RESUMO
Septate junctions (SJs) evolved as cell-cell junctions that regulate the paracellular barrier and integrity of epithelia in invertebrates. Multiple morphological variants of SJs exist specific to different epithelia and/or phyla but the biological significance of varied SJ morphology is unclear because the knowledge of the SJ associated proteins and their functions in non-insect invertebrates remains largely unknown. Here we report cell-specific expression of nine candidate SJ genes in the early life stages of the sea urchin Strongylocentrotus purpuratus. By use of in situ RNA hybridization and single cell RNA-seq we found that the expression of selected genes encoding putatively SJ associated transmembrane and cytoplasmic scaffold molecules was dynamically regulated during epithelial development in the embryos and larvae with different epithelia expressing different cohorts of SJ genes. We focused a functional analysis on SpMesh, a homolog of the Drosophila smooth SJ component Mesh, which was highly enriched in the endodermal epithelium of the mid- and hindgut. Functional perturbation of SpMesh by both CRISPR/Cas9 mutagenesis and vivo morpholino-mediated knockdown shows that loss of SpMesh does not disrupt the formation of the gut epithelium during gastrulation. However, loss of SpMesh resulted in a severely reduced gut-paracellular barrier as quantitated by increased permeability to 3-5 âkDa FITC-dextran. Together, these studies provide a first look at the molecular SJ physiology during the development of a marine organism and suggest a shared role for Mesh-homologous proteins in forming an intestinal barrier in invertebrates. Results have implications for consideration of the traits underlying species-specific sensitivity of marine larvae to climate driven ocean change.
Assuntos
Proteínas de Drosophila , Strongylocentrotus purpuratus , Animais , Strongylocentrotus purpuratus/genética , Strongylocentrotus purpuratus/metabolismo , Junções Íntimas/genética , Junções Íntimas/metabolismo , Epitélio/metabolismo , Junções Intercelulares/metabolismo , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Ouriços-do-Mar/genética , Ouriços-do-Mar/metabolismo , Larva/genética , Larva/metabolismoRESUMO
The transcriptome data of skin cells from domestic cats with brown, orange, and white coats were analyzed using a public database to investigate the possible relationship between coat color-related gene expression and squamous cell carcinoma risk, as well as the mechanism of deafness in white cats. We found that the ratio of the expression level of genes suppressing squamous cell carcinoma to that of genes promoting squamous cell carcinoma might be considerably lower than the theoretical estimation in skin cells with orange and white coats in white-spotted cat. We also found the possibility of the frequent production of KIT lacking the first exon (d1KIT) in skin cells with white coats, and d1KIT production exhibited a substantial negative correlation with the expression of SOX10, which is essential for melanocyte formation and adjustment of hearing function. Additionally, the production of d1KIT was expected to be due to the insulating activity of the feline endogenous retrovirus 1 (FERV1) LTR in the first intron of KIT by its CTCF binding sequence repeat. These results contribute to basic veterinary research to understand the relationship between cat skin coat and disease risk, as well as the underlying mechanism.
Assuntos
Surdez , Pigmentação da Pele , Animais , Gatos , RNA-Seq , Pigmentação da Pele/genética , Íntrons , Fatores de RiscoRESUMO
The update of the draft genome assembly of sea urchin, Hemicentrotus pulcherrimus, which is widely studied in East Asia as a model organism of early development, was performed using Oxford nanopore long-read sequencing. The updated assembly provided ~600-Mb genome sequences divided into 2,163 contigs with N50 = 516 kb. BUSCO completeness score and transcriptome model mapping ratio (TMMR) of the present assembly were obtained as 96.5% and 77.8%, respectively. These results were more continuous with higher resolution than those by the previous version of H. pulcherrimus draft genome, HpulGenome_v1, where the number of scaffolds = 16,251 with a total of ~100 Mb, N50 = 143 kb, BUSCO completeness score = 86.1%, and TMMR = 55.4%. The obtained genome contained 36,055 gene models that were consistent with those in other echinoderms. Additionally, two tandem repeat sequences of early histone gene locus containing 47 copies and 34 copies of all histone genes, and 185 of the homologous sequences of the interspecifically conserved region of the Ars insulator, ArsInsC, were obtained. These results provide further advance for genome-wide research of development, gene regulation, and intranuclear structural dynamics of multicellular organisms using H. pulcherrimus.
