Combinatorial expression of ebony and tan generates body color variation from nymph through adult stages in the cricket, Gryllus bimaculatus.

Insect body colors and patterns change markedly during development in some species as they adapt to their surroundings. The contribution of melanin and sclerotin pigments, both of which are synthesized from dopamine, to cuticle tanning has been well studied. Nevertheless, little is known about how insects alter their body color patterns. To investigate this mechanism, the cricket Gryllus bimaculatus, whose body color patterns change during postembryonic development, was used as a model in this study. We focused on the ebony and tan genes, which encode enzymes that catalyze the synthesis and degradation, respectively, of the precursor of yellow sclerotin N-ß-alanyl dopamine (NBAD). Expression of the G. bimaculatus (Gb) ebony and tan transcripts tended to be elevated just after hatching and the molting period. We found that dynamic alterations in the combined expression levels of Gb'ebony and Gb'tan correlated with the body color transition from the nymphal stages to the adult. The body color of Gb'ebony knockout mutants generated by CRISPR/Cas9 systemically darkened. Meanwhile, Gb'tan knockout mutants displayed a yellow color in certain areas and stages. The phenotypes of the Gb'ebony and Gb'tan mutants probably result from an over-production of melanin and yellow sclerotin NBAD, respectively. Overall, stage-specific body color patterns in the postembryonic stages of the cricket are governed by the combinatorial expression of Gb'ebony and Gb'tan. Our findings provide insights into the mechanism by which insects evolve adaptive body coloration at each developmental stage.

UVA-LED device to disinfect hydroponic nutrient solution.

The number of plant factories in which crops are cultivated in an artificial environment has been increasing every year. In cultivation techniques involving hydroponics, plants are supplied with a circulating nutrient solution, which can become contaminated by pathogens that can propagate and spread throughout plant factories. Therefore, strategies to disinfect hydroponic nutrient solutions are needed. In this study, we developed a new disinfection device equipped with an ultraviolet A (UVA) light emitting diode (LED) that can be used to disinfect hydroponic nutrient solutions in plant factories. We first evaluated the basic disinfection capability of the device and then estimated its bactericidal effect in a small scale model system. The log survival ratio was related to UVA irradiation fluence and the volume of nutrient solution. From the assay results, we devised a kinetics equation to describe the relationship between nutrient solution volume, log survival ratio, and UVA fluence. Together our results show that UVA irradiation could be used to disinfect hydroponic nutrient solutions, and the derived kinetics equations can be used to determine optimal conditions, such as nutrient solution volume, UVA irradiation, and killing activity, to develop devices that disinfect hydroponic nutrient solutions. J. Med. Invest. 65:171-176, August, 2018.

TGF-ß signaling in insects regulates metamorphosis via juvenile hormone biosynthesis.

Although butterflies undergo a dramatic morphological transformation from larva to adult via a pupal stage (holometamorphosis), crickets undergo a metamorphosis from nymph to adult without formation of a pupa (hemimetamorphosis). Despite these differences, both processes are regulated by common mechanisms that involve 20-hydroxyecdysone (20E) and juvenile hormone (JH). JH regulates many aspects of insect physiology, such as development, reproduction, diapause, and metamorphosis. Consequently, strict regulation of JH levels is crucial throughout an insect's life cycle. However, it remains unclear how JH synthesis is regulated. Here, we report that in the corpora allata of the cricket, Gryllus bimaculatus, Myoglianin (Gb'Myo), a homolog of Drosophila Myoglianin/vertebrate GDF8/11, is involved in the down-regulation of JH production by suppressing the expression of a gene encoding JH acid O-methyltransferase, Gb'jhamt In contrast, JH production is up-regulated by Decapentaplegic (Gb'Dpp) and Glass-bottom boat/60A (Gb'Gbb) signaling that occurs as part of the transcriptional activation of Gb'jhamt Gb'Myo defines the nature of each developmental transition by regulating JH titer and the interactions between JH and 20E. When Gb'myo expression is suppressed, the activation of Gb'jhamt expression and secretion of 20E induce molting, thereby leading to the next instar before the last nymphal instar. Conversely, high Gb'myo expression induces metamorphosis during the last nymphal instar through the cessation of JH synthesis. Gb'myo also regulates final insect size. Because Myo/GDF8/11 and Dpp/bone morphogenetic protein (BMP)2/4-Gbb/BMP5-8 are conserved in both invertebrates and vertebrates, the present findings provide common regulatory mechanisms for endocrine control of animal development.

