Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Association for Research in Vision and Ophthalmology (ARVO)  

Purpose: The purpose of this study was to establish and analyze a cell model of Leber congenital amaurosis type 16 (LCA16), which is caused by mutations in the KCNJ13 gene encoding Kir7.1, an inward-rectifying potassium ion channel. Methods: The two guide RNAs specific to the target sites in the KCNJ13 gene were designed and KCNJ13 knock-out (KO) human-induced pluripotent stem cells (hiPSCs) were generated using the CRISPR/Cas9 system. The KCNJ13-KO hiPSCs were differentiated into retinal pigment epithelial cells (hiPSC-RPEs). The KCNJ13-KO in hiPSC-RPEs was confirmed by immunostaining. Phagocytic activity of hiPSC-RPEs was assessed using the uptake of fluorescently labeled porcine photoreceptor outer segments (POSs). Phagocytosis-related genes in RPE cells were assessed by quantitative polymerase chain reaction. Results: Most of the translated region of the KCNJ13 gene was deleted in the KCNJ13-KO hiPSCs by the CRISPR/Cas9 system, and this confirmed that the Kir7.1 protein was not present in RPE cells induced from the hiPSCs. Expression of RPE marker genes such as BEST1 and CRALBP was retained in the wild-type (WT) and in the KCNJ13-KO hiPSC-RPE cells. However, phagocytic activity and expression of phagocytosis-related genes in the KCNJ13-null hiPSC-RPE cells were significantly reduced compared to those of WT. Conclusions: We succeeded in generating an RPE model of LCA16 using hiPSCs. We suggest that Kir7.1 is required for phagocytosis of POSs by RPE cells and that impaired phagocytosis in the absence of Kir7.1 would be involved in the retinal degeneration found in LCA16.

DOI： 10.1167/iovs.61.5.38

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Recent studies show that exposure to ultraviolet (UV) light suppresses ocular elongation, which causes myopia development. However, the specific mechanisms of this process have not been elucidated. A UV-sensor, Opsin 5 (Opn5) mRNA was shown to be present in extraretinal tissues. To test the possibility that UV-signals mediated by Opn5 would have a direct effect on the outer connective tissues of the eye, we first examined the expression patterns of a mammalian type Opn5 (Opn5m) in the late-embryonic chicken eye. Quantitative PCR showed Opn5m mRNA expression in the cornea and sclera. The anti-Opn5m antibody stained a small subset of cells in the corneal stroma and fibrous sclera. We next assessed the effect of UV-A (375 nm) irradiation on the chicken fibroblast cell line DF-1 overexpressing chicken Opn5m. UV-A irradiation for 30 min significantly increased the expression of Early growth response 1 (Egr1), known as an immediate early responsive gene, and of Matrix metalloproteinase 2 (Mmp2) in the presence of retinal chromophore 11-cis-retinal. In contrast, expression of Transforming growth factor beta 2 and Tissue inhibitor of metalloproteinase 2 was not significantly altered. These results indicate that UV-A absorption by Opn5m can upregulate the expression levels of Egr1 and Mmp2 in non-neuronal, fibroblasts. Taken together with the presence of Opn5m in the cornea and sclera, it is suggested that UV-A signaling mediated by Opn5 in the extraretinal ocular tissues could influence directly the outer connective tissues of the chicken late-embryonic eye.

DOI： 10.1016/j.bbrep.2019.100665

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Nature Publishing Group  

Most opsins are G protein-coupled receptors that utilize retinal both as a ligand and as a chromophore. Opsins' main established mechanism is light-triggered activation through retinal 11-cis-to-all-trans photoisomerization. Here we report a vertebrate non-visual opsin that functions as a Gi-coupled retinal receptor that is deactivated by light and can thermally self-regenerate. This opsin, Opn5L1, binds exclusively to all-trans-retinal. More interestingly, the light-induced deactivation through retinal trans-to-cis isomerization is followed by formation of a covalent adduct between retinal and a nearby cysteine, which breaks the retinal-conjugated double bond system, probably at the C11 position, resulting in thermal re-isomerization to all-trans-retinal. Thus, Opn5L1 acts as a reverse photoreceptor. We conclude that, like vertebrate rhodopsin, Opn5L1 is a unidirectional optical switch optimized from an ancestral bidirectional optical switch, such as invertebrate rhodopsin, to increase the S/N ratio of the signal transduction, although the direction of optimization is opposite to that of vertebrate rhodopsin.

DOI： 10.1038/s41467-018-03603-3

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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.

DOI： 10.1038/s41598-017-00088-w

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Opsin family genes encode G protein-coupled seven-transmembrane proteins that bind a retinaldehyde chromophore in photoreception. Here, we sought potential as yet undescribed avian retinal photoreceptors, focusing on Opsin 3 homologs in the chicken. We found two Opsin 3-related genes in the chicken genome: one corresponding to encephalopsin/panopsin (Opn3) in mammals, and the other belonging to the teleost multiple tissue opsin (TMT) 2 group. Bioluminescence imaging and G protein activation assays demonstrated that the chicken TMT opsin (cTMT) functions as a blue light sensor when forced-expressed in mammalian cultured cells. We did not detect evidence of light sensitivity for the chicken Opn3 (cOpn3). In situ hybridization demonstrated expression of cTMT in subsets of differentiating cells in the inner retina and, as development progressed, predominant localization to retinal horizontal cells (HCs). Immunohistochemistry (IHC) revealed cTMT in HCs as well as in small numbers of cells in the ganglion and inner nuclear layers of the post-hatch chicken retina. In contrast, cOpn3-IR cells were found in distinct subsets of cells in the inner nuclear layer. cTMT-IR cells were also found in subsets of cells in the hypothalamus. Finally, we found differential distribution of cOpn3 and cTMT proteins in specific cells of the cerebellum. The present results suggest that a novel TMT-type opsin 3 may function as a photoreceptor in the chicken retina and brain.

DOI： 10.1371/journal.pone.0163925

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Opn5 is one of the recently identified opsin groups that is responsible for nonvisual photoreception in animals. We previously showed that a chicken homolog of mammalian Opn5 (Opn5m) is a G(i)-coupled UV sensor having molecular properties typical of bistable pigments. Here we demonstrated that mammalian Opn5m evolved to be a more specialized photosensor by losing one of the characteristics of bistable pigments, direct binding of all-trans-retinal. We first confirmed that Opn5m proteins in zebrafish, Xenopus tropicalis, mouse, and human are also UV-sensitive pigments. Then we found that only mammalian Opn5m proteins lack the ability to directly bind all-trans-retinal. Mutational analysis showed that these characteristics were acquired by a single amino acid replacement at position 168. By comparing the expression patterns of Opn5m between mammals and chicken, we found that, like chicken Opn5m, mammalian Opn5m was localized in the ganglion cell layer and inner nuclear layer of the retina. However, the mouse and primate (common marmoset) opsins were distributed not in the posterior hypothalamus (including the region along the third ventricle) where chicken Opn5m is localized, but in the preoptic hypothalamus. Interestingly, RPE65, an essential enzyme for forming 11-cis-retinal in the visual cycle is expressed near the preoptic hypothalamus of the mouse and common marmoset brain but not near the region of the chicken brain where chicken Opn5m is expressed. Therefore, mammalian Opn5m may work exclusively as a short wavelength sensor in the brain as well as in the retina with the assistance of an 11-cis-retinal-supplying system.

DOI： 10.1074/jbc.M113.514075

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A mammalian type opsin 5 (neuropsin) is a recently identified ultraviolet (UV)-sensitive pigment of the retina and other photosensitive organs in birds. Two other opsin 5-related molecules have been found in the genomes of non-mammalian vertebrates. However, their functions have not been examined as yet. Here, we identify the molecular properties of a second avian opsin 5, cOpn5L2 (chicken opsin 5-like 2), and its localization in the post-hatch chicken. Spectrophotometric analysis and radionucleotide-binding assay have revealed that cOpn5L2 is a UV-sensitive bistable pigment that couples with the Gi subtype of guanine nucleotide-binding protein (G protein). As a bistable pigment, it also shows the direct binding ability to agonist all-trans-retinal to activate G protein. The absorption maxima of UV-light-absorbing and visible light-absorbing forms were 350 and 521 nm, respectively. Expression analysis showed relatively high expression of cOpn5L2 mRNA in the adrenal gland, which is not photoreceptive but an endocrine organ, while lower expression was found in the brain and retina. At the protein level, cOpn5L2 immunoreactive cells were present in the chromaffin cells of the adrenal gland. In the brain, cOpn5L2 immunoreactive cells were found in the paraventricular and supraoptic nuclei of the anterior hypothalamus, known for photoreceptive deep brain areas. In the retina, cOpn5L2 protein was localized to subsets of cells in the ganglion cell layer and the inner nuclear layer. These results suggest that the non-mammalian type opsin 5 (Opn5L2) functions as a second UV sensor in the photoreceptive organs, while it might function as chemosensor using its direct binding ability to agonist all-trans-retinal in non-photoreceptive organs such as the adrenal gland of birds.

DOI： 10.1371/journal.pone.0031534

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATL ACAD SCIENCES  

Opn5 (neuropsin) belongs to an independent group separated from the other six groups in the phylogenetic tree of opsins, for which little information of absorption characteristics and molecular properties of the members is available. Here we show that the chicken Opn5 (cOpn5m) is a UV-sensitive bistable pigment that couples with Gi subtype of G protein. The recombinant expression of cOpn5m in HEK 293s cells followed by the addition of 11-cis- and all-trans-retinal produced UV light-absorbing and visible light-absorbing forms, respectively. These forms were interconvertible by UV and visible light irradiations, respectively, indicating that cOpn5m is a bistable pigment. The absorption maxima of these forms were estimated to be 360 and 474 nm, respectively. The GTP gamma S binding assay clearly showed that the visible light-absorbing form having all-trans-retinal activates Gi type of G protein, whereas no Gt or Gq activation ability was observed. Immunohistochemical studies using an antibody against cOpn5m clearly showed that this pigment is localized within some types of amacrine cells and some cells in the ganglion cell layer of the retinas, the vast majority of cells in the pineal gland and serotonin-positive cells in the paraventricular organ. Because cOpn5m is the only UV-sensitive opsin among the opsins found so far in chicken, this study provides the molecular basis for UV reception in chicken.

DOI： 10.1073/pnas.1012498107

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The opsin gene family encodes G protein-coupled seven-transmembrane proteins that bind to a retinaldehyde chromophore for photoreception. It has been reported that opsin 5 is expressed in mammalian neural tissue, but its function has been elusive. As a first step to understand the function for opsin 5 in the developing eye, we searched for chicken opsin 5-related genes in the genome by a bioinformatic approach and isolated opsin 5 cDNA fragments from the embryonic retina by RT-PCR. We found that there are three opsin 5-related genes, designated cOpn5m (chicken opsin 5, mammalian type), cOpn5L1 (chicken opsin 5-like 1), and cOpn5L2 (chicken opsin 5-like 2), in the chicken genome. Quantitative PCR analysis has revealed that cOpn5m is the most abundant in the developing and early posthatching neural retina. In situ hybridization analysis has shown that cOpn5m is specifically expressed in subsets of differentiating ganglion cells and amacrine cells. These results suggest that the mammalian type opsin 5 may contribute to the development of these retinal cells in the chicken.

DOI： 10.1002/dvdy.21611

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The development of the eyelid requires coordinated cellular processes of proliferation, cell shape changes, migration and cell death. Mutant mice deficient in the fibroblast growth factor 10 (Fgf10) gene exhibit open-eyelids at birth. To elucidate the roles of FGF10 during eyelid formation, we examined the expression pattern of Fgf10 during eyelid formation and the phenotype of Fgf10-null eyelids in detail. Fgf10 is expressed by mesenchymal cells just beneath the protruding epidermal cells of the nascent eyelid. However, Fgf10-null epithelial cells running though the eyelid groove do not exhibit typical cuboid shape or sufficient proliferation. Furthermore, peridermal clumps are not maintained on the eyelid leading edge, and epithelial extension does not occur. At the cellular level, the accumulation of actin fibers is not observed in the mutant epithelial leading edge. The expression of activin/inhibin beta B (Act beta B/Inhbb) and transforming growth factor a (Tgfa), previously reported to be crucial for eyelid development, is down-regulated in the mutant leading edge, while the onset of sonic hedgehog (Shh) expression is delayed on the mutant eyelid margin. Explant cultures of mouse eyelid primordia shows that the open-eyelid phenotype of the mutant is reduced by exogenous FGF10 protein, and that the expression of Act beta B and Tgfa is ectopically induced in the thickened eyelid epithelium by the FGF10 protein. These results indicate a dual role of FGF10 in mouse eyelid development, for both proliferation and coordinated migration of eyelid epithelial cells by reorganization of the cytoskeleton, through the regulation of activin, TGF alpha and SHH signaling.

DOI： 10.1242/dev.01892

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

Autosomal dominant aplasia of lacrimal and salivary glands (ALSG; OMIM 180920 and OMIM 103420) is a rare condition characterized by irritable eyes and dryness of the mouth. We mapped ALSG to 5p13. 2- 5q13. 1, which coincides with the gene fibroblast growth factor 10 (FGF10). In two extended pedigrees, we identified heterozygous mutations in FGF10 in all individuals with ALSG. Fgf10(+/-) mice have a phenotype similar to ALSG, providing a model for this disorder. We suggest that haploinsufficiency for FGF10 during a crucial stage of development results in ALSG.

DOI： 10.1038/ng1507

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FGF receptor 2 isoform IIIb (FGFR2b), originally discovered as a receptor for FGF7, is known to be an important receptor in vertebrate morphogenesis, because FGFR2b null mice exhibit agenesis or dysgenesis of various organs, which undergo budding and branching morphogenesis. Since FGF7 null mice do not exhibit marked defects in organogenesis, it has been considered that other FGF(s) than FGF7 might function as a major ligand for FGFR2b during organogenesis. One of the candidate ligands is FGF10, because FGF10 binds to FGFR2b with high affinity and the formation of the limb and lung is arrested in FGF10 null mice as found in FGFR2b-deficient mice. Previous analyses of FGF10 null mice revealed that FGF10 is required for limb and lung development. To elucidate the role of FGF10 in wide-range organogenesis, we further analyzed the phenotypes of the FGF10 knockout mice. We found diverse phenotypes closely related to those for FGFR2-deficient mice, which includes the absence of thyroid, pituitary, and salivary glands, while minor defects were observed in the formation of teeth, kidneys, hair follicles, and digestive organs. These results suggest that FGF10 acts as a major ligand for FGFR2b in mouse multi-organ development. (C) 2000 Academic Press.

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The interactions between fibroblast growth factors (FGF) and their receptors have important roles in mediating mesenchymal-epithelial cell interactions during embryogenesis. In particular, Fgf10 is predicted to function as a regulator of brain, lung and limb development on the basis of its spatiotemporal expression pattern in the developing embryo. To define the role of Fgf10, we generated Fgf10-deficient mice. Fgf10(-/-) mice died at birth due to the lack of lung development. Trachea was formed, but subsequent pulmonary branching morphogenesis was disrupted. In addition, mutant mice had complete truncation of the fore- and hindlimbs. In Fgf10(-/-) embryos, limb bud formation was initiated but outgrowth of the limb buds did not occur
however, formation of the clavicles was not affected. Analysis of the expression of marker genes in the mutant limb buds indicated that the apical ectodermal ridge (AER) and the zone of polarizing activity (ZPA) did not form. Thus, we show here that Fgf10 serves as an essential regulator of lung and limb formation.

DOI： 10.1038/5096

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Animal 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.

