Tetsuya Bando

Regulation of leg size and shape by the Dachsous/Fat signalling pathway during regeneration

An amputated cricket leg regenerates all missing parts with normal size and shape, indicating that regenerating blastemal cells are aware of both their position and the normal size of the leg. However, the molecular mechanisms regulating this process remain elusive. Here, we use a cricket model to show that the Dachsous/Fat (Ds/Ft) signalling pathway is essential for leg regeneration. We found that knockdown of ft or ds transcripts by regeneration-dependent RNA interference (rdRNAi) suppressed proliferation of the regenerating cells along the proximodistal (PD) axis concomitantly with remodelling of the pre-existing stump, making the regenerated legs shorter than normal. By contrast, knockdown of the expanded (ex) or Merlin (Mer) transcripts induced over-proliferation of the regenerating cells, making the regenerated legs longer. These results are consistent with those obtained using rdRNAi during intercalary regeneration induced by leg transplantation. We present a model to explain our results in which the steepness of the Ds/Ft gradient controls growth along the PD axis of the regenerating leg.

Imaging of Transgenic Cricket Embryos Reveals Cell Movements Consistent with a Syncytial Patterning Mechanism

The mode of insect embryogenesis varies among species, reflecting adaptations to different life history strategies [1, 2]. In holometabolous insects, which include the model systems, such as the fruit fly and the red flour beetle, a large proportion of the blastoderm produces an embryo, whereas hemimetabolous embryos generally arise from a small region of the blastoderm [3]. Despite their importance in evolutionary studies, information of early developmental dynamics of hemimetabolous insects remains limited. Here, to clarify how maternal and gap gene products act in patterning the embryo of basal hemimetabolous insects, we analyzed the dynamic segmentation process in transgenic embryos of an intermediate-germ insect species, the cricket, Gryllus bimaculatus. Our data based on live imaging of fluorescently labeled embryonic cells and nuclei suggest that the positional specification of the cellular blastoderm may be established in the syncytium, where maternally derived gradients could act fundamentally in a way that is similar to that of Drosophila, namely throughout the egg. Then, the blastoderm cells move dynamically, retaining their positional information to form the posteriorly localized germ anlage. Furthermore, we find that the anterior head region of the cricket embryo is specified by orthodenticle in a cellular environment earlier than the gnathal and thoracic regions. Our findings imply that the syncytial mode of the early segmentation in long-germ insects evolved from a dynamic syncytial-to-cellular mode found in the present study, accompanied by a heterochronic shift of gap gene action.

The advent of RNA interference in Entomology

RNA interference (RNAi) is a cellular process by which an mRNA is targeted for degradation by a small interfering RNA that contains a strand complementary to a fragment of the target mRNA, resulting in sequence specific inhibition of gene expression. The discovery of RNAi enabled the use of loss‐of‐function analyses in many non‐model insects other than Drosophila to elucidate the roles of specific genes. The RNAi approach has been widely used on insects in several fields, including embryogenesis, pattern formation, reproduction, biosynthesis and behavior. The increasing availability of insect genomes has made the RNAi technique an indispensable technique for characterizing gene functions in insects. Here we review the current status of RNAi‐based experiments in insects and the applications of RNAi for species‐specific insecticides, focusing on non‐drosophilid insects. We also identify future applications for RNAi‐based studies in Entomology.

Molecular and Cellular Basis of Regeneration and Tissue Repair

Abstract.Nymphs of hemimetabolous insects such as cockroaches and crickets exhibit a remarkable capacity for regenerating complex structures from damaged legs. Until recent years, however, approaches to elucidate the molecular mechanisms underlying the leg regeneration process have been lacking. Taking the cricket Gryllus bimaculatus as a model, we found that a phenotype related to regeneration frequently appears during leg regeneration, even though no phenotype is induced by RNA interference (RNAi) in the cricket nymph, designated as regeneration-dependent RNAi. Since then, we have investigated the functions of various genes encoding signaling factors and cellular adhesion proteins like Fat and Dachsous during leg regeneration. In this review, we summarize the classical knowledge about insect leg regeneration and introduce recent advances concerning the signaling cascades required for regenerating a leg. Our results provide clues to the mechanisms of regeneration which are relevant to vertebrate systems.

Dissecting insect leg regeneration through RNA interference.

Abstract.Nymphs of hemimetabolous insects such as cockroaches and crickets exhibit a remarkable capacity for regenerating complex structures from damaged legs. Until recent years, however, approaches to elucidate the molecular mechanisms underlying the leg regeneration process have been lacking. Taking the cricket Gryllus bimaculatus as a model, we found that a phenotype related to regeneration frequently appears during leg regeneration, even though no phenotype is induced by RNA interference (RNAi) in the cricket nymph, designated as regeneration-dependent RNAi. Since then, we have investigated the functions of various genes encoding signaling factors and cellular adhesion proteins like Fat and Dachsous during leg regeneration. In this review, we summarize the classical knowledge about insect leg regeneration and introduce recent advances concerning the signaling cascades required for regenerating a leg. Our results provide clues to the mechanisms of regeneration which are relevant to vertebrate systems.