Light and abscisic acid independently regulated FaMYB10 in Fragaria × ananassa fruit.

MAIN CONCLUSION: Light and ABA independently regulated anthocyanin biosynthesis via activation of FaMYB10 expression. FaMYB10 accelerated anthocyanin synthesis of pelargonidin 3-glucoside and cyanidin 3-glucoside during strawberry fruit ripening. Light is an integral factor in fruit ripening. Ripening in non-climacteric fruit is also effected by the plant hormone abscisic acid (ABA). However, how light and/or ABA regulate fruit ripening processes, such as strawberry color development remains elusive. Results of the present study showed light and ABA regulated strawberry fruit coloration via activation of FaMYB10 expression, an R2R3 MYB transcription factor. Light exposure increased FaMYB10 transcript levels, flavonoid pathway genes, and anthocyanin content. Exogenous ABA promoted FaMYB10 expression, and anthocyanin content, accompanied by increased ABA-responsive transcript levels and flavonoid pathway genes. ABA biosynthesis inhibitor treatment, and RNAi-mediated down-regulation of the ABA biosynthetic gene (9-cis epoxycarotenoid dioxygenase: FaNCED1), and ABA receptor (magnesium chelatase H subunit: FaCHLH/ABAR) showed inverse ABA effects. Furthermore, additive effects were observed in anthocyanin accumulation under combined light and ABA, indicating independent light and ABA signaling pathways. FaMYB10 down-regulation by Agrobacterium-mediated RNA interference (RNAi) in strawberry fruits showed decreased pelargonidin 3-glucoside and cyanidin 3-glucoside levels, accompanied by consistent flavonoid pathway gene expression levels. FaMYB10 over-expression showed opposite FaMYB10 RNAi phenotypes, particularly cyanidin 3-glucoside synthesis by FaMYB10, which was correlated with FaF3'H transcript levels. These data provided evidence that light and ABA promoted FaMYB10 expression, resulting in anthocyanin accumulation via acceleration of flavonoid pathway gene expression. Finally, our results suggested FaMYB10 serves a role as a signal transduction mediator from light and ABA perception to anthocyanin synthesis in strawberry fruit.

Correlation of fibroblast growth factor 21 serum levels with metabolic parameters in Japanese subjects.

OBJECTS: Since serum level of fibroblast growth factor 21 (FGF21) has been implicated as a potential biomarker for the early detection of the metabolic syndrome and type 2 diabetes, we examined how FGF21 serum levels are correlated with metabolic parameters in Japanese subjects. METHODS: FGF21 levels were analyzed by enzyme-linked immunosorbent assays. Spearman's correlation and multiple stepwise regression analyses were used to examine the relationship between serum FGF21 and other factors. A Mann-Whitney U test was performed between the normal and high groups for triglycerides and systolic blood pressure (BP) respectively. RESULTS: By univariate correlation analysis, serum FGF21 levels were significantly associated with triglyceride levels, systolic BP, diastolic BP, pulse pressure, body mass index (BMI), age, fasting plasma glucose (FPG) levels, and total cholesterol levels. Multiple regression analysis (adjusted for age, gender, and BMI) showed that serum FGF21 levels were independently and significantly associated with triglyceride levels and systolic BP. Serum FGF21 levels were significantly higher in subjects with high triglyceride levels and high systolic BP compared with those who had normal triglyceride levels and normal systolic BP respectively. CONCLUSIONS: This study found that FGF21 levels might be a biomarker for some metabolic disorders associated with metabolic syndrome.

