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Clarifying the effect of the sampling protocol on the detection of environmental DNA (eDNA) is essential for appropriately designing biodiversity research. However, technical issues influencing eDNA detection in the open ocean, which consists of water masses with varying environmental conditions, have not been thoroughly investigated. This study evaluated the sampling effort for the metabarcoding-based detection of fish eDNA using replicate sampling with filters of different pore sizes (0.22 and 0.45 µm) in the subtropical and subarctic northwestern Pacific Ocean and Arctic Chukchi Sea. The asymptotic analysis predicted that the accumulation curves for detected taxa did not saturate in most cases, indicating that our sampling effort (7 or 8 replicates, corresponding to 10.5-40 L of filtration in total) was insufficient to fully assess the species diversity in the open ocean and that tens of replicates or a substantial filtration volume were required. The Jaccard dissimilarities between filtration replicates were comparable with those between the filter types at any site. In subtropical and subarctic sites, turnover dominated the dissimilarity, suggesting that the filter pore size had a negligible effect. In contrast, nestedness dominated the dissimilarity in the Chukchi Sea, implying that the 0.22 µm filter could collect a broader range of eDNA than the 0.45 µm filter. Therefore, the effect of filter selection on the collection of fish eDNA likely varies depending on the region. These findings highlight the highly stochastic nature of fish eDNA collection in the open ocean and the difficulty of standardizing the sampling protocol across various water masses.
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At the moment of union in fertilization, sperm and oocyte are transcriptionally silent. The ensuing onset of embryonic transcription (embryonic genome activation [EGA]) is critical for development, yet its timing and profile remain elusive in any vertebrate species. We here dissect transcription during EGA by high-resolution single-cell RNA sequencing of precisely synchronized mouse one-cell embryos. This reveals a program of embryonic gene expression (immediate EGA [iEGA]) initiating within 4 h of fertilization. Expression during iEGA produces canonically spliced transcripts, occurs substantially from the maternal genome, and is mostly downregulated at the two-cell stage. Transcribed genes predict regulation by transcription factors (TFs) associated with cancer, including c-Myc. Blocking c-Myc or other predicted regulatory TF activities disrupts iEGA and induces acute developmental arrest. These findings illuminate intracellular mechanisms that regulate the onset of mammalian development and hold promise for the study of cancer.
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Embrión de Mamíferos , Perfilación de la Expresión Génica , Masculino , Animales , Ratones , Embrión de Mamíferos/metabolismo , Regulación del Desarrollo de la Expresión Génica , Semen , Expresión Génica , Desarrollo Embrionario/genética , Mamíferos/genéticaRESUMEN
Gamete (sperm and oocyte) genomes are transcriptionally silent until embryonic genome activation (EGA) following fertilization. EGA in humans had been thought to occur around the eight-cell stage, but recent findings suggest that it is triggered in one-cell embryos, by fertilization. Phosphorylation and other post-translational modifications during fertilization may instate transcriptionally favorable chromatin and activate oocyte-derived transcription factors (TFs) to initiate EGA. Expressed genes lay on cancer-associated pathways and their identities predict upregulation by MYC and other cancer-associated TFs. One interpretation of this is that the onset of EGA, and the somatic cell trajectory to cancer, are mechanistically related: cancer initiates epigenetically. We describe how fertilization might be linked to the initiation of EGA and involve distinctive processes recapitulated in cancer.
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Embrión de Mamíferos , Semen , Animales , Masculino , Humanos , Semen/metabolismo , Embrión de Mamíferos/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Genoma , Activación Transcripcional , Desarrollo Embrionario/genética , Regulación del Desarrollo de la Expresión Génica/genética , Mamíferos/genética , Mamíferos/metabolismoRESUMEN
In human embryos, the initiation of transcription (embryonic genome activation [EGA]) occurs by the eight-cell stage, but its exact timing and profile are unclear. To address this, we profiled gene expression at depth in human metaphase II oocytes and bipronuclear (2PN) one-cell embryos. High-resolution single-cell RNA sequencing revealed previously inaccessible oocyte-to-embryo gene expression changes. This confirmed transcript depletion following fertilization (maternal RNA degradation) but also uncovered low-magnitude upregulation of hundreds of spliced transcripts. Gene expression analysis predicted embryonic processes including cell-cycle progression and chromosome maintenance as well as transcriptional activators that included cancer-associated gene regulators. Transcription was disrupted in abnormal monopronuclear (1PN) and tripronuclear (3PN) one-cell embryos. These findings indicate that human embryonic transcription initiates at the one-cell stage, sooner than previously thought. The pattern of gene upregulation promises to illuminate processes involved at the onset of human development, with implications for epigenetic inheritance, stem-cell-derived embryos, and cancer.