DOI： 10.1016/j.jmb.2023.168319

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The vertebrate body comprises four distinct cell populations: cells derived from (1) ectoderm, (2) mesoderm, (3) endoderm, and (4) neural crest cells, often referred to as the fourth germ layer. Neural crest cells arise when the neural plate edges fuse to form a neural tube, which eventually develops into the brain and spinal cord. To date, the embryonic origin of exocrine glands located in the head and neck remains under debate. In this study, transgenic TRiCK mice were used to investigate the germinal origin of the salivary and lacrimal glands. TRiCK mice express fluorescent proteins under the regulatory control of Sox1, T/Brachyury, and Sox17 gene expressions. These genes are representative marker genes for neuroectoderm (Sox1), mesoderm (T), and endoderm (Sox17). Using this approach, the cellular lineages of the salivary and lacrimal glands were examined. We demonstrate that the salivary and lacrimal glands contain cells derived from all three germ layers. Notably, a subset of Sox1-driven fluorescent cells differentiated into epithelial cells, implying their neural crest origin. Also, these Sox1-driven fluorescent cells expressed high levels of stem cell markers. These cells were particularly pronounced in duct ligation and wound damage models, suggesting the involvement of neural crest-derived epithelial cells in regenerative processes following tissue injury. This study provides compelling evidence clarifying the germinal origin of exocrine glands and the contribution of neural crest-derived cells within the glandular epithelium to the regenerative response following tissue damage.

DOI： 10.3390/ijms241813692

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Opsins are universal photosensitive proteins in animals. Vertebrates have a variety of opsin genes for visual and non-visual photoreceptions. Analysis of the gene structures shows that most opsin genes have introns in their coding regions. However, teleosts exceptionally have several intron-less opsin genes which are presumed to have been duplicated by an RNA-based gene duplication mechanism, retroduplication. Among these retrogenes, we focused on the Opn4 (melanopsin) gene responsible for non-image-forming photoreception. Many teleosts have five Opn4 genes including one intron-less gene, which is speculated to have been formed from a parental intron-containing gene in the Actinopterygii. In this study, to reveal the evolutionary history of Opn4 genes, we analyzed them in teleost (zebrafish and medaka) and non-teleost (bichir, sturgeon and gar) fishes. Our synteny analysis suggests that the intron-less Opn4 gene emerged by retroduplication after branching of the bichir lineage. In addition, our biochemical and histochemical analyses showed that, in the teleost lineage, the newly acquired intron-less Opn4 gene became abundantly used without substantial changes of the molecular properties of the Opn4 protein. This stepwise evolutionary model of Opn4 genes is quite similar to that of rhodopsin genes in the Actinopterygii. The unique acquisition of rhodopsin and Opn4 retrogenes would have contributed to the diversification of the opsin gene repertoires in the Actinopterygii and the adaptation of teleosts to various aquatic environments.

DOI： 10.1016/j.jbc.2023.104899

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Opsins are photosensitive G protein-coupled receptor proteins and are classified into visual and nonvisual receptors. Opn5L1 is a nonvisual opsin that binds all-trans retinal as a chromophore. A unique feature of Opn5L1 is that the protein exhibits a photocyclic reaction upon photoexcitation. Determining the chromophore structures of intermediates in the photocycle is essential for understanding the functional mechanism of Opn5L1. A previous study revealed that a long-lived intermediate in the photocycle cannot activate the G protein and forms a covalent bond between the retinal chromophore and a nearby cysteine residue. However, the position of this covalent bond in the chromophore remains undetermined. Here, we report a resonance Raman study on isotopically labeled samples in combination with density functional theory calculations and reveal that the 11th carbon atom of the chromophore of the intermediate forms a covalent linkage to the cysteine residue. Furthermore, vibrational assignments based on the isotopic substitutions and density functional theory calculations suggested that the Schiff base of the intermediate is deprotonated. The chromophore structure determined in the present study well explains the mechanism of the photocyclic reaction, which is crucial to the photobiological function of Opn5L1.

DOI： 10.1021/acs.jpcb.2c08780

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media {LLC}  

<jats:title>Abstract</jats:title><jats:sec>
<jats:title>Background</jats:title>
<jats:p>Oral care with gel is a common method for preventing aspiration in high-risk patients. An oral care gel is used to clean and moisturize the oral cavity. However, the effects of gel care on the oral bacteria remain unclear. In this pilot study, we described a matching transformation system (MA-T) for elderly high-risk patients. MA-T is an on-demand aqueous chlorine dioxide solution that provides excellent safety and has various antimicrobial activities, even in the presence of abundant organic compounds. This study investigated the effects of MA-T gel in patients requiring nursing care.</jats:p>
</jats:sec><jats:sec>
<jats:title>Materials and methods</jats:title>
<jats:p>Patients who were hospitalized for nursing care were included in this study. No drugs and foods were administered orally. Oral bacteria and intraoral humidity were examined by daily care using MA-T gel. Moreover, oral membranous substances were analyzed and material from the oral cavity was cultured on selective media for identifying opportunistic organisms.</jats:p>
</jats:sec><jats:sec>
<jats:title>Results</jats:title>
<jats:p>Membranous substances were present in the oral cavities of all patients. The number of bacteria decreased, and oral moisture improved, after treatment with MA-T gel. Moreover, oral humidity was also controlled with the continued use of MA-T gel. MA-T gels should be used not only for professional care but also on a daily basis for better oral care. Furthermore, the results of bacterial cultures showed that MA-T controls the propagation of opportunistic bacterial infections.</jats:p>
</jats:sec><jats:sec>
<jats:title>Conclusion</jats:title>
<jats:p>Membranous substances may be observed in the oral cavity of individuals requiring nursing care for tube feeding. The results of this pilot study suggest that MA-T, a novel disinfectant, can be used for oral care in the elderly to reduce the risk of aspiration-pneumonia.</jats:p>
</jats:sec>

DOI： 10.1186/s12903-023-02779-5

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Cysteinyl leukotriene receptor 1 (CysLTR1) is a G protein-coupled receptor for the inflammatory lipid mediators cysteinyl leukotrienes, which are involved in smooth muscle constriction, vascular permeability, and macrophage chemokine release. The Cysltr1 gene encoding CysLTR1 is expressed in the macrophage lineage, including osteoclasts, and the CysLTR1 antagonist Montelukast has been shown to suppress the formation of osteoclasts. However, it currently remains unclear whether CysLTR1 is involved in osteoclast differentiation and bone loss. Therefore, to clarify the role of CysLTR1 in osteoclastogenesis and pathological bone loss, we herein generated CysLTR1 loss-of-function mutant mice by disrupting the cysltr1 gene using the CRISPR-Cas9 system. These mutant mice had a frameshift mutation resulting in a premature stop codon (Cysltr1 KO) or an in-frame mutation causing the deletion of the first extracellular loop (Cysltr1^Δ105). Bone marrow macrophages (BMM) from these mutant mice lost the intracellular flux of calcium in response to leukotriene D₄, indicating that these mutants completely lost the activity of CysLTR1 without triggering genetic compensation. However, disruption of the Cysltr1 gene did not suppress the formation of osteoclasts from BMM in vitro. We also demonstrated that the CysLTR1 antagonist Montelukast suppressed the formation of osteoclasts without functional CysLTR1. On the other hand, disruption of the Cysltr1 gene partially suppressed the formation of osteoclasts stimulated by leukotriene D₄ and did not inhibit that by glutathione, functioning as a substrate in the synthesis of cysteinyl leukotrienes. Disruption of the Cysltr1 gene did not affect ovariectomy-induced osteoporosis or lipopolysaccharide-induced bone resorption. Collectively, these results suggest that the CysLT-CysLTR1 axis is dispensable for osteoclast differentiation in vitro and pathological bone loss, while the leukotriene D₄-CysTR1 axis is sufficient to stimulate osteoclast formation. We concluded that the effects of glutathione and Montelukast on osteoclast formation were independent of CysLTR1.

DOI： 10.1371/journal.pone.0277307

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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.

DOI： 10.3390/jdb10040045

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The role of Dickkopf-3 (Dkk3)/REIC (The Reduced Expression in Immortalized Cells), a Wnt-signaling inhibitor, in male reproductive physiology remains unknown thus far. To explore the functional details of Dkk3/REIC in the male reproductive process, we studied the Dkk3/REIC knock-out (KO) mouse model. By examining testicular sections and investigating the sperm characteristics (count, vitality and motility) and ultrastructure, we compared the reproductive features between Dkk3/REIC-KO and wild-type (WT) male mice. To further explore the underlying molecular mechanism, we performed RNA sequencing (RNA-seq) analysis of testicular tissues. Our results showed that spermiation failure existed in seminiferous tubules of Dkk3/REIC-KO mice, and sperm from Dkk3/REIC-KO mice exhibited inferior motility (44.09 ± 8.12% vs. 23.26 ± 10.02%, p < 0.01). The Ultrastructure examination revealed defects in the sperm fibrous sheath of KO mice. Although the average count of Dkk3/REIC-KO epididymal sperm was less than that of the wild-types (9.30 ± 0.69 vs. 8.27 ± 0.87, ×106), neither the gap (p > 0.05) nor the difference in the sperm vitality rate (72.83 ± 1.55% vs. 72.50 ± 0.71%, p > 0.05) were statistically significant. The RNA-seq and GO (Gene Oncology) enrichment results indicated that the differential genes were significantly enriched in the GO terms of cytoskeleton function, cAMP signaling and calcium ion binding. Collectively, our research demonstrates that Dkk3/REIC is involved in the process of spermiation, fibrous sheath integrity maintenance and sperm motility of mice.

DOI： 10.3390/genes13020285

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Opsins are G protein-coupled receptors specialized for photoreception in animals. Opn5 is categorized in an independent opsin group and functions for various non-visual photoreceptions. Among vertebrate Opn5 subgroups (Opn5m, Opn5L1 and Opn5L2), Opn5m and Opn5L2 bind 11-cis retinal to form a UV-sensitive resting state, which is inter-convertible with the all-trans retinal bound active state by photoreception. Thus, these opsins are characterized as bistable opsins. To assess the molecular basis of the UV-sensitive bistable property, we introduced comprehensive mutations at Thr188, which is well conserved among these opsins. The mutations in Opn5m drastically hampered 11-cis retinal incorporation and the bistable photoreaction. Moreover, T188C mutant Opn5m exclusively bound all-trans retinal and thermally self-regenerated to the original form after photoreception, which is similar to the photocyclic property of Opn5L1 bearing Cys188. Therefore, the residue at position 188 underlies the UV-sensitive bistable property of Opn5m and contributes to the diversification of vertebrate Opn5 subgroups.

DOI： 10.1038/s42003-022-03010-x

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Authorship：Last author,　Corresponding author   Publishing type：Research paper (scientific journal)   Publisher：The Company of Biologists  

Hemimetabolous insects, such as the two-spotted cricket Gryllus bimaculatus, can recover lost tissues in contrast to the limited regenerative abilities in 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-seq during leg regeneration. Among 11 Toll genes in the Gryllus genome, expression of Gb'Toll2-1, Gb'Toll2-2, and Gb'Toll2-5 was upregulated during regeneration. RNA interference (RNAi) of Gb'Toll, Gb'Toll2-1, Gb'Toll2-2, Gb'Toll2-3, or Gb'Toll2-4 produced regeneration defects in more than 50% of crickets. RNAi of Gb'Toll2-2 decreased the ratios of S and M phase cells, expression of JAK/STAT signalling genes, and accumulation of plasmatocytes in the blastema. Depletion of plasmatocytes in crickets using clodronate also produced regeneration defects, along with reduced 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.

DOI： 10.1242/dev.199916

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

The uncoupling protein 1 (UCP1) gene is known to be highly expressed in brown adipose tissue (BAT) that functions in thermogenesis. It has been shown that UCP1 mRNA is localized to the mouse adrenal gland, but its significance remains elusive. To explore how UCP1 expression in the adrenal gland is regulated, we generated a reporter knock-in mouse in which the GFP gene was inserted into the UCP1 locus using CRISPR-Cas9 system. Firstly, we confirmed by Western blot analysis UCP1-driven GFP protein expression in interscapular BAT of the knock-in mice kept at 4 °C. Immunohistochemistry showed that GFP protein was detected in the adrenal gland of the knock-in mice. More intense GFP expression was observed in the adrenal medulla than in the cortex of the reporter mice irrespectively of cold exposure. Immunohistochemistry using anti-UCP1 antibody, as well as Western blot analysis verified UCP1 protein expression in the wild-type adrenal medulla. These results suggest that the mouse adrenal gland is a novel organ expressing UCP1 protein and its expression is not upregulated by cold exposure.

DOI： 10.1016/j.bbrc.2021.06.013

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Dickkopf 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.

DOI： 10.18926/AMO/59950

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

REIC (reduced expression in immortalized cells) has been identified as a gene whose expression was reduced in immortalized cultured cells. The REIC gene is identical to Dickkopf-3 (Dkk3), which encodes a secreted glycoprotein belonging to the Dkk family. Previously, we showed that Dkk3 protein is present in the mouse adrenal medulla. However, its role in this tissue has not been elucidated. To explore it, we performed electron microscopic (EM) studies and RNA-sequencing (RNA-seq) analysis on Dkk3-null adrenal glands. EM studies showed that the number of dense core secretory vesicles were significantly reduced and empty vesicles were increased in the medulla endocrine cells. Quantitative PCR (qPCR) analysis showed relative expression levels of chromogranin A (Chga) and neuropeptide Y (Npy) were slightly but significantly reduced in the Dkk3-null adrenal glands. From the result of RNA-seq analysis as a parallel study, we selected three of the downregulated genes, uncoupled protein-1 (Ucp1), growth arrest and DNA-damage-inducible 45 gamma (Gadd45g), and Junb with regard to the estimated expression levels. In situ hybridization confirmed that these genes were regionally expressed in the adrenal gland. However, expression levels of these three genes were not consistent as revealed by qPCR. Thus, Dkk3 maintains the integrity of secreting vesicles in mouse adrenal medulla by regulating the expression of Chga and Npy.

DOI： 10.1007/s00441-019-03113-8

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Recent progress in whole genome sequencing has revealed that animals have various kinds of opsin genes for photoreception. Among them, most opsin genes have introns in their coding regions. However, it has been known for a long time that teleost retinas express intron-less rhodopsin genes, which are presumed to have been formed by retroduplication from an ancestral intron-containing rhodopsin gene. In addition, teleosts have an intron-containing rhodopsin gene (exo-rhodopsin) exclusively for pineal photoreception. In this study, to unravel the evolutionary origin of the two teleost rhodopsin genes, we analyzed the rhodopsin genes of non-teleost fishes in the Actinopterygii. The phylogenetic analysis of full-length sequences of bichir, sturgeon and gar rhodopsins revealed that retroduplication of the rhodopsin gene occurred after branching of the bichir lineage. In addition, analysis of the tissue distribution and the molecular properties of bichir, sturgeon and gar rhodopsins showed that the abundant and exclusive expression of intron-containing rhodopsin in the pineal gland and the short lifetime of its meta II intermediate, which leads to optimization for pineal photoreception, were achieved after branching of the gar lineage. Based on these results, we propose a stepwise evolutionary model of teleost intron-containing and intron-less rhodopsin genes.

DOI： 10.1038/s41598-019-47028-4

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In the version of the paper initially published, Fig. 5a was inadvertently duplicated and presented as both Fig. 5a and 5f, and the correct image for Fig. 5f was omitted. Images of the original and corrected figure panels are shown in the correction notice.

DOI： 10.1038/s41588-019-0396-9

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Chronic inflammation associated with bone tissues often destructs bones, which is essentially performed by osteoclasts in the presence of immunoregulatory molecules. Hence, regulating osteoclastogenesis is crucial to develop therapeutics for bone-destructive inflammatory diseases. It is believed that reactive oxygen species (ROS) are involved in receptor activator of NF-κB (RANK) ligand (RANKL)-induced osteoclast differentiation, and, therefore, glutathione (GSH), the most abundant endogenous antioxidant, suppresses osteoclast differentiation and bone resorption by RANKL. Interestingly, GSH also contributes to inflammatory responses, and the effects of GSH on osteoclast differentiation and bone destruction under inflammatory conditions have not yet been determined. Here, we investigated how GSH affects inflammatory cytokine-stimulated osteoclast differentiation in vitro and in a mouse model of inflammatory bone destruction. We found that GSH significantly promoted TNFα-stimulated osteoclast formation, while an inhibitor of GSH synthesis, buthionine sulfoximine, suppressed it. GSH facilitated the nuclear localisation of the nuclear factor of activated T cells c1 (NFATc1) protein, a master regulator of osteoclastogenesis, as well as the expression of osteoclast marker genes in a dose-dependent manner. N-acetylcysteine, a substrate of GSH synthesis, also stimulated osteoclast formation and NFATc1 nuclear localisation. GSH did not suppress cell death after osteoclast differentiation. In mouse calvaria injected with lipopolysaccharide, GSH treatment resulted in a fivefold increase in the osteolytic lesion area. These results indicate that GSH accelerates osteoclast differentiation and inflammatory bone destruction, suggesting GSH appears to be an important molecule in the mechanisms responsible for inflammatory bone destruction by osteoclasts.