Phototropin 2 is involved in blue light-induced anthocyanin accumulation in Fragaria x ananassa fruits.

Anthocyanins are widespread, essential secondary metabolites in higher plants during color development in certain flowers and fruits. In strawberries, anthocyanins are also key contributors to fruit antioxidant capacity and nutritional value. However, the effects of different light qualities on anthocyanin accumulation in strawberry (Fragaria x ananassa, cv. Sachinoka) fruits remain elusive. In the present study, we showed the most efficient increase in anthocyanin content occurred by blue light irradiation. Light sensing at the molecular level was investigated by isolation of two phototropin (FaPHOT1 and FaPHOT2), two cryptochrome (FaCRY1 and FaCRY2), and two phytochrome (FaPHYA and FaPHYB) homologs. Expression analysis revealed only FaPHOT2 transcripts markedly increased depending on fruit developmental stage, and a corresponding increase in anthocyanin content was detected. FaPHOT2 knockdown resulted in decreased anthocyanin content; however, overexpression increased anthocyanin content. These findings suggested blue light induced anthocyanin accumulation, and FaPHOT2 may play a role in sensing blue light, and mediating anthocyanin biosynthesis in strawberry fruits. This is the first report to find a relationship between visible light sensing, and color development in strawberry fruits.

Transglutaminase-mediated in situ hybridization (TransISH) system: a new methodology for simplified mRNA detection.

Detection and localization of specific DNA or RNA sequences in cells and tissues are of great importance for biological research, diagnosis, and environmental monitoring. However, the most common procedure for in situ hybridization employs laborious immunostaining techniques. In the present study, we report proof-of-concept for a new RNA-enzyme conjugated probe for the detection of mRNA on tissue sections with a simple procedure. An RNA probe modified with a specific dipeptide substrate of transglutaminase was prepared. Alkaline phosphatase was then covalently and site-specifically combined to the dipeptide-labeled RNA using microbial transglutaminase. The new RNA probe labeled with alkaline phosphatase was validated by in situ hybridization (ISH) and proved to be a sensitive and sequence specific probe for mRNA detection in tissues. The new transglutaminase-mediated ISH (TransISH) strategy is free from antigen-antibody reaction, leads to one-step signal amplification after hybridization, and thus will be widely applicable for highly sensitive nucleic acid detection.

Cricket body size is altered by systemic RNAi against insulin signaling components and epidermal growth factor receptor.

A long-standing problem of developmental biology is how body size is determined. In Drosophila melanogaster, the insulin/insulin-like growth factor (I/IGF) and target of rapamycin (TOR) signaling pathways play important roles in this process. However, the detailed mechanisms by which insect body growth is regulated are not known. Therefore, we have attempted to utilize systemic nymphal RNA interference (nyRNAi) to knockdown expression of insulin signaling components including Insulin receptor (InR), Insulin receptor substrate (chico), Phosphatase and tensin homologue (Pten), Target of rapamycin (Tor), RPS6-p70-protein kinase (S6k), Forkhead box O (FoxO) and Epidermal growth factor receptor (Egfr) and observed the effects on body size in the Gryllus bimaculatus cricket. We found that crickets treated with double-stranded RNA (dsRNA) against Gryllus InR, chico, Tor, S6k and Egfr displayed smaller body sizes, while Gryllus FoxO nyRNAi-ed crickets exhibited larger than normal body sizes. Furthermore, RNAi against Gryllus chico and Tor displayed slow growth and RNAi against Gryllus chico displayed longer lifespan than control crickets. Since no significant difference in ability of food uptake was observed between the Gryllus chico(nyRNAi) nymphs and controls, we conclude that the adult cricket body size can be altered by knockdown of expressions of Gryllus InR, chico, Tor, S6k, FoxO and Egfr by systemic RNAi. Our results suggest that the cricket is a promising model to study mechanisms underlying controls of body size and life span with RNAi methods.

