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One of most challenging issues in tumor immunology is a better understanding of the dynamics in the accumulation of myeloid-derived suppressor cells (MDSCs) in the tumor microenvironment (TIME), as this would lead to the development of new cancer therapeutics. Here, we show that translationally controlled tumor protein (TCTP) released by dying tumor cells is an immunomodulator crucial to full-blown MDSC accumulation in the TIME. We provide evidence that extracellular TCTP mediates recruitment of the polymorphonuclear MDSC (PMN-MDSC) population in the TIME via activation of Toll-like receptor-2. As further proof of principle, we show that inhibition of TCTP suppresses PMN-MDSC accumulation and tumor growth. In human cancers, we find an elevation of TCTP and an inverse correlation of TCTP gene dosage with antitumor immune signatures and clinical prognosis. This study reveals the hitherto poorly understood mechanism of the MDSC dynamics in the TIME, offering a new rationale for cancer immunotherapy. Cell death in the context of cancer therapies is often associated with immunogenicity. Taniguchi and colleagues instead find that release of the cellular protein TCTP following cell death triggers an immunosuppressive pathway in the tumor microenvironment.

This study aims at identifying transcriptional targets of FruitlessBM (FruBM), which represents the major isoform of male-specific FruM transcription factors that induce neural sexual dimorphisms. A promoter of the axon-guidance factor gene robo1 carries the 16-bp palindrome motif Pal1, to which FruM binds. Our genome-wide search for Pal1-homologous sequences yielded ~200 candidate genes. Among these, CG17716 potentially encodes a transmembrane protein with extracellular immunoglobulin (Ig)-like domains similar to Robo1. Indeed, FruBM overexpression reduced CG17716 mRNA and protein expression. In the fru-expressing mAL neuron cluster exhibiting sexual dimorphism, we found that CG17716 knockdown in female neurons completely transformed all neurites to the male-type. Conversely, CG17716 overexpression suppressed male-specific midline crossing of fru-expressing sensory axons. We renamed CG17716 teiresias (tei) based on this feminizing function. We hypothesize that Tei interacts with other Ig superfamily transmembrane proteins, including Robo1, to feminize the neurite patterns in females, whereas FruBM represses tei transcription in males., Sato, Ito, and Yamamoto report the identification of a Fruitless target gene, teiresias (tei), required for neuronal feminization in Drosophila. They find that tei is responsible for specifying the sex-specific structure of neurites and propose that the Tei protein forms a complex with robo1, conferring the ligand specificity on the heteromeric receptor complex.

The fruitless gene of Drosophila produces multiple protein isoforms, which are classified into two major classes, sex-specific Fru proteins (FruM) and non-sex specific proteins (FruCOM). Whereas FruM proteins are expressed in ∼2000 neurons to masculinize their structure and function, little is known about FruCOM's roles. As an attempt to obtain clues to the roles of FruCOM, we compared expression patterns of FruCOM and FruM in the central nervous system at the late larval stage. We found that nearly all neuroblasts express FruCOM but not FruM, whereas a subset of ganglion mother cells and differentiated neurons express FruM but not FruCOM. It is inferred that FruCOM proteins support fundamental stem cell functions, contrasting to FruM proteins, which play major roles in sex-specific differentiation of neurons.

Recent genetic studies have indicated relationships between gene mutations and colon cancer phenotypes. However, how physical properties of tumor cells are changed by genetic alterations has not been elucidated. We examined genotype-defined mouse intestinal tumor-derived cells using a high-speed scanning ion conductance microscope (HS-SICM) that can obtain high-resolution live images of nano-scale topography and stiffness. The tumor cells used in this study carried mutations in Apc (A), Kras (K), Tgfbr2 (T), Trp53 (P), and Fbxw7 (F) in various combinations. Notably, high-metastatic cancer-derived cells carrying AKT mutations (AKT, AKTP, and AKTPF) showed specific ridge-like morphology with active membrane volume change, which was not found in low-metastatic and adenoma-derived cells. Furthermore, the membrane was significantly softer in the metastatic AKT-type cancer cells than other genotype cells. Importantly, a principal component analysis using RNAseq data showed similar distributions of expression profiles and physical properties, indicating a link between genetic alterations and physical properties. Finally, the malignant cell-specific physical properties were confirmed by an HS-SICM using human colon cancer-derived cells. These results indicate that the HS-SICM analysis is useful as a novel diagnostic strategy for predicting the metastatic ability of cancer cells.

Musculoskeletal and psychological complaints have increased with the widespread use of visual display terminals, and musculoskeletal pain is known to be closely related to stress. One method of experimentally inducing persistent muscle pain is repeated cold stress (RCS), and animals exposed to such stress exhibit a dysfunction in the descending pain inhibitory system. Acetaminophen (N-acetyl-p-aminophenol; APAP) is widely used to relieve several types of pain, including musculoskeletal pain, and is available as an OTC drug. However, the mechanism underlying its analgesic action has not yet been fully elucidated. In this study, we compared the analgesic effect of APAP on RCS-induced muscular hyperalgesia with those of other analgesics to identify its mechanism of action. The daily oral administration of APAP significantly suppressed the decrease in the mechanical withdrawal threshold caused by RCS, similar to the results for neurotropin but not for the cyclooxygenase inhibitor ibuprofen (IBP). Moreover, the intrathecal administration of antagonists of the 5-hydroxytryptamine (5-HT)

While epigamic traits likely evolve via sexual selection, the mechanism whereby internal sexual dimorphism arises remains less well understood. Seeking clues as to how the internal sexual dimorphism evolved, we compared the abdominal musculature of 41 Drosophila montium group species, to determine whether any of these species carry a male-specific muscle of Lawrence (MOL). Our quantitative analysis revealed that the size of a sexually dimorphic MOL analog found in 19 montium group species varied widely from species to species, suggesting the gradual evolution of this sexually dimorphic neuromuscular trait. We attempted the ancestral state reconstitution for the presence or absence of the neuromuscular sexual dimorphism in the A5 segment; the neuromuscular sexual dimorphism existed in an old ancestor of the montium group, which was lost in some of the most recent common ancestors of derived lineages, and subsequently some species regained it. This loss-and-gain history was not shared by evolutionary changes in the courtship song pattern, even though both traits were commonly regulated by the master regulator male-determinant protein FruM. It is envisaged that different sets of FruM target genes may serve for shaping the song and MOL characteristics, respectively, and, as a consequence, each phenotypic trait underwent a distinct evolutionary path.