DOI： 10.1080/10715762.2018.1563782

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DOI： 10.1111/dgd.12560

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Pinopsin is the opsin most closely related to vertebrate visual pigments on the phylogenetic tree. This opsin has been discovered among many vertebrates, except mammals and teleosts, and was thought to exclusively function in their brain for extraocular photoreception. Here, we show the possibility that pinopsin also contributes to scotopic vision in some vertebrate species. Pinopsin is distributed in the retina of non-teleost fishes and frogs, especially in their rod photoreceptor cells, in addition to their brain. Moreover, the retinal chromophore of pinopsin exhibits a thermal isomerization rate considerably lower than those of cone visual pigments, but comparable to that of rhodopsin. Therefore, pinopsin can function as a rhodopsin-like visual pigment in the retinas of these lower vertebrates. Since pinopsin diversified before the branching of rhodopsin on the phylogenetic tree, two-step adaptation to scotopic vision would have occurred through the independent acquisition of pinopsin and rhodopsin by the vertebrate lineage.

DOI： 10.1038/s42003-018-0164-x

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

Dickkopf-related protein 3 (Dkk3) is the third member of the Dkk gene family and identical to the gene, whose expression was reduced in immortalized cells. Therefore, its another name is reduced expression in immortalized cells. Since the intratumoral introduction of Dkk3 inhibits tumor growth in mouse models of cancers, Dkk3 is likely a tumor suppressor gene. However, the functions of Dkk3 in vivo remain unclear. As the first step to decipher the physiological roles of this gene, we examined the expression pattern of Dkk3 in various tissues from adult mice. In situ hybridization showed that Dkk3 mRNA was detected in the brain, retina, heart, gastrointestinal tract, adrenal glands, thymus, prostate glands, seminal vesicles, testes, and ovaries in a regionally specific manner. Furthermore, we raised anti-mouse Dkk3 antibody and performed immunohistochemistry. Cytoplasmic localization of Dkk3 protein was observed in the cells of the adrenal medulla, while Dkk3 immunoreactivity was observed in the lumen of the stomach and intestine, implying that the Dkk3 protein may be secreted into the lumen of the gastrointestinal tract. These results suggest that Dkk3 has pleiotropic roles for a secretory glycoprotein that acts primarily in the gastrointestinal tract, thymus, endocrine and reproductive organs of the mouse.

DOI： 10.1007/s10735-016-9703-2

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Part of collection (book)   Publisher：Springer Japan  

The hemimetabolous insect, Gryllus bimaculatus, has two compound eyes that begin to form in the embryo and increase in size five- to sixfolds during nymphal development. Retinal stemlike cells reside in the anteroventral proliferation zone (AVPZ) of the nymphal compound eye and proliferate to increase retinal progenitors, which then differentiate to form new ommatidia in the anterior region of the eye. Here, we introduce the morphology and development of the cricket eye first, and then we focus on the roles of retinal determination genes (RDGs) such as eyes absent (eya) and sine oculis (so) in Gryllus eye formation and growth. Since the principal function of the eye is photoreception, we finally summarize opsin photopigments in this species, broadening the roles of photoreception.

DOI： 10.1007/978-4-431-56478-2_4

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Language：Japanese   Publisher：(一社)日本骨代謝学会  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATL ACAD SCIENCES  

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.

DOI： 10.1073/pnas.1600612113

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PUBLIC LIBRARY SCIENCE  

Opn5 is a group within the opsin family of proteins that is responsible for visual and non-visual photoreception in animals. It consists of several subgroups, including Opn5m, the only subgroup containing members found in most vertebrates, including mammals. In addition, recent genomic information has revealed that some ray-finned fishes carry paralogous genes of Opn5m while other fishes have no such genes. Here, we report the molecular properties of the opsin now called Opn5m2 and its distributions in both the retina and brain. Like Opn5m, Opn5m2 exhibits UV light-sensitivity when binding to 11-cis-retinal and forms a stable active state that couples with Gi subtype of G protein. However, Opn5m2 does not bind all-trans-retinal and exhibits exclusive binding to 11-cis-retinal, whereas many bistable opsins, including fish Opn5m, can bind directly to all-trans-retinal as well as 11-cis-retinal. Because medaka fish has lost the Opn5m2 gene from its genome, we compared the tissue distribution patterns of Opn5m in medaka fish, zebrafish, and spotted gar, in addition to the distribution patterns of Opn5m2 in zebrafish and spotted gar. Opn5m expression levels showed a gradient along the dorsal-ventral axis of the retina, and preferential expression was observed in the ventral retina in the three fishes. The levels of Opn5m2 showed a similar gradient with preferential expression observed in the dorsal retina. Opn5m expression was relatively abundant in the inner region of the inner nuclear layer, while Opn5m2 was expressed in the outer edge of the inner nuclear layer. Additionally, we could detect Opn5m expression in several brain regions, including the hypothalamus, of these fish species. Opn5m2 expression could not be detected in zebrafish brain, but was clearly observed in limited brain regions of spotted gar. These results suggest that ray-finned fishes can generally utilize UV light information for non-image-forming photoreception in a wide range of cells in the retina and brain.

DOI： 10.1371/journal.pone.0155339

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：OKAYAMA UNIV MED SCHOOL  

Human congenital anomalies provide information that contributes to the understanding of developmental mechanisms. Here we report bilateral optic nerve aplasia (ONA) with microphthalmia in the autopsy of the cadaver of a 70-year-old Japanese female. The gross anatomical inspection of the brain showed a cotton thread-like cord in the presumed location of the optic nerve tract or chiasm. Histologically, no neural retina, optic nerve bundle or retinal central vessels were formed in the eye globe, and the retinal pigment cells formed rosettes. The cornea, iris, and lens were also histologically abnormal. Immunohistochemically, no retinal cells expressed beta III tubulin, and Pax6-immunoreactive cells were present in the ciliary non-pigmented epithelial cells. This case of ONA could be attributed to the agenesis of retinal projection neurons as a sequel to the disruption of neural retina development. The neural retina formation would coordinate the proper development of ocular tissues.

DOI： 10.18926/AMO/54192

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER ASSOC CANCER RESEARCH  

One promising method to visualize cancer cells is based on the detection of the fluorescent photosensitizer protoporphyrin IX (PpIX) synthesized from 5-aminolevulinic acid (ALA), but this method cannot be used in cancers that exhibit poor PpIX accumulation. PpIX appears to be pumped out of cancer cells by the ABC transporter G2 (ABCG2), which is associated with multidrug resistance. Genistein is a phytoestrogen that appears to competitively inhibit ABCG2 activity. Therefore, we investigated whether genistein can promote PpIX accumulation in human lung carcinoma cells. Here we report that treatment of A549 lung carcinoma cells with genistein or a specific ABCG2 inhibitor promoted ALA-mediated accumulation of PpIX by approximately 2-fold. ABCG2 depletion and overexpression studies further revealed that genistein promoted PpIX accumulation via functional repression of ABCG2. After an extended period of genistein treatment, a significant increase in PpIX accumulation was observed in A549 cells (3.7-fold) and in other cell lines. Systemic preconditioning with genistein in a mouse xenograft model of lung carcinoma resulted in a 1.8-fold increase in accumulated PpIX. Long-term genistein treatment stimulated the expression of genes encoding enzymes involved in PpIX synthesis, such as porphobilinogen deaminase, uroporphyrinogen decarboxylase, and protoporphyrinogen oxidase. Accordingly, the rate of PpIX synthesis was also accelerated by genistein pretreatment. Thus, our results suggest that genistein treatment effectively enhances ALA-induced PpIX accumulation by preventing the ABCG2-mediated efflux of PpIX from lung cancer cells and may represent a promising strategy to improve ALA-based diagnostic approaches in a broader set of malignancies.

DOI： 10.1158/0008-5472.CAN-15-1484

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INTRODUCTION: Insects show daily behavioral rhythms controlled by an endogenous oscillator, the circadian clock. The rhythm synchronizes to daily light-dark cycles (LD) and changes waveform in association with seasonal change in photoperiod. RESULTS: To explore the molecular basis of the photoperiod-dependent changes in circadian locomotor rhythm, we investigated the role of a chromatin modifier, Enhancer of zeste (Gb'E(z)), in the cricket, Gryllus bimaculatus. Under a 12 h:12 h LD (LD 12:12), Gb'E(z) was constitutively expressed in the optic lobe, the site of the biological clock; active phase (α) and rest phase (ρ) were approximately 12 h in duration, and α/ρ ratio was approximately 1.0. When transferred to LD 20:4, the α/ρ ratio decreased significantly, and the Gb'E(z) expression level was significantly reduced at 6 h and 10 h after light-on, as was reflected in the reduced level of trimethylation of histone H3 lysine 27. This change was associated with change in clock gene expression profiles. The photoperiod-dependent changes in α/ρ ratio and clock gene expression profiles were prevented by knocking down Gb'E(z) by RNAi. CONCLUSIONS: These results suggest that histone modification by Gb'E(z) is involved in photoperiodic modulation of the G. bimaculatus circadian rhythm.

DOI： 10.1186/s40851-016-0042-7

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Language：Japanese   Publisher：(公社)日本生化学会  

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：COMPANY OF BIOLOGISTS LTD  

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.

DOI： 10.1242/dev.122598

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

Cricket nymphs have the remarkable ability to regenerate a functional leg following amputation, indicating that the regenerating blastemal cells contain information for leg morphology. However, the molecular mechanisms that underlie regeneration of leg patterns remain poorly understood. Here, we analyzed phenotypes of the tibia and tarsus (three tarsomeres) obtained by knockdown with regeneration-dependent RNA interference (rdRNAi) against Gryllus dachshund (Gb'dac) and Distal-less (Gb'Dll). We found that depletion of Gb'Dll mRNA results in loss of the tarsal segments, while rdRNAi against Gb'dac shortens the tibia at the two most distal tarsomeres. These results indicate that Gb'Dll expression is indispensable for formation of the tarsus, while Gb'dac expression is necessary for elongation of the tibia and formation of the most proximal tarsomere. These findings demonstrate that mutual transcriptional regulation between the two is indispensable for formation of the tarsomeres, whereas Gb'dac is involved in determination of tibial size through interaction with Gb'ds/Gb'ft.

DOI： 10.1038/srep08387

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What determines organ size has been a long-standing biological question. Lawrence et al. (2008) proposed the steepness hypothesis suggesting that the protocadherin Dachsous/Fat (Ds/Ft) system may provide some measure of dimension to the cells in relation to the gradient. In this paper we extended the model as a means of interpreting experimental results in cricket leg regeneration. We assumed that (1) Ds/Ft trans-heterodimers or trans-homodimers are redistributed during cell division, and (2) growth would cease when a differential of the dimer across each cell decreases to a certain threshold. We applied our model to simulate the results obtained by leg regeneration experiments in a cricket model. The results were qualitatively consistent with the experimental data obtained for cricket legs by RNA interference methodology. Using our extended steepness model, we provided a molecular-based explanation for leg size determination even in intercalary regeneration and for organ size determination.

DOI： 10.1038/srep04335

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

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.

DOI： 10.1111/dgd.12074

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Authorship：Last author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：OKAYAMA UNIV MED SCHOOL  

Ever since protoporphyrin IX (PpIX) was discovered to accumulate preferentially in cancer cells after 5-aminolevulinic acid (ALA) treatment, photodynamic treatment or therapy (PDT) has been developed as an exciting new treatment option for cancer patients. However, the level of PpIX accumulation in oral cancer is fairly low and insufficient for PDT. Ferrochelatase (FECH) and ATP-binding cassette transporter G2 (ABCG2) are known to regulate PpIX accumulation. In addition, serum enhances PpIX export by ABCG2. We investigated here whether and how inhibitors of FECH and ABCG2 and their combination could improve PpIX accumulation and PDT efficacy in an oral cancer cell line in serum-containing medium. ABCG2 inhibitor and the combination of ABCG2 and FECH inhibitors increased PpIX in the presence of fetal bovine serum (FBS) in an oral cancer cell line. Analysis of ABCG2 gene silencing also revealed the involvement of ABCG2 in the regulation of PpIX accumulation. Inhibitors of FECH and ABCG2, and their combination increased the efficiency of ALA-PDT even in the presence of FBS. ALA-PDT-induced cell death was accompanied by apoptotic events and lipid peroxidation. These results suggest that accumulation of PpIX is determined by the activities of ABCG2 and FECH and that treatment with a combination of their inhibitors improves the efficacy of PDT for oral cancer, especially in the presence of serum.

DOI： 10.18926/AMO/50408

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：COMPANY OF BIOLOGISTS LTD  

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.

DOI： 10.1242/dev.084590

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：COMPANY OF BIOLOGISTS LTD  

How the eye forms has been one of the fundamental issues in developmental biology. The retinal anlage first appears as the optic vesicle (OV) evaginating from the forebrain. Subsequently, its distal portion invaginates to form the two-walled optic cup, which develops into the outer pigmented and inner neurosensory layers of the retina. Recent work has shown that this optic-cup morphogenesis proceeds as a self-organizing activity without any extrinsic molecules. However, intrinsic factors that regulate this process have not been elucidated. Here we show that a LIM-homeobox gene, Lhx1, normally expressed in the proximal region of the nascent OV, induces a second neurosensory retina formation from the outer pigmented retina when overexpressed in the chicken OV. Lhx2, another LIM-homeobox gene supposed to be involved in early OV formation, could not substitute this function of Lhx1, while Lhx5, closely related to Lhx1, could replace it. Conversely, knockdown of Lhx1 expression by RNA interference resulted in the formation of a small or pigmented vesicle. These results suggest that the proximal region demarcated by Lhx1 expression permits OV development, eventually dividing the two retinal domains. (C) 2012. Published by The Company of Biologists Ltd.

DOI： 10.1242/bio.20121396

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

Hemimetabolous, or incompletely metamorphosing, insects are phylogenetically relatively basal and comprise many pests. However, the absence of a sophisticated genetic model system, or targeted gene-manipulation system, has limited research on hemimetabolous species. Here we use zinc-finger nuclease and transcription activator-like effector nuclease technologies to produce genetic knockouts in the hemimetabolous insect Gryllus bimaculatus. Following the microinjection of mRNAs encoding zinc-finger nucleases or transcription activator-like effector nucleases into cricket embryos, targeting of a transgene or endogenous gene results in sequence-specific mutations. Up to 48% of founder animals transmit disrupted gene alleles after zinc-finger nucleases microinjection compared with 17% after microinjection of transcription activator-like effector nucleases. Heterozygous offspring is selected using mutation detection assays that use a Surveyor (Cel-I) nuclease, and subsequent sibling crosses create homozygous knockout crickets. This approach is independent from a mutant phenotype or the genetic tractability of the organism of interest and can potentially be applied to manage insect pests using a non-transgenic strategy.

DOI： 10.1038/ncomms2020

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In the cricket Gryllus bimaculatus, a hemimetabolous insect, the compound eyes begin to form in the embryo and increase 56 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 Gbeya resulted in loss of the embryonic eye. In the nymphal eye, RNAi against Gbeya or Gbso impaired retinal morphology by apparently transforming cornea structures into head cuticle. These results imply that Gbeya and Gbso are essential for the differentiation of the retinal progenitor cells and maintaining retinal structures during eye development.

