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.American Chemical Society (ACS)Acta Medica Okayama0002-78632025Optogenetic Cancer Therapy Using the Light-Driven Outward Proton Pump Rhodopsin Archaerhodopsin-3 (AR3)ENShinNakaoGraduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityKeiichiKojimaGraduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityKeitaSatoGraduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityNaoyaKemmotsuGraduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityHideyoOhuchiGraduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityYosukeTogashiGraduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityYukiSudoGraduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityMedicines used for cancer treatment often cause serious side effects by damaging normal cells due to nonspecific diffusion. To address this issue, we previously developed an optical method to induce apoptotic cell death via intracellular pH alkalinization using the outward proton pump rhodopsin, Archaerhodopsin-3 (AR3) in various noncancer model cells in vitro and in vivo. In this study, we applied this method to cancer cells and tumors to evaluate its potential as an anticancer therapeutic strategy. First, we confirmed that AR3-expressing murine cancer cell lines (MC38, B16F10) showed apoptotic cell death upon green light irradiation, as indicated by increased levels of cell death and apoptosis-related markers. Next, we established stable AR3-expressing MC38 and B16F10 cells by using viral vectors. When these AR3-expressing cells were subcutaneously transplanted into C57BL/6 mice, the resulting tumors initially grew at a rate comparable to that of control tumors lacking AR3 expression or light stimulation. However, upon green light irradiation, AR3-expressing tumors exhibited either a marked reduction in size or significantly suppressed growth, accompanied by the induction of apoptosis signals and decreased proliferation signals. These results demonstrate that AR3-mediated cell death has potent antitumor effects both in vitro and in vivo. This optical method thus holds promise as a novel cancer therapy with potentially reduced side effects.No potential conflict of interest relevant to this article was reported.Elsevier BVActa Medica Okayama0021-925830172025A repertoire of visible light–sensitive opsins in the deep-sea hydrothermal vent shrimp Rimicaris hybisae110291ENYuyaNagataGraduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityNorioMiyamotoInstitute for Extra-Cutting-Edge Science and Technology Avant-Garde Research (X-Star), Japan Agency for Marine-Earth Science and Technology (JAMSTEC)KeitaSatoFaculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityYosukeNishimuraResearch Center for Bioscience and Nanoscience (CeBN), Research Institute for Marine Resources Utilization, Japan Agency for Marine-Earth Science and Technology (JAMSTEC)YukiTaniokaSchool of Pharmaceutical Sciences, Okayama UniversityYujiYamanakaSchool of Pharmaceutical Sciences, Okayama UniversitySusumuYoshizawaAtmosphere and Ocean Research Institute, The University of TokyoKutoTakahashiGraduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityKoheiObayashiDepartment of Biology, Graduate School of Science, Kobe UniversityHisaoTsukamotoDepartment of Biology, Graduate School of Science, Kobe UniversityKenTakaiInstitute for Extra-Cutting-Edge Science and Technology Avant-Garde Research (X-Star), Japan Agency for Marine-Earth Science and Technology (JAMSTEC)HideyoOhuchiFaculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityTakahiroYamashitaDepartment of Biophysics, Graduate School of Science, Kyoto UniversityYukiSudoFaculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityKeiichiKojimaFaculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityUnlike terrestrial environments, where humans reside, there is no sunlight in the deep sea. Instead, dim visible light from black-body radiation and bioluminescence illuminates hydrothermal vent areas in the deep sea. A deep-sea hydrothermal vent shrimp, Rimicaris hybisae, is thought to detect this dim light using its enlarged dorsal eye; however, the molecular basis of its photoreception remains unexplored. Here, we characterized the molecular properties of opsins, universal photoreceptive proteins in animals, found in R. hybisae. Transcriptomic analysis identified six opsins: three Gq-coupled opsins, one Opn3, one Opn5, and one peropsin. Functional analysis revealed that five of these opsins exhibited light-dependent G protein activity, whereas peropsin exhibited the ability to convert all-trans-retinal to 11-cis-retinal like photoisomerases. Notably, all the R. hybisae opsins, including Opn5, convergently show visible light sensitivity (around 457–517 nm), whereas most opsins categorized as Opn5 have been demonstrated to be UV sensitive. Mutational analysis revealed that the unique visible light sensitivity of R. hybisae Opn5 is achieved through the stabilization of a protonated Schiff base by a counterion residue at position 83 (Asp83), which differs from the position identified in other opsins. These findings suggest that the vent shrimp R. hybisae has adapted its photoreceptive devices to dim deep-sea hydrothermal light by selectively maintaining a repertoire of visible light–sensitive opsins, including the uniquely tuned Opn5.No potential conflict of