Transglutaminase-mediated synthesis of a DNA-(enzyme)n probe for highly sensitive DNA detection.

A new synthetic strategy for DNA-enzyme conjugates with a novel architecture was explored using a natural cross-linking catalyst, microbial transglutaminase (MTG). A glutamine-donor substrate peptide of MTG was introduced at the 5-position on the pyrimidine of deoxyuridine triphosphate to prepare a DNA strand with multiple glutamine-donor sites by polymerase chain reaction (PCR). A substrate peptide that contained an MTG-reactive lysine residue was fused to the N terminus of a thermostable alkaline phoshatase from Pyrococcus furiosus (PfuAP) by genetic engineering. By combining enzymatically the substrate moieties of MTG introduced to the DNA template and the recombinant enzyme, a DNA-(enzyme)(n) conjugate with 1:n stoichiometry was successfully obtained. The enzyme/DNA ratio of the conjugate increased as the benzyloxycarbonyl-L-glutaminylglycine (Z-QG) moiety increased in the DNA template. The potential utility of the new conjugate decorated with signaling enzymes was validated in a dot blot hybridization assay. The DNA-(enzyme)(n) probe could clearly detect 10(4) copies of the target nucleic acid with the complementary sequence under harsh hybridization conditions, thereby enabling a simple detection procedure without cumbersome bound/free processes associated with a conventional hapten-antibody reaction-based DNA-detection system.

Conjugation of enzymes on RNA probes through Cu(I) catalyzed alkyne-azide cycloaddition.

Northern and Southern blots are the most commonly used techniques for the confirmation of presence and expression of target genes. Molecular tools available for this purpose include radioisotope-, enzyme- and hapten-labeled nucleic acid probes. In particular, the use of enzyme-labeled probes are easy and safe, and do not require bound/free processes after hybridization associated with an antibody-based detection system. However, there are few approaches that enable the post-transcriptional modification of RNA with enzymes or proteins. In this study, we applied the Cu(I)-catalyzed [3 + 2] azide-alkyne cycloaddition (CuAAC) reaction to the labeling of an RNA strand with enzymes. The C-5 position of UTP was modified with an alkyne group and alkyne-bearing RNA was prepared by in vitro transcription using T7 RNA polymerase. Surface amino groups of bacterial alkaline phosphatase (BAP) were randomly derivatized with azide groups at different modification ratios. The CuAAC reaction occurred selectively between the alkyne-modified RNA and the azide-modified enzyme. The RNA probe conjugated with BAP using this technique could detect a specific RNA by dot blot northern hybridization.

Loss-of-function analyses of the fragile X-related and dopamine receptor genes by RNA interference in the cricket Gryllus bimaculatus.

In order to explore a possibility that the cricket Gryllus bimaculatus would be a useful model to unveil molecular mechanisms of human diseases, we performed loss-of-function analyses of Gryllus genes homologous to human genes that are responsible for human disorders, fragile X mental retardation 1 (fmr1) and Dopamine receptor (DopR). We cloned cDNAs of their Gryllus homologues, Gb'fmr1, Gb'DopRI, and Gb'DopRII, and analyzed their functions with use of nymphal RNA interference (RNAi). For Gb'fmr1, three major phenotypes were observed: (1) abnormal wing postures, (2) abnormal calling song, and (3) loss of the circadian locomotor rhythm, while for Gb'DopRI, defects of wing posture and morphology were found. These results indicate that the cricket has the potential to become a novel model system to explore human neuronal pathogenic mechanisms and to screen therapeutic drugs by RNAi.

Systemic RNA interference for the study of learning and memory in an insect.