DOI： 10.1111/j.1440-169X.2011.01325.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE SOC PLANT CELL & MOLECULAR BIOLOGY  

Traditional methods used to study strawberry ripening-related gene function are time-consuming, and require at least 15 months from initiating the transformation experiment until the first ripe fruits are available for analysis. To accelerate data acquisition during gene function studies, we explored a transient assay method that employs an Agrobacterium-mediated RNAi (AmRNAi) technique in post-harvest strawberry fruit, Fragaria x ananassa (Fa) cv. Sachinoka, a Japanese cultivar. Our results showed that artificial white light induced strong expression of Fa'chalcone synthase (Fa'CHS), Fa'chalcone isomerase (Fa'CHI), and Fa'flavonoid 3'-hydroxylase orthologues (Fa'F3'H) in post-harvest fruit. Fa'CHS and Fa'F3'H function was subsequently examined by performing AmRNAi with post-harvest fruit. Although reduction of light-induced Fa'F3'H expression by AmRNAi resulted in no significant change in anthocyanin content, reduction of Fa'CHS significantly decreased anthocyanin levels, and up-regulated Fa'F3'H levels. Our results are consistent with previous data indicating that while CHS is required for anthocyanin accumulation during late stage strawberry fruit maturation, Fa'F3'H is not required. The novel system described here enabled gene function data to be available within 10 days of initiating the incubation period following in filtration. therefore, we conclude our system is a valuable tool to elucidate the molecular mechanisms underlying light-induced ripening of strawberry fruit.

DOI： 10.5511/plantbiotechnology.12.0406a

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In submammalian animals including chicks, the retina contains oligodendrocytes (OLs), and axons in the optic fiber layer are wrapped with compact myelin within the retina; however, the expression of myelin genes in the chick retina has not been demonstrated yet. In the present study, we examined the expression of three myelin genes (proteolipid protein, PLP; myelin basic protein. MBP; cyclic nucleotide phosphodiesterase, CNP) and PLP in the developing chick retina, in comparison to the localization of Mueller cells. In situ hybridization demonstrated that all three myelin genes began to be expressed at E14 in the chick embryo retina. They are mostly restricted to the ganglion cell layer and the optic fiber layer, with a few exceptions in the inner nuclear layer where Mueller cells reside; however. PLP mRNA+ cells do not express glutamine synthetase, or vice versa. The present results elucidate that myelin genes are expressed only by OLs that are mostly localized in the innermost layer of the developing chick retina. (C) 2011 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.gep.2011.08.002

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

VA/VAL opsin is one of the four kinds of nonvisual opsins that are closely related to vertebrate visual pigments in the phylogenetic tree of opsins. Previous studies indicated that among these opsins, parapinopsin and pinopsin exhibit molecular properties similar to those of invertebrate bistable visual pigments and vertebrate visual pigments, respectively. Here we show that VA/VAL opsin exhibits molecular properties intermediate between those of parapinopsin and pinopsin. VAL opsin from Xenopus tropicalis was expressed in cultured cells, and the pigment with an absorption maximum at 501 nm was reconstituted by incubation with 11-cis-retinal. Light irradiation of this pigment caused cis-to-trans isomerization of the chromophore to form a state having an absorption maximum in the visible region. This state has the ability to activate Gi and Gt types of G proteins. Therefore, the active state of VAL opsin is a visible light-absorbing intermediate, which probably has a protonated retinylidene Schiff base as its chromophore, like the active state of parapinopsin. However, this state was apparently photoinsensitive and did not show reverse reaction to the original pigment, unlike the active state of parapinopsin, and instead similar to that of pinopsin. Furthermore, the Gi activation efficiency of VAL opsin was between those of pinopsin and parapinopsin. Thus, the molecular properties of VA/VAL opsin give insights into the mechanism of conversion of the molecular properties from invertebrate to vertebrate visual pigments.

DOI： 10.1021/bi201212z

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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.

DOI： 10.1111/j.1440-169X.2011.01291.x

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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&apos;Delta) and Notch (Gb&apos;Notch) genes in the hemimetabolous, intermediate-germ cricket Gryllus bimaculatus. We found that Gb&apos;Delta and Gb&apos;Notch were expressed in developing legs, and that RNAi silencing of Gb&apos;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&apos;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&apos;Notch was uniformly expressed in early germbands. RNAi knockdown of Gb&apos;Delta or Gb&apos;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.

DOI： 10.1242/dev.060681

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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&apos;Lft), which modulates Ds/Ft signaling in Drosophila, is involved in leg regeneration. Gb&apos;lft is expressed in regenerating legs, and RNAi against Gb&apos;lft (Gb&apos;lft RNAi) suppressed blastema cell hyperproliferation caused by Gb&apos;ft(RNAi) or Gb&apos;ds(RNAi) but enhanced that caused by Gb&apos;kibra(RNAi) or Gb&apos;warts(RNAi). In Gb&apos;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&apos;lft(RNAi) nymphs, suggesting that Gb&apos;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. Developmental Dynamics 240:1440-1453, 2011. (C) 2011 Wiley-Liss, Inc.

DOI： 10.1002/dvdy.22647

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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. Developmental Dynamics 240:1028-1041, 2011. (C) 2011 Wiley-Liss, Inc.

DOI： 10.1002/dvdy.22590

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Autotaxin (ATX) is a lysophospholipid-generating exoenzyme expressed in embryonic and adult neural tissues. We previously showed that ATX is expressed in the neural organizing centers, anterior head process, and midbrain-hindbrain boundary (MHB). To elucidate the role of ATX during neural development, here we examined the neural phenotypes of ATX-deficient mice. Expression analysis of neural marker genes revealed that lateral expansion of the rostral forebrain is reduced and establishment of the MHB is compromised as early as the late headfold stage in ATX mutant embryos. Moreover, ATX mutant embryos fail to complete cranial neural tube closure. These results indicate that ATX is essential for cranial neurulation and MHB establishment. Developmental Dynamics 240:413-421, 2011. (C) 2011 Wiley-Liss, Inc.

DOI： 10.1002/dvdy.22543

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Autotaxin (ATX) is a lysophospholipid-generating exoenzyme expressed in embryonic and adult neural tissues. We previously showed that ATX is expressed in the neural organizing centers, anterior head process, and midbrain-hindbrain boundary (MHB). To elucidate the role of ATX during neural development, here we examined the neural phenotypes of ATX-deficient mice. Expression analysis of neural marker genes revealed that lateral expansion of the rostral forebrain is reduced and establishment of the MHB is compromised as early as the late headfold stage in ATX mutant embryos. Moreover, ATX mutant embryos fail to complete cranial neural tube closure. These results indicate that ATX is essential for cranial neurulation and MHB establishment. © 2011 Wiley-Liss, Inc.

DOI： 10.1002/dvdy.22543

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Small interfering RNA (siRNA)-mediated silencing of gene expression is rapidly becoming a powerful tool for molecular therapy. However, the rapid degradation of siRNAs and their limited duration of activity require efficient delivery methods. Atelocollagen (ATCOL)-mediated administration of siRNAs is a promising approach to disease treatment, including muscular atrophy. Herein, we report that ATCOL-mediated systemic administration of a myostatin-targeting siRNA into a caveolin-3-deficient mouse model of limb-girdle muscular dystrophy 1C (LGMD1C) induced a marked increase in muscle mass and a significant recovery of contractile force. These results provide evidence that ATCOL-mediated systemic administration of siRNAs may be a powerful therapeutic tool for disease treatment, including muscular atrophy.

DOI： 10.1111/j.1440-169X.2010.01221.x

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Silencing gene expression by small interfering RNAs (siRNAs) has become a powerful tool for the genetic analysis of many animals. However, the rapid degradation of siRNA and the limited duration of its action in vivo have called for an efficient delivery technology. Here, we describe that siRNA complexed with a synthetic collagen poly(Pro-Hyp-Gly) (SYCOL) is resistant to nucleases and is efficiently transferred into cells in vitro and in vivo, thereby allowing long-term gene silencing in vivo. We found that the SYCOL-mediated local application of siRNA targeting myostatin, coding a negative regulator of skeletal muscle growth, in mouse skeletal muscles, caused a marked increase in the muscle mass within a few weeks after application. Furthermore, in vivo administration of an anti-luciferase siRNA/SYCOL complex partially reduced luciferase expression in xenografted tumors in vivo. These results indicate a SYCOL-based non-viral delivery method could be a reliable simple approach to knockdown gene expression by RNAi in vivo as well as in vitro.

DOI： 10.1111/j.1440-169X.2010.01206.x

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The diencephalon is the embryonic anlagen of the higher integration centers of the brain. Recent studies have elucidated how the cells in the rostral diencephalon acquire their regional identities. However, the understanding of the mechanisms under which the caudal diencephalon is formed is still limited. Here we focus on the role of Autotaxin (ATX), a lysophospholipid-generating exoenzyme, whose mRNA is detected in the caudal diencephalon. RNA interference against ATX altered the expression pattern of Pax6-regualted genes, Tcf4, Lim1, and En1, implying that ATX is required for the maintenance of the regional identity of the caudal diencephalon and the diencephalon-mesencephalon boundary (DMB). Furthermore, ATX-RNAi inhibited neuroepithelial cell proliferation on both sides of the DMB. We propose a dual role of ATX in chick brain development, in which ATX not only contributes to the formation of caudal diencephalon as a short-range signal, but also regulates the growth of mesencephalon as a long-range signal. Developmental Dynamics 239:2647-2658, 2010. (C) 2010 Wiley-Liss, Inc.

DOI： 10.1002/dvdy.22403

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：CELL PRESS  

The mode of insect embryogenesis varies among species, reflecting adaptations to different lite 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 syncytiurn, 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.

DOI： 10.1016/j.cub.2010.07.044

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In order to explore a possibility that the cricket Gryllus bimaculatus would be a useful model to unveil molecular mechanisms of human diseases, we performed loss-of-function analyses of Gryllus genes homologous to human genes that are responsible for human disorders, fragile X mental retardation 1 (fmr1) and Dopamine receptor (DopR). We cloned cDNAs of their Gryllus homologues, Gb&apos;fmr1, Gb&apos;DopRI, and Gb&apos;DopRII, and analyzed their functions with use of nymphal RNA interference (RNAi). For Gb&apos;fmr1, three major phenotypes were observed: (1) abnormal wing postures, (2) abnormal calling song, and (3) loss of the circadian locomotor rhythm, while for Gb&apos;DopRI, defects of wing posture and morphology were found. These results indicate that the cricket has the potential to become a novel model system to explore human neuronal pathogenic mechanisms and to screen therapeutic drugs by RNAi. Developmental Dynamics 238. 2025-2033, 2009. (C) 2009 Wiley-Liss, Inc.

DOI： 10.1002/dvdy.22029

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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.

DOI： 10.1242/dev.035204

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

The mechanisms underlying retinal cell diversification are crucial to proper neural development. Fibroblast growth factor 19 (Fgf19) is expressed by developing horizontal cells (HCs) in the chicken retina. Although there are two major HC subtypes, axon-bearing and axon-less, the precise subtype expressing Fgf19 remains uncertain. Here we characterize Fgf19-expressing cells by co-labeling with antibodies against Lim1 (LIM homeodomain 1, or Lhx1), Islet1, and Prox1 (prospero-related homeobox 1) which are axon-bearing HC, axon-less HC, and pan-HC markers, respectively. We found that a subset of Fgr19-expressing cells was positive for Prox1 and Lim1 in the vitread neuroepithelium at embryonic day 4 (E4). By E9, the majority of Fgf19-expressing cells became positive for Prox1 and Lim1 prior to arrival at the prospective HC layer. In contrast, Fgf19-expressing cells did not overlap with the Islet1-positive population at any stage examined. These results suggest that Fgf19 is expressed by the early migratory horizontal precursors, and later by the presumptive axon-bearing HCs. (C) 2009 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.gep.2009.02.007

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Lysophospholipids (LPs) such as lysophosphatidic acid (LPA) and sphingosine-1-phosphate (SIP) are known to mediate various biological responses, including cell proliferation, migration, and differentiation. To better understand the role of these lipids in mammalian early development, we applied whole-mount in situ hybridization techniques to E8.5 to E12.5 mouse embryos. We determined the expression patterns of the following LP receptor genes, which belong to the G protein-coupled receptor (GPCR) family: EDG1 to EDG8 (S1P(1) to S1P(5) and LPA(1) to LPA(3)), LPA(4) (GPR23 /P2Y9), and LPA(5) (GPR92). We found that the S1P/LPA receptor genes exhibit overlapping expression patterns in a variety of organ primordia, including the developing brain and cardiovascular system, presomitic mesoderm and somites, branchial arches, and limb buds. These results suggest that multiple receptor systems for LPA/S1P lysophospholipids may be functioning during organogenesis. Developmental Dynamics 237.3280-3294, 2008. (C) 2008 Wiley-Liss, Inc.

DOI： 10.1002/dvdy.21736

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RNA interference (RNAi) offers a novel therapeutic strategy based on the highly specific and efficient silencing of a target gene. Since it relies on small interfering RNAs (siRNAs), a major issue is the delivery of therapeutically active siRNAs into the target tissue/target cells in vivo. For safety reasons, strategies based on vector delivery may be of only limited clinical use. The more desirable approach is to directly apply active siRNAs in vivo. Here, we report the effectiveness of in vivo siRNA delivery into skeletal muscles of normal or diseased mice through nanoparticle formation of chemically unmodified siRNAs with atelocollagen (ATCOL). ATCOL-mediated local application of siRNA targeting myostatin, a negative regulator of skeletal muscle growth, in mouse skeletal muscles or intravenously, caused a marked increase in the muscle mass within a few weeks after application. These results imply that ATCOL-mediated application of siRNAs is a powerful tool for future therapeutic use for diseases including muscular atrophy.

DOI： 10.1038/gt.2008.24

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

The specification of germ cells during embryogenesis is an important issue in the development of metazoans. In insects, the mode of germ cell specification appears to be highly variable among species and molecular data are not sufficient to provide an evolutionary perspective to this issue. Expression of vasa can be used as a germ line marker. Here, we report the isolation of a vasa-like gene in a hemimetabolous insect, the cricket Gryllus bimaculatus (Gb'vas), and its expression patterns during oogenesis and embryogenesis. Gb'vas is preferentially expressed in the germarium and the expression of Gb'vas is detectable throughout vitellogenesis including mature eggs subjected to oviposition, suggesting that Gb'vas is maternally contributed to the cricket eggs. The zygotic expression of Gb'vas appears to start at the mid blastoderm stage in the posterior region of the egg, expanding in a developing germ anlage. In early germbands, an intense expression of Gb'vas is restricted to the posterior end. In later embryos, Gb'vas expression extends over the whole body and then distinctly localized to the embryonic gonad at the stage immediately before hatching. These results suggest that, in the cricket, germ cells are specified early in development at the posterior end of an early germband, as proposed by Heymons (1895) based on cytological criteria.