Assuntos
Genoma , Animais , Genoma/genética , Hemicentrotus/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodosRESUMO
Sea urchins have been used as model organisms in developmental biology research and the genomes of several sea urchin species have been sequenced. Recently, genome editing technologies have become available for sea urchins, and methods for gene knockout using the CRISPRCas9 system have been established. Heliocidaris crassispina is an important marine fishery resource with edible gonads. Although H. crassispina has been used as a biological research material, its genome has not yet been published, and it is a non-model sea urchin for molecular biology research. However, as recent advances in genome editing technology have facilitated genome modification in non-model organisms, we applied genome editing using the CRISPR-Cas9 system to H. crassispina. In this study, we targeted genes encoding ETS transcription factor (HcEts) and pigmentation-related polyketide synthase (HcPks1). Gene fragments were isolated using primers designed by inter-specific sequence comparisons within Echinoidea. When Ets gene was targeted using two sgRNAs, one successfully introduced mutations and impaired skeletogenesis. In the Pks1 gene knockout, when two sgRNAs targeting the close vicinity of the site corresponding to the target site that showed 100% mutagenesis efficiency of the Pks1 gene in Hemicentrotus pulcherrimus, mutagenesis was not observed. However, two other sgRNAs targeting distant sites efficiently introduced mutations. In addition, Pks1 knockout H. crassispina exhibited an albino phenotype in the pluteus larvae and adult sea urchins after metamorphosis. This indicates that the CRISPRCas9 system can be used to modify the genome of the non-model sea urchin H. crassispina.
Assuntos
Anthocidaris , Animais , Anthocidaris/genética , Sistemas CRISPR-Cas , RNA Guia de Sistemas CRISPR-Cas , Técnicas de Inativação de Genes , Ouriços-do-Mar/genética , Edição de Genes/métodosRESUMO
BACKGROUND: Nasal congestion in allergic rhinitis (AR) is caused by vascular hyperpermeability and vascular relaxation of the nasal mucosa. We previously detected high levels of a lipoxygenation metabolite of dihomogammalinolenic acid, 15-hydroxy-8Z,11Z,13E-eicosatrienoic acid (15-HETrE) in the nasal lavage fluid of AR model mice. Here, we investigated the effects of 15-HETrE on vascular functions associated with nasal congestion. METHODS: We measured 15-HETrE levels in the nasal lavage fluid of ovalbumin-induced AR model mice and nasal discharge of patients with AR. We also assessed nasal congestion and vascular relaxation in mice. Vascular contractility was investigated using isolated mouse aortas. RESULTS: Five ovalbumin challenges increased 15-HETrE levels in AR model mice. 15-HETrE was also detected in patients who exhibiting AR-related symptoms. Intranasal administration of 15-HETrE elicited dyspnea-related behavior and decreased the nasal cavity volume in mice. Miles assay and whole-mount immunostaining revealed that 15-HETrE administration caused vascular hyperpermeability and relaxation of the nasal mucosa. Intravital imaging demonstrated that 15-HETrE relaxed the ear vessels that were precontracted via thromboxane receptor stimulation. Moreover, 15-HETrE dilated the isolated mouse aortas, and this effect was attenuated by K+ channel inhibitors and prostaglandin D2 (DP) and prostacyclin (IP) receptor antagonists. Additionally, vasodilatory effects of 15-HETrE were accompanied by an increase in intracellular cAMP levels. CONCLUSIONS: Our results indicate that 15-HETrE, whose levels are elevated in the nasal cavity upon AR, can be a novel lipid mediator that exacerbates nasal congestion. Moreover, it can stimulate DP and IP receptors and downstream K+ channels to dilate the nasal mucosal vasculature.