RNA interference (RNAi) is a powerful technique for the study of molecular mechanisms underlying many biological processes, including brain functions. Among methods for RNAi, systemic administration of double-stranded RNA (systemic RNAi) is the most convenient for basic research as well as medical application, but it has yielded only limited success. To our knowledge, systemic RNAi has not been achieved for the study of learning and memory in any animals. Here we demonstrate successful systemic RNAi of the NOS gene coding for nitric oxide synthase, which, as we previously suggested, plays a critical role in the formation of olfactory long-term memory (LTM), in the nymphal cricket Gryllus bimaculatus. In situ hybridization demonstrated a high level of expression of NOS in a subset of Kenyon cells of the mushroom body, which is known to participate in olfactory learning and memory, in addition to some neurons around the antenna lobe and the base of the optic lobe. Injection of NOS double-stranded RNA (dsRNA) into the haemolymph completely impaired 1-day memory retention, although 30 min retention was unaffected. This impairment was fully rescued by injection of an NO donor, NOR3, thus suggesting that the effect of NOS dsRNA is through inhibition of NOS. Inhibition of NOS had no effects on recall of LTM. The results demonstrate that silencing of NOS expression by systemic RNAi impairs LTM formation. Systemic RNAi will become a useful method for study of the molecular mechanisms of learning and memory.

Common coding variant in the TCF7L2 gene and study of the association with type 2 diabetes in Japanese subjects.

Genetic variants of the transcription factor 7-like 2 (TCF7L2) gene affect the risk of type 2 diabetes in populations with multiple ethnic groups. However, a comprehensive survey of this gene has not been done for a Japanese population. Thus, we conducted this gene-based association study, in which the common genetic variants were analyzed. Using 24 Japanese type 2 diabetic subjects, we first screened a 9.5 kb region, which included the entire coding sequence, to assess potential functional variants of TCF7L2. Sequencing revealed a common coding variant (Pro477Thr) in exon 14 of TCF7L2 that was not enrolled in the public SNP database. Nineteen SNPs and the microsatellite DG10S478 were genotyped across the gene in 2,877 unrelated Japanese subjects. This independent screen identified the previously reported rs7903146 with a strongest association (allele P = 0.0001, odds ratio = 1.59 [95% confidence interval 1.25-2.01]), but there was no significant association between Pro477Thr and type 2 diabetes (allele P = 0.64). Expression of the Pro477Thr variant did not alter TCF7L2 expression in 30 lymphoblast cells. Although a genotypic effect of Pro477Thr on expression of TCF7L2 was not apparent, Pro477Thr was identified as a common variant of TCF7L2 in 2,877 Japanese subjects. Further functional studies are required to determine the possible effect of this coding variant on type 2 diabetes.

Transgenic expression of a mutated cyclin-dependent kinase 4 (CDK4/R24C) in pancreatic beta-cells prevents progression of diabetes in db/db mice.

In an attempt to rectify the hyperglycemic state in obese insulin resistant db/db mice, a transgenic line was generated (db/db-CDK4(R24C)) that expresses a constitutively active form of cyclin-dependent kinase 4 (CDK4/R24C) under the control of the insulin promoter. Compared with non-transgenic db/db littermates, adult db/db-CDK4(R24C) mice show near-complete glycemic normalization and improved plasma lipid concentrations, but are also more susceptible to weight gain and have significantly lower plasma adiponection levels. They have striking islet hypertrophy and beta-cell hyperplasia, and retain an insulin secretory response during the glucose tolerance test. We examined the expression of several key regulatory transcription factor genes involved in lipid and glucose metabolism in insulin target tissues of db/db-CDK4(R24C) as well as db/db mice, and found that the expression levels of members of the peroxisome proliferator-activated receptor (PPAR) family are highly associated with metabolic alterations in a gene- and tissue-specific manner. We show for the first time that the Ppar-delta in skeletal muscle and white adipose tissues is transcriptionally down-regulated in db/db mice. The db/db-CDK4(R24C) mice present a novel model of leptin-resistant obesity with compensatory hyperinsulinemia and normalized blood glucose levels, and thus may be useful for future studies that aim to dissect relationships between insulin and leptin signaling.

EGFR signaling is required for re-establishing the proximodistal axis during distal leg regeneration in the cricket Gryllus bimaculatus nymph.