Behavior is a readout of neural function. Therefore, any difference in behavior among different species is, in theory, an outcome of interspecies diversification in the structure and/or function of the nervous system. However, the neural diversity underlying the species-specificity in behavioral traits and its genetic basis have been poorly understood. In this article, we discuss potential neural substrates for species differences in the courtship pulse song frequency and mating partner choice in the Drosophila melanogaster subgroup. We also discuss possible neurogenetic mechanisms whereby a novel behavioral repertoire emerges based on the study of nuptial gift transfer, a trait unique to D. subobscura in the genus Drosophila. We found that the conserved central circuit composed primarily of fruitless-expressing neurons (the fru-circuit) serves for the execution of courtship behavior, whereas the sensory pathways impinging onto the fru-circuit or the motor pathways downstream of the fru-circuit are susceptible to changes associated with behavioral species differences.

In Drosophila, some neurons develop sex-specific neurites that contribute to dimorphic circuits for sex-specific behavior. As opposed to the idea that the sexual dichotomy in transcriptional profiles produced by a sex-specific factor underlies such sex differences, we discovered that the sex-specific cleavage confers the activity as a sexual-fate inducer on the pleiotropic transcription factor Longitudinals lacking (Lola). Surprisingly, Fruitless, another transcription factor with a master regulator role for courtship circuitry formation, directly binds to Lola to protect its cleavage in males. We also show that Lola cleavage involves E3 ubiquitin ligase Cullin1 and 26S proteasome. Our work adds a new dimension to the study of sex-specific behavior and its circuit basis by unveiling a mechanistic link between proteolysis and the sexually dimorphic patterning of circuits. Our findings may also provide new insights into potential causes of the sex-biased incidence of some neuropsychiatric diseases and inspire novel therapeutic approaches to such disorders.

Environmental stress is a major factor that affects courtship behavior and evolutionary fitness. Although mature virgin females of Drosophila melanogaster usually accept a courting male to mate, they may not mate under stressful conditions. Above the temperature optimal for mating (20-25 °C), copulation success of D. melanogaster declines with increasing temperature although we observed vigorous courtship attempts by males, and no copulation takes place at temperatures over 36 °C. We attempted to identify the sensory pathway for detecting heat threat that drives a female to escape rather than to engage in mating that detects hot temperature and suppresses courtship behavior. We found that the artificial activation of warmth-sensitive neurons ('hot cells') in the antennal arista of females completely abrogates female copulation success even at permissive temperatures below 32 °C. Moreover, mutational loss of the GR28b.d thermoreceptor protein caused females to copulate even at 36 °C. These results indicate that antennal hot cells provide the input channel for detecting the high ambient temperature in the control of virgin female mating under stressful conditions.

We developed a new paradigm for quantitative analysis of courtship behavior in flies, Fly Motion-detector with an Actuator-Coupled Stimulator (FlyMacs), in which the stimulation of a fly with a moving visual target and recording of induced behaviors are automated under computer control. We employ FlyMacs for the identification of motion features that trigger specific courtship elements in Drosophila subobscura, whose mating is suggested to be strongly vision dependent. A female abdomen attached to the actuator, when moved in an appropriate pattern, evokes in the test male tapping-like foreleg motions, midleg swing and proboscis extension, which are considered to be elementary actions in male courtship behavior. Tapping is primarily induced when the target is moving, whereas midleg swing and proboscis extension are most frequently observed after the target stops moving. In contrast to midleg swing, which tends to occur immediately after target cessation (∼3000 ms), the incidence of proboscis extension gradually increases with time after target cessation, reaching a plateau at 3000 ms. The results suggest that tapping, midleg swing and proboscis extension are each induced by different movement features of the visual target. These findings do not support the view that a single key stimulus induces the entire courtship ritual. Rather, courtship behaviors in D. subobscura are correlated with movement and position of the target, which suggests that D. subobscura uses sensory information to pattern its courtship.

Colorectal cancer (CRC) is the third leading cause of cancer-related deaths worldwide. Several genome sequencing studies have provided comprehensive CRC genomic datasets. Likewise, in our previous study, we performed genome-wide Sleeping Beauty transposon-based mutagenesis screening in mice and provided comprehensive datasets of candidate CRC driver genes. However, functional validation for most candidate CRC driver genes, which were commonly identified from both human and mice, has not been performed. Here, we describe a platform for functionally validating CRC driver genes that utilizes CRISPR-Cas9 in mouse intestinal tumor organoids and human CRC-derived organoids in xenograft mouse models. We used genetically defined benign tumor-derived organoids carrying 2 frequent gene mutations (Apc and Kras mutations), which act in the early stage of CRC development, so that we could clearly evaluate the tumorigenic ability of the mutation in a single gene. These studies showed that Acvr1b, Acvr2a, and Arid2 could function as tumor suppressor genes (TSGs) in CRC and uncovered a role for Trp53 in tumor metastasis. We also showed that co-occurrent mutations in receptors for activin and transforming growth factor-β (TGF-β) synergistically promote tumorigenesis, and shed light on the role of activin receptors in CRC. This experimental system can also be applied to mouse intestinal organoids carrying other sensitizing mutations as well as organoids derived from other organs, which could further contribute to identification of novel cancer driver genes and new drug targets.

Males of the Drosophila melanogaster mutant croaker (cro) generate a polycyclic pulse song dissimilar to the monocyclic songs typical of wild-type males during courtship. However, cro has not been molecularly mapped to any gene in the genome. We demonstrate that cro is a mutation in the gene encoding the Calmodulin-binding transcription factor (Camta) by genetic complementation tests with chromosomal deficiencies, molecular cloning of genomic fragments that flank the cro-mutagenic P-insertion, and phenotypic rescue of the cro mutant phenotype by Camta+-encoding cDNA as well as a BAC clone containing the gene for Camta. We further show that knockdown of the Camta-encoding gene phenocopies cro mutant songs when targeted to a subset of fruitless-positive neurons that include the mcALa and AL1 clusters in the brain. cro-GAL4 and an anti-Camta antibody labeled a large number of brain neurons including mcALa. We conclude that the Camta-encoding gene represents the cro locus, which has been implicated in a species-specific difference in courtship songs between D. sechellia and simulans., Author summary Selecting a suitable mate is a prerequisite for successful breeding in organisms. Indeed, the animals instinctively distinguish a conspecific partner from individuals of other species, yet the mechanism underlying such species-recognition remains largely unknown. In choosing a conspecific male as a mate, fruit fly females rely on a male-derived auditory signal, love song, which is generated by a series of unilateral wing vibration by the male. We study how the males produce love song that is unique to the species. We particularly focus on croaker (cro) mutants, whose males generate distorted love song. Our molecular analysis reveals that the cro mutation inhibits expression of the gene encoding a protein called Calmodulin-binding transcription factor (Camta) and that an introduction of the Camta-encoding DNA into the genome of cro mutants allows the mutant male to sing a normal song. Therefore, the Camta protein is an essential component for love song generation by males. We further show that knockdown of Camta only in tens of specific neurons in the brain is sufficient for inducing the cro mutant phenotype. This study paves the way for unraveling the mechanistic basis for female-male communications in conspecific mating.