Assuntos
Modelos Animais de Doenças , Rinite Alérgica , Animais , Camundongos , Rinite Alérgica/metabolismo , Humanos , Masculino , Mucosa Nasal/metabolismo , Mucosa Nasal/efeitos dos fármacos , Mucosa Nasal/irrigação sanguínea , Ácidos Hidroxieicosatetraenoicos/metabolismo , Feminino , Obstrução Nasal/metabolismo , Permeabilidade Capilar/efeitos dos fármacos , Ovalbumina , Vasodilatação/efeitos dos fármacos , Líquido da Lavagem NasalRESUMO
Gastrulation is a universal process in the morphogenesis of many animal embryos. Although morphological and molecular events in gastrulation have been well studied, the mechanical driving forces and underlying regulatory mechanisms are not fully understood. Here, we investigated the gastrulation of embryos of a sea urchin, Hemicentrotus pulcherrimus, which involves the invagination of a single-layered vegetal plate into the blastocoel. We observed that omeprazole, a proton pump inhibitor capable of perturbing the left-right asymmetry of sea urchin embryo, induced "partial exogastrulation" where the secondary invagination proceeds outward. During early gastrulation, intracellular apical-basal polarity of F-actin distribution in vegetal half was higher than those in animal half, while omeprazole treatment disturbed the apical-basal polarity of F-actin distribution in vegetal half. Furthermore, gastrulation stopped and even partial exogastrulation did not occur when F-actin polymerization or degradation in whole embryo was partially inhibited via RhoA or YAP1 knockout. A mathematical model of the early gastrulation reproduced the shapes of both normal and exogastrulating embryos using cell-dependent cytoskeletal features based on F-actin. Additionally, such cell position-dependent intracellular F-actin distributions might be regulated by intracellular pH distributions. Therefore, apical-basal polarity of F-actin distribution disrupted by omeprazole may induce the partial exogastrulation via anomalous secondary invagination.
Assuntos
Actinas , Gástrula , Actinas/metabolismo , Animais , Embrião não Mamífero , Gástrula/metabolismo , Morfogênese , Omeprazol/metabolismo , Omeprazol/farmacologia , Ouriços-do-MarRESUMO
CCCTC-binding factor (CTCF), an insulator protein with 11 zinc fingers, is enriched at the boundaries of topologically associated domains (TADs) in eukaryotic genomes. In this study, we isolated and analyzed the cDNAs encoding HpCTCF, the CTCF homolog in the sea urchin Hemicentrotus pulcherrimus, to investigate its expression patterns and functions during the early development of sea urchin. HpCTCF contains nine zinc fingers corresponding to fingers 2-10 of the vertebrate CTCF. Expression pattern analysis revealed that HpCTCF mRNA was detected at all developmental stages and in the entire embryo. Upon expressing the HpCTCF-GFP fusion protein in early embryos, we observed its uniform distribution within interphase nuclei. However, during mitosis, it disappeared from the chromosomes and subsequently reassembled on the chromosome during telophase. Moreover, the morpholino-mediated knockdown of HpCTCF resulted in mitotic arrest during the morula to blastula stage. Most of the arrested chromosomes were not phospholylated at serine 10 of histone H3, indicating that mitosis was arrested at the telophase by HpCTCF depletion. Furthermore, impaired sister chromatid segregation was observed using time-lapse imaging of HpCTCF-knockdown embryos. Thus, HpCTCF is essential for mitotic progression during the early development of sea urchins, especially during the telophase-to-interphase transition. However, the normal development of pluteus larvae in CRISPR-mediated HpCTCF-knockout embryos suggests that disruption of zygotic HpCTCF expression has little effect on embryonic and larval development.
