Katsuyuki Miyawaki

Involvement of Wingless/Armadillo signaling in the posterior sequential segmentation in the cricket, Gryllus bimaculatus (Orthoptera), as revealed by RNAi analysis

In insects, there are two different modes of segmentation. In the higher dipteran insects (like Drosophila), their segmentation takes place almost simultaneously in the syncytial blastoderm. By contrast, in the orthopteran insects (like Schistocerca (grasshopper)), the anterior segments form almost simultaneously in the cellular blastoderm and then the remaining posterior part elongates to form segments sequentially from the posterior proliferative zone. Although most of their orthologues of the Drosophila segmentation genes may be involved in their segmentation, little is known about their roles. We have investigated segmentation processes of Gryllus bimaculatus, focusing on its orthologues of the Drosophila segment-polarity genes, G. bimaculatus wingless (Gbwg), armadillo (Gbarm) and hedgehog (Gbhh). Gbhh and Gbwg were observed to be expressed in the each anterior segment and the posterior proliferative zone. In order to know their roles, we used RNA interference (RNAi). We could not observed any significant effects of RNAi for Gbwg and Gbhh on segmentation, probably due to functional replacement by another member of the corresponding gene families. Embryos obtained by RNAi for Gbarm exhibited abnormal anterior segments and lack of the abdomen. Our results suggest that GbWg/GbArm signaling is involved in the posterior sequential segmentation in the G. bimaculatus embryos, while Gbwg, Gbarm and Gbhh are likely to act as the segment-polarity genes in the anterior segmentation similarly as in Drosophila.

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

AbstractMany 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.

Correlation of expression patterns of homothorax, dachshund, and Distal-less with the proximodistal segmentation of the cricket leg bud

We describe the expression pattern of Gryllus homothorax (Gbhth) and dachshund (Gbdac), a cricket homologue of Drosophila homothorax and dachshund, together with localization of Distal-less or Extradenticle protein during leg development. We correlated their expression patterns with the morphological segmentation of the leg bud. The boundary of Gbhth/GbDll subdivision is correlated with the segment boundary of the future trochanter/femur at early stages. Gbdac expression subdivides the leg bud into the presumptive femur and more distal region. During the leg proximodistal formation, although the early expression patterns of GbDll, Gbdac, and Gbhth significantly differ from those of Drosophila imaginal disc, their expression patterns in the fully segmented Gryllus leg were similar to those in the Drosophila late third instar disc.

Involvement of hedgehog, wingless, and dpp in the initiation of proximodistal axis formation during the regeneration of insect legs, a verification of the modified boundary model

To understand the mechanism of regeneration, many experiments have been carried out with hemimetabolous insects, since their nymphs possess the ability to regenerate amputated legs. We first succeeded in observing expression patterns of hedgehog, wingless (wg), and decapentaplegic (dpp) during leg regeneration of the cricket Gryllus bimaculatus. The observed expression patterns were essentially consistent with the predictions derived from the boundary model modified by Campbell and Tomlinson (CTBM). Thus, we concluded that the formation of the proximodistal axis of a regenerating leg is triggered at a site where ventral wg-expressing cells abut dorsal dpp-expressing cells in the anteroposterior (A/P) boundary, as postulated in the CTBM.

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.

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.

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

AbstractMain conclusionLight and ABA independently regulated anthocyanin biosynthesis via activation ofFaMYB10expression. 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.

Expression patterns of aristaless in developing appendages of Gryllus bimaculatus (cricket).

We report the isolation and expression patterns of aristaless (al), a paired-type homeobox gene, of Gryllus bimaculatus (Gb), a hemimetabola model insect. Gryllus al (Gbal) is expressed in the most distal region of developing labrum, antenna, mandible, maxilla, labium, leg, cercus, and hindgut. Gbal is also expressed in the proximal region, corresponding to the presumptive coxopodite, of the developing antenna, mandible, maxilla, labium, and leg, but not in the developing labrum, cercus, and hindgut. During development of the leg, expression of Gbal changes dynamically with the progress in leg segmentation: Gbal is expressed in order in the presumptive pretarsus, coxa, femur, tibia and tarsus before appearance of morphological segmentation.

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.

piggyBac‐mediated somatic transformation of the two‐spotted cricket, Gryllus bimaculatus

Transgenic insects have been artificially produced to study functions of interesting developmental genes, using insect transposons such as piggyBac. In the case of the cricket, however, transgenic animals have not yet been successfully artificially produced. In the present study, we examined whether the piggyBac transposon functions as a tool for gene delivery in embryos of Gryllus bimaculatus. We used either a piggyBac helper plasmid or a helper RNA synthesized in vitro as a transposase source. An excision assay revealed that the helper RNA was more effective in early Gryllus eggs to transpose a marker gene of eGFP than the helper plasmid containing the piggyBac transposase gene driven by the G. bimaculatus actin3/4 promoter. Further, only when the helper RNA was used, somatic transformation of the embryo with the eGFP gene was observed. These results suggest that the piggyBac system with the helper RNA may be effective for making transgenic crickets.