Toll signalling promotes blastema cell proliferation during cricket leg regeneration via insect macrophages.

Hemimetabolous insects, such as the two-spotted cricket Gryllus bimaculatus, can recover lost tissues, in contrast to the limited regenerative abilities of human tissues. Following cricket leg amputation, the wound surface is covered by the wound epidermis, and plasmatocytes, which are insect macrophages, accumulate in the wound region. Here, we studied the function of Toll-related molecules identified by comparative RNA sequencing during leg regeneration. Of the 11 Toll genes in the Gryllus genome, expression of Toll2-1, Toll2-2 and Toll2-5 was upregulated during regeneration. RNA interference (RNAi) of Toll, Toll2-1, Toll2-2, Toll2-3 or Toll2-4 produced regeneration defects in more than 50% of crickets. RNAi of Toll2-2 led to a decrease in the ratio of S- and M-phase cells, reduced expression of JAK/STAT signalling genes, and reduced accumulation of plasmatocytes in the blastema. Depletion of plasmatocytes in crickets using clodronate also produced regeneration defects, as well as fewer proliferating cells in the regenerating legs. Plasmatocyte depletion also downregulated the expression of Toll and JAK/STAT signalling genes in the regenerating legs. These results suggest that Spz-Toll-related signalling in plasmatocytes promotes leg regeneration through blastema cell proliferation by regulating the Upd-JAK/STAT signalling pathway.

Evolutionarily conserved function of the even-skipped ortholog in insects revealed by gene knock-out analyses in Gryllus bimaculatus.

Comparing the developmental mechanisms of segmentation among insects with different modes of embryogenesis provides insights on how the function of segmentation genes evolved. Functional analysis of eve by genetic mutants shows that the Drosophila pair-rule gene, even-skipped (eve), contributes to initial segmental patterning. However, eve orthologs tends to have diverse functions in other insects. To compare the evolutionary functional divergence of this gene, we evaluated eve function in a phylogenetically basal insect, the cricket Gryllus bimaculatus. To investigate the phenotypic effects of eve gene knock-out, we generated CRISPR/Cas9 system-mediated mutant strains of the cricket. CRISPR/Cas9 mutagenesis of multiple independent sites in the eve coding region revealed that eve null mutant embryos were defective in forming the gnathal, thoracic, and abdominal segments, consequently shortening the anterior-posterior axis. In contrast, the structures of the anterior and posterior ends (e.g., antenna, labrum, and cercus) formed normally. Hox gene expression in the gnathal, thoracic, and abdominal segments was detected in the mutant embryos. Overall, this study showed that Gryllus eve plays an important role in embryonic elongation and the formation of segmental boundaries in the gnathal to abdominal region of crickets. In the light of studies on other species, the eve function shown in Gryllus might be ancestral in insects.

Involvement of the scalloped gene in morphogenesis of the wing margin via regulating cell growth in a hemimetabolous insect Gryllus bimaculatus.

The acquisition of wings was a key event in insect evolution. As hemimetabolous insects were the first group to acquire functional wings, establishing the mechanisms of wing formation in this group could provide useful insights into their evolution. In this study, we aimed to elucidate the expression and function of the gene scalloped (sd), which is involved in wing formation in Drosophila melanogaster, and in Gryllus bimaculatus mainly during postembryonic development. Expression analysis showed that sd is expressed in the tergal edge, legs, antennae, labrum, and cerci during embryogenesis and in the distal margin of the wing pads from at least the sixth instar in the mid to late stages. Because sd knockout caused early lethality, nymphal RNA interference experiments were performed. Malformations were observed in the wings, ovipositor, and antennae. By analyzing the effects on wing morphology, it was revealed that sd is mainly involved in the formation of the margin, possibly through the regulation of cell proliferation. In conclusion, sd might regulate the local growth of wing pads and influence wing margin morphology in Gryllus.

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.

Leg regeneration is epigenetically regulated by histone H3K27 methylation in the cricket Gryllus bimaculatus.