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Embrión de Mamíferos , Genoma Humano , Blastocisto , Embrión de Mamíferos/metabolismo , Desarrollo Embrionario/genética , Regulación del Desarrollo de la Expresión Génica , Humanos , OocitosRESUMEN
In mammalian genomes, differentially methylated regions (DMRs) and histone marks including trimethylation of histone 3 lysine 27 (H3K27me3) at imprinted genes are asymmetrically inherited to control parentally-biased gene expression. However, neither parent-of-origin-specific transcription nor imprints have been comprehensively mapped at the blastocyst stage of preimplantation development. Here, we address this by integrating transcriptomic and epigenomic approaches in mouse preimplantation embryos. We find that seventy-one genes exhibit previously unreported parent-of-origin-specific expression in blastocysts (nBiX: novel blastocyst-imprinted expressed). Uniparental expression of nBiX genes disappears soon after implantation. Micro-whole-genome bisulfite sequencing (µWGBS) of individual uniparental blastocysts detects 859 DMRs. We further find that 16% of nBiX genes are associated with a DMR, whereas most are associated with parentally-biased H3K27me3, suggesting a role for Polycomb-mediated imprinting in blastocysts. nBiX genes are clustered: five clusters contained at least one published imprinted gene, and five clusters exclusively contained nBiX genes. These data suggest that early development undergoes a complex program of stage-specific imprinting involving different tiers of regulation.
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Blastocisto/metabolismo , Impresión Genómica/genética , Histonas/metabolismo , Alelos , Animales , Blastocisto/citología , Metilación de ADN , Desarrollo Embrionario/genética , Femenino , Expresión Génica , Células Germinativas/metabolismo , Estratos Germinativos/metabolismo , Haploidia , Masculino , Metilación , Ratones , Células Madre Embrionarias de Ratones/metabolismo , Familia de Multigenes , Sitio de Iniciación de la TranscripciónRESUMEN
Diagnosis of possible neurosarcoidosis (NS) involves assessment of clinical presentation suggestive of NS and exclusion of other diagnoses, whereas its definitive diagnosis is challenging and it requires positive nervous system histology. Due to an exceptionally low incidence, limited data are available on the optimal therapeutic options for NS. NS is commonly associated with other sarcoidosis forms; however, the patients with NS experience more frequent relapses than with other organs sarcoidosis. While corticosteroid therapy is considered as the first-line of treatment for NS, secondary treatment options with immunosuppressive agents such as methotrexate, azathioprine, mycophenolate mofetil and tumor necrosis factor (TNF)-α inhibitors are considered based on the severity of NS manifestations and the disease status.
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Enfermedades del Sistema Nervioso Central , Sarcoidosis , Humanos , Inmunosupresores/uso terapéutico , RecurrenciaRESUMEN
Cells comprise mechanically active matter that governs their functionality, but intracellular mechanics are difficult to study directly and are poorly understood. However, injected nanodevices open up opportunities to analyse intracellular mechanobiology. Here, we identify a programme of forces and changes to the cytoplasmic mechanical properties required for mouse embryo development from fertilization to the first cell division. Injected, fully internalized nanodevices responded to sperm decondensation and recondensation, and subsequent device behaviour suggested a model for pronuclear convergence based on a gradient of effective cytoplasmic stiffness. The nanodevices reported reduced cytoplasmic mechanical activity during chromosome alignment and indicated that cytoplasmic stiffening occurred during embryo elongation, followed by rapid cytoplasmic softening during cytokinesis (cell division). Forces greater than those inside muscle cells were detected within embryos. These results suggest that intracellular forces are part of a concerted programme that is necessary for development at the origin of a new embryonic life.