DOI： 10.1007/s00427-008-0226-z

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Nymphs of hemimetabolous insects, such as cockroaches and crickets, possess functional legs with a remarkable capacity for epimorphic regeneration. In this study, we have focused on the role of epidermal growth factor receptor (EGFR) signaling in regeneration of a nymphal leg in the cricket Gryllus bimaculatus. We performed loss-of-function analyses with a Gryllus Egfr homolog (Gb'Egfr) and nymphal RNA interference (RNAi). After injection of double-stranded RNA for Gb'Egfr in the body cavity of the third instar cricket nymph, amputation of the leg at the distal tibia resulted in defects of normal distal regeneration. The regenerated leg lacked the distal tarsus and pretarsus. This result indicates that EGFR signaling is required for distal leg patterning in regeneration during the nymphal stage of the cricket. Furthermore, we demonstrated that EGFR signaling acts downstream of the canonical Wnt/Wg signaling and regulates appendage proximodistal (PD) patterning genes aristaless and dachshund during regeneration. Our results suggest that EGFR signaling influences positional information along the PD axis in distal leg patterning of insects, regardless of the leg formation mode. (C) 2008 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.ydbio.2008.04.002

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL PUBLISHING  

Fgf19 is known to be expressed in the developing chicken eye but its functions during retinal development have remained elusive. Since Fgf19 is expressed in the dorsal portion of the optic cup, it is intriguing to know whether FGF19 is required for expression of dorso-ventral morphogenetic genes in the eye. To clarify this, expression patterns of Tbx5 and Vax were examined in the developing eye after in ovo RNA interference targeted against Fgf19. Quantitative polymerase chain reaction (PCR) analysis showed that the short-hairpin RNAs (shRNAs) targeted against Fgf19 could reduce its expression in the eye to less than 50% of a relative amount of mRNA, compared with contralateral or untreated control eyes. However, no obvious alteration in expression domains of Tbx5 or Vax was observed. Misexpression of Tbx5 or Tbx5-RNAi did not alter the Fgf19 expression either. Furthermore, although Fgf19 is expressed in the central retina before neurogenesis occurs, beta 3-tubulin, a marker for early retinal differentiation was still detected in the central retina after knockdown of Fgf19. Thus, knockdown of Fgf19 supports no obvious regulations between Fgf19 and Tbx5, or exhibits no phenotypes that perturb early retinal differentiation.

DOI： 10.1111/j.1440-169x.2008.00996.x

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The cricket Gryllus bimaculatus is a typical hemimetabolous intemediate germ insect, in which the processes of segmentation and appendage formation differ from those in Drosophila, a holometabolous long germ insect. In order to compare their developmental mechanisms, we have focused on Gryllus orthologs of the Drosophila developmental regulatory genes and studied their functions. Here, we report a functional analysis of the Gryllus ortholog of extradenticle (Gb'exd) using embryonic and parental RNA interference (RNAi) techniques. We found the following: (1) RNAi suppression of Gb'exd results in the deletion or fusion of body segments. Especially the head was often very severely affected. This gap-like phenotype may be related to reduced expression of the gap genes hunchback and Kruppel in early RNAi germbands. (2) In the appendages, several segments (podomeres) were fused. (3) Head appendages including the antenna were transformed to a leg-like structure consisting of at least one proximal podomere as well as several tarsomeres. The defects in appendages are reminiscent of the phenotype caused by large exd clones in Drosophila antennal discs. These findings led us to the conclusion that (1) Gb'exd is required for segment patterning in the gnathal to abdominal region, acting in a gap gene-like manner in the anterior region. (2) Gb'exd plays important roles in formation of the appendages and the determination of their identities, acting as a regulatory switch that chooses between the fates of head appendages versus the appendage ground state. Although functions of Gbexd in appendage patterning appear fundamentally conserved between Gryllus and Drosophila, its role in body segmentation may differ from that of Drosophila exd. (c) 2007 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.ydbio.2007.09.060

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In order to observe three-dimensional (3D) expression patterns of genes in whole animals, whole organs, or whole tissues, in situ hybridization (ISH) of many sections must be carried out and then used to construct a 3D image. For this purpose, we have developed an automatic microtome to prepare tissue sections with an adhesive film. We used commercially available film suitable for sectioning and ISH. We constructed a microtome and, after adherence of the film to a paraffin-embedded tissue block, cut the block with a blade to prepare sections on film. Then, the sections-on-film were automatically set in a plastic frame that was the same size as a conventional glass slide. With this automatic microtome, tissue sections can be made for ISH or immunohistochemistry in addition to conventional hematoxylin and eosin staining without specific training. We demonstrate that we can construct 3D images of gene expression patterns obtained by ISH on sections prepared with this automatic microtome. We have designated this method as &apos;Film Tomography (FITO)&apos;.

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We examined the expression pattern of melanopsin-like (cOpn4m) and VA opsin-like (cVAL) genes during chicken development. Two types of cOpn4m transcripts, distinct in their carboxyl terminals were found, as is the case for the chicken melanopsin (cOpn4) reported previously. The expression of cOpn4m was restricted to the developing retina, specifically to a subset of developing amacrine cells from embryonic day 10. VA opsin is one of the non-canonical opsins, reported to exist in fish so far. In this study, an aberrant type of VA opsin-like (cVAL) cDNA was isolated from chicken embryonic neural tissues. The expression of cVAL was observed in the ventral region of the developing brain and neural tube; however, specific signals for cVAL could not be detected in the developing retina. These results indicate that the additional melanopsin in avian identifies a subset of developing amacrine cells in the retina and that the aberrant transcript of the VA opsin-like gene are present during neural tube development in the chicken. (c) 2007 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.modgep.2007.06.001

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DOI： 10.1002/dvdy.21158

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-LISS  

Autotaxin (ATX), or nucleotide pyrophosphatase-phosphodiesterase 2, is a secreted lysophospholipase D that generates bioactive phospholipids that act on G protein-coupled receptors. Here we show the expression patterns of the ATX gene in mouse and chicken embryos. ATX has a dynamic spatial and temporal expression pattern in both species and the expression domains during neural development are quite distinct from each other. Murine ATX (mATX) is expressed immediately rostral to the midbrain-hindbrain boundary, whereas chicken ATX (cATX) is expressed in the diencephalon and later in the parencephalon-synencephalon boundary. In the neural tube, cATX is expressed in the alar plate in contrast to mATX in the floor plate. ATX is also expressed in the hindbrain and various organ primordia such as face anlagen and skin appendages of the mouse and chicken. These results suggest conserved and non-conserved roles for ATX during neural development and organogenesis in these species.

DOI： 10.1002/dvdy.21119

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Academic Press Inc.  

Developmental mechanisms of segmentation appear to be varied among insects in spite of their conserved body plan. Although the expression patterns of the segment polarity genes in all insects examined imply well conserved function of this class of genes, expression patterns and function of the pair-rule genes tend to exhibit diversity. To gain further insights into the evolution of the segmentation process and the role of pair-rule genes, we have examined expression and function of an ortholog of the Drosophila pair-rule gene even-skipped (eve) in a phylogenetically basal insect, Gryllus bimaculatus (Orthoptera, intermediate germ cricket). We find that Gryllus eve (Gb'eve) is expressed as stripes in each of the prospective gnathal, thoracic, and abdominal segments and as a broad domain in the posterior growth zone. Dynamics of stripe formation vary among Gb'eve stripes, representing one of the three modes, the segmental, incomplete pair-rule, and complete pair-rule mode. Furthermore, we find that RNAi suppression of Gb'eve results in segmentation defects in both anterior and posterior regions of the embryo. Mild depletion of Gb'eve shows a pair-rule-like defect in anterior segments, while stronger depletion causes a gap-like defect showing deletion of anterior and posterior segments. These results suggest that Gb'eve acts as a pair-rule gene at least during anterior segmentation and also has segmental and gap-like functions. Additionally, Gb'eve may be involved in the regulation of hunchback and Krüppel expression. Comparisons with eve functions in other species suggest that the Gb'eve function may represent an intermediate state of the evolution of pair-rule patterning by eve in insects. © 2006 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.ydbio.2006.11.003

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL PUBLISHING  

The cricket Gryllus bimaculatus is a hemimetabolous insect whose nymphs posses the ability to regenerate amputated legs. Previously, we showed that Gryllus orthologues of Drosophila hedgehog (Gb'hh), wingless (Gb'wg) and decapentaplegic (Gb'dpp) are expressed during leg regeneration and play essential roles in the establishment of the proximal-distal axis. Here, we examined their roles during intercalary regeneration: when a distally amputated tibia with disparate positional values is placed next to a proximally amputated host, intercalary growth occurs in order to regenerate the missing part. In this process, we examined expression patterns of Gb'hh and Gb'wg. We found that expressions of Gb'hh and Gb'wg were induced in a regenerate and the host proximal to the amputated region, but not in the grafted donor distal to the regenerate. This directional induction occurs even in the reversed intercalation. Because these results are consistent with a distal-to-proximal respecification of the regenerate, Gb'wg may be involved in the re-establishment of the positional values in the regenerate. Furthermore, we found that no regeneration occurs when Gb'armadillo (the orthologue of beta-catenin) was knocked down by RNA interference. These results indicate that the canonical Wnt/Wingless signaling pathway is involved in the process of leg regeneration and determination of positional information in the leg segment.

DOI： 10.1111/j.1440-169x.2007.00915.x

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Follistatin-related gene (FLRG) encodes a secretory glycoprotein that has characteristic cysteine-rich follistatin domains. FLRG protein binds to and neutralizes several transforming growth factor-β (TGF-β) superfamily members, including myostatin (MSTN), which is a potent negative regulator of skeletal muscle mass. We have previously reported that FLRG was abundantly expressed in fetal and adult mouse heart. In this study, we analyzed the expression of FLRG mRNA during mouse heart development. FLRG mRNA was continuously expressed in the embryonic heart, whereas it was very low in skeletal muscles. By contrast, MSTN mRNA was highly expressed in embryonic skeletal muscles, whereas the expression of MSTN mRNA was rather low in the heart. In situ hybridization and immunohistochemical analysis revealed that FLRG expressed in smooth muscle of the aorta and pulmonary artery, valve leaflets of mitral and tricuspid valves, and cardiac muscles in the ventricle of mouse embryonic heart. However, MSTN was expressed in very limited areas, such as valve leaflets of pulmonary and aortic valves, the top of the ventricular and atrial septa. Interestingly, the expression of MSTN was complementary to that of FLRG, especially in the valvular apparatus. Biochemical analyses with surface plasmon resonance biosensor and reporter assays demonstrated that FLRG hardly dissociates from MSTN and activin once it bound to them, and efficiently inhibits these activities. Our results suggest that FLRG could function as a negative regulator of activin family members including MSTN during heart development.

DOI： 10.2152/jmi.54.276

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In the long-germband insect Drosophila, all body segments and posterior terminal structures, including the posterior gut and anal pads, are specified at the blastoderm stage. In short- and intermediate-germband insects, however, posterior segments are sequentially produced from the posterior growth zone, a process resembling somitogenesis in vertebrates, and invagination of the posterior gut starts after anteroposterior (AP) axial elongation from the growth zone. The mechanisms underlying posterior segmentation and terminal patterning in these insects are poorly understood. In order to elucidate these mechanisms, we have investigated the roles of the Brachyurylbrachyenteron (Bra/byn) homolog in the intermediate-germ band cricket Gryllus bimaculatus. Loss-of-function analysis by RNA interference (RNAi) revealed that Gryllus byn (Gb'byn) is not required for AP axial elongation or normal segment formation, but is required for specification of the posterior gut. We also analyzed Gryllus caudal (Gb'cad) RNAi embryos using in situ hybridization with a Gb'byn probe, and found that Gb'cad is required for internalization of the posterior gut primordium, in addition to AP axial elongation. These results suggest that the functions of byn and cad in posterior terminal patterning are highly conserved in Gryllus and Drosophila despite their divergent posterior patterning. Moreover, because it is thought that the progressive growth of the AP axis from the growth zone, controlled by a genetic program involving Cdx/cad and Bra/byn, might be ancestral to bilaterians, our data suggest that the function of Bra/byn in this process might have been lost in insects.

DOI： 10.1242/dev.02646

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In Drosophila, a long germ insect, segmentation occurs simultaneously across the entire body. In contrast, in short and intermediate germ insects, the anterior segments are specified during the blastoderm stage, while the remaining posterior segments are specified during later stages. In Drosophila embryos, the transcriptional factors coded by gap genes, such as Krüppel, diffuse in the syncytial environment and regulate the expression of other gap, pair-rule, and Hox genes. To understand the segmentation mechanisms in short and intermediate germ insects, we investigated the role of Kr ortholog (Gb'Kr) in the development of the intermediate germ insect Gryllus bimaculatus. We found that Gb'Kr is expressed in a gap pattern in the prospective thoracic region after cellularization of the embryo. To determine the function of Gb'Kr in segmentation, we analyzed knockdown phenotypes using RNA interference (RNAi). Gb'Kr RNAi depletion resulted in a gap phenotype in which the posterior of the first thoracic through seventh abdominal segments were deleted. Analysis of the expression patterns of Hox genes in Gb'Kr RNAi embryos indicated that regulatory relationships between Hox genes and Kr in Gryllus differ from those in Oncopeltus, another intermediate germ insect. Furthermore, we found that Gb'Kr regulates expression minimally of hunchback and even-skipped, directly or indirectly, in the prospective thoracic region. Our findings suggest that Gb'Kr is a gap gene that acts in the cellular environment and is required for segmentation in the thoracic and abdominal regions through the regulation of gap and pair-rule gene expression. © 2006 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.ydbio.2005.12.057

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL PUBLISHING  

Mutant mice deficient in the fibroblast growth factor 10 (Fgf10) gene exhibit an eye-open phenotype at birth. It has previously been shown that FGF10 has a dual role in proliferation and migration during the early and later stages of eyelid development, respectively. To verify the role of FGF10 during eyelid closure, explant culture of Fgf10-null eyelid anlagen was performed, by which it was examined whether or not exogenous FGF10 could rescue the expression of activin beta B and transforming growth factor alpha, known to be required for eyelid closure. We found that the expression of these genes was markedly induced while that of Shh or Ptch1, Ptch2 was not. We also observed the distribution of filamentous actin (F-actin) after FGF10 application in the mutant eyelid explant, finding that the FGF10 protein induced F-actin accumulation. We further examined filopodia of the eyelid leading edge cells, finding the length of the filopodia was significantly reduced in the mutant. These results verify that FGF10 promotes eyelid closure through activating activin and TGF alpha-EGFR signaling.

DOI： 10.1111/j.1440-169x.2006.00869.x

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Mouse, rat and human molars begin to form root after the completion of crown formation. In these teeth, fibroblast growth factor (Fgf) 10 disappears in the transitional stage from crown formation to root. By contrast, rodent incisors and vole molars demonstrate continuous growth, owing to the formation and maintenance of a stem cell compartment by the constant expression of Fgf10. To clarify the relationship between root formation and disappearance of Fgf10, we carried out two experiments for the loss and gain of Fgf10 function. First, we examined postnatal growth in the incisors of Fgf10-deficient mice, which have the defect of a dental epithelial stem cell compartment referred to as 'apical bud', after implantation under the kidney capsule. The growth at the labial side in the mutant mice mimics the development of limited-growth teeth. 5'-Bromo-2'-deoxyuridine (BrdU) labeling and cytokeratin (CK) 14 and Notch2 immunostaining suggested that the inhibition of inner enamel epithelium growth and the more-active proliferation of the outer enamel epithelium and/or stellate reticulum result in Hertwig's epithelial root sheath formation. Second, we examined the effects of Fgf10 overexpression in the transitional stage of molar germs, which led to the formation of apical bud involving in the inhibition of HERS formation. Taken together, these results suggest that the disappearance of Fgf10 signaling leads to the transition from crown to root formation, owing to the loss of a dental epithelial stem cell compartment.

DOI： 10.1242/dev.02307

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-LISS  

Vertebrate melanopsin is a photopigment in the eye, required for photoentrainment. Melanopsin is more closely related to opsin proteins found in invertebrates, than to the other photo-pigments. Although the invertebrate melanopsin-like protein is localized in rhabdomeric photoreceptors in the invertebrate eye, it has been shown to be expressed in a subset of retinal ganglion cells in the mouse and in horizontal cells in the frog, indicating its diversified expression pattern in vertebrates. Here we show that two types of melanopsin transcripts are expressed in the developing chicken retina. Melanopsin is firstly expressed by a small subset of ganglion cells, and then prominently expressed by horizontal cells and later by bipolar cells in the developing chicken retina. This suggests that a subset of ganglion, horizontal, and bipolar cells in the chicken retina may have rhabdomeric properties in their origins.

