MDPI AGActa Medica Okayama1422-00672752026Fgf10 Gene Dosage from a Single Allele Is Insufficient for Forming Multilayered Epithelial Cells in the Murine Lacrimal Gland2113ENShioriIkedaDepartment of Cytology and Histology, Faculty of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama UniversityKeitaSatoDepartment of Cytology and Histology, Faculty of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama UniversityYukiTajikaDepartment of Radiological Technology, Gumma Prefectural College of Health SciencesHirofumiFujitaDepartment of Cytology and Histology, Faculty of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama UniversityTetsuyaBandoDepartment of Cytology and Histology, Faculty of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama UniversityTsutomuNohnoDepartment of Cytology and Histology, Faculty of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama UniversitySatoruMiyaishiDepartment of Legal Medicine, Faculty of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama UniversityHideyoOhuchiDepartment of Cytology and Histology, Faculty of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama UniversityMutations in the fibroblast growth factor 10 (FGF10) gene in humans cause aplasia of the lacrimal and salivary glands (ALSG). In patients with ALSG, heterozygous loss-of-function mutations are found, and FGF10 haploinsufficiency results in the absence of these secretory organs. Lacrimal glands (LGs) are formed through epithelial thickening, budding, and branching morphogenesis. To compare the variable phenotypes of the Fgf10+/| Harderian glands (HGs) previously reported, we examined the development of LGs in wild-type (WT), Fgf10+/|, and Fgf10-null mice. Pax6 immunostaining was performed to visualize the LG primordia from embryonic day 15.5 (E15.5) onwards. In situ hybridization of the genes encoding the epithelial receptor of FGF10, FGFR2b, and its other ligands was performed to determine their potential involvement in LG development. LG primordia were not observed in Fgf10+/| mice bilaterally at E16.5 or later stages. At E15.5, budding from the developing conjunctival epithelium (CE) was observed in a small fraction of the Fgf10+/| LG primordia. In contrast, the Fgf10-null CE failed to promote budding. Among Fgf1, Fgf3, Fgf7, Fgf10, and Fgf22, Fgf10 was expressed in the mesenchyme surrounding developing LG epithelial cells, whereas Fgf1 was expressed in the LG epithelium of WT mice. Fgf7 was initially expressed in the mesenchyme surrounding the nascent LG epithelium, but its expression subsequently became diffused. Thus, we conclude that among the FGFR2b ligands, initial LG formation is dependent on the mesenchymal factors FGF10 and FGF7, and FGF1 is likely to function as an epithelial factor in the LG primordia. A single allele of Fgf10 was found to be insufficient to support the budding process during LG morphogenesis.No potential conflict of interest relevant to this article was reported.The Company of BiologistsActa Medica Okayama0950-1991152222025ROS produced by Dual oxidase regulate cell proliferation and haemocyte migration during leg regeneration in the cricketdev204763ENMisaOkumura-HironoDepartment of Cytology and Histology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesTetsuyaBandoDepartment of Cytology and Histology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesYoshimasaHamadaDepartment of Cytology and Histology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesMotooArakiDepartment of Urology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesHideyoOhuchiDepartment of Cytology and Histology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesMany animals regenerate lost body parts through several signalling pathways; however, the triggers that initiate regeneration remain unclear. In the present study, we focused on the role of reactive oxygen species (ROS) produced by the NADPH oxidase Dual oxidase (Duox) during cricket leg regeneration. The results showed that ROS levels were upregulated during leg regeneration and decreased by DuoxRNAi. In DuoxRNAi nymphs, wound closure and scab formation were incomplete 2 days after amputation, and hypertrophy occurred in the distal region of the regenerating legs at 5 days after amputation. In addition, the hypertrophic phenotype was induced by DuoxARNAi and NADPH oxidase inhibitor treatment. During hypertrophy, haemocytes, including plasmatocytes, oenocytoids and granulocytes, accumulated. Proliferation of haemocytes in regenerating legs was not increased by DuoxRNAi; however, haemocyte accumulation was regulated by the Spatzle (Spz) family molecules, which are Toll receptor ligands. As