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Embrión de Mamíferos/citología , Desarrollo Embrionario/fisiología , Animales , Fenómenos Biomecánicos , Femenino , Masculino , Ratones , Análisis de la Célula IndividualAsunto(s)
Epidídimo , Espermatozoides , Animales , Desarrollo Embrionario , Masculino , Ratones , ARNRESUMEN
BACKGROUND: Assist use of inhaled short-acting beta 2 agonists (SABAs) is reportedly effective for preventing shortness of breath on exertion in chronic obstructive pulmonary disease (COPD) patients. However, it is unclear what strategy would be useful for improving physical activity in such patients. The aim is to investigate the effects of assisted use of SABA (procaterol) on physical activity in Japanese COPD patients targeting patient-specific restrictions in daily behavior. METHODS: Fourteen patients with stable COPD (age: 72.1±1.5, %FEV1: 55.6±4.5%) were asked to inhale 20⯵g of procaterol 15â¯minutes before patient-specific daily physical activity that had been identified as limited by a questionnaire and document their usage in a diary. Physical activity was measured using a triaxial accelerometer and the results were collected every month for 2 months. In the first month, a clinician assessed whether inhalation of SABA was appropriate based on a usage diary and coached patients to conduct adequate assist use of SABA for limited physical activity. RESULTS: The strategy significantly improved the physical activity level, assessed using the values of the metabolic equivalents (METs) multiplied by physical activity endurance, at ≥3.0 METs (p<0.05), and physical activity endurance at ≥2.5 and ≥3.0 METs, (p<0.05, p<0.05, respectively). The degree of improvement of physical activity level was significantly positively correlated with the baseline %FVC and %FEV1 (p<0.05, p<0.05, respectively). CONCLUSIONS: Assist use of SABA targeting patient-specific restrictions, particularly when better lung function is still preserved, could be a useful approach for improving physical activity in patients with COPD.
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Agonistas de Receptores Adrenérgicos beta 2/administración & dosificación , Ejercicio Físico , Tutoría , Procaterol/administración & dosificación , Enfermedad Pulmonar Obstructiva Crónica/fisiopatología , Enfermedad Pulmonar Obstructiva Crónica/terapia , Administración por Inhalación , Anciano , Pueblo Asiatico , Disnea/etiología , Disnea/prevención & control , Femenino , Humanos , Masculino , Resistencia Física , Enfermedad Pulmonar Obstructiva Crónica/complicaciones , Resultado del TratamientoRESUMEN
Bacterial artificial chromosomes (BACs) offer a means of manipulating gene expression and tagging gene products in the mammalian genome without the need to alter endogenous gene structure and risk deleterious phenotypic consequences. However, for a BAC clone to be useful for such purposes it must be shown to contain all the regulatory elements required for normal gene expression and allow phenotypic rescue in the absence of an endogenous gene. Here, we report identification of a functional BAC containing Gadd45g, a gene implicated in DNA repair, DNA demethylation and testis determination in mice and exhibiting a broad pattern of embryonic expression. Mouse fetuses lacking the endogenous Gadd45g gene undergo normal testis development in the presence of the Gadd45g BAC transgene. Moreover, a survey of embryonic Gadd45g expression from the BAC reveals that all reported sites of expression are maintained. This functional BAC can now be used for subsequent manipulation of the Gadd45g gene with the confidence that regulatory elements required for embryonic expression, including testis determination, are present. We describe the generation and characterisation of a Gadd45g-mCherry fluorescent reporter exhibiting strong expression in developing gonads and neural tissue, recapitulating endogenous gene expression, as evidence of this.
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Cromosomas Artificiales Bacterianos , Regulación del Desarrollo de la Expresión Génica , Ingeniería Genética , Péptidos y Proteínas de Señalización Intracelular/genética , Secuencias Reguladoras de Ácidos Nucleicos , Testículo/crecimiento & desarrollo , Transgenes , Animales , Masculino , Ratones , Ratones Transgénicos , Testículo/metabolismo , Proteinas GADD45RESUMEN
Multiple applications of genome editing by CRISPR-Cas9 necessitate stringent regulation and Cas9 variants have accordingly been generated whose activity responds to small ligands, temperature or light. However, these approaches are often impracticable, for example in clinical therapeutic genome editing in situ or gene drives in which environmentally-compatible control is paramount. With this in mind, we have developed heritable Cas9-mediated mammalian genome editing that is acutely controlled by the cheap lysine derivative, Lys(Boc) (BOC). Genetic code expansion permitted non-physiological BOC incorporation such that Cas9 (Cas9BOC) was expressed in a full-length, active form in cultured somatic cells only after BOC exposure. Stringently BOC-dependent, heritable editing of transgenic and native genomic loci occurred when Cas9BOC was expressed at the onset of mouse embryonic development from cRNA or Cas9BOC transgenic females. The tightly controlled Cas9 editing system reported here promises to have broad applications and is a first step towards purposed, spatiotemporal gene drive regulation over large geographical ranges.