interest relevant to this article was reported.MDPIActa Medica Okayama2221-37591222024Harderian Gland Development and Degeneration in the Fgf10-Deficient Heterozygous Mouse16ENShioriIkedaDepartment of Cytology and Histology, Medical School, Okayama UniversityKeitaSatoDepartment of Cytology and Histology, Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityHirofumiFujitaDepartment of Cytology and Histology, Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityHitomiOno-MinagiDepartment of Cytology and Histology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversitySatoruMiyaishiDepartment of Legal Medicine, Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityTsutomuNohnoDepartment of Cytology and Histology, Medical School, Okayama UniversityHideyoOhuchiDepartment of Cytology and Histology, Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityThe mouse Harderian gland (HG) is a secretory gland that covers the posterior portion of the eyeball, opening at the base of the nictitating membrane. The HG serves to protect the eye surface from infection with its secretions. Mice open their eyelids at about 2 weeks of age, and the development of the HG primordium mechanically opens the eye by pushing the eyeball from its rear. Therefore, when HG formation is disturbed, the eye exhibits enophthalmos (the slit-eye phenotype), and a line of Fgf10(+/-) heterozygous loss-of-function mice exhibits slit-eye due to the HG atrophy. However, it has not been clarified how and when HGs degenerate and atrophy in Fgf10(+/-) mice. In this study, we observed the HGs in embryonic (E13.5 to E19), postnatal (P0.5 to P18) and 74-week-old Fgf10(+/-) mice. We found that more than half of the Fgf10(+/-) mice had markedly degenerated HGs, often unilaterally. The degenerated HG tissue had a melanized appearance and was replaced by connective tissue, which was observed by P10. The development of HGs was delayed or disrupted in the similar proportion of Fgf10(+/-) embryos, as revealed via histology and the loss of HG-marker expression. In situ hybridization showed Fgf10 expression was observed in the Harderian mesenchyme in wild-type as well as in the HG-lacking heterozygote at E19. These results show that the Fgf10 haploinsufficiency causes delayed or defective HG development, often unilaterally from the unexpectedly early neonatal period.No potential conflict of interest relevant to this article was reported.Springer Science and Business Media LLCActa Medica Okayama0302-766X39122022The medaka mutant deficient in eyes shut homolog exhibits opsin transport defects and enhanced autophagy in retinal photoreceptors249267ENKeitaSatoDepartment of Cytology and Histology, Faculty of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama UniversityYangLiuDepartment of Cytology and Histology, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama UniversityTakahiroYamashitaDepartment of Biophysics, Graduate School of Science, Kyoto UniversityHideyoOhuchiDepartment of Cytology and Histology, Faculty of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama UniversityEyes shut homolog (EYS) encodes a proteoglycan and the human mutation causes retinitis pigmentosa type 25 (RP25) with progressive retinal degeneration. RP25 most frequently affects autosomal recessive RP patients with many ethnic backgrounds. Although studies using RP models have facilitated the development of therapeutic medications, Eys has been lost in rodent model animals. Here we examined the roles for Eys in the maintenance of photoreceptor structure and function by generating eys-null medaka fish using the CRISPR-Cas9 system. Medaka EYS protein was present near the connecting cilium of wild-type photoreceptors, while it was absent from the eys|/| retina. The mutant larvae exhibited a reduced visual motor response compared with wild-type. In contrast to reported eys-deficient zebrafish at the similar stage, no retinal cell death was detected in the 8-month post-hatching (8-mph) medaka eys mutant. Immunohistochemistry showed a significant reduction in the length of cone outer segments (OSs), retention of OS proteins in the inner segments of photoreceptors, and abnormal filamentous actin network at the base of cone OSs in the mutant retina by 8 mph. Electron microscopy revealed aberrant structure of calyceal processes, numerous vesiculation and lamellar interruptions, and autophagosomes in the eys-mutant cone photoreceptors. In situ hybridization showed an autophagy component gene, gabarap, was ectopically expressed in the eys-null retina. These results suggest eys is required for regeneration of OS, especially of cone photoreceptors, and transport of OS proteins by regulating actin filaments. Enhanced autophagy may delay the progression of retinal degeneration when lacking EYS in the medaka retina.No potential conflict of interest relevant to this article was reported. ElsevierActa Medica Okayama1083-351X29982023Mammalian type opsin 5 preferentially activates G14 in Gq-type G proteins triggering intracellular calcium response105020ENKeitaSatoDepartment of Cytology and Histology, Faculty of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama UniversityTakahiroYamashitaDepartment of Biophysics, Graduate School of Science, Kyoto UniversityHideyoOhuchiDepartment of Cytology and Histology, Faculty of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama UniversityMammalian