The fruitless (fru) locus was originally defined by a male sterile mutation that promotes male‐to‐male courtship while suppressing male‐to‐female courtship in Drosophila melanogaster. The fru promoter‐1 pre‐RNA generates a set of BTB‐zinc finger family FruM proteins expressed exclusively in the male neurons, leading to the formation of sexual dimorphisms in neurons via male‐specific neuroblast proliferation, male‐specific neural survival, male‐specific neuritegenesis or male‐specific arbor patterning. Such a wide spectrum of phenotypic effects seems to result from chromatin modifications, in which FruBM recruits Bonus, Histone deacetylase 1 (HDAC1) and/or Heterochromatin protein 1a (HP1a) to ~130 target sites. One established FruBM transcriptional target is the axon guidance protein gene robo1. Multiple transcriptional regulator‐binding sites are nested around the FruBM‐binding site, and mediate sophisticated modulation of the repressor activity of FruBM. FruBM also binds to the Lola‐Q transcriptional repressor to protect it from proteasome‐dependent degradation in male but not female neurons as FruBM exists only in male neurons, leading to the formation of sexually dimorphic neural structures. These findings shed light on the multilayered network of transcription regulation orchestrated by the master regulator FruBM., Drosophila Fruitless regulates proteolysis to produce sex‐specific Lola isoforms that specify neuronal sex types.

Desaturase1 (desat1) is one of the few genes known to be involved in the two complementary aspects of sensory communication - signal emission and signal reception - in

Summary Wolbachia , endosymbiotic bacteria prevalent in invertebrates, manipulate their hosts in a variety of ways: they induce cytoplasmic incompatibility, male lethality, male-to-female transformation, and parthenogenesis. However, little is known about the molecular basis for host manipulation by these bacteria. In Drosophila melanogaster , Wolbachia infection makes otherwise sterile Sex - lethal ( Sxl ) mutant females capable of producing mature eggs. Through a functional genomic screen for Wolbachia genes with growth-inhibitory effects when expressed in cultured Drosophila cells, we identified the gene WD1278 encoding a novel protein we call toxic manipulator of oogenesis (TomO), which phenocopies some of the Wolbachia effects in Sxl mutant D. melanogaster females. We demonstrate that TomO enhances the maintenance of germ stem cells (GSCs) by elevating Nanos (Nos) expression via its interaction with nos mRNA, ultimately leading to the restoration of germ cell production in Sxl mutant females that are otherwise without GSCs.

Dent's disease is characterized by defective endocytosis in renal proximal tubules (PTs) and caused by mutations in the 2Cl(-)/H(+) exchanger, CLC-5. However, the pathological role of endosomal acidification in endocytosis has recently come into question. To clarify the mechanism of pathogenesis for Dent's disease, we examined the effects of a novel gating glutamate mutation, E211Q, on CLC-5 functions and endosomal acidification. In Xenopus oocytes, wild-type (WT) CLC-5 showed outward-rectifying currents that were inhibited by extracellular acidosis, but E211Q and an artificial pure Cl(-) channel mutant, E211A, showed linear currents that were insensitive to extracellular acidosis. Moreover, depolarizing pulse trains induced a robust reduction in the surface pH of oocytes expressing WT CLC-5 but not E211Q or E211A, indicating that the E211Q mutant functions as a pure Cl(-) channel similar to E211A. In HEK293 cells, E211A and E211Q stimulated endosomal acidification and hypotonicity-inducible vacuolar-type H(+)-ATPase (V-ATPase) activation at the plasma membrane. However, the stimulatory effects of these mutants were reduced compared with WT CLC-5. Furthermore, gene silencing experiments confirmed the functional coupling between V-ATPase and CLC-5 at the plasma membrane of isolated mouse PTs. These results reveal for the first time that the conversion of CLC-5 from a 2Cl(-)/H(+) exchanger into a Cl(-) channel induces Dent's disease in humans. In addition, defective endosomal acidification as a result of insufficient V-ATPase activation may still be important in the pathogenesis of Dent's disease.

Among the sensory modalities involved in controlling mating behavior in

By genetic manipulations, we study the roles played by insulin-producing cells (IPCs) in the brain and their target, the corpora allata (CA), for reproductive dormancy in female Drosophila melanogaster, which is induced by exposing them to a combination of low temperature (11°C), short-day photoperiod (10L:14D) and starvation (water only) for 7 days immediately after eclosion (dormancy-inducing conditions). Artificial inactivation of IPCs promotes, whereas artificial activation impedes, the induction of reproductive dormancy. A transcriptional reporter assay reveals that the IPC activity is reduced when the female flies are exposed to dormancy-inducing conditions. The photoperiod sensitivity of reproductive dormancy is lost in pigment-dispersing factor (pdf), but not cry, mutants, suggesting that light input to IPCs is mediated by pdf-expressing neurons. Genetic manipulations to upregulate and downregulate insulin signaling in the CA, a pair of endocrine organs that synthesize the juvenile hormone (JH), decrease and increase the incidence of reproductive dormancy, respectively. These results demonstrate that the IPC-CA axis constitutes a key regulatory pathway for reproductive dormancy.

Although genetic diversity within a population is suggested to improve population-level fitness and productivity, the existence of these effects is controversial because empirical evidence for an ecological effect of genetic diversity and the underlying mechanisms is scarce and incomplete. Here, we show that the natural single-gene behavioural polymorphism ( Rover and sitter ) in Drosophila melanogaster has a positive effect on population fitness. Our simple numerical model predicted that the fitness of a polymorphic population would be higher than that expected with two monomorphic populations, but only under balancing selection. Moreover, this positive diversity effect of genetic polymorphism was attributable to a complementarity effect, rather than to a selection effect. Our empirical tests using the behavioural polymorphism in D. melanogaster clearly supported the model predictions. These results provide direct evidence for an ecological effect of genetic diversity on population fitness and its condition dependence.