Nymphs of hemimetabolous insects, such as cockroaches and crickets, possess functional legs with a remarkable capacity for epimorphic regeneration. In this study, we have focused on the role of epidermal growth factor receptor (EGFR) signaling in regeneration of a nymphal leg in the cricket Gryllus bimaculatus. We performed loss-of-function analyses with a Gryllus Egfr homolog (Gb'Egfr) and nymphal RNA interference (RNAi). After injection of double-stranded RNA for Gb'Egfr in the body cavity of the third instar cricket nymph, amputation of the leg at the distal tibia resulted in defects of normal distal regeneration. The regenerated leg lacked the distal tarsus and pretarsus. This result indicates that EGFR signaling is required for distal leg patterning in regeneration during the nymphal stage of the cricket. Furthermore, we demonstrated that EGFR signaling acts downstream of the canonical Wnt/Wg signaling and regulates appendage proximodistal (PD) patterning genes aristaless and dachshund during regeneration. Our results suggest that EGFR signaling influences positional information along the PD axis in distal leg patterning of insects, regardless of the leg formation mode.

SNPs in the KCNJ11-ABCC8 gene locus are associated with type 2 diabetes and blood pressure levels in the Japanese population.

Many genetic association studies support a contribution of genetic variants in the KCNJ11-ABCC8 gene locus to type 2 diabetes (T2D) susceptibility in Caucasians. In non-Caucasian populations, however, there have been only a few association studies, and discordant results were obtained. Herein, we selected a total of 31 SNPs covering a 211.3-kb region of the KCNJ11-ABCC8 locus, characterized the patterns of linkage disequilibrium (LD) and haplotype structure, and performed a case-control association study in a Japanese population consisting of 909 T2D patients and 893 control subjects. We found significant associations between eight SNPs, including the KCNJ11 E23K and ABCC8 S1369A variants, and T2D. These disease-associated SNPs were genetically indistinguishable because of the presence of strong LD, as found previously in Caucasians. For the KCNJ11 E23K variant, the most significant association was obtained under a dominant genetic model (OR 1.32, 95% CI 1.09-1.60, P = 0.004). A meta-analysis of East Asian studies, comprising a total of 3,357 T2D patients (77.4% Japanese) and 2,836 control subjects (77.8% Japanese), confirmed the significant role of the KCNJ11 E23K variant in T2D susceptibility. Furthermore, we found evidence suggesting that the KCNJ11 E23K genotype is independently associated with higher blood-pressure levels.

Molecular characterization of GDD1/TMEM16E, the gene product responsible for autosomal dominant gnathodiaphyseal dysplasia.

The human GDD1/TMEM16E gene has been found to be mutated in gnathodiaphyseal dysplasia, an unusual skeletal syndrome with autosomal dominant inheritance. The molecular and biochemical function(s) of GDD1 protein has not yet been elucidated. In this study, we examined the murine GDD1 gene expression pattern during embryonic development, and characterized the cellular and tissue localizations of its gene product using a GDD1-specific antibody. In the developing embryos, GDD1 mRNA expression was principally associated with differentiating and developing somites, with a highly complex spatiotemporal pattern that involved the myotomal and sclerotomal lineages of somites. Biochemical studies indicated that GDD1 protein is an integral membrane glycoprotein that resides predominantly in intracellular vesicles. Immunohistochemical analysis showed a high level of murine GDD1 protein expression in cardiac and skeletal muscle tissues, and in growth-plate chondrocytes and osteoblasts in bone. These observations suggest diverse cellular role(s) of GDD1 in the development of musculoskeletal system.

even-skipped has gap-like, pair-rule-like, and segmental functions in the cricket Gryllus bimaculatus, a basal, intermediate germ insect (Orthoptera).