Hemimetabolous insects such as the cricket Gryllus bimaculatus regenerate lost tissue parts using blastemal cells, a population of dedifferentiated proliferating cells. The expression of several factors that control epigenetic modification is upregulated in the blastema compared with differentiated tissue, suggesting that epigenetic changes in gene expression might control the differentiation status of blastema cells during regeneration. To clarify the molecular basis of epigenetic regulation during regeneration, we focused on the function of the Gryllus Enhancer of zeste [Gb'E(z)] and Ubiquitously transcribed tetratricopeptide repeat gene on the X chromosome (Gb'Utx) homologues, which regulate methylation and demethylation of histone H3 lysine 27 (H3K27), respectively. Methylated histone H3K27 in the regenerating leg was diminished by Gb'E(z)(RNAi) and was increased by Gb'Utx(RNAi). Regenerated Gb'E(z)(RNAi) cricket legs exhibited extra leg segment formation between the tibia and tarsus, and regenerated Gb'Utx(RNAi) cricket legs showed leg joint formation defects in the tarsus. In the Gb'E(z)(RNAi) regenerating leg, the Gb'dac expression domain expanded in the tarsus. By contrast, in the Gb'Utx(RNAi) regenerating leg, Gb'Egfr expression in the middle of the tarsus was diminished. These results suggest that regulation of the histone H3K27 methylation state is involved in the repatterning process during leg regeneration among cricket species via the epigenetic regulation of leg patterning gene expression.

Bone morphogenetic protein signaling in distal patterning and intercalation during leg regeneration of the cricket, Gryllus bimaculatus.

The cricket, Gryllus bimaculatus, is a classic model of leg regeneration following amputation. We previously demonstrated that Gryllus decapentaplegic (Gb'dpp) is expressed during leg regeneration, although it remains unclear whether it is essential for this process. In this study, double-stranded RNA targeting the Smad mathers-against-dpp homolog, Gb'mad, was used to examine the role of bone morphogenetic protein (BMP) signaling in the leg regeneration process of Gryllus bimaculatus. RNA interference (RNAi)-mediated knockdown of Gb'mad led to a loss of tarsus regeneration at the most distal region of regenerating leg segments. Moreover, we confirmed that the phenotype obtained by knockdown of Dpp type I receptor, Thick veins (Gb'tkv), closely resembled that observed for Gb'mad RNAi crickets, thereby suggesting that the BMP signaling pathway is indispensable for the initial stages of tarsus formation. Interestingly, knockdown of Gb'mad and Gb'tkv resulted in significant elongation of regenerating tibia along the proximodistal axis compared with normal legs. Moreover, our findings indicate that during the regeneration of tibia, the BMP signaling pathway interacts with Dachsous/Fat (Gb'Ds/Gb'Ft) signaling and dachshund (Gb'dac) to re-establish positional information and regulate determination of leg size. Based on these observations, we discuss possible roles for Gb'mad in the distal patterning and intercalation processes during leg regeneration in Gryllus bimaculatus.

Analysis of RNA-Seq data reveals involvement of JAK/STAT signalling during leg regeneration in the cricket Gryllus bimaculatus.

In the cricket Gryllus bimaculatus, missing distal parts of the amputated leg are regenerated from the blastema, a population of dedifferentiated proliferating cells that forms at the distal tip of the leg stump. To identify molecules involved in blastema formation, comparative transcriptome analysis was performed between regenerating and normal unamputated legs. Components of JAK/STAT signalling were upregulated more than twofold in regenerating legs. To verify their involvement, Gryllus homologues of the interleukin receptor Domeless (Gb'dome), the Janus kinase Hopscotch (Gb'hop) and the transcription factor STAT (Gb'Stat) were cloned, and RNAi was performed against these genes. Gb'dome(RNAi), Gb'hop(RNAi) and Gb'Stat(RNAi) crickets showed defects in leg regeneration. Blastema expression of Gb'cyclinE was decreased in the Gb'Stat(RNAi) cricket compared with that in the control. Hyperproliferation of blastema cells caused by Gb'fat(RNAi) or Gb'warts(RNAi) was suppressed by RNAi against Gb'Stat. The results suggest that JAK/STAT signalling regulates blastema cell proliferation during leg regeneration.