SummaryThe aim of the present study was to determine the effects of increased dietary intake and high fat diet (HFD) in mice on artificial oocyte activation by using puromycin or roscovitine. Six-week-old mice were fed as either a control diet group, an increased dietary intake group or an HFD group for 4 weeks. Oocytes were obtained following superovulation and were divided into three treatment groups (no activation treatment, calcium ionophore and puromycin treatment, and calcium ionophore and roscovitine treatment) and were incubated for 4 h. Retrieved oocytes and numbers of oocytes activated as assessed by morphological changes were compared among the three treatment groups. The proportion of degenerated oocytes in HFD mice was significantly higher than that in control diet mice. The rates of activation in oocytes treated with roscovitine were 90.3% in control diet mice, 89.8% in increased dietary intake mice and 67.9% in HFD mice. The rate of activation in oocytes treated with roscovitine in HFD mice was significantly lower than the rates in control diet mice and increased dietary intake mice. The rates of activation in oocytes treated with puromycin were 90.6% in control diet mice, 94.0% in increased dietary intake mice and 71.4% in HFD mice, and the rate of activation in oocytes treated with puromycin in HFD mice was significantly lower than the rates in control diet mice and increased dietary intake mice. HFD-induced obesity deteriorated induction of oocyte activation by roscovitine or puromycin in mice.

Lysosomal storage disorders are characterized by progressive accumulation of undigested macromolecules within the cell due to lysosomal dysfunction. 573C10 is a Schwann cell line derived from a mouse model of Niemann-Pick type C disease-1, NPC (−/−). Under serum-starved conditions, NPC (−/−) cells manifested impaired autophagy accompanied by an increase in the amount of p62 and lysosome enlargement. Addition of L-leucine to serum-starved NPC (−/−) cells ameliorated the enlargement of lysosomes and the p62 accumulation. Similar autophagy defects were observed in NPC (−/−) cells even without serum starvation upon the knockdown of Spinster-like 1 (SPNS1), a putative transporter protein thought to function in lysosomal recycling. Conversely, SPNS1 overexpression impeded the enlargement of lysosomes, p62 accumulation and mislocalization of the phosphorylated form of the mechanistic Target of rapamycin in NPC (−/−) cells. In addition, we found a reduction in endogenous SPNS1 expression in fibroblasts derived from NPC-1 patients compared with normal fibroblasts. We propose that SPNS1-dependent L-leucine export across the lysosomal membrane is a key step for triggering autophagy, and that this mechanism is impaired in NPC-1.

In fruit flies, the male-specific fruitless (fru) gene product FruBM plays a central role in establishing the neural circuitry for male courtship behavior by orchestrating the transcription of genes required for the male-type specification of individual neurons. We herein identify the core promoter recognition factor gene Trf2 as a dominant modifier of fru actions. Trf2 knockdown in the sexually dimorphic mAL neurons leads to the loss of a male-specific neurite and a reduction in male courtship vigor. TRF2 forms a repressor complex with FruBM, strongly enhancing the repressor activity of FruBM at the promoter region of the robo1 gene, whose function is required for inhibiting the male-specific neurite formation. In females that lack FruBM, TRF2 stimulates robo1 transcription. Our results suggest that TRF2 switches its own role from an activator to a repressor of transcription upon binding to FruBM, thereby enabling the ipsilateral neurite formation only in males., fruitless (fru) is an important sex-determinant gene that controls the expression of neuroanatomical sex differences in Drosophila. Here the authors report that a core-promoter factor, TRF2, suppresses a male-type neurite specification through direct interaction with FruBM isoform at the robo1 target gene promoter.

It remains an enigma how the nervous system of different animal species produces different behaviors. We studied the neural circuitry for mating behavior in Drosophila subobscura, a species that displays unique courtship actions not shared by other members of the genera including the genetic model D. melanogaster, in which the core courtship circuitry has been identified. We disrupted the D. subobscura fruitless (fru) gene, a master regulator for the courtship circuitry formation in D. melanogaster, resulting in complete loss of mating behavior. We also generated fru(soChrimV), which expresses the optogenetic activator Chrimson fused with a fluorescent marker under the native fru promoter. The fru-labeled circuitry in D. subobscura visualized by fru(soChrimV) revealed differences between females and males, optogenetic activation of which in males induced mating behavior including attempted copulation. These findings provide a substrate for neurogenetic dissection and manipulation of behavior in non-model animals, and will help to elucidate the neural basis for behavioral diversification. SIGNIFICANCE STATEMENT How did behavioral specificity arise during evolution? Here we attempted to address this question by comparing the parallel genetically definable neural circuits controlling the courtship behavior of Drosophila melanogaster, a genetic model, and its relative, D. subobscura, which exhibits a courtship behavioral pattern unique to it, including nuptial gift transfer. We found that the subobscura fruitless circuit, which is required for male courtship behavior, was slightly but clearly different from its melanogaster counterpart, and that optogenetic activation of this circuit induced subobscura-specific behavior, i.e., regurgitating crop contents, a key element of transfer of nuptial gift. Our study will pave the way for determining how and which distinctive cellular elements within the fruitless circuit determine the species-specific differences in courtship behavior.

Drosophila Btk29A is a Tec family nonreceptor tyrosine kinase, the ortholog of which causes X-linked agammaglobulinemia in humans when mutant. In Btk29A

Neurogenetic analyses in the fruit fly Drosophila melanogaster revealed that gendered behaviors, including courtship, are underpinned by sexually dimorphic neural circuitries, whose development is directed in a sex-specific manner by transcription factor genes, fruitless (fru) and doublesex (dsx), two core members composing the sex-determination cascade. Via chromatin modification the Fru proteins translated specifically in the male nervous system lead the fru-expressing neurons to take on the male fate, as manifested by their male-specific survival or male-specific neurite formations. One such male-specific neuron group, P1, was shown to be activated when the male taps the female abdomen. Moreover, when artificially activated, P1 neurons are sufficient to induce the entire repertoire of the male courtship ritual. These studies provide a conceptual framework for understanding how the genetic code for innate behavior can be embodied in the neuronal substrate.

Wnt–β-Catenin in Germ Cells The Wnt–β-catenin pathway contributes to many signaling mechanisms during organismal development and carcinogenesis by regulating both transcription and cell adhesion. Hamada-Kawaguchi et al. (p. 294 ) demonstrate that this pathway must be activated in ovarian somatic cells to stop proliferation of germ cells in Drosophila. Phosphorylation of a tyrosine residue on β-catenin by the tyrosine kinase Btk turns on signaling in the niche cells by promoting transcriptional activity of β-catenin. Failure in this process resulted in ovarian tumors in the flies.