Assuntos
Hemicentrotus , Ouriços-do-Mar , Animais , Fator de Ligação a CCCTC/genética , Fator de Ligação a CCCTC/metabolismo , Ouriços-do-Mar/genética , Histonas/metabolismo , Núcleo CelularRESUMO
We seek to manipulate gene function here through CRISPR-Cas9 editing of cis-regulatory sequences, rather than the more typical mutation of coding regions. This approach would minimize secondary effects of cellular responses to nonsense mediated decay pathways or to mutant protein products by premature stops. This strategy also allows for reducing gene activity in cases where a complete gene knockout would result in lethality, and it can be applied to the rapid identification of key regulatory sites essential for gene expression. We tested this strategy here with genes of known function as a proof of concept, and then applied it to examine the upstream genomic region of the germline gene Nanos2 in the sea urchin, Strongylocentrotus purpuratus. We first used CRISPR-Cas9 to target established genomic cis-regulatory regions of the skeletogenic cell transcription factor, Alx1, and the TGF-ß signaling ligand, Nodal, which produce obvious developmental defects when altered in sea urchin embryos. Importantly, mutation of cis-activator sites (Alx1) and cis-repressor sites (Nodal) result in the predicted decreased and increased transcriptional output, respectively. Upon identification of efficient gRNAs by genomic mutations, we then used the same validated gRNAs to target a deadCas9-VP64 transcriptional activator to increase Nodal transcription directly. Finally, we paired these new methodologies with a more traditional, GFP reporter construct approach to further our understanding of the transcriptional regulation of Nanos2, a key gene required for germ cell identity in S. purpuratus. With a series of reporter assays, upstream Cas9-promoter targeted mutagenesis, coupled with qPCR and in situ RNA hybridization, we concluded that the promoter of Nanos2 drives strong mRNA expression in the sea urchin embryo, indicating that its primordial germ cell (PGC)-specific restriction may rely instead on post-transcriptional regulation. Overall, we present a proof-of-principle tool-kit of Cas9-mediated manipulations of promoter regions that should be applicable in most cells and embryos for which CRISPR-Cas9 is employed.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes/métodos , Regulação da Expressão Gênica no Desenvolvimento , Loci Gênicos , Regiões Promotoras Genéticas/genética , Strongylocentrotus purpuratus/embriologia , Strongylocentrotus purpuratus/genética , Animais , Animais Geneticamente Modificados , Proteína 9 Associada à CRISPR/genética , Embrião não Mamífero/metabolismo , Desenvolvimento Embrionário/genética , Expressão Gênica , Técnicas de Inativação de Genes , Células Germinativas/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Proteína Nodal/genética , Proteína Nodal/metabolismo , RNA Guia de Cinetoplastídeos/genética , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Transcrição Gênica/genéticaRESUMO
Urinary tetranor-PGDM is a useful diagnostic biomarker for food allergy which often affects infants. We attempted to extract and measure urinary tetranor-PGDM absorbed in polymer of diapers. We applied CaCl2 to the collected polymer, determined the adequate time length of shaking the polymer to release urine, and measured tetranor-PGDM in the extracted urine. This procedure provided high linearity and recovery rate in tetranor-PGDM measurement. We also found that urinary tetranor-PGDM was stable for 24 h at 4°C in diapers. This method can be useful to monitor the food allergic condition of non-toilet trained children.