Developmental mechanisms of segmentation appear to be varied among insects in spite of their conserved body plan. Although the expression patterns of the segment polarity genes in all insects examined imply well conserved function of this class of genes, expression patterns and function of the pair-rule genes tend to exhibit diversity. To gain further insights into the evolution of the segmentation process and the role of pair-rule genes, we have examined expression and function of an ortholog of the Drosophila pair-rule gene even-skipped (eve) in a phylogenetically basal insect, Gryllus bimaculatus (Orthoptera, intermediate germ cricket). We find that Gryllus eve (Gb'eve) is expressed as stripes in each of the prospective gnathal, thoracic, and abdominal segments and as a broad domain in the posterior growth zone. Dynamics of stripe formation vary among Gb'eve stripes, representing one of the three modes, the segmental, incomplete pair-rule, and complete pair-rule mode. Furthermore, we find that RNAi suppression of Gb'eve results in segmentation defects in both anterior and posterior regions of the embryo. Mild depletion of Gb'eve shows a pair-rule-like defect in anterior segments, while stronger depletion causes a gap-like defect showing deletion of anterior and posterior segments. These results suggest that Gb'eve acts as a pair-rule gene at least during anterior segmentation and also has segmental and gap-like functions. Additionally, Gb'eve may be involved in the regulation of hunchback and Krüppel expression. Comparisons with eve functions in other species suggest that the Gb'eve function may represent an intermediate state of the evolution of pair-rule patterning by eve in insects.

Expression and distribution of Gpr119 in the pancreatic islets of mice and rats: predominant localization in pancreatic polypeptide-secreting PP-cells.

The GPR119 was recently shown to be activated by oleoylethanolamide (OEA), a naturally occurring bioactive lipid with hypophagic and anti-obesity effects. In this study, we have cloned and characterized its murine counterpart, Gpr119. The full-length cDNA contained an open reading frame of 1008bp encoding a 335-amino acid protein. The genomic organization of Gpr119 was unique, having a 3'-untranslated second exon that was also involved in an alternative splicing event. Gene expression analyses confirmed its specific expressions in pancreatic islets and two endocrine cell-lines, MIN6 and alphaTC1. Immunohistochemistry and double-immunofluorescence studies using a specific antibody revealed the predominant Gpr119 localization in pancreatic polypeptide (PP)-cells of islets. No definitive evidence of Gpr119-immunoreactivity in adult beta- or alpha-cells was obtained. The Gpr119 mRNA levels were elevated in islets of obese hyperglycemic db/db mice as compared to control islets, suggesting a possible involvement of this receptor in the development of obesity and diabetes.

brachyenteron is necessary for morphogenesis of the posterior gut but not for anteroposterior axial elongation from the posterior growth zone in the intermediate-germband cricket Gryllus bimaculatus.

In the long-germband insect Drosophila, all body segments and posterior terminal structures, including the posterior gut and anal pads, are specified at the blastoderm stage. In short- and intermediate-germband insects, however, posterior segments are sequentially produced from the posterior growth zone, a process resembling somitogenesis in vertebrates, and invagination of the posterior gut starts after anteroposterior (AP) axial elongation from the growth zone. The mechanisms underlying posterior segmentation and terminal patterning in these insects are poorly understood. In order to elucidate these mechanisms, we have investigated the roles of the Brachyury/brachyenteron (Bra/byn) homolog in the intermediate-germband cricket Gryllus bimaculatus. Loss-of-function analysis by RNA interference (RNAi) revealed that Gryllus byn (Gb'byn) is not required for AP axial elongation or normal segment formation, but is required for specification of the posterior gut. We also analyzed Gryllus caudal (Gb'cad) RNAi embryos using in situ hybridization with a Gb'byn probe, and found that Gb'cad is required for internalization of the posterior gut primordium, in addition to AP axial elongation. These results suggest that the functions of byn and cad in posterior terminal patterning are highly conserved in Gryllus and Drosophila despite their divergent posterior patterning. Moreover, because it is thought that the progressive growth of the AP axis from the growth zone, controlled by a genetic program involving Cdx/cad and Bra/byn, might be ancestral to bilaterians, our data suggest that the function of Bra/byn in this process might have been lost in insects.