Yasunari Kayashima

Autocrine regulation of ecdysone synthesis by β3-octopamine receptor in the prothoracic gland is essential for Drosophila metamorphosis

Significance Metamorphosis is an important biological process by which animals alter their body structures to become sexually mature adults. We discovered that tyramine signaling through the β3-octopamine receptor plays an essential role in producing the steroid hormone ecdysone, which is critical for metamorphosis. Based on our observations, we propose that monoamine signaling acts downstream of a body size checkpoint that allows metamorphosis to occur only when a critical body weight is attained during larval development and nutrients are sufficiently abundant. This work also provides a new perspective on an evolutionarily conserved monoaminergic regulation of steroid hormone production during developmental transitions such as metamorphosis. This study provides a new understanding of how metamorphosis is coordinately regulated by nutritional conditions and developmental timing. In Drosophila, pulsed production of the steroid hormone ecdysone plays a pivotal role in developmental transitions such as metamorphosis. Ecdysone production is regulated in the prothoracic gland (PG) by prothoracicotropic hormone (PTTH) and insulin-like peptides (Ilps). Here, we show that monoaminergic autocrine regulation of ecdysone biosynthesis in the PG is essential for metamorphosis. PG-specific knockdown of a monoamine G protein-coupled receptor, β3-octopamine receptor (Octβ3R), resulted in arrested metamorphosis due to lack of ecdysone. Knockdown of tyramine biosynthesis genes expressed in the PG caused similar defects in ecdysone production and metamorphosis. Moreover, PTTH and Ilps signaling were impaired by Octβ3R knockdown in the PG, and activation of these signaling pathways rescued the defect in metamorphosis. Thus, monoaminergic autocrine signaling in the PG regulates ecdysone biogenesis in a coordinated fashion on activation by PTTH and Ilps. We propose that monoaminergic autocrine signaling acts downstream of a body size checkpoint that allows metamorphosis to occur when nutrients are sufficiently abundant.

Gene Duplication of endothelin 3 Is Closely Correlated with the Hyperpigmentation of the Internal Organs (Fibromelanosis) in Silky Chickens

During early development in vertebrates, pluripotent cells are generated from the neural crest and migrate according to their presumptive fate. In birds and mammals, one of the progeny cells, melanoblasts, generally migrate through a dorsolateral route of the trunk region and differentiate to melanocytes. However, Silky is an exceptional chicken in which numerous melanoblasts travel via a ventral pathway and disperse into internal organs. Finally, these ectopic melanocytes induce heavy dermal and visceral melanization known as Fibromelanosis (Fm). To identify the genetic basis of this phenotype, we confirmed the mode of inheritance of Fm as autosomal dominant and then performed linkage analysis with microsatellite markers and sequence-tagged site markers. Using 85 backcross progeny from crossing Black Minorca chickens (BM-C) with F1 individuals between White Silky (WS) and BM-C Fm was located on 10.2–11.7 Mb of chicken chromosome 20. In addition, we noticed a DNA marker that all Silky chickens and the F1 individuals showed heterozygous genotyping patterns, suggesting gene duplication in the Fm region. By quantitative real-time PCR assay, Silky line-specific gene duplication was detected as an ∼130-kb interval. It contained five genes including endothelin 3 (EDN3), which encoded a potent mitogen for melanoblasts/melanocytes. EDN3 with another three of these duplicated genes in Silky chickens expressed almost twofold of those in BM-C. Present results strongly suggest that the increase of the expression levels resulting from the gene duplication in the Fm region is the trigger of hypermelanization in internal organs of Silky chickens.

Freeze-Dried Royal Jelly Maintains Its Developmental and Physiological Bioactivity in Drosophila melanogaster

Royal jelly (RJ), a honeybee-derived product, has been found to possess developmental and physiological bioactivity in the fruit fly, Drosophila melanogaster, but little is known about the in vivo bioactivity of freeze-dried RJ (FDRJ) powder, which is another form of RJ processed for human use. To address this, we used Drosophila as a model animal to examine the effects of FDRJ in multicellular organisms. When flies were reared on food supplemented with FDRJ, the developmental time from larva to adult was shortened, the adult male lifespan was prolonged, and female fecundity was increased without any significant morphological alterations. Moreover, the expression of dilp5, an insulin-like peptide, its receptor InR, and the nutrient sensing molecule TOR, the target of rapamycin, was significantly increased in FDRJ-fed female flies as compared with ones reared on standard and on protein-enriched food. These findings suggest that like RJ, FDRJ maintains its bioactivity even after processing from RJ, what is expected to have bioactivity for multicellular organisms, including humans.

Expression of β-adrenergic-like Octopamine Receptors during Drosophila Development

An invertebrate biogenic amine, octopamine, plays diverse roles in multiple physiological processes (e.g. neurotransmitter, neuromodulator, and circulating neurohormone). Octopamine is thought to function by binding to G-protein-coupled receptors. In Drosophila, three &bgr;-adrenergic-like octopamine receptors (Oct&bgr;1R, Oct&bgr;2R, and Oct&bgr;3R) have been identified. We investigated the expression of three Oct&bgr;R genes in embryos, larvae, and adults. These Oct&bgr;Rs showed distinct expression patterns in the central nervous system (CNS) throughout development, and Oct&bgr;3R expression was evident in an endocrine organ, the ring gland, in larvae. In larvae, Oct&bgr;1R, Oct&bgr;2R, and Oct&bgr;3R were expressed in salivary glands and imaginal discs, Oct&bgr;2R and Oct&bgr;3R in midgut, and Oct&bgr;3R in gonads. In adult, besides in the CNS, each Oct&bgr;R was strongly expressed in ovary and testis. Our findings provide a basis for understanding the mechanisms by which Oct&bgr;Rs mediate multiple diverse octopaminergic functions during development.