Assuntos
Fraldas Infantis , Hipersensibilidade Alimentar/diagnóstico , Extração Líquido-Líquido/métodos , Prostaglandina D2/análogos & derivados , Biomarcadores/urina , Cloreto de Cálcio , Pré-Escolar , Humanos , Lactente , Polímeros , Prostaglandina D2/isolamento & purificação , Prostaglandina D2/urina , Temperatura , Fatores de TempoRESUMO
The nuclear positioning and chromatin dynamics of eukaryotic genes are closely related to the regulation of gene expression, but they have not been well examined during early development, which is accompanied by rapid cell cycle progression and dynamic changes in nuclear organization, such as nuclear size and chromatin constitution. In this study, we focused on the early development of the sea urchin Hemicentrotus pulcherrimus and performed three-dimensional fluorescence in situ hybridization of gene loci encoding early histones (one of the types of histone in sea urchin). There are two non-allelic early histone gene loci per sea urchin genome. We found that during the morula stage, when the early histone gene expression levels are at their maximum, interchromosomal interactions were often formed between the early histone gene loci on separate chromosomes and that the gene loci were directed to locate to more interior positions. Furthermore, these interactions were associated with the active transcription of the early histone genes. Thus, such dynamic interchromosomal interactions may contribute to the efficient synthesis of early histone mRNA during the morula stage of sea urchin development.
Assuntos
Desenvolvimento Embrionário/genética , Histonas/genética , Ouriços-do-Mar/genética , Transcrição Gênica , Animais , Núcleo Celular/genética , Cromatina/genética , Regulação da Expressão Gênica no Desenvolvimento , Genoma , Hibridização in Situ Fluorescente , Ouriços-do-Mar/crescimento & desenvolvimentoRESUMO
Sea urchins are used as a model organism for research on developmental biology and gene regulatory networks during early development. Gene knockdown by microinjection of morpholino antisense oligonucleotide (MASO) has been used to analyze gene function in early sea urchin embryos. However, as the effect of MASO is not long lasting, it is impossible to perturb genes expressed during late development by MASO. Recent advances in genome editing technologies have enabled gene modification in various organisms. We previously reported genome editing in the sea urchin Hemicentrotus pulcherrimus using zinc-finger nuclease (ZFN) and transcription activator-like effector nuclease (TALEN); however, the efficiencies of these technologies were not satisfactory. Here, we applied clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated nuclease 9 (Cas9) technology to knock out the Pks1 gene in H. pulcherrimus. When sgRNAs targeting Pks1, which is required for the biosynthesis of larval pigment, were microinjected into fertilized eggs with SpCas9 mRNA, high-efficiency mutagenesis was achieved within 24 hr post fertilization and SpCas9/sgRNA-injected pluteus larvae had an albino phenotype. One of the sgRNAs yielded 100% mutagenesis efficiency, and no off-target effect was detected. In addition, the albino phenotype was maintained in juvenile sea urchins after metamorphosis, and the knockout sea urchins survived for at least one year and grew to albino adult sea urchins. These findings suggest that knockout adult sea urchins were successfully established and the CRISPR-Cas9 system is a feasible method for analyzing gene functions from late developmental to adult stage.
Assuntos
Proteína 9 Associada à CRISPR , Sistemas CRISPR-Cas , Ouriços-do-Mar/embriologia , Ouriços-do-Mar/genética , Animais , Edição de Genes/métodos , Técnicas de Inativação de Genes/métodosRESUMO
Homozygous mutations in the glucocerebrosidase (GBA) gene result in Gaucher disease (GD), the most common lysosomal storage disease. Recent genetic studies have revealed that GBA mutations confer a strong risk for sporadic Parkinson's disease (PD). To investigate how GBA mutations cause PD, we generated GBA nonsense mutant (GBA-/-) medaka that are completely deficient in glucocerebrosidase (GCase) activity. In contrast to the perinatal death in humans and mice lacking GCase activity, GBA-/- medaka survived for months, enabling analysis of the pathological progression. GBA-/- medaka displayed the pathological phenotypes resembling human neuronopathic GD including infiltration of Gaucher cell-like cells into the brains, progressive neuronal loss, and microgliosis. Detailed pathological findings represented lysosomal abnormalities in neurons and alpha-synuclein (α-syn) accumulation in axonal swellings containing autophagosomes. Unexpectedly, disruption of α-syn did not improve the life span, formation of axonal swellings, neuronal loss, or neuroinflammation in GBA-/- medaka. Taken together, the present study revealed GBA-/- medaka as a novel neuronopathic GD model, the pahological mechanisms of α-syn accumulation caused by GCase deficiency, and the minimal contribution of α-syn to the pathogenesis of neuronopathic GD.
