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1.
Dev Growth Differ ; 65(6): 348-359, 2023 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-37310211

RESUMO

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


Assuntos
Desenvolvimento Embrionário , Gryllidae , Proteínas de Insetos , Fatores de Transcrição , Asas de Animais , Animais , Ciclo Celular , Proliferação de Células , Desenvolvimento Embrionário/genética , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Asas de Animais/embriologia , Asas de Animais/metabolismo , Gryllidae/embriologia , Gryllidae/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
2.
Dev Biol ; 485: 1-8, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35196518

RESUMO

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


Assuntos
Proteínas de Drosophila , Gryllidae , Sequência de Aminoácidos , Animais , Padronização Corporal/genética , Drosophila/genética , Proteínas de Drosophila/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Gryllidae/genética , Gryllidae/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Insetos/genética , Insetos/metabolismo , Interferência de RNA , Fatores de Transcrição/metabolismo
3.
Sci Rep ; 11(1): 23806, 2021 12 13.
Artigo em Inglês | MEDLINE | ID: mdl-34903813

RESUMO

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


Assuntos
Edição de Genes/métodos , Mutação , Suínos/genética , Transfecção/métodos , Animais , Blastocisto/efeitos dos fármacos , Blastocisto/metabolismo , Sistemas CRISPR-Cas , Edição de Genes/veterinária , Lipídeos/farmacologia , Miostatina/genética , Miostatina/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Transfecção/veterinária
4.
Commun Biol ; 4(1): 733, 2021 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-34127782

RESUMO

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


Assuntos
Evolução Molecular , Genoma de Inseto/genética , Gryllidae/genética , Insetos/genética , Animais , Metilação de DNA , Elementos de DNA Transponíveis/genética , Feminino , Genes de Insetos/genética , Masculino , Filogenia , Sequências Repetitivas de Ácido Nucleico/genética , Análise de Sequência de DNA
5.
Philos Trans R Soc Lond B Biol Sci ; 374(1783): 20190225, 2019 10 14.
Artigo em Inglês | MEDLINE | ID: mdl-31438810

RESUMO

Juvenile hormones and the genetic interaction between the transcription factors Krüppel homologue 1 (Kr-h1) and Broad (Br) regulate the transformation of insects from immature to adult forms in both types of metamorphosis (holometaboly with a pupal stage versus hemimetaboly with no pupal stage); however, knowledge about the exact instar in which this occurs is limited. Using the hemimetabolous cricket Gryllus bimaculatus (Gb), we demonstrate that a genetic interaction occurs among Gb'Kr-h1, Gb'Br and the adult-specifier transcription factor Gb'E93 from the sixth to final (eighth) nymphal instar. Gb'Kr-h1 and Gb'Br mRNAs were strongly expressed in the abdominal tissues of sixth instar nymphs, with precocious adult moults being induced by Gb'Kr-h1 or Gb'Br knockdown in the sixth instar. The depletion of Gb'Kr-h1 or Gb'Br upregulates Gb'E93 in the sixth instar. By contrast, Gb'E93 knockdown at the sixth instar prevents nymphs transitioning to adults, instead producing supernumerary nymphs. Gb'E93 also represses Gb'Kr-h1 and Gb'Br expression in the penultimate nymphal instar, demonstrating its important role in adult differentiation. Our results suggest that the regulatory mechanisms underlying the pupal transition in holometabolous insects are evolutionarily conserved in hemimetabolous G. bimaculatus, with the penultimate and final nymphal periods being equivalent to the pupal stage. This article is part of the theme issue 'The evolution of complete metamorphosis'.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Gryllidae/crescimento & desenvolvimento , Proteínas de Insetos/genética , Metamorfose Biológica , Fatores de Transcrição/genética , Animais , Gryllidae/genética , Proteínas de Insetos/metabolismo , Ninfa/genética , Ninfa/crescimento & desenvolvimento , Pupa/genética , Pupa/crescimento & desenvolvimento , Fatores de Transcrição/metabolismo
6.
Mol Immunol ; 106: 127-134, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30597474