the exoskeleton of DuoxRNAi nymphs was thinner than that of the control, excessive haemocyte accumulation can cause hypertrophy in DuoxRNAi nymphs. Thus, Duox-derived ROS are involved in wound healing and haemocyte accumulation through the Spz/Toll signalling pathway during leg regeneration in crickets.No potential conflict of interest relevant to this article was reported.Oxford University Press (OUP)Acta Medica Okayama1365-2672136102025Gut dysbiosis allows foodborne salmonella colonization in edible crickets: a probiotic strategy for enhanced food safetylxaf217ENShumaTsujiDepartment of Medical Laboratory Science, Okayama University Graduate School of Health SciencesOsamuMatsushitaDepartment of Bacteriology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesJumpeiUchiyamaDepartment of Bacteriology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesKenjiYokotaDepartment of Medical Laboratory Science, Okayama University Graduate School of Health SciencesTetsuyaBandoDepartment of Cytology and Histology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesHideyoOhuchiDepartment of Cytology and Histology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesKazuyoshiGotohDepartment of Medical Laboratory Science, Okayama University Graduate School of Health SciencesAims: Edible insects, including crickets, represent a promising protein source, yet concerns over foodborne pathogens limit consumer acceptance. This study investigated whether gut microbiota modulates colonization by Salmonella enterica subsp. enterica serovar Enteritidis (SE) in the two-spotted cricket (Gryllus bimaculatus).<br>
Methods and Results: Under standard conditions, SE was undetectable in crickets despite prolonged exposure; however, antibiotic-induced dysbiosis enabled stable SE colonization. Long-read 16S rRNA sequencing revealed significant microbiota shifts, notably a reduction in Lactococcus garvieae. In vitro assays showed strong inhibitory effects of L. garvieae against SE, and supplementation of dysbiotic crickets with L. garvieae reduced SE colonization by ∼1000-fold.<br>
Conclusions: The native cricket gut microbiota, especially L. garvieae, plays a protective role against SE colonization. Enhancing beneficial gut bacteria could mitigate pathogen risks and promote edible insects as a sustainable protein.No potential conflict of interest relevant to this article was reported.MDPIActa Medica Okayama2221-37591042022Involvement of a Basic Helix-Loop-Helix Gene BHLHE40 in Specification of Chicken Retinal Pigment Epithelium45ENToshikiKinuhataDepartment of Cytology and Histology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesKeitaSatoDepartment of Cytology and Histology, Okayama University Faculty of Medicine, Dentistry and Pharmaceutical SciencesTetsuyaBandoDepartment of Cytology and Histology, Okayama University Faculty of Medicine, Dentistry and Pharmaceutical SciencesTaroMitoBio-Innovation Research Center, Tokushima UniversitySatoruMiyaishiDepartment of Legal Medicine, Okayama University Faculty of Medicine, Dentistry and Pharmaceutical SciencesTsutomuNohnoDepartment of Cytology and Histology, Okayama University Medical SchoolHideyoOhuchiDepartment of Cytology and Histology, Okayama University Faculty of Medicine, Dentistry and Pharmaceutical SciencesThe 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.No potential conflict of interest relevant to this article was reported.The Company of BiologistsActa Medica Okayama0950-199114982021Toll signalling promotes blastema cell proliferation during cricket leg regeneration via insect macrophagesdev199916ENTetsuyaBandoDepartment of Cytology and Histology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesMisaOkumuraDepartment of Cytology and Histology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesYukiBandoFaculty of Medicine, Okayama University Medical SchoolMarouHagiwaraFaculty of Medicine, Okayama University Medical SchoolYoshimasaHamadaDepartment of Cytology and Histology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesYoshiyasuIshimaruDivision of Bioscience and Bioindustry, Graduate School of Technology, Industrial and Social Sciences, Tokushima UniversityTaroMitoDivision of Bioscience and Bioindustry, Graduate School of Technology, Industrial and Social Sciences, Tokushima UniversityEriKawaguchiDivision of Biological Science, Graduate School of Science, Kyoto UniversityTakeshiInoueDivision of Biological Science, Graduate School of Science, Kyoto UniversityKiyokazuAgataDivision of Biological Science, Graduate School of Science, Kyoto UniversitySumihareNojiDivision of Bioscience and Bioindustry, Graduate School of Technology, Industrial