DOI： 10.1002/dvdy.20600

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Fibroblast growth factor (FGF) signaling is crucial for the induction and growth of the ear, a sensory organ that involves intimate tissue interactions. Here, we report the abnormality of Fgf10 null ear and the identification of a cis-regulatory element directing otic expression of Fgf10. In Fgf10 null inner ears, we found that the initial development of semicircular, vestibular, and cochlear divisions is roughly normal, after which there are abnormalities of semicircular canal/cristae and vestibular development. The mutant semicircular disks remain without canal formation by the perinatal stage. To elucidate regulation of the Fgf10 expression during inner ear development, we isolated a 6.6-kb fragment of its 5'-upstream region and examined its transcriptional activity with transgenic mice, using a lacZ-reporter system. From comparison of the mouse sequences of the 6.6-kb fragment with corresponding sequences of the human and chicken Fgf10, we identified a 0.4-kb enhancer sequence that drives Fgf10 expression in the developing inner ear. The enhancer sequences have motifs for many homeodomain-containing proteins (e.g., Prx, Hox, Nkx), in addition to POU-domain factors (e.g., Brn3), zinc-finger transcription factors (e.g., GATA-binding factors), TCF/LEF-1, and a SALAD-interacting protein. Thus, FGF10 signaling is dispensable for specification of otic compartment identity but is required for hollowing the semicircular disk. Furthermore, the analysis of a putative inner ear enhancer of Fgf10 has disclosed a complicated regulation of Fgf10 during inner ear development by numerous transcription factors and signaling pathways. (c) 2005 Wiley-Liss, Inc.

DOI： 10.1002/dvdy.20319

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In short and intermediate germ insects, only the anterior segments are specified during the blastoderm stage, leaving the posterior segments to be specified later, during embryogenesis, which differs from the segmentation process in Drosophila, long germ insect. To elucidate the segmentation mechanisms of short and intermediate germ insects, we have investigated the orthologs of the Drosophila segmentation genes in a phylogenetically basal, intermediate germ insect, Gryllus bimaculatus (Gb). Here, we have focused on its hunchback ortholog (Gb'hb), because Drosophila hb functions as a gap gene during anterior segmentation, referred as a canonical function. Gb'hb is expressed in a gap pattern during the early stages of embryogenesis, and later in the posterior growth zone. By means of embryonic and parental RNA interference for Gb'hb, we found the following: (1) Gb'hb regulates Hox gene expression to specify regional identity in the anterior region, as observed in Drosophila and Oncopeltus
(2) Gb'hb controls germband morphogenesis and segmentation of the anterior region, probably through the pair-rule gene, even-skipped at least
(3) Gb'hb may act as a gap gene in a limited region between the posterior of the prothoracic segment and the anterior of the mesothoracic segment
and (4) Gb'hb is involved in the formation of at least seven abdominal segments, probably through its expression in the posterior growth zone, which is not conserved in Drosophila. These findings suggest that Gb'hb functions in a non-canonical manner in segment patterning. A comparison of our results with the results for other derived species revealed that the canonical hb function may have evolved from the non-canonical hb functions during evolution.

DOI： 10.1242/dev.01784

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL PUBLISHING ASIA  

The fibroblast growth factor (FGF) family is known to be involved in vertebrate eye development. However, distinct roles of individual FGF members during eye development remain largely elusive. Here, we show a detailed expression pattern of Fgf19 in chick lens development. Fgf19 expression initiated in the forebrain, and then became restricted to the distal portion of the optic vesicle abutting the future lens placode, where FGF receptor 4 (Fgfr4), a receptor for FGF19, was expressed. Fgf8, a positive regulator for L-Maf, was expressed in a portion of the optic vesicle. To examine the role of FGF19 signaling during early eye development, Fgf19 was misexpressed near the presumptive lens ectoderm; however, no alteration in the expression of lens marker genes was observed. Conversely, a secreted form of FGFR4 was misexpressed to inhibit an FGF19 signal, resulting in the induction of L-Maf expression. To further define the relationship between L-Maf and Fgf19, L-Maf misexpression was performed, resulting in ectopic induction of Fgf19 expression by Hamburger and Hamilton's stage 12/13. Furthermore, misexpression of Fgf8 induced Fgf19 expression in addition to L-Maf. These results suggest that FGF19-FGFR4 signaling plays a role in early lens development in collaboration with FGF8 signaling and L-Maf transcriptional system.

DOI： 10.1111/j.1440-169X.2005.00795.x

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We have studied embryogenesis of the two-spotted cricket Gryllus bimaculatus as an example of a hemimetabolous, intermediate germ insect, which is a phylogenetically basal insect and may retain primitive features. We observed expression patterns of the orthologs of the Drosophila homeotic genes, Sex combs reduced (Scr), Antennapedia (Antp), Ultrabithorax (Ubx) and abdominal-A (abd-A) during embryogenesis and compared the expression patterns of these genes with the more basal thysanuran insect, Thermobia domestica (the firebrat), and the derived higher dipteran insect, Drosophila melanogaster. Although Scr is expressed commonly in the presumptive posterior maxillary and labial segment in all three insects, the thoracic expression domains vary. Antp is expressed similarly in the three thoracic segments, the limbs, and the anterior abdominal region among these three insects. The early Antp expression in the firebrat and cricket obeys a segmental register in all three thoracic segments, while in Drosophila its initial expression appears in parasegments 4 and 6. Ubx is expressed in the metathoracic (T3) and abdominal segments similarly in the three insects, whereas the expression pattern in the T3 leg differs among them. abd-A is expressed in the posterior compartment of the first abdominal segment and the remaining abdominal segments in all three insects, although its posterior border varies among them. © 2004 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.modgep.2004.12.006

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Early embryogenesis of the two-spotted cricket Gryllus bimaculatus was examined by scanning electron microscopy and several fluorescence staining methods, with special reference to these four issues: (i) the location of micropyles
(ii) the transfer of the female pronucleus following meiosis
(iii) the timing of cellularization
and (iv) the process of the germ primordium formation. Between two and four micropyles lie in the mid-ventral region of the egg. The egg nucleus is at the mid-dorsal periphery of the new laid egg, and meiosis resumes and is completed there. The female pronucleus moves to the mid-ventral side, and fertilization occurs there. Energid starts to proliferate and migrates to the periphery of the egg, initiating blastoderm formation. Actin caps surround each superficial nucleus. Cellularization occurs during the blastoderm stage. At a late blastoderm stage, nuclei aggregate in both the posterolateral patch-like regions of the egg to form a germ primordium. The germ primordium looks like a pair of dumbbells. Both the patches shift towards the ventral side and fuse into a germ primordium. The germ primordium contracts to produce a clearly delineated germ band. Observations on distribution patterns of F-actin indicate that, all through the process, the germ primordium retains that unity, and is not separated into two parts.

DOI： 10.1111/j.1440-169x.2005.00786.x

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Although the molecular mechanisms directing anteroposterior patterning of the Drosophila embryo (long-germband mode) are well understood, how these mechanisms were evolved from an ancestral mode of insect embryogenesis remains largely unknown. In order to gain insight into mechanisms of evolution in insect embryogenesis, we have examined the expression and function of the orthologue of Drosophila caudal (cad) in the intermediate-germband cricket Gryllus bimaculatus. We observed that a posterior (high) to anterior (low) gradient in the levels of Gryllus bimaculatus cad (Gb′ cad) transcript was formed in the early-stage embryo, and then Gb′ cad was expressed in the posterior growth zone until the posterior segmentation was completed. Reduction of Gb′ cad expression level by RNA interference resulted in deletion of the gnathum, thorax, and abdomen in embryos, remaining only anterior head. We found that the gnathal and thoracic segments are formed by Gb′ cad probably through the transcriptional regulation of gap genes including Gb′ hunchback and Gb′ Krüppel. Furthermore, Gb′ cad was found to be involved in the posterior elongation, acting as a downstream gene in the Wingless/Armadillo signalling pathways. These findings indicate that Gb′ cad does not function as it does in Drosophila, suggesting that regulatory and functional changes of cad occurred during insect evolution. Since Wnt/Cdx pathways are involved in the posterior patterning of vertebrates, such mechanisms may be conserved in animals that undergo sequential segmentation from the posterior growth zone. © 2004 Elsevier Ireland Ltd. All rights reserved.

DOI： 10.1016/j.mod.2004.10.001

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We report that Gryllus bimaculatus dachshund (Gbdac), a cricket homologue of Drosophila dachshund (Dmdac), is expressed in the developing eye and brain. During brain development, Gbdac was first expressed in the medial head region, corresponding to a part of developing protocephalic region, and expressed in the primordial and adult Kenyon cells. During eye development, Gbdac was first expressed in the lateral head region, becoming to the eye primordium and a part of the deutocerebrum. Then, Gbdac was expressed in the posterior region of the eye primordium, prior to the formation of compound eyes. The expression domain shifted to the anterior domain concomitantly with the movement of morphogenetic furrows. Gbdac was also expressed in the developing optic lobes during differentiation of the retina. These expression patterns were compared with those of Dmdac. We found that although developmental processes of the Gryllus eye and brain differ from those of the Drosophila ones, the expression patterns of Gbdac are essentially similar to those of the Dmdac.

DOI： 10.1016/j.modgep.2004.03.010

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

The normal development of eyes relies on proper signaling through Fibroblast growth factor (FGF) receptors, but the source and identity of cognate ligands have remained largely unknown. We have found that Fgf19 is expressed in the developing chicken retina. In situ hybridization discloses dynamic expression patterns for Fgf19 in the optic vesicle, lens primordia and retinal horizontal cells. Overall expression pattern of Fgf19 during chicken embryogenesis was also examined: Fgf19 is expressed in the regions associated with cranial placodes induction, boundary regions of rhombomeres, somites, specific groups of neural cells in midbrain, hindbrain, and those derived from epibranchial placodes, and the apical ectodermal ridge of limb buds. Expression pattern of the Fgf19-orthologous gene Fgf15 was further examined in the mouse developing eye. Fgf15 is expressed in the optic vesicle, a subset of progenitor cells of neural retina, and emerging ganglion and amacrine cells during retinogenesis. (C) 2004 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.modgep.2004.04.005

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Transgenic insects have been artificially produced to study functions of interesting developmental genes, using insect transposons such as piggyBac. In the case of the cricket, however, transgenic animals have not yet been successfully artificially produced. In the present study, we examined whether the piggyBac transposon functions as a tool for gene delivery in embryos of Gryllus bimaculatus. We used either a piggyBac helper plasmid or a helper RNA synthesized in vitro as a transposase source. An excision assay revealed that the helper RNA was more effective in early Gryllus eggs to transpose a marker gene of eGFP than the helper plasmid containing the piggyBac transposase gene driven by the G. bimaculatus actin3/4 promoter. Further, only when the helper RNA was used, somatic transformation of the embryo with the eGFP gene was observed. These results suggest that the piggyBac system with the helper RNA may be effective for making transgenic crickets.

DOI： 10.1111/j.1440-169x.2004.00751.x

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In insects, there are two different modes of segmentation. In the higher dipteran insects (like Drosophila), their segmentation takes place almost simultaneously in the syncytial blastoderm. By contrast, in the orthopteran insects (like Schistocerca (grasshopper)), the anterior segments form almost simultaneously in the cellular blastoderm and then the remaining posterior part elongates to form segments sequentially from the posterior proliferative zone. Although most of their orthologues of the Drosophila segmentation genes may be involved in their segmentation, little is known about their roles. We have investigated segmentation processes of Gryllus bimaculatus, focusing on its orthologues of the Drosophila segment-polarity genes, G. bimaculatus wingless (Gbwg), armadillo (Gbarm) and hedgehog (Gbhh). Gbhh and Gbwg were observed to be expressed in the each anterior segment and the posterior proliferative zone. In order to know their roles, we used RNA interference (RNAi). We could not observed any significant effects of RNAi for Gbwg and Gbhh on segmentation, probably due to functional replacement by another member of the corresponding gene families. Embryos obtained by RNAi for Gbarm exhibited abnormal anterior segments and lack of the abdomen. Our results suggest that GbWg/GbArm signaling is involved in the posterior sequential segmentation in the G. bimaculatus embryos, while Gbwg, Gbarm and Gbhh are likely to act as the segment-polarity genes in the anterior segmentation similarly as in Drosophila. © 2004 Elsevier Ireland Ltd. All rights reserved.

DOI： 10.1016/j.mod.2004.01.002

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In insects, egg activation is known to occur in vivo and independently of fertilization, but its mechanisms are poorly understood. To gain understanding of these mechanisms, an attempt was made to activate the egg of Gryllus bimaculatus in vitro. It was found that meiosis resumed and was completed in unfertilized eggs treated with hypotonic buffer. Early developmental processes in activated, unfertilized eggs were investigated and compared with those in fertilized eggs. Mitosis did not progress, resulting in formation of anucleate cytoplasmic islands (pseudoenergids). Development in the activated, unfertilized eggs stopped at this stage and both yolk subdivision and cellularization did not occur. To elucidate the role of the nucleus in the developmental process to the syncytial stage in fertilized eggs, eggs were treated with aphidicolin to inhibit DNA polymerization. It was found that pseudoenergids also formed in these aphidicolin-treated fertilized eggs. These results demonstrate that pseudoenergids can increase in number independently of nuclei, suggesting that the cytoplasm rather than the nucleus plays the primary role in development to the syncytial stage in G. bimaculatus.

DOI： 10.1034/j.1600-0854.2004.00679.x

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Fibroblast Growth Factor (FGF) signaling is known to play an important role during cutaneous development. To elucidate the role of FGF10 during whisker formation, we examined the expression of Fgf10 in normal developing whiskers and phenotypes of Fgf10-deficient whiskers. Fgf10 is first expressed in the maxillary process, lateral and medial nasal processes, then in the mesenchymal cells underneath the future whisker placodes, and in the surrounding mesenchyme of developing whiskers. Fgf10-null whiskers exhibit a significant decrease in number and their structure is disorganized as revealed by scanning electron microscopy. Hair follicle marker genes such as Sonic hedgehog, Patched, and Patched 2 are aberrantly expressed in the mutant whiskers. Thus, FGF10 is required for proper whisker development mediated by SHH signaling in the mouse. (C) 2003 Elsevier Science (USA). All rights reserved.

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DOI： 10.1046/j.1440-169X.2002.00654.x

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DOI： 10.1016/S0925-4773(02)00052-7

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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.

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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.

DOI： 10.1016/S0925-4773(02)00020-5

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Mouse incisors are regenerative tissues that grow continuously throughout life. The renewal of dental epithelium-producing enamel matrix and/or induction of dentin formation by mesenchymal cells is performed by stem cells that reside in cervical loop of the incisor apex. However, little is known about the mechanisms of stem cell compartment formation. Recently, a mouse incisor was used as a model to show that fibroblast growth factor (FGF) 10 regulates mitogenesis and fate decision of adult stem cells. To further illustrate the role of FGF10 in the formation of the stem cell compartment during tooth organogenesis, we have analyzed incisor development in Fgf10-deficient mice and have examined the effects of neutralizing anti-FGF10 antibody on the developing incisors in organ cultures. The incisor germs of FGF10-null mice proceeded to cap stage normally. However, at a later stage, the cervical loop was not formed. We found that the absence of the cervical loop was due to a divergence in Fgf10 and Fgf3 expression patterns at E16. Furthermore, we estimated the growth of dental epithelium from incisor explants of FGF10-null mice by organ culture. The dental epithelium of FGF10-null mice showed limited growth, although the epithelium of wild-type mice appeared to grow normally. In other experiments, a functional disorder of FGF10, caused by a neutralizing anti-FGF10 antibody, induced apoptosis in the cervical loop of developing mouse incisor cultures. However, recombinant human FGF10 protein rescued the cervical loop from apoptosis. Taken together, these results suggest that FGF10 is a survival factor that maintains the stem cell population in developing incisor germs.