type opsin 5 (Opn5m), a UV-sensitive G protein-coupled receptor opsin highly conserved in vertebrates, would provide a common basis for UV sensing from lamprey to humans. However, G protein coupled with Opn5m remains controversial due to variations in assay conditions and the origin of Opn5m across different reports. Here, we examined Opn5m from diverse species using an aequorin luminescence assay and G alpha-KO cell line. Beyond the commonly studied major G alpha classes, G alpha q, G alpha 11, G alpha 14, and G alpha 15 in the Gq class were individually investigated in this study, as they can drive distinct signaling pathways in addition to a canonical calcium response. UV light triggered a calcium response via all the tested Opn5m proteins in 293T cells, which was abolished by Gq-type G alpha deletion and rescued by cotransfection with mouse and medaka Gq-type G alpha proteins. Opn5m preferentially activated G alpha 14 and close relatives. Mutational analysis implicated specific regions, including alpha 3-beta 5 and alpha G-alpha 4 loops, alpha G and alpha 4 helices, and the extreme C terminus, in the preferential activation of G alpha 14 by Opn5m. FISH revealed co-expression of genes encoding Opn5m and G alpha 14 in the scleral cartilage of medaka and chicken eyes, supporting their physiological coupling. This suggests that the preferential activation of G alpha 14 by Opn5m is relevant for UV sensing in specific cell types.No potential conflict of interest relevant to this article was reported.ElsevierActa Medica Okayama0022-283643652024Molecular Property, Manipulation, and Potential Use of Opn5 and Its Homologs168319ENKeitaSatoDepartment of Cytology and Histology, Faculty of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama UniversityHideyoOhuchiDepartment of Cytology and Histology, Faculty of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama UniversityAnimal opsin is a G-protein coupled receptor (GPCR) and binds retinal as a chromophore to form a photopigment. The Opsin 5 (Opn5) group within the animal opsin family comprises a diverse array of related proteins, such as Opn5m, a protein conserved across all vertebrate lineages including mammals, and other members like Opn5L1 and Opn5L2 found in non-mammalian vertebrate genomes, and Opn6 found in non-therian vertebrate genomes, along with Opn5 homologs present in invertebrates. Although these proteins collectively constitute a single clade within the molecular phylogenetic tree of animal opsins, they exhibit markedly distinct molecular characteristics in areas such as retinal binding properties, photoreaction, and G-protein coupling specificity. Based on their molecular features, they are believed to play a significant role in physiological functions. However, our understanding of their precise physiological functions and molecular characteristics is still developing and only partially realized. Furthermore, their unique molecular characteristics of Opn5-related proteins suggest a high potential for their use as optogenetic tools through more specialized manipulations. This review intends to encapsulate our current understanding of Opn5, discuss potential manipulations of its molecular attributes, and delve into its prospective utility in the burgeoning field of animal opsin optogenetics.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.WileyActa Medica Okayama0021-99675292021The Opsin 3/Teleost multiple tissue opsin system: mRNA localization in the retina and brain of medaka (Oryzias latipes) 24842516ENKeitaSatoDepartment of Cytology and Histology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityKhine NweNweDepartment of Cytology and Histology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityHideyoOhuchiDepartment of Cytology and Histology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityThe photoreceptor protein, opsin, is one of the major components for vision and photoreceptive function in animals. Although many opsins have been discovered from animal genomes, only a few nonimage]forming functions mediated by opsins have been identified. Understanding the mRNA distribution of photoreceptor proteins is one crucial step in uncovering their photoreceptive function in animals. Here, we focus on the medaka fish (Oryzias latipes) Opsin 3 (Opn3)/Teleost multiple opsin (Tmt) system, which constitutes a separate phylogenetic group, having putative blue light photoreceptors for nonimage]forming functions. In medaka, there is one opn3 and five tmt]opsin orthologs. The expression pattern of the opn3/tmt]opsins in the retina and brain was investigated by in situ hybridization. mRNAs for opn3/tmt]opsins were distributed in the retinal ganglion cells as well as interneurons and specific brain nuclei. Specifically, hybridization signals were observed in the glutamate decarboxylase 1 (gad1)]expressing amacrine cells for opn3, tmt1a, tmt1b, and tmt2, in the caudal lobe of the cerebellum for tmt1b and tmt2, in the cranial nerve nuclei for opn3, tmt1a, tmt1b, tmt2, and in the rostral pars distalis (adenohypophysis) for opn3. These expression patterns suggest that blue light sensing in the fish retina and brain may be involved in the integration of visual inputs, vestibular function, somatosensation, motor outputs, and pituitary endocrine regulation.No potential conflict of interest relevant to this article was reported.