass culture methods for both adults and their progeny made daily observation possible and allowed the constant spawning of animals collected from the wild, at least in the summer season. Our culture method saves labor in maintenance and is easily set up without any specific demands except for running seawater, though still required to better survival rate and spawning control. Lancelet populations maintained in the laboratory can promote studies on these animals across disciplines and especially contribute to elucidation of the evolutionary history of chordates.

Neuronal patterning of the tubular collar cord is highly conserved among enteropneusts but dissimilar to the chordate neural tube.

A tubular nervous system is present in the deuterostome groups Chordata (cephalochordates, tunicates, vertebrates) and in the non-chordate Enteropneusta. However, the worm-shaped enteropneusts possess a less complex nervous system featuring only a short hollow neural tube, whereby homology to its chordate counterpart remains elusive. Since the majority of data on enteropneusts stem from the harrimaniid Saccoglossus kowalevskii, putative interspecific variations remain undetected resulting in an unreliable ground pattern that impedes homology assessments. In order to complement the missing data from another enteropneust family, we investigated expression of key neuronal patterning genes in the ptychoderid Balanoglossus misakiensis. The collar cord of B. misakiensis shows anterior Six3/6 and posterior Otx + Engrailed expression, in a region corresponding to the chordate brain. Neuronal Nk2.1/Nk2.2 expression is absent. Interestingly, we found median Dlx and lateral Pax6 expression domains, i.e., a condition that is reversed compared to chordates. Comparative analyses reveal that adult nervous system patterning is highly conserved among the enteropneust families Harrimaniidae, Spengelidae and Ptychoderidae. BmiDlx and BmiPax6 have no corresponding expression domains in the chordate brain, which may be indicative of independent acquisition of a tubular nervous system in Enteropneusta and Chordata.

Effects of low-intensity pulsed ultrasound on osteoclasts: Analysis with goldfish scales as a model of bone.

The effects of low-intensity pulsed ultrasound (LIPUS) on osteoclastogenesis were examined using fish scales that had both osteoclasts and osteoblasts. The binding of the receptor activator of NF-κB ligand (RANKL) in osteoblasts to the receptor activator of NF-κB (RANK) in osteoclasts induced osteoclastogenesis. Therefore, we focused on RANK/RANKL signaling. After 6 h of incubation following LIPUS treatment, mRNA expression of RANKL increased significantly. Resulting from the increased RANKL mRNA level, the expression of transcription-regulating factors significantly increased after 6 h of incubation, and then the mRNA expression of functional genes was significantly up-regulated after 12 h of incubation. However, the mRNA expression of osteoprotegerin (OPG), which is known as an osteoclastogenesis inhibitory factor, also significantly increased after 6 h of incubation and tended to further increase after 12 h of incubation. At 24 h of incubation, osteoclastic functional genes' mRNA expression decreased to the level of the control. Furthermore, we performed an in vivo experiment with goldfish. Two weeks after daily LIPUS exposure, osteoclastic marker enzymes tended to decrease while osteoblastic marker enzymes were activated. The regeneration rate of the LIPUS-treated scales was significantly higher than that of the control scales. Thus, LIPUS moderately activates osteoclasts and induces bone formation.