Behavioral changes in evolution have attracted the attention of many evolutionary biologists. Closely related species, or even individuals from different populations within a species, often exhibit remarkably different behaviors. Such behavioral diversification has been implicated as a cause of speciation in some cases, yet the mechanisms that produce and maintain these changes remain largely unknown. Drosophila melanogaster, an outstanding model organism with which to explore the causal link among the gene, neural circuitry, and behavior, provides an excellent entry point for a comparative approach to the origin of behavioral diversification with a single-gene and single-cell resolution. Here we survey studies that attempted to reveal the mechanistic bases for behavioral changes potentially associated with speciation, and we discuss the successfully identified candidate genes or neurons involved in such events. Although evolutionary developmental biology (evo-devo), a field devoted to the developmental and molecular basis of phenotypic diversity, has mostly been focused on morphological traits, the extension of this evo-devo approach to behavioral diversity will provide a comprehensive understanding of the genome-environment interactions underlying adaptive evolution.

The fruitless (fru) gene in Drosophila has been proposed to play a master regulator role in the formation of neural circuitries for male courtship behavior, which is typically considered to be an innate behavior composed of a fixed action pattern as generated by the central pattern generator. However, recent studies have shed light on experience-dependent changes and sensory-input-guided plasticity in courtship behavior. For example, enhanced male-male courtship, a fru mutant "hallmark," disappears when fru-mutant males are raised in isolation. The fact that neural fru expression is induced by neural activities in the adult invites the supposition that Fru as a chromatin regulator mediates experience-dependent epigenetic modification, which underlies the neural and behavioral plasticity.

Previous comparative studies suggest that the requirement for Nodal in epiblast and hypoblast development is unique to mammalians. Expression of anterior visceral endoderm (AVE) genes in the visceral endoderm and of their orthologs in the hypoblast may be unique to mammalians and avians, and is absent in the reptilian hypoblast. Axis formation in reptiles is signaled by the formation of the posterior marginal epiblast (PME), which expresses a series of primitive streak genes. To assess the phylogenetic origin of Nodal and AVE gene expression and axis formation in amniotes, we examined marker gene expression in gray short-tailed opossum, a metatherian.Nodal was expressed in neither epiblast nor hypoblast of opossum embryos. No AVE genes were expressed in the opossum hypoblast. Attainment of polarity in the embryonic disk was signaled by Nodal, Wnt3a, Fgf8, and Bra expression in the PME at 8.5 days post-coitus.Nodal expression in epiblast or hypoblast may be unique to eutherians. AVE gene expression in visceral endoderm and hypoblast may have been independently acquired in eutherian and avian lineages. PME formation appears to be the event that signals axis formation in reptilian and metatherian embryos, and thus may be an ancestral characteristic of basal amniotes. Developmental Dynamics 245:1176-1188, 2016. © 2016 Wiley Periodicals, Inc.

Drosophila platonic (plt) males court females, but fail to copulate. Here we show that plt is an allele of scribbler (sbb), a BMP signalling component. sbb knockdown in larvae leads to the loss of approximately eight serotonergic neurons, which express the sex-determinant protein Doublesex (Dsx). Genetic deprivation of serotonin (5-HT) from dsx-expressing neurons results in copulation defects. Thus, sbb+ is developmentally required for the survival of a specific subset of dsx-expressing neurons, which support the normal execution of copulation in adults by providing 5-HT. Our study highlights the conserved involvement of serotonergic neurons in the control of copulatory mechanisms and the key role of BMP signalling in the formation of a sex-specific circuitry., Drosophila platonic (plt) mutant males court with females but fail to copulate. Here, the authors find plt is an allele of scribbler and may disrupt courtship behaviour via developmental disruption of a subgroup of serotonergic Doublesex+ neurons in the abdominal ganglion.

The employment of gliding in aquatic animals as a means of conserving energy has been theoretically predicted and discussed for decades. Several studies have shown that some species glide, whereas others do not. Freshwater fish species that widely inhabit both lentic and lotic environments are thought to be able to adapt to fluctuating flow conditions in terms of locomotion. In adapting to the different functional demands of lentic and lotic environments on fish energetics, physostomous (open swim bladder) fish may optimise their locomotion and activity by controlling their net buoyancy; however, few buoyancy studies have been conducted on physostomous fish in the wild. We deployed accelerometers on free-ranging channel catfish

A male Drosophila that is not successful in courtship will reduce his courtship efforts in the next encounter with a female. This courtship suppression persists for more than 1 h in wild-type males. The Btk29A(ficP) mutant males null for the Btk29A type 2 isoform, a fly homolog of the nonreceptor tyrosine kinase Btk, show no courtship suppression, while Btk29A hypomorphic males exhibit a rapid decline in courtship suppression, leading to its complete loss within 30 min. The males of a revertant stock or Btk29A(ficP) males that are also mutant for parkas, a gene encoding the presumptive negative regulator of Btk29A, exhibit normal courtship suppression. Since another behavioral assay has shown that Btk29A(ficP) mutants are sensitization-defective, we hypothesize that the mutant flies are unable to maintain the neural excitation state acquired by experience, resulting in the rapid loss of courtship suppression.

Atrogin-1 and MuRF1, muscle-specific ubiquitin ligases, and autophagy play a role in protein degradation in muscles. We hypothesized that branched-chain amino acids (BCAAs) may decrease atrogin-1, MuRF1, and autophagy, and may have a protective effect on disuse muscle atrophy. To test this hypothesis, we selected hindlimb suspension (HS)-induced muscle atrophy as a model of disuse muscle atrophy because it is an established model to investigate the effects of decreased muscle activity. Sprague-Dawley male rats were assigned to 4 groups: control, HS (14 days), oral BCAA administration (600 mg/[kg day], 22.9% L-isoleucine, 45.8% L-leucine, and 27.6% L-valine), and HS and BCAA administration. After 14 days of the treatment, muscle weights and protein concentrations, cross-sectional area (CSA) of the muscle fibers, atrogin-1 and MuRF1 proteins, and microtubule-associated protein 1 light chain 3 II/I (ratio of LC3 II/I) were measured. Hindlimb suspension significantly reduced soleus muscle weight and CSA of the muscle fibers. Branched-chain amino acid administration partly but significantly reversed the HS-induced decrease in CSA. Hindlimb suspension increased atrogin-1 and MuRF1 proteins, which play a pivotal role in various muscle atrophies. Branched-chain amino acid attenuated the increase in atrogin-1 and MuRF1 in soleus muscles. Hindlimb suspension significantly increased the ratio of LC3 II/I, an indicator of autophagy, whereas BCAA did not attenuate the increase in the ratio of LC3 II/I. These results indicate the possibility that BCAA inhibits HS-induced muscle atrophy, at least in part, via the inhibition of the ubiquitin-proteasome pathway. Oral BCAA administration appears to have the potential to prevent disuse muscle atrophy.