Gene knockout by targeted mutagenesis in a hemimetabolous insect, the two-spotted cricket Gryllus bimaculatus, using TALENs.

Hemimetabolous, or incompletely metamorphosing, insects are phylogenetically basal. These insects include many deleterious species. The cricket, Gryllus bimaculatus, is an emerging model for hemimetabolous insects, based on the success of RNA interference (RNAi)-based gene-functional analyses and transgenic technology. Taking advantage of genome-editing technologies in this species would greatly promote functional genomics studies. Genome editing using transcription activator-like effector nucleases (TALENs) has proven to be an effective method for site-specific genome manipulation in various species. TALENs are artificial nucleases that are capable of inducing DNA double-strand breaks into specified target sequences. Here, we describe a protocol for TALEN-based gene knockout in G. bimaculatus, including a mutant selection scheme via mutation detection assays, for generating homozygous knockout organisms.

Ancestral functions of Delta/Notch signaling in the formation of body and leg segments in the cricket Gryllus bimaculatus.

Delta/Notch signaling controls a wide spectrum of developmental processes, including body and leg segmentation in arthropods. The various functions of Delta/Notch signaling vary among species. For instance, in Cupiennius spiders, Delta/Notch signaling is essential for body and leg segmentation, whereas in Drosophila fruit flies it is involved in leg segmentation but not body segmentation. Therefore, to gain further insight into the functional evolution of Delta/Notch signaling in arthropod body and leg segmentation, we analyzed the function of the Delta (Gb'Delta) and Notch (Gb'Notch) genes in the hemimetabolous, intermediate-germ cricket Gryllus bimaculatus. We found that Gb'Delta and Gb'Notch were expressed in developing legs, and that RNAi silencing of Gb'Notch resulted in a marked reduction in leg length with a loss of joints. Our results suggest that the role of Notch signaling in leg segmentation is conserved in hemimetabolous insects. Furthermore, we found that Gb'Delta was expressed transiently in the posterior growth zone of the germband and in segmental stripes earlier than the appearance of wingless segmental stripes, whereas Gb'Notch was uniformly expressed in early germbands. RNAi knockdown of Gb'Delta or Gb'Notch expression resulted in malformation in body segments and a loss of posterior segments, the latter probably due to a defect in posterior growth. Therefore, in the cricket, Delta/Notch signaling might be required for proper morphogenesis of body segments and posterior elongation, but not for specification of segment boundaries.

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.

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.

Functional analysis of the role of eyes absent and sine oculis in the developing eye of the cricket Gryllus bimaculatus.

In the cricket Gryllus bimaculatus, a hemimetabolous insect, the compound eyes begin to form in the embryo and increase 5-6 fold in size during the postembryonic development of the nymphal stage. Retinal stem cells in the anteroventral proliferation zone (AVPZ) of the nymphal eye proliferate to increase retinal progenitors, which then differentiate to form new ommatidia in the anterior region of the eye. However, mechanisms underlying this type of eye formation have not been well elucidated yet. Here, we found that the homologues of the retinal determination transcription factor genes of eyes absent (eya) and sine oculis (so) are expressed during the cricket embryonic eye formation. eya is also expressed intensely in the AVPZ of the nymphal eye. To explore their functions, we performed knockdown by RNA interference (RNAi). Knockdown of Gb'eya resulted in loss of the embryonic eye. In the nymphal eye, RNAi against Gb'eya or Gb'so impaired retinal morphology by apparently transforming cornea structures into head cuticle. These results imply that Gb'eya and Gb'so are essential for the differentiation of the retinal progenitor cells and maintaining retinal structures during eye development.