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Mouse incisors have a cervical loop that gives rise to dental epithelium in the apical region of the tooth germ, in contrast to molars. In a study of formation of the stem-cell compartment, we focused on expression patterns of fibroblast growth factor (Fgf) 10 and Fgf3 in developing mice incisors. At E14, Fgf10 and Fgf3 were coexpressed in the dental papilla. After E16 mesenchymal cells underlying cervical loop expressed Fgf10 but not Fgf3. To illustrate the role of FGF10, we analyzed incisor development of Fgf10-deficient mice. The germs of FGF10-null mice proceeded to cap stage normally. However, at a later stage, the cervical loop was not formed. Functional disorder of FGF10 by a neutralizing anti-FGF10 antibody induced apoptosis in the cervical loop of incisor explants. Recombinant FGF10 rescued the cervical loop from apoptosis. These results show that FGF10 maintains the stem-cell compartment in the developing incisor tooth germ.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：U B C PRESS  

Fibroblast growth factor 10 (FGF10) is known to be expressed in limb mesenchymal cells and to function as a mesenchymal signaling factor involved in epithelial-mesenchymal interactions during limb development. To elucidate regulation of Fgf10 expression, we isolated the promoter region of Fgf10 containing its 2.0 kb upstream 5'-fragment from the initiation codon and its 0.9 kb downstream fragment. Transcriptional activity of the fragment was examined with transgenic mice, using a lacZ-reporter system. Although no significant expression of the reporter gene was observed for the 0.2 kb 5'-fragment, expression was detected in the apical ectodermal ridge of the limb bud and developing cartilage of the limb for the 2.0 kb and 0.7 kb 5'-fragments, respectively. From comparison of the mouse sequences of the 2.0 kb fragment with corresponding sequences of human and chicken Fgf10, we identified 17 conserved putative enhancer motifs for AER expression and other unidentified expressions. For limb cartilage expression, we found putative enhancer sequences conserved among the three species in the 0.7 kb 5'-fragment. In the fragment, three DNA binding motifs were identified in the mouse and human sequence, although they are not conserved in the corresponding chicken sequence.

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Myostatin, which is a member of the TGF-β superfamily, is a negative regulator of skeletal muscle formation. Double-muscled Piedmontese cattle have a C313Y mutation in myostatin and show increased skeletal muscle mass which resulted from an increase of myofiber number (hyperplasia) without that of myofiber size (hypertrophy). To examine whether this mutation in myostatin gene affects muscle development in a dominant negative manner, we generated transgenic mice overexpressing the mutated gene. The transgenic mice exhibited dramatic increases in the skeletal muscle mass resulting from hyperplasia without hypertrophy. In contrast, it has been reported that a myostatin mutated at its cleavage site produces hypertrophy without hyperplasia in the muscle. Thus, these results suggest that (1) the myostatin containing the missense mutation exhibits a dominant negative activity and that (2) there are two types in the dominant negative form of myostatin, causing either hypertrophy or hyperplasia. © 2002 Elsevier Science (USA). All rights reserved.

DOI： 10.1016/S0006-291X(02)00209-7

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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. © 2002 Elsevier Science Ireland Ltd. All rights reserved.

DOI： 10.1016/S0925-4773(01)00584-6

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Expression of four BMP antagonist genes, noggin, chordin, gremlin and Follistatin, was examined during chick feather development. Although expression of noggin and chordin was not detected, gremlin and Follistatin were expressed differentially in feather buds. The differential expression patterns of gremlin and Follistatin change dynamically from the nascent inter-feather bud region to the posterior domain of the feather bud. Copyright © 2001 Elsevier Science Ireland Ltd.

DOI： 10.1016/S0925-4773(00)00525-6

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

We have isolated a chick cDNA for p63, a member of the p53 transcription factor family. This cDNA encodes a protein of 582 amino acids for an α isoform in the C-terminal region, while lacking the N-terminal transactivation domain. The chick p63 gene is first expressed in the prospective cutaneous ectoderm at stage 6 and later in the developing epithelia. The p63 expression is intense in specialized epithelial structures, such as apical ectodermal ridge of the limb bud, epithelia of branchial arches and feather buds. Furthermore, we have found that the transcripts are detected in the interdigital epithelium, intersomite epithelium, and epaxial dermamyotome. (C) 2001 Elsevier Science Ireland Ltd.

DOI： 10.1016/S0925-4773(00)00504-9

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Insects can be grouped into mainly two categories, holometabolous and hemimetabolous, according to the extent of their morphological change during metamorphosis. The three thoracic legs, for example, are known to develop through two overtly different pathways: holometabolous insects make legs through their imaginal discs, while hemimetabolous legs develop from their leg buds. Thus, how the molecular mechanisms of leg development differ from each other is an intriguing question. In the holometabolous long-germ insect, these mechanisms have been extensively studied using Drosophila melanogaster, However, little is known about the mechanism in the hemimetabolous insect. Thus, we studied leg development of the hemimetabolous short-germ insect, Gryllus bimaculatus (cricket), focusing on expression patterns of the three key signaling molecules, hedgehog (hh), wingless (wg) and decapentaplegic (dpp), which are essential during leg development in Drosophila, In Gryllus embryos, expression of hh is restricted in the posterior half of each leg bud, while dpp and wg are expressed in the dorsal and ventral sides of its anteroposterior (A/P) boundary, respectively. Their expression patterns are essentially comparable with those of the three genes in Drosophila leg imaginal discs, suggesting the existence of the common mechanism for leg pattern formation. However, we found that expression pattern of dpp was significantly divergent among Glyllus, Schistocerca (grasshopper) and Drosophila embryos, while expression patterns of hh and wg are conserved. Furthermore, the divergence was found between the pro/mesothoracic and metathoracic Gryllus leg buds, These observations imply that the divergence in the dpp expression pattern may correlate with diversity of leg morphology.

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To elucidate roles of fibroblast growth factors (FGF)18 during vertebrate development, we examined expression patterns of Fgf18 in chick embryos and observed effects of FGF18 protein on the Hensen's node, isthmus, and limb buds. Fgf18 is expressed on the right side of the node before the expression of Fgf8 starts. FGF18 protein can induce expression of Fgf8 on the left side of the node, indicating involvement of both FGFs in specification of left-right asymmetry. In the developing brain, Fgf18 is expressed in the isthmus, following the Fgf8 expression. Since Fgf18 is induced ectopically during formation of the second midbrain by FGF8 protein, both FGFs also elaborate midbrain development. In the limb bud, Fgf18 is expressed in the mesenchyme and ectopic application of FGF18 protein inhibits bone growth in the limb. FGF18 is thus likely an endogenous ligand of FGF receptor 3, whose mutation causes bone dysplasia in humans. These results demonstrate that the FGF18-FGF8 signaling is involved in various organizing activities and the signaling hierarchies between FGF18 and FGF8 seem to change during patterning of different structures. Copyright (C) 2000 Elsevier Science Ireland Ltd.

DOI： 10.1016/S0925-4773(00)00331-2

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Pitx2, a bicoid-related homeobox gene, is involved in Rieger's syndrome and the left-right (L-R) asymmetrical pattern formation in body plan. In order to define the genomic structure and roles of Pitx2, we analyzed the genomic structure and generated Pitx2-deficient mice with the lacZ gene in the homeobox-containing exon of Pitx2. We were able to show that among three isoforms of Pitx2, Pitx2c shows asymmetrical expression whereas Pitx2a, Pitx2b and Pitx2c show symmetrical expression. In Pitx2(-/-) embryos there was an increase in mesodermal cells in the distal end of the left lateral body wall and an amnion continuous with the lateral body wall thickened in its mesodermal layer. These changes resulted in a failure of ventral body wall closure. In lung and heart in which Pitx2 is expressed asymmetrically, right pulmonary isomerism, atrioventricular canals with prominent swelling, and juxtaposition of the atrium were detected. The hearts failed to develop tricuspid and mitral valves and a common atrioventricular valve forms. Further, dysgenesis of the Pitx2(-/-) extraocular muscle and thickening of the mesothelial layer of cornea were observed in the ocular system where Pitx2 is expressed symmetrically, and these resulted in enophthalmos. The present study shows that Pitx2 expressed in various sites participates in morphogenesis through three types of actions: the involvement of asymmetric Pitx2 expression in the entire morphogenetic process of L-R asymmetric organs
the involvement of asymmetric Pitx2 expression in the regional morphogenesis of asymmetric organs
and finally the involvement of symmetric Pitx2 expression in the regional morphogenesis of symmetric organs.

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL SCIENCE ASIA  

The limb bud has a thickened epithelium at the dorsal-ventral boundary, the apical ectodermal ridge (AER), which sustains limb outgrowth and patterning. A secreted molecule fibroblast growth factor (FGF)10 is involved in inducing Fgf8 expression in the prospective AER and mutual interaction between mesenchymal FGF10 and FGF8 in the AER is essential for limb outgrowth. A secreted factor Wnt7a and a homeobox protein Lmx1 are involved in the dorsal patterning of the limb, whereas a homeobox protein Engrailed 1 (En1) is involved in the dorsal-ventral patterning as well as AER formation. Radical fringe (R-fng), a vertebrate homolog of Drosophila fringe was also found to elaborate AER formation in chicks. However, little is known about the molecular interactions between these factors during AER formation. The present study clarified the relationship between FGF10, Wnt7a, Lmx1, R-fng and En1 during limb development using a foil-barrier insertion experiment. It was found that a foil-barrier inserted into the chick prospective wing mesenchyme lateral to the mesonephric duct blocks AER induction. This experiment was expanded by implanting Fgf10-expressing cells lateral to the barrier and examined whether FGF10 could rescue the expression of the limb-patterning genes reported in AER formation. It was found that FGF10 is sufficient to induce Fgf8 expression in the ectoderm of the foil-inserted limb bud, concomitantly with R-fng and En1 expression. However, FGF10 could not rescue the expression of the dorsal marker genes, Wnt7a or Lmx1. Thus, it is suggested that epithelial factors of En1 and R-fng can induce Fgf8 expression in the limb ectoderm in cooperation with a mesenchymal factor FGF10. Some factor(s) other than FGF10, possibly from the paraxial structures medial to the limb mesoderm, is responsible for the initial dorsal-ventral specification of the limb bud.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Academic Press Inc.  

During vertebrate limb development, the apical ectodermal ridge (AER) plays a vital role in both limb initiation and distal outgrowth of the limb bud. In the early chick embryo the prelimb bud mesoderm induces the AER in the overlying ectoderm. However, the direct inducer of the AER remains unknown. Here we report that FGF7 and FGF10, members of the fibroblast growth factor family, are the best candidates for the direct inducer of the AER. FGF7 induces an ectopic AER in the flank ectoderm of the chick embryo in a different manner from FGF1, -2, and -4 and activates the expression of Fgf8, an AER marker gene, in a cultured flank ectoderm without the mesoderm. Remarkably, FGF7 and FGF10 applied in the back induced an ectopic AER in the dorsal median ectoderm. Our results suggest that FGF7 and FGF10 directly induce the AER in the ectoderm both of the flank and of the dorsal midline and that these two regions have the competence for AER induction. Formation of the AER of the dorsal median ectoderm in the chick embryo is likely to appear as a vestige of the dorsal fin of the ancestors.

DOI： 10.1006/dbio.1999.9290

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

We have isolated chick rax/rx cDNAs, cRaxL (chick Rax/Rx-like) and cRax, (chick Rax) and examined their expression patterns during early eye and brain development. The cRaxL cDNA encodes a 225 amino acid protein that is most closely related to the zebrafish Rx1 and Rx2. The cRax cDNA encodes a 317 amino acid protein, which shares higher homology with the Xenopus Rx. In addition to the home- odomain, the octapeptide and paired tail domains are conserved between the cRax and other vertebrate Rax/Rx, while cRaxL lacks the octapeptide containing N-terminal region which is conserved among all other members of the rax/rx gene family identified so far. The chick rax/rx genes are expressed in overlapping domains in the anterior neural ectoderm which corresponds to the forebrain and retina field, and later in the optic vesicle. cRax mRNA can be detected earlier than cRaxL prior to the formation of the notochord and its expression domain appears broader than that of cRaxL. (C) 1999 Elsevier Science Ireland Ltd. All rights reserved.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Academic Press Inc.  

FGF-10 is a mesenchymal factor affecting epithelial cells during pattern formation. However, the expression and physiological role of FGF-10 in adults remains to be elucidated. We examined the expression of FGF-10 mRNA in a variety of adult rat tissues, and found to be most abundant in white adipose tissue. In white adipose tissue, FGF-10 mRNA was expressed in preadipocytes but not in mature adipocytes. The expression in white adipose tissue during postnatal development was also examined. The expression level was low at postnatal day 10 (P10). However, FGF-10 mRNA was abundantly detected later on (P28 and P48) when white adipose tissue growth was stimulated. We also examined the activity of recombinant FGF-10 for primary rat preadipocytes. FGF-10 showed significant mitogenic activity for primary preadipocytes, but did not affect the differentiation of preadipocytes. The expression profile of FGF-10 mRNA and the activity of FGF-10 reported here indicate that FGF-10, a unique secreted factor produced in white adipose tissue, acts as a growth factor for preadipocytes in white adipose tissues.

DOI： 10.1006/bbrc.1999.0594

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The mes-metencephalic boundary (isthmus) has been suggested to act as an organizer in the development of the optic tectum. Pax-5 was cloned as a candidate for regulator of the organizing center. Isthmus-specific expression of Pax-5 and analogy with the genetic cascade in Drosophila suggest that Pax- 5 may be at a higher hierarchical position in the gene regulation cascade of tectum development. To examine this possibility, a gain-of-function experiment on Pax-5 was carried out. In ovo electroporation on E2 chick brain with the eucaryotic expression vector that encodes chick Pax-5 cDNA was used. Not only was a considerable amount of Pax-5 expressed ectopically in the transfected brain, but irregular bulging of the neuroepithelium was induced in the diencephalon and mesencephalon. At Pax-5 misexpressing sites, uptake of BrdU was increased. Histological examination of E7 transfected brain revealed that Pax-5 caused transdifferentiation of diencephalon into the tectum-like structure. In the bulges of the E7 mesencephalon, differentiation of laminar structure was repressed when compared to the normal side. In transfected embryos, En-2, Wnt-1 and Fgf8 were up-regulated ectopically, and Otx2 was down-regulated in the diencephalon to mesencephalon. Moreover, Ephrin-A2, which is expressed specifically in the tectum with a gradient highest at the caudal end, is suggested to be involved in pathfinding of the retinal fibers, and was induced in the bulges. When the mouse Fgf8 expression vector was electroporated, Pax-5 and chick Fgf8 were also induced ectopically. These results suggest that Pax-5, together with Fgf8, hold a higher position in the genetic hierarchy of the isthmus organizing center and regulate its activity.

DOI： 10.1046/j.1440-169x.1999.00401.x

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DOI： 10.1016/S0167-4781(98)00255-3

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Wiley-Liss Inc.  

FGF 10 and FGF8, which are reciprocally expressed by the mesoderm and AER of the developing limb bud, have been implicated in limb initiation, outgrowth, and patterning. FGF 10 and FGF8 signal through the FGFR2b and FGFR2c alternative splice isoforms, respectively [Ornitz DM, et al. 1996. J Biol Chem 271:15292-15297
Igarashi M, et al. 1998. J Biol Chem 273:13230- 13235]. A paracrine signaling loop model has been proposed whereby FGF 10 expressed by limb mesoderm signals via ectodermally restricted FGFR2b to regulate FGF8 expression by the apical ectoderm
in turn, FGF8 signals via mesodermally restricted FGFR2c to maintain FGF 10 expression [Ohuchi H, et al. 1997. Development 124:2235-2244
Xu X, et al. 1998. Development 125:753- 765]. To explore this model, we have examined FGFR2b and FGFR2c mRNA expression, using isoform-specific probes during the early stages of development of the chick limb when limb initiation, AER induction, and outgrowth are occurring. We have found that FGFR2b is expressed by limb ectoderm, including the AER, consistent with paracrine signaling of FGF 10. By contrast, FGFR2c is expressed by both mesoderm and ectoderm, indicating that FGF8 has the potential to function in an autocrine as well as paracrine fashion. Indeed, as the limb grows out in response to the AER, FGFR2c expression attenuates in the mesoderm of the progress zone, but is maintained in the AER itself, arguing against exclusive paracrine signaling of FGF8 during limb outgrowth. We also report that transcripts for FGF 10, FGFR2b, and FGFR2c are expressed normally in the limb buds of limbless mutant embryos, which fail to form an AER and do not express FGF8. Furthermore, we detect no mutations in exons specific for the FGFR2c or FGFR2b isoforms in limbless embryos. Since gene targeting has shown that expression of FGF8 in limb ectoderm depends on FGF 10 [Min H, et al. 1998. Genes Dev 12:3156-3161
Sekine K, et al. 1999. Nature Genet 21:138-141], these results indicate that the product of the limbless gene is required for FGF 10 to induce expression of FGF8.