Courtship and aggression are induced in a mutually exclusive manner in male Drosophila melanogaster, which quickly chooses one of these behavioral repertoires to run depending on whether the encountered conspecific is a female or male, yet the neural mechanism underlying this decision making remains obscure. By targeted excitation and synaptic blockage in a subset of brain neurons, we demonstrate here that the fruitless (fru)-negative subfraction (∼20 cells) of a doublesex-positive neural cluster, pC1, acts as the aggression-triggering center whereas the fru-positive subfraction (∼20 cells) of pC1 acts as the courtship-triggering center, and that the mutually exclusive activation of these two centers is attained by a double-layered inhibitory switch composed of two fru single-positive clusters, LC1 and mAL. To our knowledge, this is the first report to unravel the cellular identity of the neural switch that governs the alternative activation of aggression and courtship in the animal kingdom.

Sexual recognition among individuals is crucial for the reproduction of animals. In Drosophila, like in many other animals, pheromones are suggested to play an important role in conveying information about an individual, such as sex, maturity and mating status. Sex-specific cuticular hydrocarbon components are thought to be major sex pheromones in Drosophila, and are postulated to act through the gustatory system, since they are mostly non-volatile chemicals. However, very little is known about the molecular and neural bases of gustatory pheromone reception. So far, a few putative gustatory receptors, including Gr32a and Gr68a, have been implicated in courtship behavior. Here, we examine another putative gustatory receptor, Gr39a, which shares a cluster with both Gr32a and Gr68a in a molecular phylogeny of the gustatory receptor family, for its potential role in courtship behavior. The Gr39a gene produces four isoforms through alternative splicing of different 5'-most exons. A quantitative real-time PCR analysis showed that the expression levels of all four splice variants of Gr39a were reduced in a fly line in which a P element was inserted into the Gr39a locus. Homozygous and hemizygous males for the P-element insertion, as well as males in which Gr39a was knocked down by RNAi, all showed reduced courtship levels toward females. The courtship levels returned to normal when the P element was excised out. A close analysis of courtship behavior of the mutant males revealed that the average duration of a continuous courtship bout was significantly shorter in the mutants than in the wild type. The results suggest that Gr39a has a role in sustaining courtship behavior in males, possibly through the reception of a stimulating arrestant pheromone.

Overactivation of poly(ADP-ribose) polymerase-1 (PARP-1) has been demonstrated to result in various stress-related diseases, including diabetes mellitus. Deficiency of cellular nicotinamide adenine dinucleotide (NAD(+)) content, consumed by PARP-1 to add ADP-ribose moieties onto target proteins, contributes to pathophysiological conditions. Adenosine thiamine triphosphate (AThTP) exists in small amounts in mammals; however, the function(s) of this metabolite remains unresolved. The structure of AThTP resembles NAD(+). Recent experimental studies demonstrate beneficial impacts of high-dose thiamine treatment of diabetic complications. These findings have led us to hypothesize that AThTP may modulate the activity of PARP-1. We have chemically synthesized AThTP and evaluated the effect of AThTP on recombinant PARP-1 enzyme activity. AThTP inhibited the PARP-1 activity at 10 µM, and a structural model of the PARP-1-AThTP complex highlighted the AThTP binding site. The results provide new insights into the pharmacological importance of AThTP as an inhibitor of PARP-1.

Prop1 activates POU1F1 (Pit-1) gene expression, which in turn stimulates GH, PRL, TSHβ and GHRH receptor gene expressions. Therefore the patients with Prop1 mutation show GH, PRL, and TSH deficiency. The mutation of Prop1 is a major abnormality causing combined pituitary hormone deficiency (CPHD). However, DNA-binding and activating functions of mutant Prop1 have not been examined fully because Prop1-binding elements (PBEs) in human POU1F1 gene were not identified until 2008. The aim of this study is to test DNA-binding and transcriptional activities of two mutant Prop1s (W194XProp1 and S156insTProp1, both of them were found in the patients with CPHD) whose mutation is located in putative transactivating domain but not in DNA-binding domain. W194XProp1 showed a marked DNA-binding to PBE as well as a consensus element of paired-like transcription factors (PRDQ9). Activating function for POU1F1 reporter genes expression was lost or decreased in W194XProp1 but still preserved for PRDQ9 reporter gene. S156insTProp1 did not bind PBE but bound PRDQ9. Consistent with the result, S156insTProp1 did not stimulate POU1F1 reporter gene but stimulated PRDQ9 reporter gene. These results support the inference that W194XProp1 is unable to increase POU1F1 gene expression by the defect of transactivating domain and that S156insTProp1 is unable to increase due to the loss of DNA-binding activity. DNA-binding domain that has been assumed is not sufficient to provide full DNA-binding activity of Prop1 and transactivating domain of Prop1 is likely to affect DNA binding to PBE.

The ELAV family of RNA-binding proteins is involved in various aspects of the post-transcriptional regulation of gene expression, from alternative splicing to translation. The members of this family have been shown to interact with each other and have been suggested to function as homo- and/or hetero-multimers. However, the functional interactions among them have not been demonstrated in vivo. In this study, we examined the genetic interaction between elav and Rbp9, two of the three genes encoding ELAV-family proteins in Drosophila. Mutants of both elav and Rbp9 showed shorter life spans than the control, with elav showing a shorter life span than Rbp9. The survival curve of elav-Rbp9 double-mutant flies was indistinguishable from that of elav single-mutant flies, suggesting that both mutations affect longevity through the same pathway. Considering the fact that both genes are co-expressed in adult neurons, we hypothesize that ELAV and Rbp9 cooperate to maintain the functional integrity of the adult nervous system.