and Social Sciences, Tokushima UniversityHideyoOhuchiDepartment of Cytology and Histology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesHemimetabolous 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.No potential conflict of interest relevant to this article was reported.BMCActa Medica Okayama2056-306X612020The role of clockwork orange in the circadian clock of the cricket Gryllus bimaculatus12ENYasuakiTomiyamaGraduate School of Natural Science and Technology, Okayama UniversityTsugumichiShinoharaGraduate School of Natural Science and Technology, Okayama UniversityMiraiMatsukaGraduate School of Natural Science and Technology, Okayama UniversityTetsuyaBandoGraduate School of Medicine, Dentistry and Pharmaceutical Science, Okayama UniversityTaroMitoGraduate School of Technology, Industrial and Social Sciences, Tokushima UniversityKenjiTomiokaGraduate School of Natural Science and Technology, Okayama UniversityThe circadian clock generates rhythms of approximately 24 h through periodic expression of the clock genes. In insects, the major clock genes period (per) and timeless (tim) are rhythmically expressed upon their transactivation by CLOCK/CYCLE, with peak levels in the early night. In Drosophila, clockwork orange (cwo) is known to inhibit the transcription of per and tim during the daytime to enhance the amplitude of the rhythm, but its function in other insects is largely unknown. In this study, we investigated the role of cwo in the clock mechanism of the cricket Gryllus bimaculatus. The results of quantitative RT-PCR showed that under a light/dark (LD) cycle, cwo is rhythmically expressed in the optic lobe (lamina-medulla complex) and peaks during the night. When cwo was knocked down via RNA interference (RNAi), some crickets lost their locomotor rhythm, while others maintained a rhythm but exhibited a longer free-running period under constant darkness (DD). In cwo(RNAi) crickets, all clock genes except for cryptochrome 2 (cry2) showed arrhythmic expression under DD; under LD, some of the clock genes showed higher mRNA levels, and tim showed rhythmic expression with a delayed phase. Based on these results, we propose that cwo plays an important role in the cricket circadian clock.No potential conflict of interest relevant to this article was reported.Public Library of ScienceActa Medica Okayama1932-620315102020Fgf10-CRISPR mosaic mutants demonstrate the gene dose-related loss of the accessory lobe and decrease in the number of alveolar type 2 epithelial cells in mouse lunge0240333ENMunenoriHabutaDepartment of Cytology and Histology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesAkihiroYasueDepartment of Orthodontics and Dentofacial Orthopedics, Institute of Biomedical Sciences, Tokushima University Graduate SchoolKen-Ichi T.SuzukiDepartment of Mathematical and Life Sciences, Hiroshima UniversityHirofumiFujitaDepartment of Cytology and Histology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesKeitaSatoDepartment of Cytology and Histology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesHitomiKonoDepartment of Cytology and Histology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesAyukoTakayamaCenter for the Development of New Model Organisms, National Institute for Basic BiologyTetsuyaBandoDepartment of Cytology and Histology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesSatoruMiyaishiDepartment of Legal Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesSeiichiOyadomariDivision of Molecular Biology, Institute of Advanced Medical Sciences, Tokushima UniversityEijiTanakaDepartment of Orthodontics and Dentofacial Orthopedics, Institute of Biomedical Sciences, Tokushima University Graduate SchoolHideyoOhuchiDepartment of Cytology and Histology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesCRISPR/Cas9-mediated gene editing often generates founder generation (F0) mice that exhibit somatic mosaicism in the targeted gene(s). It has been known thatFibroblast growth factor 10(Fgf10)-null mice exhibit limbless and lungless phenotypes, while intermediate limb phenotypes (variable defective limbs) are observed in theFgf10-CRISPR F0 mice. However, how the lung phenotype in theFgf10-mosaic mutants is related to the limb phenotype and genotype has not been investigated. In this study, we examined variable lung phenotypes in theFgf10-targeted F0 mice to determine if the lung phenotype was correlated with percentage of functionalFgf10genotypes. Firstly, according to a previous report,Fgf10-CRISPR F0 embryos on embryonic day 16.5 (E16.5) were classified into three types: type I, no limb; type II, limb defect; and type III, normal limbs. Cartilage