DOI： 10.1002/(SICI)1520-6408(1999)25:4<331::AID-DVG7>3.0.CO;2-U

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Signaling molecules such as Activin, Sonic hedgehog, Nodal, Lefty, and Vg1 have been found to be involved in determination of left-right (L-R) asymmetry in the chick, mouse, or frog. However, a common signaling pathway has not yet been identified in vertebrates. We report that Pitx2, a bicoid- type homeobox gene expressed asymmetrically in the left lateral plate mesoderm, may be involved in determination of L-R asymmetry in both mouse and chick. Since Pitx2 appears to be downstream of lefty-1 in the mouse pathway, we examined whether mouse Lefty proteins could affect the expression of Pitx2 in the chick. Our results indicate that a common pathway from lefty-1 to Pitx2 likely exists for determination of L-R asymmetry in vertebrates.

DOI： 10.1016/S0092-8674(00)81473-7

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：COMPANY OF BIOLOGISTS LTD  

Mesenchyme Fork Head-1 (MFH-1) is a forkhead (also called winged helix) transcription factor defined by a common 100-amino acid DNA-binding domain, MFH-1 is expressed in non-notochordal mesoderm in the prospective trunk region and in cephalic neural-crest and cephalic mesoderm-derived mesenchymal cells in the prechordal region of early embryos, Subsequently, strong expression is localized in developing cartilaginous tissues, kidney and dorsal aortas, To investigate the developmental roles of MFH-1 during embryogenesis, mice lacking the MFH-1 locus were generated by targeted mutagenesis, MFH-1-deficient mice died embryonically and perinatally, and exhibited interrupted aortic arch and skeletal defects in the neurocranium and the vertebral column. Interruption of the aortic arch seen in the mutant mice was the same as in human congenital anomalies. These results suggest that MFH-1 has indispensable roles during the extensive remodeling of the aortic arch in neural-crest-derived cells and in skeletogenesis in cells derived from the neural crest and the mesoderm.

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Pax-6 is a member of the vertebrate Pax gene family, which is structurally related to the Drosophila pair-rule gene, paired. In mammals, Pax-6 is expressed in several discrete domains of the developing CNS and has been implicated in neural development, although its precise role remains elusive. We found a novel Small eye rat strain (rSey2) with phenotypes similar to mouse and rat Small eye. Analyses of the Pax-6 gene revealed one base (C) insertion in an exon encoding the region downstream of the paired box of the Pax-6 gene, resulting in generation of truncated protein due to the frame shift. To explore the roles of Pax-6 in neural development, we searched for abnormalities in the nervous system in rSey2 homozygous embryos. rSey2/rSey2, exhibited abnormal development of motor neurons in the hindbrain. The Islet-1-positive motor neurons were generated just ventral to the Pax-6-expressing domain both in the wild-type and mutant embryos. However, two somatic motor (SM) nerves, the abducent and hypoglossal nerves, were missing in homozygous embryos, By retrograde and anterograde labeling, we found no SM-type axonogenesis (ventrally growing) in the mutant postotic hindbrain, though branchiomotor and visceral motor (BM/VM)-type axons (dorsally growing) were observed within the neural tube. To discover whether the identity of these motor neuron subtypes was changed in the mutant, we examined expression of LIM homeobox genes, Islet-1, Islet-2 and Lim-3. At the postotic levels of the hindbrain, SM neurons expressed all the three LIM genes, whereas BM/VM-type neurons were marked by Islet-1 only. In the Pax-6 mutant hindbrain, Islet-2 expression was specifically missing, which resulted in the loss of the cells harboring the postotic hindbrain SM-type LIM code (Islet-1 + Islet-2 + Lim-3). Furthermore, we found that expression of Wnt-7b, which overlapped with Pax-6 in the ventrolateral domain of the neural tube, was also specifically missing in the mutant hindbrain, while it remained intact in the dorsal non-overlapping domain. These results strongly suggest that Pax-6 is involved in the specification of subtypes of hindbrain motor neurons, presumably through the regulation of Islet-2 and Wnt-7b expression.

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We investigated the role of Sonic hedgehog (SHH) in osteoblast differentiation and bone formation. The numbers of ALP-positive cells in the mouse fibroblastic cell line C3H10T1/2 and the mouse osteoblastic cell line MC3T3-E1 were increased by co-culture with chicken fibroblasts transfected with chicken Shh cDNA encoding amino-terminal peptide (Shh-N). The conditioned medium of Shh-N-RCAS-transfected chicken fibroblast cultures also significantly increased ALP activity in both C3H10T1/2 and MC3T3-E1 cells. Intramuscular transplantation of Shh-N-RCAS-transfected chicken fibroblasts into athymic mice induced ectopic bone formation. These results indicate that SHH induces osteoblast differentiation and ectopic bone formation.

DOI： 10.1016/S0014-5793(97)00014-8

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Language：English   Publisher：Zoological Society of Japan  

A million of insect species have been identified so far, displaying a staggering variety of adult morphologies. To elucidate mechanism how such insect morphologies are developed at a molecular level, we investigated developmental process of the two-spotted cricket, Gryllus bimaculatus, as a typical hemimetabola, and compared with that of Drosophila as an extensively studied typical holometabola. We analyzed expression patterns of homeobox genes of engrailed (en) and Distal-less (Dll) during development. In early embryos, en is expressed in the posterior compartments of body segments and developing appendages, while Dll is expressed in the distal region corresponding to the telopodite of developing appendages. Interestingly, these expression patterns are very similar to those observed in Drosophila imaginal discs. In the case of Dll, we found that its expression pattern, which is similar to each other in various appendages at early stages, changes in progress with elongation and segmentation, depending on the type of appendages. Late expression patterns of DII are classified into three types : Dll expression in the entire region of the antenna, in a distal region of the cercus, and in distal and middle regions of the leg, maxillary and labial palpus, indicating that Dll expression patterns are closely related to segmentation patterns of the appendages. Furthermore, since Dll is intensely expressed in both sides of the femur-tibia articulation of the leg, we considered that Dll is involved in positioning of articulation during the late appendage development. Hence, our results indicated that although common molecules are involved in development of insect appendages, the variety of the morphologies depends on pattern and timing of their expressions.

DOI： 10.2108/zsj.14.115

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Other Link： http://dl.ndl.go.jp/info:ndljp/pid/10861262

Authorship：Lead author   Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：JAPAN BIOCHEMICAL SOC  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-LISS  

A new gene, nel, was isolated from a 9-day-old chick embryonic cDNA library. The gene encodes a protein of 835 amino acids (93,407 M(r)) consisting of two hydrophobic domains presumed to be the signal and transmembrane sequences, a histidine rich domain, two repeats of a cysteine rich structure similar to the C-terminal domain of von Willebrand factor, five EGF-like repeats, and again two repeats of the cysteine rich sequence similar to the C-terminal domain of von Willebrand factor in the presumed cytoplasmic domain. The expression of the nel gene was studied by Northern blot and in situ hybridization analyses of chick embryos. The mRNA of the gene was found in all tissues of 10- and 17-day-old embryos by Northern blot hybridization. Among the tissues examined, the level in the brain was highest and increased with age. After hatching, gene expression was retained in the brain at about the same level found in old embryos, increased in the retina, and disappeared from the other tissues. In situ hybridization with a nel gene probe revealed that the gene was strongly expressed in neural tissues such as brain, spinal cord, and dorsal root ganglia of early embryos. Gene expression was observed in the mantle layer of the neurepithelium of the brain and of the spinal cord. Gene expression in early embryos was not restricted to the neural tissues, but was also detected in the cells around cartilage, myocardium, lung mesenchymal cells, and in the liver, etc. One band of about 4.5 Kb mRNA was detected in all tissues and stages by Northern blot hybridization analysis. The possible function of the gene is discussed. (C) 1995 Wiley-Liss, Inc.

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In the vertebrate embryo, only somatopleural cells in the limb-forming region are released from the mesodermal layer and undergo outgrowth from the embryonic body to form the limb bud. Molecular signals which regulate limb bud induction are unknown to date. In the present study we examined the ability of fibroblast growth factor (FGF) to induce limb bud formation in chicken embryos. A replication-defective retrovirus encoding FGF type 4 with a reporter, bacterial β-galactosidase, was microinjected into lateral plate mesoderm inside and outside limb-forming regions. Effects of the ectopic and precocious expression of FGF were assessed at various stages after infection. Here we report that somatic mesodermal cells in both flank and limb-forming regions can respond to FGF and induce limb bud-like outgrowth. The supernumerary limb bud induced within a limb-forming region differentiated into extralimb structures. These results strongly suggest potential roles of FGF signaling for induction of limb bud formation. © 1995 Academic Press. All rights reserved.

DOI： 10.1006/dbio.1995.1053

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Vertebrate limb morphogenesis has been investigated as a model system of pattern formation. Recent results suggest that peptide growth factors are involved in the limb morphogenesis. Members of the fibroblast growth factor family stimulate proliferation of the limb mesenchymal cells, while members of the transforming growth factor (TGF)-β family, in contrast, inhibit the proliferation. The antagonistic effect between the two families is probably essential for establishment of both anteroposterior and proximodistal axes. The Wnt and TGF-β families may be related to morphogenesis of the dorsoventral axis. The three families are also involved in chondrogenesis, osteogenesis, and feather formation. In this review, possible roles of such growth factors and their receptors in morphogenesis of the limb are discussed.

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Expression patterns of three fibroblast growth factor receptor genes, FGFR1 (cek1), FGFR2 (cek3) and FGFR3 (cek2), were observed in limb and feather morphogenesis. Expression of FGFR1 was observed in the mesoderm of limb bud, in the mesenchyme just underneath the feather placode, and then in the anterior mesenchyme of the feather bud. While expression of FGFR2 was observed in both surface ectoderm and mesenchymal aggregates corresponding to the future bones of the limb, the mesenchyme between the feather placode, and surface ectoderm of feather buds. Expression of FGFR3 was observed rather ubiquitously over mesoderm of limb and feather buds. Differential expression of these FGF receptor genes suggested that differential roles of these receptors in epithelia-mesenchymal interactions of limb and feather morphogenesis.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING CO  

The rat small eye strain (rSey) lacks eyes and nose in the homozygote, an is similar the mouse Sey strain with mutations in the Pax-6 gene. We isolated Pax-6 cDNA clones from an rSey homozygote library, and found an internal deletion of about 600 basepairs in the serine/threonine-rich domain. At the genomic level, a single base (G) insertion in an exon generates an abnormal 5' donor splice site, thereby producing the truncated mRNA. Anterior midbrain crest cells in the homozygous rSey embryos reached the eye rudiments but did not migrate any further to the nasal rudiments, suggesting that the Pax-6 gene is involved in conducting migration of neural crest cells from the anterior midbrain.

DOI： 10.1038/ng0493-299

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Other Link： http://orcid.org/0000-0003-0548-6452

Language：Japanese   Publishing type：Research paper (scientific journal)  

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Language：Japanese   Publishing type：Research paper (scientific journal)  

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：WILEY-LISS, INC  

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Authorship：Lead author,　Corresponding author   Language：English  

The eye is a sensory organ that primarily captures light and provides the sense of sight, as well as delivering non-visual light information involving biological rhythms and neurophysiological activities to the brain. Since the early 1990s, rapid advances in molecular biology have enabled the identification of developmental genes, genes responsible for human congenital diseases, and relevant genes of mutant animals with various anomalies. In this review, we first look at the development of the eye, and we highlight seminal reports regarding archetypal gene defects underlying three developmental ocular disorders in humans: (1) holoprosencephaly (HPE), with cyclopia being exhibited in the most severe cases; (2) microphthalmia, anophthalmia, and coloboma (MAC) phenotypes; and (3) anterior segment dysgenesis (ASDG), known as Peters anomaly and its related disorders. The recently developed methods, such as next-generation sequencing and genome editing techniques, have aided the discovery of gene mutations in congenital eye diseases and gene functions in normal eye development. Finally, we discuss Pax6-genome edited mosaic eyes and propose that somatic mosaicism in developmental gene mutations should be considered a causal factor for variable phenotypes, sporadic cases, and de novo mutations in human developmental disorders.

DOI： 10.1111/cga.12304

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Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

<p>Opsins are animal photoreceptor proteins that use retinal as their chromophore (ligand) and are classified into several groups based on sequence similarity. Opn5 forms one phylogenetically separate opsin group and includes three vertebrate-specific subgroups, Opn5m, Opn5L1 and Opn5L2. In this article, we introduce highly diversified functionalities of vertebrate Opn5. Three Opn5 subgroups show characteristic spectral sensitivity, preference for retinal isomers and photoreaction process, and work as bistable photoreceptor, retinal chemoreceptor or unidirectional photoreceptor. Such broad spectrum of functionalities may be attributed to optimization for respective physiological functions.</p>

DOI： 10.2142/biophys.59.132

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Other Link： http://search.jamas.or.jp/link/ui/2019266724

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (bulletin of university, research institution)  

DOI： 10.4044/joma.125.229

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Other Link： http://ousar.lib.okayama-u.ac.jp/51892

Language：English   Publishing type：Book review, literature introduction, etc.   Publisher：OKAYAMA UNIV MED SCHOOL  

A central issue of evolutionary developmental biology is how the eye is diverged morphologically and functionally. However, the unifying mechanisms or schemes that govern eye diversification remain unsolved. In this review, I first introduce the concept of evolutionary developmental biology of the eye with a focus on photoreception, the fundamental property of retinal cells. Second, I summarize the early development of vertebrate eyes and the role of a homeobox gene, Lhx1, in subdivision of the retina into 2 domains, the neural retina and retinal pigmented epithelium of the optic primordium. The 2 retinal domains are essential components of the eye as they are found in such prototypic eyes as the extant planarian eye. Finally, I propose the presence of novel retinal cell subtypes with photosensory functions based on our recent work on atypical photopigments (opsins) in vertebrates. Since human diseases are attributable to the aberration of various types of cells due to alterations in gene expression, understanding the precise mechanisms of cellular diversification and unraveling the molecular profiles of cellular subtypes are essential to future regenerative medicine.

DOI： 10.18926/AMO/51064

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Language：English   Publisher：ETHIS COMMUNICATINS, INC  

Various congenital anomalies of the eyelids can result from abnormal tissue proliferation, fusion, and reopening of the eyelids. Therefore, it is important to study the molecular mechanisms underlying eyelid development, focusing on cell behaviors. Mammalian eyelid development occurs in four steps: specification, growth, epithelial fusion, and reopening. Epithelial-mesenchymal interactions are fundamental processes during eyelid formation, and epithelial factors functioning within the eyelid epithelium are also critical. Studies with mutant and genetically modified mice have revealed that various signaling pathways and transcriptional factors are involved in mouse eyelid development. In this review, eyelid morphogenetic factors or pathways are described, as revealed by their mutant phenotype, eye-open at birth (EOB). These include FGFR2b-FGF10, EGFR-ERK, MEKK-JNK, BMP, Shh, Wnt, GPR48, Jun, Forkhead, and Grainyhead.

DOI： 10.1016/j.jtos.2012.07.005

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Language：English   Publishing type：Book review, literature introduction, etc.   Publisher：WILEY-BLACKWELL  

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.

DOI： 10.1111/j.1479-8298.2010.00408.x

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Language：Japanese  

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