RESUMO

Food scarcity is a serious problem for many developing as well as developed countries. Edible insects have attracted attention recently as a novel food source. Crickets are especially high in nutritional value and easy to breed and harvest. In this study, we evaluated the risk of allergic reactions associated with cricket consumption in individuals with crustacean allergy. We evaluated food allergy risk in the consumption of Gryllus bimaculatus (cricket) in patients with shrimp allergy, using enzyme-linked immunosorbent assay (ELISA) and IgE crosslinking-induced luciferase expression assay (EXiLE). Sera from individuals with shrimp allergy (positive for shrimp-specific IgE by ImmunoCAP (>0.35 UA/mL; n = 9) or without shrimp allergy (negative for shrimp-specific IgE; n = 6) were obtained. There was a strong correlation between shrimp- and Gryllus-specific IgE levels obtained by ELISA (rs = 0.99; P < 0.001). The binding of shrimp-specific IgE on shrimp allergen was dose-dependently inhibited by Gryllus allergen (0-1.0 mg/mL). There was a strong correlation between shrimp- and Gryllus-specific IgE responses, as assessed by EXiLE assays (rs = 0.89; P < 0.001). We determined that a protein of approximately 40 kDa reacted with the positive, but not negative, sera for shrimp-specific IgE by ImmunoCAP. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis identified the major allergen in shrimp and Gryllus to be tropomyosin. Our data suggest that the cricket allergen has the potential to induce an allergic reaction in individuals with crustacean allergy. Therefore, allergy risk and shrimp-specific IgE levels should be considered before consumption of cricket meal.


Assuntos
Alérgenos/imunologia , Gryllidae/imunologia , Imunoglobulina E/imunologia , Hipersensibilidade a Frutos do Mar/imunologia , Frutos do Mar , Adolescente , Adulto , Animais , Criança , Pré-Escolar , Reações Cruzadas , Feminino , Humanos , Imunoglobulina E/sangue , Masculino , Hipersensibilidade a Frutos do Mar/sangue
7.
Nat Commun ; 9(1): 1255, 2018 03 28.
Artigo em Inglês | MEDLINE | ID: mdl-29593298

RESUMO

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.


Assuntos
Opsinas/química , Células Fotorreceptoras de Vertebrados/química , Animais , Galinhas , Cromatografia Líquida de Alta Pressão , Fator Xa/química , Células HEK293 , Humanos , Hibridização In Situ , Luz , Masculino , Células Fotorreceptoras , Ligação Proteica , Proteínas Recombinantes/química , Regeneração , Retinaldeído/metabolismo , Rodopsina/química , Transdução de Sinais , Vitamina A/química , Xenopus/metabolismo
8.
PLoS One ; 11(11): e0163925, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27861495

RESUMO

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.


Assuntos
Encéfalo/metabolismo , Retina/metabolismo , Opsinas de Bastonetes/metabolismo , Animais , Encéfalo/citologia , Cálcio/metabolismo , Cerebelo/citologia , Cerebelo/metabolismo , Galinhas , Éxons , Expressão Gênica , Genômica , Hipotálamo/citologia , Hipotálamo/metabolismo , Íntrons , Luz , Família Multigênica , Filogenia , Transporte Proteico , Transporte de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Retina/citologia , Células Horizontais da Retina/fisiologia , Opsinas de Bastonetes/genética
9.
Proc Natl Acad Sci U S A ; 113(20): 5634-9, 2016 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-27140602

RESUMO

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.


Assuntos
Gryllidae/crescimento & desenvolvimento , Proteínas de Insetos/fisiologia , Hormônios Juvenis/biossíntese , Metamorfose Biológica , Transdução de Sinais/fisiologia , Fator de Crescimento Transformador beta/fisiologia , Sequência de Aminoácidos , Animais , Proteínas de Drosophila/fisiologia , Interferência de RNA , RNA Mensageiro/análise , Fator de Crescimento Transformador beta/química , Fator de Crescimento Transformador beta/genética
10.
J Biol Chem ; 289(7): 3991-4000, 2014 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-24403072

RESUMO

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 Gi-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.


Assuntos
Encéfalo/metabolismo , Evolução Molecular , Proteínas de Membrana/metabolismo , Mutação de Sentido Incorreto , Opsinas/metabolismo , Retina/metabolismo , Raios Ultravioleta , Substituição de Aminoácidos , Animais , Callithrix , Embrião de Galinha , Humanos , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos ICR , Opsinas/genética , Retinaldeído/genética , Retinaldeído/metabolismo , Xenopus , Peixe-Zebra , cis-trans-Isomerases/genética , cis-trans-Isomerases/metabolismo
11.
Biol Open ; 1(11): 1083-93, 2012 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-23213388

RESUMO

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.

12.
PLoS One ; 7(2): e31534, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22348098

RESUMO

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.


Assuntos
Aves/fisiologia , Opsinas/fisiologia , Células Fotorreceptoras de Vertebrados/fisiologia , Glândulas Suprarrenais , Animais , Galinhas , Luz , Células Fotorreceptoras de Vertebrados/química , Raios Ultravioleta
13.
Proc Natl Acad Sci U S A ; 107(51): 22084-9, 2010 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-21135214

RESUMO

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 GTPγS 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.