Wolbachia is an endosymbiont prevalent in arthropods. To maximize its transmission thorough the female germline, Wolbachia induces in infected hosts male-to-female transformation, male killing, parthenogenesis, and cytoplasmic incompatibility, depending on the host species and Wolbachia strain involved. However, the molecular mechanisms underlying these host manipulations by Wolbachia remain largely unknown. The Wolbachia strain wMel, an inhabitant of Drosophila melanogaster, impairs host oogenesis only when transplanted into a heterologous host, for example, Drosophila simulans. We found that egg polarity defects induced by wMel infection in D. simulans can be recapitulated in the natural host D. melanogaster by transgenic overexpression of a variant of the Wolbachia protein Toxic manipulator of oogenesis (TomO), TomOwMel∆HS , in the female germline. RNA immunoprecipitation assays demonstrated that TomO physically associates with orb mRNA, which, as a result, fails to interact with the translation repressor Cup. This leads to precocious translation of Orb, a posterior determinant, and thereby to the misspecification of oocytes and accompanying polarity defects. We propose that the ability of TomO to bind to orb mRNA might provide a means for Wolbachia to enter the oocyte located at the posterior end of the egg chamber, thereby accomplishing secure maternal transmission thorough the female germline.

Hawaiian Drosophila offer an excellent model for adaptive evolution. More than 500 species are reported in Hawaiian islands, and there is considerable diversity in behavior and morphology. Such diversity is mainly driven by sexual selection. In this study qualitative and quantitative chemical compositions of cuticular hydrocarbons (CHCs) in 138 flies belonging to 27 Hawaiian Drosophila species, picture-winged and non picture-winged, were analyzed regarding sexual dimorphism, differences in saturation, branching position, and lengths of CHCs. We found significant variation in the CHC patterns. In several subgroups, new species show decreases in unsaturated hydrocarbons, and gradual increases in branched compounds, monomethylalkanes and dimethylalkanes, not commonly found in Drosophila. Moreover, branching positions gradually shifted towards internal carbons, and chain lengths increased in the new species. The long-term evolution of CHCs in the light of the recent evolutionary migration and adaptation history of Hawaiian Drosophila species along the developing archipelago was discussed.

The toxic manipulator of oogenesis (TomO) protein has been identified in the wMel strain of Wolbachia that symbioses with the vinegar fly Drosophila melanogaster, as a protein that affects host reproduction. TomO protects germ stem cells (GSCs) from degeneration, which otherwise occurs in ovaries of host females that are mutant for the gene Sex-lethal (Sxl). We isolated the TomO homologs from wPip, a Wolbachia strain from the mosquito Culex quinquefasciatus. One of the homologs, TomOwPip 1, exerted the GSC rescue activity in fly Sxl mutants when lacking its hydrophobic stretches. The GSC-rescuing action of the TomOwPip 1 variant was ascribable to its abilities to associate with Nanos (nos) mRNA and to enhance Nos protein expression. The analysis of structure-activity relationships with TomO homologs and TomO deletion variants revealed distinct modules in the protein that are each dedicated to different functions, i.e., subcellular localization, nos mRNA binding or Nos expression enhancement. We propose that modular reshuffling is the basis for structural and functional diversification of TomO protein members.

金沢大学理工研究域数物科学系, Elucidating origin, composition, size, and lifetime of microdomains in biological membranes remains a major issue for the understanding of cell biology. For lipid domains, the lack of a direct access to the behaviour of samples at the mesoscopic scale has constituted for long a major obstacle to their characterization, even in simple model systems made of immiscible binary mixtures. By its capacity to image soft surfaces with a resolution that extends from the molecular to the microscopic level, in air as well as under liquid, atomic force microscopy (AFM) has filled this gap and has become an inescapable tool in the study of the surface topography of model membrane domains, the first essential step for the understanding of biomembranes organization. In this review we mainly focus on the type of information on lipid microdomains in model systems that only AFM can provide. We will also examine how AFM can contribute to understand data acquired by a variety of other techniques and present recent developments which might open new avenues in model and biomembrane AFM applications. © 2009 Elsevier B.V. All rights reserved.

The spin gene was first identified by its mutant phenotype, which is characterized by extremely strong mate refusal by females in response to male courtship in Drosophila. Spin mutants are also known to be accompanied by a remarkable reduction in programmed cell death in the reproductive and nervous systems. To better understand the molecular functions of spin, we searched for its genetic modifiers. Forced expression of spin(+) in somatic cells as driven by ptc-Gal4 in the testis resulted in the invasion of mature sperm into the anterior testes tip, which is otherwise occupied only by immature germ cells. To obtain genes that modulate spin's effect, the gain-of-function spin phenotype was observed in the presence of a chromosome harboring an EP or GS P-element insertion, which initiates transcription of the genomic sequence neighboring the insertion site. We isolated th and emc as suppressors of spin and atg8a as a gene that reproduces the spin phenotype on its own. th encodes Inhibitor of apoptosis-1, and mammalian Id genes homologous to emc are known to inhibit apoptosis. atg8a encodes a protein essential for autophagy. These results suggest that spin promotes cell death mechanisms that are regulated negatively by th and emc and positively by atg8a.

Flat substrate surfaces are a key to successful imaging of biological macromolecules by atomic force microscopy (AFM). Although usable substrate surfaces have been prepared for still imaging of immobilized molecules, surfaces that are more suitable have recently been required for dynamic imaging to accompany the progress of the scan speed of AFM. In fact, the stateof-the-art high-speed AFM has achieved temporal resolution of 30 ms, a capacity allowing us to trace molecular processes played by biological macromolecules. Here, we characterize three types of streptavidin two-dimensional crystals as substrates, concerning their qualities of surface roughness, uniformity, stability, and resistance to nonspecific protein adsorption. These crystal surfaces are commonly resistant to nonspecific protein adsorption, but exhibit differences in other properties to some extent. These differences must be taken into consideration, but these crystal surfaces are still useful for dynamic AFM imaging, as demonstrated by observation of calcium-induced changes in calmodulin, GroES binding to GroEL, and actin polymerization on the surfaces. © 2009 by the Biophysical Society.

Temperature affects the physiology, behavior, and evolution of organisms. We conducted mutagenesis and screens for mutants with altered temperature preference in Drosophila melanogaster and identified a cryophilic (cold-seeking) mutant, named atsugari ( atu ). Reduced expression of the Drosophila ortholog of dystroglycan (DmDG) induced tolerance to cold as well as preference for the low temperature. A sustained increase in mitochondrial oxidative metabolism caused by the reduced expression of DmDG accounted for the cryophilic phenotype of the atu mutant. Although most ectothermic animals do not use metabolically produced heat to regulate body temperature, our results indicate that their thermoregulatory behavior is closely linked to rates of mitochondrial oxidative metabolism and that a mutation in a single gene can induce a sustained change in energy homeostasis and the thermal responses.

