Yoshiko Inoue

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.

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.

Expression patterns of hedgehog, wingless, and decapentaplegic during gut formation of Gryllus bimaculatus (cricket).

We observed expression patterns of hedgehog (hh), wingless (wg), and decapentaplegic (dpp) during gut development of Gryllus bimaculatus (the cricket), a typical hemimetabolous insect, and compared with those observed in Drosophila, a typical holometabolous insect. Gryllus hh(Gbhh) and Gbwg are expressed in both foregut and hindgut, while Gbdpp is expressed only in the hindgut: at the boundaries between the small and large intestine, and between the large intestine and rectum. Although the expression patterns of Gbhh and Gbwg are essentially comparable to those observed in Drosophila, the expression pattern of Gbdpp differs from those of the Drosophila dpp.

Extrachromosomal transposition of the transposable element Minos in embryos of the cricket Gryllus bimaculatus.

Effective germline transformation of insects has been shown to depend on the right choice of transposon system and selection marker. In this study the promoter region of a Gryllus cytoplasmic actin (GbA3/4) gene was isolated and characterized, and was used to drive the expression of Minos transposase in embryos of the cricket Gryllus bimaculatus. Active Minos transposase was produced in these embryos as monitored through established transposon excision and interplasmid transposition assays. In contrast, Drosophila melanogaster hsp70 promoter, previously used to express Minos transposase in a number of insect species and insect cell lines, failed to produce any detectable Minos transposase activity, as recorded by using the very sensitive transposon excision assay. In addition, the GbA3/4 promoter was found to drive expression of enhanced green fluorescent protein (eGFP) predominantly in vitellophages of the developing Gryllus eggs when a plasmid carrying a GbA3/4 promoter‐eGFP fusion gene was transiently injected into embryos. These results strongly support the use of Minos transposons marked with the GbA3/4 promoter‐eGFP for the genetic transformation of this emerging model insect species.

Formation of New Organizing Regions by Cooperation of hedgehog, wingless, and dpp in Regeneration of the Insect Leg; a Verification of the Boundary Model

Meinhardt [15] proposed the boundary model (BM) to explain pattern formation in developmental subfields. One of applications of his model was to explain formation of supernumerary legs after amputating a leg and reimplanting it onto a contralateral stump. The boundary model was cemented by Campbell and Tomlinson [6]. Their model (CTBM) postulates that key genes during leg development such as hedgehog (hh),wingless (wg), and decapentaplegic (dpp) are also involved in leg regeneration and 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 (AP) boundary. To verify this model, we experimentally examined whether the model would fit our results obtained during the leg regen-eration of a cricket Gryllus bimaculatus. Since the expression patterns of the three genes can be predicted in a regenerating leg by the model, we observed corresponding expression patterns in supernumerary legs formed when the distal part of an amputated leg was grafted onto the contralateral leg stump so as to reverse the AP polarity. Since our observations were essentially consistent with the C’l’BM. we concluded that we were able to verify the BM by our regeneration experiments.