Assuntos
Células Amácrinas/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Retinaldeído/metabolismo , Opsinas de Bastonetes/metabolismo , Raios Ultravioleta , Animais , Galinhas , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/efeitos da radiação , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/genética , Guanosina 5'-O-(3-Tiotrifosfato)/farmacologia , Células HEK293 , Humanos , Retinaldeído/genética , Opsinas de Bastonetes/genética
14.
Dev Dyn ; 239(10): 2647-58, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20737506

RESUMO

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.


Assuntos
Diencéfalo/embriologia , Mesencéfalo/embriologia , Complexos Multienzimáticos/metabolismo , Animais , Embrião de Galinha , Eletroporação , Imunofluorescência , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Hibridização In Situ , Marcação In Situ das Extremidades Cortadas , Complexos Multienzimáticos/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa
15.
Dev Dyn ; 237(7): 1910-22, 2008 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-18570255

RESUMO

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.


Assuntos
Proteínas Aviárias/genética , Regulação da Expressão Gênica no Desenvolvimento , Opsinas/genética , Retina/metabolismo , Células Amácrinas/metabolismo , Sequência de Aminoácidos , Animais , Proteínas Aviárias/classificação , Proteínas Aviárias/metabolismo , Embrião de Galinha , Galinhas , Imunofluorescência , Perfilação da Expressão Gênica , Hibridização In Situ , Modelos Genéticos , Dados de Sequência Molecular , Opsinas/classificação , Opsinas/metabolismo , Filogenia , Retina/citologia , Retina/embriologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos
16.
Dev Growth Differ ; 50(3): 159-68, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18312426

RESUMO

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, beta3-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.


Assuntos
Proteínas do Olho/biossíntese , Fatores de Crescimento de Fibroblastos/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Interferência de RNA , Retina/embriologia , Proteínas com Domínio T/biossíntese , Tubulina (Proteína)/biossíntese , Animais , Células Cultivadas , Embrião de Galinha , Eletroporação , Proteínas do Olho/genética , Fatores de Crescimento de Fibroblastos/antagonistas & inibidores , Fatores de Crescimento de Fibroblastos/biossíntese , Fatores de Crescimento de Fibroblastos/genética , Morfogênese/genética , Reação em Cadeia da Polimerase , RNA Mensageiro/biossíntese , RNA Interferente Pequeno/genética , Retina/metabolismo , Proteínas com Domínio T/genética , Transgenes , Tubulina (Proteína)/genética
17.
Gene Expr Patterns ; 7(7): 746-53, 2007 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-17631423

RESUMO

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.


Assuntos
Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Sistema Nervoso/embriologia , Retina/embriologia , Opsinas de Bastonetes/biossíntese , Sequência de Aminoácidos , Animais , Sequência de Bases , Embrião de Galinha , Galinhas , Dados de Sequência Molecular , Crista Neural/embriologia , Crista Neural/metabolismo , Retina/metabolismo , Homologia de Sequência de Aminoácidos , Distribuição Tecidual
18.
Dev Dyn ; 234(3): 783-90, 2005 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-16217736

RESUMO

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.


Assuntos
Retina/citologia , Retina/metabolismo , Células Bipolares da Retina/metabolismo , Células Ganglionares da Retina/metabolismo , Células Horizontais da Retina/metabolismo , Pigmentos da Retina/metabolismo , Opsinas de Bastonetes/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Diferenciação Celular , Embrião de Galinha , Dados de Sequência Molecular , RNA Mensageiro/genética , Retina/embriologia , Células Bipolares da Retina/citologia , Células Ganglionares da Retina/citologia , Células Horizontais da Retina/citologia , Células Horizontais da Retina/embriologia , Opsinas de Bastonetes/química , Opsinas de Bastonetes/genética
19.
Dev Dyn ; 233(1): 177-87, 2005 May.
Artigo em Inglês | MEDLINE | ID: mdl-15765517

RESUMO

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 SMAD-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.


Assuntos
Orelha Interna/embriologia , Fatores de Crescimento de Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Animais , Sequência de Bases , Orelha Interna/anormalidades , Orelha Interna/anatomia & histologia , Fator 10 de Crescimento de Fibroblastos , Fatores de Crescimento de Fibroblastos/genética , Humanos , Camundongos , Dados de Sequência Molecular , Mutação , Regiões Promotoras Genéticas , Transdução de Sinais/fisiologia
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