Assuntos
Axônios/metabolismo , Doença de Gaucher/genética , Glucosilceramidase/deficiência , Oryzias/genética , alfa-Sinucleína/metabolismo , Animais , Axônios/ultraestrutura , Modelos Animais de Doenças , Doença de Gaucher/metabolismo , Doença de Gaucher/patologia , Glucosilceramidase/genética , Oryzias/metabolismo , Fagossomos/metabolismoRESUMO
Left-right asymmetry of bilaterian animals is established during early development. In mice, frogs and fishes, the ciliated left-right organizer plays an essential role in establishing bilateral asymmetry, and leftward flow of extracellular fluid generated by ciliary motion results in Nodal activity on the left side. However, H(+) /K(+) -ATPase activity is also involved in the determination of left-right asymmetry in a variety of animals, and it has been thought to be an ancestral mechanism in deuterostomes. In sea urchin, the determination of the left-right asymmetry based on H(+) /K(+) -ATPase activity was already clarified, but it remains to be uncovered whether ciliary motion is involved in the left-right asymmetry of the embryo. Here, we show evidence that ciliary motion is involved in the establishment of left-right asymmetry of sea urchin embryo. Furthermore, we show that the initial cilia generated on small micromeres during the early stage of embryogenesis may be involved in this process. These results suggest that the cilia-mediated mechanism for the determination of left-right asymmetry may be acquired at the base of the deuterostomes.
Assuntos
Ouriços-do-Mar/embriologia , Animais , Cílios , Embrião não Mamífero , Desenvolvimento Embrionário , Ligantes da Sinalização Nodal/metabolismo , Ouriços-do-Mar/metabolismoRESUMO
Xenopus laevis tadpoles can completely regenerate their appendages, such as tail and limbs, and therefore provide a unique model to decipher the molecular mechanisms of organ regeneration in vertebrates. Epigenetic modifications are likely to be involved in this remarkable regeneration capacity, but they remain largely unknown. To examine the involvement of histone modification during organ regeneration, we generated transgenic X. laevis ubiquitously expressing a fluorescent modification-specific intracellular antibody (Mintbody) that is able to track histone H3 lysine 9 acetylation (H3K9ac) in vivo through nuclear enhanced green fluorescent protein (EGFP) fluorescence. In embryos ubiquitously expressing H3K9ac-Mintbody, robust fluorescence was observed in the nuclei of somites. Interestingly, H3K9ac-Mintbody signals predominantly accumulated in nuclei of regenerating notochord at 24 h postamputation following activation of reactive oxygen species (ROS). Moreover, apocynin (APO), an inhibitor of ROS production, attenuated H3K9ac-Mintbody signals in regenerating notochord. Our results suggest that ROS production is involved in acetylation of H3K9 in regenerating notochord at the onset of tail regeneration. We also show this transgenic Xenopus to be a useful tool to investigate epigenetic modification, not only in organogenesis but also in organ regeneration.
Assuntos
Histonas/metabolismo , Proteínas de Xenopus/metabolismo , Acetilação , Animais , Animais Geneticamente Modificados , Desenvolvimento Embrionário , Código das Histonas , Espécies Reativas de Oxigênio/metabolismo , Regeneração , Cauda/fisiologia , Xenopus laevisRESUMO
Genome editing with programmable site-specific nucleases is an emerging technology that enables the manipulation of targeted genes in many organisms and cell lines. Since the development of the CRISPR-Cas9 system in 2012, genome editing has rapidly become an indispensable technology for all life science researchers, applicable in various fields. In this seminar, we will introduce the basics of genome editing and focus on the recent development of genome editing tools and technologies for the modification of various organisms and discuss future directions of the genome editing research field, from basic to medical applications.