In 2003, we examined the chromosomes of grass voles at an illegal dumpsite at the Aomori-lwate prefectural boundary. In subsequent years, from 2003-2006, we surveyed the chromosomes of four species of small mammals, namely, the Japanese grass vole (Microtus montebelli), the large Japanese field mouse (Apodemus speciosus), the small Japanese field mouse (A. argenteus), and the greater Japanese shrew mole (Urotrichus talpoides). Each annual survey revealed, both on a yearly basis and during the entire period in question, that the frequencies of breaks and gaps in chromosomes of M. montebelli were significantly higher at the dumpsite than on the outskirts and in controls, suggesting that grass voles at the dumpsite have been subject to continuous genotoxic effects since the establishment of the dumpsite. We also ascertained that grass voles are much more susceptible to chromosomal damage than field mice and shrew moles, which had very low levels of chromosomal aberrations at the dumpsite, on the outskirts of the dumpsite, and in controls. Our four-year survey revealed two variants of M. montebelli from the dumpsite with M6 fission (2n=31), two variants of A. speciosus from the outskirts with XO monosomy (2n=47, XO), and a variant of A. speciosus from the dumpsite with situs inversus. Our analysis confirms our previously proposed hypothesis that M. montebelli might be useful as an indicator species for genotoxic assessment of below-ground pollution by industrial waste at illegal dumpsites.

The effect of amino acid on muscle protein degradation remains unclear. Recent studies have elucidated that proteolysis in catabolic conditions occurs through ubiquitin-proteasome proteolysis pathway and that muscle-specific ubiquitin ligases (atrogin-1 and MuRF1) play an important role in protein degradation. In the present study, we examined the direct effect of 5 mM amino acids (leucine, isoleucine, valine, glutamine and arginine) on atrogin-1 and MuRF1 levels in C2C12 muscle cells and the involved intracellular signal transduction pathway. Leucine, isoleucine and valine suppressed atrogin-1 and MuRF1 mRNA levels (approximately equal to 50%) at 6 and 24 h stimulations. Arginine showed a similar effect except at 24 h-treatment for atrogin-1 mRNA. However, glutamine failed to reduce atrogin-1 and MuRF1 mRNA levels. The inhibitory effect of leucine, isoleucine or arginine on atrogin-1 mRNA level was reversed by rapamycin, although wortmannin did not reverse the effect. PD98059 and HA89 reduced basal atrogin-1 level without influencing the inhibitory effects of those amino acids. The inhibitory effect of leucine, isoleucine or arginine on MuRF1 mRNA levels was not reversed by rapamycin. Taken together, these findings indicated that leucine, isoleucine and arginine decreased atrogin-1 mRNA levels via mTOR and that different pathways were involved in the effect of those amino acids on MuRF1 mRNA levels.

SummaryBiologists postulate that sexual dimorphism in the brain underlies gender differences in behavior, yet direct evidence for this has been sparse. We identified a male-specific, fruitless (fru)/doublesex (dsx)-coexpressing neuronal cluster, P1, in Drosophila. The artificial induction of a P1 clone in females effectively provokes male-typical behavior in such females even when the other parts of the brain are not masculinized. P1, located in the dorsal posterior brain near the mushroom body, is composed of 20 interneurons, each of which has a primary transversal neurite with extensive ramifications in the bilateral protocerebrum. P1 is fated to die in females through the action of a feminizing protein, DsxF. A masculinizing protein Fru is required in the male brain for correct positioning of the terminals of P1 neurites. Thus, the coordinated actions of two sex determination genes, dsx and fru, confer the unique ability to initiate male-typical sexual behavior on P1 neurons.

Prophet of Pit-1 (Prop1) is a transcription factor that regulates Pit-1 gene expression. Because Pit-1 regulates the differentiation of pituitary cells and the expressions of GH, prolactin and TSHβ genes, Prop1 mutation results in combined pituitary hormone deficiency in humans. However, Prop1-binding sites in human Pit-1 gene and the mechanism leading to combined pituitary hormone deficiency have remained unclear. In this study, we identified and analyzed Prop1-binding elements of the human Pit-1 gene. Prop1 stimulated the expression of the reporter plasmid containing Pit-1 gene from translation start site to −1340 dose dependently in GH3 cells. The activation by Prop1 was observed in GH3 and TtT/GF cells but not COS7, HeLa, JEG3, and HuH7 cells. Deletion analysis of Pit-1 gene showed that the Prop1-responsive elements were present within the −257-bp region. Within the −257-bp region, there are four elements similar to consensus sequence of paired-like transcription factors. Because Prop1 is a member of paired-like transcription factors, we assessed the elements. EMSA and transient transfection assay using the mutation of the elements revealed that the element from −63 to −53 (the proximal Prop1 binding element) was essential to Prop1-binding and Prop1-induced activation of Pit-1 reporter plasmid. A region at −8kb of human Pit-1 gene is similar to the distal region containing Prop1-binding elements in mouse Pit-1 gene. We showed the region functioned as an enhancer. Furthermore, chromatin immunoprecipitation assay showed that the proximal element could bind Prop1 in vivo cultured cells. Taken together, these findings indicated the novel functioning binding elements of Prop1 in human Pit-1 gene.

Recent reports suggest that Atrogin-1 and MuRF1, E3 ubiquitin ligases, play a pivotal role in muscle atrophy. In the present study, effect of Growth Hormone Releasing Peptide-2 (GHRP-2), a GH secretagogue receptor (GHS-R) agonist, on the expressions of Atrogin-1 and MuRF1 in vivo rat muscles was examined. Dexamethasone administration increased Atrogin-1 mRNA level in rat soleus muscle. The increased mRNA level of Atrogin-1 was significantly attenuated by GHRP-2. In addition, GHRP-2 decreased MuRF1 mRNA level irrespective of the presence of dexamethasone. Although IGF-I is a well-known protective factor for muscle atrophy, GHRP-2 did not influence plasma IGF-I levels and IGF-I mRNA levels in muscles. To clarify a direct effect of GHRP-2, differentiated C2C12 myocytes were used. Ten micrometer dexamethasone increased both Atrogin-1 and MuRF1 mRNA levels in C2C12 cells. GHRP-2 attenuated dexamethasone-induced expression of them dose-dependently and decreased the basal level of MuRF1 mRNA. The suppressive effect on the expressions of Atrogin-1 and MuRF1 by GHRP-2 was blocked by [ d -Lys 3 ]-GHRP-6, a GHS-R1a blocker, suggesting the effect of GHRP-2 was mediated through GHS-R1a. Taken together, GHRP-2 directly attenuates Atrogin-1 and MuRF1 mRNA levels through ghrelin receptors in myocytes.