and bone staining showed that limb truncations were observed in the girdle, (type I), stylopodial, or zeugopodial region (type II). Deep sequencing of theFgf10-mutant genomes revealed that the mean proportion of codons that encode putative functional FGF10 was 8.3 +/- 6.2% in type I, 25.3 +/- 2.7% in type II, and 54.3 +/- 9.5% in type III (mean +/- standard error of the mean) mutants at E16.5. Histological studies showed that almost all lung lobes were absent in type I embryos. The accessory lung lobe was often absent in type II embryos with other lobes dysplastic. All lung lobes formed in type III embryos. The number of terminal tubules was significantly lower in type I and II embryos, but unchanged in type III embryos. To identify alveolar type 2 epithelial (AECII) cells, known to be reduced in theFgf10-heterozygous mutant, immunostaining using anti-surfactant protein C (SPC) antibody was performed: In the E18.5 lungs, the number of AECII was correlated to the percentage of functionalFgf10genotypes. These data suggest theFgf10gene dose-related loss of the accessory lobe and decrease in the number of alveolar type 2 epithelial cells in mouse lung. Since dysfunction of AECII cells has been implicated in the pathogenesis of parenchymal lung diseases, theFgf10-CRISPR F0 mouse would present an ideal experimental system to explore it.No potential conflict of interest relevant to this article was reported.Okayama University Medical SchoolActa Medica Okayama0386-300X7432020Dkk3/REIC, an N-glycosylated Protein, Is a Physiological Endoplasmic Reticulum Stress Inducer in the Mouse Adrenal Gland199208ENHirofumiFujitaDepartment of Cytology and Histology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesTetsuyaBandoDepartment of Cytology and Histology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesSeiichiOyadomariDivision of Molecular Biology, Institute for Genome Research, University of TokushimaKazuhikoOchiaiDepartment of Basic Science, School of Veterinary Nursing and Technology, Faculty of Veterinary Science, Nippon Veterinary and Life Science UniversityMasamiWatanabeDepartment of Urology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesHiromiKumonInnovation Center Okayama for Nanobio-Targeted Therapy, Okayama UniversityHideyoOhuchiDepartment of Cytology and Histology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesOriginal Article10.18926/AMO/59950Dickkopf 3 (Dkk3) is a secreted protein belonging to the Dkk family and encoded by the orthologous gene of REIC. Dkk3/REIC is expressed by mouse and human adrenal glands, but the understanding of its roles in this organ is still limited. To determine the functions of Dkk3 in the mouse adrenal gland, we first identified that the mouse Dkk3 protein is N-glycosylated in the adrenal gland as well as in the brain. We performed proteome analysis on adrenal glands from Dkk3-null mice, in which exons 5 and 6 of the Dkk3 gene are deleted. Twodimensional polyacrylamide gel electrophoresis of adrenal proteins from wild-type and Dkk3-null mice revealed 5 protein spots whose intensities were altered between the 2 genotypes. Mass spectrometry analysis of these spots identified binding immunoglobulin protein (BiP), an endoplasmic reticulum (ER) chaperone. To determine whether mouse Dkk3 is involved in the unfolded protein response (UPR), we carried out a reporter assay using ER-stress responsive elements. Forced expression of Dkk3 resulted in the induction of distinct levels of reporter expression, showing the UPR initiated by the ER membrane proteins of activating transcription factor 6 (ATF6) and inositol-requring enzyme 1 (IRE1). Thus, it is possible that Dkk3 is a physiological ER stressor in the mouse adrenal gland.No potential conflict of interest relevant to this article was reported.Company of BiologistsActa Medica Okayama0950-1991142172015Leg regeneration is epigenetically regulated by histone H3K27 methylation in the cricket Gryllus bimaculatus29162927ENYoshimasaHamadaTetsuyaBandoTaroNakamuraYoshiyasuIshimaruTaroMitoSumihareNojiKenjiTomiokaHideyoOhuchiHemimetabolous 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'UtxRNAi. Regenerated Gb'E(z)RNAi cricket legs exhibited extra leg segment formation between the tibia and tarsus, and regenerated Gb'UtxRNAi 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'UtxRNAi 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.No potential conflict of interest relevant to this article was reported.Okayama UniversityActa Medica Okayama162015Epigenetics research traces how crickets restore lost legsENTetsuyaBandoNo potential conflict of interest relevant to this article was reported.