Assuntos
Edição de Genes , Genoma , Animais , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , DNA/genética , HumanosRESUMO
To understand complex biological systems, such as the development of multicellular organisms, it is important to characterize the gene expression dynamics. However, there is currently no universal technique for targeted insertion of reporter genes and quantitative imaging in multicellular model systems. Recently, genome editing using zinc-finger nucleases (ZFNs) has been reported in several models. ZFNs consist of a zinc-finger DNA-binding array with the nuclease domain of the restriction enzyme FokI and facilitate targeted transgene insertion. In this study, we successfully inserted a GFP reporter cassette into the HpEts1 gene locus of the sea urchin, Hemicentrotus pulcherrimus. We achieved this insertion by injecting eggs with a pair of ZFNs for HpEts1 with a targeting donor construct that contained â¼1-kb homology arms and a 2A-histone H2B-GFP cassette. We increased the efficiency of the ZFN-mediated targeted transgene insertion by in situ linearization of the targeting donor construct and cointroduction of an mRNA for a dominant-negative form of HpLig4, which encodes the H. pulcherrimus homolog of DNA ligase IV required for error-prone nonhomologous end joining. We measured the fluorescence intensity of GFP at the single-cell level in living embryos during development and found that there was variation in HpEts1 expression among the primary mesenchyme cells. These findings demonstrate the feasibility of ZFN-mediated targeted transgene insertion to enable quantification of the expression levels of endogenous genes during development in living sea urchin embryos.
Assuntos
Embrião não Mamífero/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Genes Reporter/genética , Ouriços-do-Mar/genética , Dedos de Zinco/genética , Sequência de Aminoácidos , Animais , DNA Ligase Dependente de ATP , DNA Ligases/genética , Desoxirribonucleases de Sítio Específico do Tipo II/genética , Embrião não Mamífero/citologia , Embrião não Mamífero/embriologia , Proteínas de Fluorescência Verde/genética , Mesoderma/citologia , Mesoderma/fisiologia , Biologia Molecular/métodos , Dados de Sequência Molecular , Fenótipo , Cultura Primária de Células , Ouriços-do-Mar/embriologia , Transgenes/genéticaRESUMO
Nanos is a translational regulator required for the survival and maintenance of primordial germ cells during embryogenesis. Three nanos homologs are present in the genome of the sea urchin Strongylocentrotus purpuratus (Sp), and each nanos mRNA accumulates specifically in the small micromere (sMic) lineage. We found that a highly conserved element in the 3' UTR of nanos2 is sufficient for reporter expression selectively in the sMic lineage: microinjection into a Sp fertilized egg of an RNA that contains the GFP open reading frame followed by Sp nanos2 3'UTR leads to selective reporter enrichment in the small micromeres in blastulae. The same result was seen with nanos2 from the sea urchin Hemicentrotus pulcherrimus (Hp). In both species, the 5'UTR alone is not sufficient for the sMic localization but it always increased the sMic reporter enrichment when present with the 3'UTR. We defined an element conserved between Hp and Sp in the nanos2 3'UTR which is necessary and sufficient for protein enrichment in the sMic, and refer to it as GNARLE (Global Nanos Associated RNA Lability Element). We also found that the nanos2 3'UTR is essential for the selective RNA retention in the small micromeres; GNARLE is required but not sufficient for this process. These results show that a combination of selective RNA retention and translational control mechanisms instills nanos accumulation uniquely in the sMic lineage.