Regulation of leg size and shape: involvement of the Dachsous-fat signaling pathway.

How limb size and shape is regulated is a long-standing question in developmental and regeneration biology. Recently, the protocadherin Dachsous-Fat (Ds-Ft) signaling pathway has been found to be essential for wing development of the fly and leg regeneration of the cricket. The Ds-Ft signaling pathway is linked to the Warts-Hippo (Wts-Hpo) signaling pathway, leading to cell proliferation. Several lines of evidence have suggested that the Wts-Hpo signaling pathway is involved in the control of organ size, and that this pathway is regulated by Ds-Ft and Merlin-Expanded, which are linked to morphogens such as decapentaplegic/bone morphogenic protein, Wingless/Wnt, and epidermal growth factor. Here we review recent progress in elucidating mechanisms controlling leg size and shape in insects and vertebrates, focusing on the Ds-Ft signaling pathway. We also introduce a working model, Ds-Ft steepness model, to explain how steepness of the Ds-Ft gradient controls leg size along the proximodistal axis.

Lowfat, a mammalian Lix1 homologue, regulates leg size and growth under the Dachsous/Fat signaling pathway during tissue regeneration.

In the cricket Gryllus bimaculatus, missing distal parts of amputated legs are regenerated from blastemas based on positional information. The Dachsous/Fat (Ds/Ft) signaling pathway regulates blastema cell proliferation and positional information along the longitudinal axis during leg regeneration. Herein, we show that the Gryllus homologue of Lowfat (Gb'Lft), which modulates Ds/Ft signaling in Drosophila, is involved in leg regeneration. Gb'lft is expressed in regenerating legs, and RNAi against Gb'lft (Gb'lft(RNAi)) suppressed blastema cell hyperproliferation caused by Gb'ft(RNAi) or Gb'ds(RNAi) but enhanced that caused by Gb'kibra(RNAi) or Gb'warts(RNAi). In Gb'lft(RNAi) nymphs, missing parts of amputated legs were regenerated, but the length of the regenerated legs was shortened depending on the position of the amputation. Both normal and reversed intercalary regeneration occurred in Gb'lft(RNAi) nymphs, suggesting that Gb'Lft is involved in blastema cell proliferation and longitudinal leg regeneration under the Ds/Ft signaling pathway, but it is not required for intercalary regeneration.

A hemimetabolous wing development suggests the wing origin from lateral tergum of a wingless ancestor.

The origin and evolution of the novel insect wing remain enigmatic after a century-long discussion. The mechanism of wing development in hemimetabolous insects, in which the first functional wings evolved, is key to understand where and how insect wings evolutionarily originate. This study explored the developmental origin and the postembryonic dramatic growth of wings in the cricket Gryllus bimaculatus. We find that the lateral tergal margin, which is homologous between apterygote and pterygote insects, comprises a growth organizer to expand the body wall to form adult wing blades in Gryllus. We also find that Wnt, Fat-Dachsous, and Hippo pathways are involved in the disproportional growth of Gryllus wings. These data provide insights into where and how insect wings originate. Wings evolved from the pre-existing lateral terga of a wingless insect ancestor, and the reactivation or redeployment of Wnt/Fat-Dachsous/Hippo-mediated feed-forward circuit might have expanded the lateral terga.

Cricket: The third domesticated insect.

Many researchers are using crickets to conduct research on various topics related to development and regeneration in addition to brain function, behavior, and biological clocks, using advanced functional and perturbational technologies such as genome editing. Recently, crickets have also been attracting attention as a food source for the next generation of humans. In addition, crickets are increasingly being used as disease models and biological factories for pharmaceuticals. Cricket research has thus evolved over the last century from use primarily in highly important basic research, to use in a variety of applications and practical uses. These insects are now a state-of-the-art model animal that can be obtained and maintained in large quantities at low cost. We therefore suggest that crickets are useful as a third domesticated insect for scientific research, after honeybees and silkworms, contributing to the achievement of global sustainable development goals.