The Binding of Multiple Nuclear Receptors to a Single Regulatory Region Is Important for the Proper Expression of EDG84A in Drosophila melanogaster

Nuclear receptor transcription factor family members share target sequence similarity; however, little is known about how these factors exert their specific regulatory control. Here, we examine the mechanism regulating the expression of the Drosophila EDG84A gene, a target gene of the orphan nuclear receptor βFTZ-F1, as a model to study the cooperative behavior among nuclear receptors. We show that the three nuclear receptors βFTZ-F1, DHR3, and DHR39 bind to a common element in the EDG84A promoter. The expression level of the EDG84A promoter-lacZ reporter genes in DHR39-induced and mutant animals, respectively, suggests that DHR39 works as a repressor. The activity of a reporter gene carrying a mutation preventing DHR3 binding was reduced in ftz-f1 mutants and rescued by the induced expression of βFTZ-F1, suggesting that DHR3 and βFTZ-F1 activate the EDG84A gene in a redundant manner. A reporter gene carrying a mutation that abolishes DHR39 and FTZ-F1 binding was prematurely expressed, and the expression level of the reporter gene carrying a mutation preventing DHR3 binding was reduced. These findings suggest that the temporal expression of this gene is mainly controlled by βFTZ-F1 but that the binding of DHR3 is also important. Comparison of the binding site sequence among Drosophila species suggests that DHR3 binding ability was gained after the melanogaster subgroup evolved, and this ability may contribute to the robust expression of this gene. These results show the complicated regulatory mechanisms utilized by multiple nuclear receptors to properly regulate the expression of their target gene through a single target site.

Tea polyphenols ameliorate fat storage induced by high-fat diet in Drosophila melanogaster

Background Polyphenols in tea are considered beneficial to human health. However, many such claims of their bioactivity still require in vitro and in vivo evidence. Results Using Drosophila melanogaster as a model multicellular organism, we assess the fat accumulation-suppressing effects of theaflavin (TF), a tea polyphenol; epitheaflagallin (ETG), which has an unknown function; and epigallocatechin gallate (EGCg), a prominent component of green tea. Dietary TF reduced the malondialdehyde accumulation related to a high-fat diet in adult flies. Other physiological and genetic responses induced by the high-fat diet, such as lipid accumulation in the fat body and expression of lipid metabolism-related genes, were ameliorated by the addition of TF, ETG, and EGCg, in some cases approaching respective levels without high-fat diet exposure. Continuous ingestion of the three polyphenols resulted in a shortened lifespan. Conclusion We provide evidence in Drosophila that tea polyphenols have a fat accumulation-suppressing effect that has received recent attention. We also suggest that tea polyphenols can provide different desirable biological activities depending on their composition and the presence or absence of other chemical components.

Effect of a Dipeptide-Enriched Diet in an Adult Drosophila melanogaster Laboratory Strain

Here we present free amino acid profiles for Drosophila melanogaster adults. Imidazol dipeptides anserine and carnosine, which are abundant in mammalian muscle tissue, are not present in Drosophila. Dipeptide-enriched food altered the amino acid balance, suggesting that the free amino acid content is nutrition-dependent and probably mediated by dipeptides.

A heteroallelic Drosophila insulin-like receptor mutant and its use in validating physiological activities of food constituents.

Here we report an additional Drosophila transheterozygote InR(GS15311)/InR(GS50346) carrying two different P-element-inducible alleles of insulin-like receptor gene (InR). InR(GS15311)/InR(GS50346) flies exhibit the following phenotypes previously reported in InR and insulin/IGF-1 signaling (IIS) pathway-related gene mutants: small bodies, developmental delay, shortened lifespan, and increased fasting resistance. All of these characteristics are shared among flies carrying mutated genes implicated in the pathway. This heteroallelic combination exhibited fertility but resulted in male semilethality, while females were viable and grew into adults. Furthermore, an experimental model employing the InR(GS15311)/InR(GS50346) strain confirmed negligible involvement of royal jelly in IIS. Thus, the heteroallelic InR mutant, discovered in this study, will serve as a good model for multiple purposes: investigating the IIS mechanisms; identifying and validating the ingredients that prevent type II diabetes; and screening of food constituents associated with IIS.

The Combined Effects of Genetic Variation in the CNDP1 and CNDP2 Genes and Dietary Carbohydrate and Carotene Intake on Obesity Risk

Background/Aims: It is possible that carnosinase (CNDP1) and cellular nonspecific dipeptidase (CNDP2) have important roles in protecting cells and tissues against the damage of oxidative stress. Oxidative stress and subsequent inflammation are key factors in the development of common chronic metabolic diseases, such as obesity. We aimed to investigate the combined effects of genetic variations in CNDP1 and CNDP2 and dietary carbohydrate and carotene intake on obesity risk. Methods: A total of 1,059 Japanese men were randomly selected from participants who visited a medical center for routine medical checkups. We analyzed the relationships between the genotypes of 4 single-nucleotide polymorphisms (SNPs) (rs12605520, rs7244647, rs4891558, and rs17089368) in the CNDP1/CNDP2 locus and body mass index or prevalence of obesity/overweight taking into account dietary carbohydrate and carotene intake. Results: We found that 2 SNPs (rs7244647 in CNDP1 and rs4891558 in CNDP2) were associated with obesity risk. In addition, these associations were observed only in the group with high carbohydrate and low carotene intake but not in the group with low carbohydrate and high carotene intake. Conclusions: Our findings indicate that the combination of genetic variations in CNDP1 and CNDP2 and dietary carbohydrate/carotene intake modulate obesity risk.