Assuntos
Regiões 3' não Traduzidas/genética , Proteínas de Transporte/genética , Linhagem da Célula/genética , Células Germinativas/citologia , Hemicentrotus/citologia , Hemicentrotus/genética , Strongylocentrotus purpuratus/citologia , Strongylocentrotus purpuratus/genética , Animais , Pareamento de Bases/genética , Sequência de Bases , Proteínas de Transporte/metabolismo , Sequência Conservada/genética , Genes Reporter , Dados de Sequência Molecular , Nucleotídeos/genética , Biossíntese de Proteínas/genética , Sequências Reguladoras de Ácido Ribonucleico/genética , Deleção de SequênciaRESUMO
Genome editing with engineered nucleases such as zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) has been reported in various animals. We previously described ZFN-mediated targeted mutagenesis and insertion of reporter genes in sea urchin embryos. In this study, we demonstrate that TALENs can induce mutagenesis at specific genomic loci of sea urchin embryos. Injection of TALEN mRNAs targeting the HpEts transcription factor into fertilized eggs resulted in the impairment of skeletogenesis. Sequence analyses of the mutations showed that deletions and/or insertions occurred at the HpEts target site in the TALEN mRNAs-injected embryos. The results suggest that targeted gene disruption using TALENs is feasible in sea urchin embryos.
Assuntos
Endodesoxirribonucleases/metabolismo , Marcação de Genes/métodos , Mutagênese Sítio-Dirigida/métodos , Ouriços-do-Mar/embriologia , Ouriços-do-Mar/genética , Animais , Sequência de Bases , Endodesoxirribonucleases/genética , Dados de Sequência Molecular , RNA Mensageiro/genéticaRESUMO
Genome editing is a cutting-edge technology that enables to modify the target gene using programmable site-specific nucleases, such as TALENs and CRISPR/Cas9. Currently, cell and animal models of human diseases have been competitively created throughout the world, because genome editing technology paved the way for genetic modifications even in cells and organisms that had been difficult to manipulate the genome. In this review, we introduce the basic principles and current situations of genome editing with programmable nucleases.
Assuntos
Sistemas CRISPR-Cas , Marcação de Genes/métodos , Genoma/genética , Animais , Sequência de Bases , Sistemas CRISPR-Cas/genética , Quebras de DNA de Cadeia Dupla , Modelos Animais de Doenças , Técnicas de Introdução de Genes/métodos , Técnicas de Silenciamento de Genes/métodos , Dados de Sequência Molecular , Replicação Viral/genéticaRESUMO
Introduction: The intestinal barrier plays a crucial role in distinguishing foods from toxins. Prostaglandin D2 (PGD2) is one of the lipid-derived autacoids synthesized from cell membrane-derived arachidonic acid. We previously reported that pharmacological stimulation of PGD2 receptor, D prostanoid 1 (DP1) attenuated the symptoms of azoxymethane/dextran sodium sulfate-induced colitis and ovalbumin-induced food allergy in mouse models. These observations suggested that DP1 stimulation protects the intestinal barrier. The present study aimed to uncover the effects of DP1 stimulation on intestinal barrier function and elucidate the underlying mechanisms. Materials and methods: Intestinal permeability was assessed in mice by measuring the transfer of orally administered fluorescein isothiocyanate-dextran (40 kDa) into the blood. The DP1 agonist BW245C (1 mg/kg) was administered 10 min prior to dextran administration. The intestinal permeability was confirmed using the ex vivo everted sac method. Tight junction integrity was evaluated in vitro by measuring the transepithelial electrical resistance (TER) in the human intestinal epithelial cell line Caco-2. Mucus secretion was assessed by observing Alcian Blue-stained intestinal sections. Results: Pharmacological DP1 stimulation reduced intestinal permeability both in vivo and ex vivo. Immunohistochemical staining showed that DP1 was strongly expressed on the apical side of the epithelial cells. DP1 stimulation did not affect TER in vitro but induced mucus secretion from goblet cells. Mucus removal by a mucolytic agent N-acetyl-l-cysteine canceled the inhibition of intestinal permeability by DP1 stimulation. Conclusion: These observations suggest that pharmacological DP1 stimulation decreases intestinal permeability by stimulating mucus secretion.