Involvement of a Basic Helix-Loop-Helix Gene BHLHE40 in Specification of Chicken Retinal Pigment Epithelium.

The first event of differentiation and morphogenesis in the optic vesicle (OV) is specification of the neural retina (NR) and retinal pigment epithelium (RPE), separating the inner and outer layers of the optic cup, respectively. Here, we focus on a basic helix-loop-helix gene, BHLHE40, which has been shown to be expressed by the developing RPE in mice and zebrafish. Firstly, we examined the expression pattern of BHLHE40 in the developing chicken eye primordia by in situ hybridization. Secondly, BHLHE40 overexpression was performed with in ovo electroporation and its effects on optic cup morphology and expression of NR and RPE marker genes were examined. Thirdly, we examined the expression pattern of BHLHE40 in LHX1-overexpressed optic cup. BHLHE40 expression emerged in a subset of cells of the OV at Hamburger and Hamilton stage 14 and became confined to the outer layer of the OV and the ciliary marginal zone of the retina by stage 17. BHLHE40 overexpression in the prospective NR resulted in ectopic induction of OTX2 and repression of VSX2. Conversely, BHLHE40 was repressed in the second NR after LHX1 overexpression. These results suggest that emergence of BHLHE40 expression in the OV is involved in initial RPE specification and that BHLHE40 plays a role in separation of the early OV domains by maintaining OTX2 expression and antagonizing an NR developmental program.

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.

Insights into the genomic evolution of insects from cricket genomes.

Most of our knowledge of insect genomes comes from Holometabolous species, which undergo complete metamorphosis and have genomes typically under 2 Gb with little signs of DNA methylation. In contrast, Hemimetabolous insects undergo the presumed ancestral process of incomplete metamorphosis, and have larger genomes with high levels of DNA methylation. Hemimetabolous species from the Orthopteran order (grasshoppers and crickets) have some of the largest known insect genomes. What drives the evolution of these unusual insect genome sizes, remains unknown. Here we report the sequencing, assembly and annotation of the 1.66-Gb genome of the Mediterranean field cricket Gryllus bimaculatus, and the annotation of the 1.60-Gb genome of the Hawaiian cricket Laupala kohalensis. We compare these two cricket genomes with those of 14 additional insects and find evidence that hemimetabolous genomes expanded due to transposable element activity. Based on the ratio of observed to expected CpG sites, we find higher conservation and stronger purifying selection of methylated genes than non-methylated genes. Finally, our analysis suggests an expansion of the pickpocket class V gene family in crickets, which we speculate might play a role in the evolution of cricket courtship, including their characteristic chirping.

Generation of mutant pigs by lipofection-mediated genome editing in embryos.

The specificity and efficiency of CRISPR/Cas9 gene-editing systems are determined by several factors, including the mode of delivery, when applied to mammalian embryos. Given the limited time window for delivery, faster and more reliable methods to introduce Cas9-gRNA ribonucleoprotein complexes (RNPs) into target embryos are needed. In pigs, somatic cell nuclear transfer using gene-modified somatic cells and the direct introduction of gene editors into the cytoplasm of zygotes/embryos by microinjection or electroporation have been used to generate gene-edited embryos; however, these strategies require expensive equipment and sophisticated techniques. In this study, we developed a novel lipofection-mediated RNP transfection technique that does not require specialized equipment for the generation of gene-edited pigs and produced no detectable off-target events. In particular, we determined the concentration of lipofection reagent for efficient RNP delivery into embryos and successfully generated MSTN gene-edited pigs (with mutations in 7 of 9 piglets) after blastocyst transfer to a recipient gilt. This newly established lipofection-based technique is still in its early stages and requires improvements, particularly in terms of editing efficiency. Nonetheless, this practical method for rapid and large-scale lipofection-mediated gene editing in pigs has important agricultural and biomedical applications.