Tissue expression of four troponin I genes and their molecular interactions with two troponin C isoforms in Caenorhabditis elegans.

Gene duplication is a major genetic event that can produce multiple protein isoforms. Comparative sequence and functional analysis of related gene products can provide insights into protein family evolution. To characterize the Caenorhabditis elegans troponin I family, we analyzed gene structures, tissue expression patterns and RNAi phenotypes of four troponin I isoforms. Tissue expression patterns were determined using lacZ/gfp/rfp reporter gene assays. The tni‐1, tni‐2/unc‐27 and tni‐3 genes, each encoding a troponin I isoform, are uniquely expressed in body wall, vulval and anal muscles but at different levels; tni‐4 was expressed solely in the pharynx. Expressing tni‐1 and ‐2 gene RNAi caused motility defects similar to unc‐27 (e155) mutant, a tni‐2 null allele. The tni‐3 RNAi expression produced egg laying defects while the tni‐4 RNAi caused arrest at gastrulation. Overlay analyses were used to assay interactions between the troponin I and two troponin C isoforms. The three body wall troponin I isoforms interacted with body wall and pharyngeal troponin C isoforms; TNI‐4 interacted only with pharyngeal troponin C. Our results suggest the body wall genes have evolved following duplication of the pharynx gene and provide important data about gene duplication and functional differentiation of nematode troponin I isoforms.

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 chiconyRNAi 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.

Relationship between somatic mosaicism of Pax6 mutation and variable developmental eye abnormalities—an analysis of CRISPR genome-edited mouse embryos

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) system is a rapid gene-targeting technology that does not require embryonic stem cells. To demonstrate dosage effects of the Pax6 gene on eye formation, we generated Pax6-deficient mice with the CRISPR/Cas system. Eyes of founder embryos at embryonic day (E) 16.5 were examined and categorized according to macroscopic phenotype as class 1 (small eye with distinct pigmentation), class 2 (pigmentation without eye globes), or class 3 (no pigmentation and no eyes). Histologically, class 1 eyes were abnormally small in size with lens still attached to the cornea at E16.5. Class 2 eyes had no lens and distorted convoluted retinas. Class 3 eyes had only rudimentary optic vesicle-like tissues or histological anophthalmia. Genotyping of neck tissue cells from the founder embryos revealed somatic mosaicism and allelic complexity for Pax6. Relationships between eye phenotype and genotype were developed. The present results demonstrated that development of the lens from the surface ectoderm requires a higher gene dose of Pax6 than development of the retina from the optic vesicle. We further anticipate that mice with somatic mosaicism in a targeted gene generated by CRISPR/Cas-mediated genome editing will give some insights for understanding the complexity in human congenital diseases that occur in mosaic form.

The expression of LIM-homeobox genes, Lhx1 and Lhx5, in the forebrain is essential for neural retina differentiation.

Elucidating the mechanisms underlying eye development is essential for advancing the medical treatment of eye‐related disorders. The primordium of the eye is an optic vesicle (OV), which has a dual potential for generation of the developing neural retina and retinal pigment epithelium. However, the factors that regulate the differentiation of the retinal primordium remain unclear. We have previously shown that overexpression of Lhx1 and Lhx5, members of the LIM‐homeobox genes, induced the formation of a second neural retina from the presumptive pigmented retina of the OV. However, the precise timing of Lhx1 expression required for neural retina differentiation has not been clarified. Moreover, RNA interference of Lhx5 has not been previously reported. Here, using a modified electroporation method, we show that, Lhx1 expression in the forebrain around stage 8 is required for neural retina formation. In addition, we have succeeded in the knockdown of Lhx5 expression, resulting in conversion of the neural retina region to a pigment vesicle‐like tissue, which indicates that Lhx5 is also required for neural retina differentiation, which correlates temporally with the activity of Lhx1. These results suggest that Lhx1 and Lhx5 in the forebrain regulate neural retina differentiation by suppressing the development of the retinal pigment epithelium, before the formation of the OV.

Molecular and cellular basis of regeneration and tissue repair: Dissecting insect leg regeneration through RNA interference

Abstract.Nymphs of hemimetabolous insects such as cockroaches and crickets exhibit a remarkable capacity for regenerating complex structures from damaged legs. Until recent years, however, approaches to elucidate the molecular mechanisms underlying the leg regeneration process have been lacking. Taking the cricket Gryllus bimaculatus as a model, we found that a phenotype related to regeneration frequently appears during leg regeneration, even though no phenotype is induced by RNA interference (RNAi) in the cricket nymph, designated as regeneration-dependent RNAi. Since then, we have investigated the functions of various genes encoding signaling factors and cellular adhesion proteins like Fat and Dachsous during leg regeneration. In this review, we summarize the classical knowledge about insect leg regeneration and introduce recent advances concerning the signaling cascades required for regenerating a leg. Our results provide clues to the mechanisms of regeneration which are relevant to vertebrate systems.