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Proteína 9 Asociada a CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Edición Génica/métodos , Animales , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Femenino , Regulación de la Expresión Génica/genética , Código Genético/genética , Vectores Genéticos/genética , Lisina/análogos & derivados , Masculino , Ratones , Ratones Transgénicos/embriología , ARN Guía de Kinetoplastida/genéticaRESUMEN
This corrects the article DOI: 10.1038/srep07621.
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Sperm are highly differentiated and the activities that reprogram them for embryonic development during fertilization have historically been considered unique to the oocyte. We here challenge this view and demonstrate that mouse embryos in the mitotic cell cycle can also directly reprogram sperm for full-term development. Developmentally incompetent haploid embryos (parthenogenotes) injected with sperm developed to produce healthy offspring at up to 24% of control rates, depending when in the embryonic cell cycle injection took place. This implies that most of the first embryonic cell cycle can be bypassed in sperm genome reprogramming for full development. Remodelling of histones and genomic 5'-methylcytosine and 5'-hydroxymethylcytosine following embryo injection were distinct from remodelling in fertilization and the resulting 2-cell embryos consistently possessed abnormal transcriptomes. These studies demonstrate plasticity in the reprogramming of terminally differentiated sperm nuclei and suggest that different epigenetic pathways or kinetics can establish totipotency.
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Reprogramación Celular , Haploidia , Mitosis/fisiología , Espermatozoides/fisiología , Animales , Desarrollo Embrionario , Femenino , Masculino , Ratones , Partenogénesis , Embarazo , Inyecciones de Esperma Intracitoplasmáticas , CigotoRESUMEN
Following fertilization in mammals, paternal genomic 5-methyl-2'-deoxycytidine (5 mC) content is thought to decrease via oxidation to 5-hydroxymethyl-2'-deoxycytidine (5 hmC). This reciprocal model of demethylation and hydroxymethylation is inferred from indirect, non-quantitative methods. We here report direct quantification of genomic 5 mC and 5 hmC in mouse embryos by small scale liquid chromatographic tandem mass spectrometry (SMM). Profiles of absolute 5 mC levels in embryos produced by in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) were almost identical. By 10 h after fertilization, 5 mC levels had declined by ~40%, consistent with active genomic DNA demethylation. Levels of 5 mC in androgenotes (containing only a paternal genome) and parthenogenotes (containing only a maternal genome) underwent active 5 mC loss in the first 6 h, showing that both parental genomes can undergo demethylation independently. We found no evidence for net loss of 5 mC 10-48 h after fertilization, implying that any passive 'demethylation' following DNA replication was balanced by active 5 mC maintenance methylation. However, levels of 5 mC declined during development after 48 h, to 1% (measured as a fraction of G-residues) in blastocysts (~96 h). 5 hmC levels were consistently low (<0.2% of G-residues) throughout development in normal diploid embryos. This work directly quantifies the dynamics of global genomic DNA modification in mouse preimplantation embryos, suggesting that SMM will be applicable to other biomedical situations with limiting sample sizes.
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Blastocisto/metabolismo , Metilación de ADN , Espectrometría de Masas , Animales , Desoxicitidina/análogos & derivados , Embrión no Mamífero , Femenino , Fertilización In Vitro , Meiosis/genética , Ratones , Mitosis/genética , Oocitos/metabolismo , Inyecciones de Esperma IntracitoplasmáticasRESUMEN
Mammalian genomes can be edited by injecting pronuclear embryos with Cas9 cRNA and guide RNA (gRNA) but it is unknown whether editing can also occur during the onset of embryonic development, prior to pronuclear embryogenesis. We here report Cas9-mediated editing during sperm-induced meiotic exit and the initiation of development. Injection of unfertilized, mouse metaphase II (mII) oocytes with Cas9 cRNA, gRNA and sperm enabled efficient editing of transgenic and native alleles. Pre-loading oocytes with Cas9 increased sensitivity to gRNA ~100-fold. Paternal allelic editing occurred as an early event: single embryo genome analysis revealed editing within 3 h of sperm injection, coinciding with sperm chromatin decondensation during the gamete-to-embryo transition but prior to pronucleus formation. Maternal alleles underwent editing after the first round of DNA replication, resulting in mosaicism. Asymmetric editing of maternal and paternal alleles suggests a novel strategy for discriminatory targeting of parental genomes.
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Ingeniería Genética/métodos , Genoma , Meiosis , Oocitos/metabolismo , Animales , Replicación del ADN/genética , Ratones , Ratones Transgénicos , Inyecciones de Esperma Intracitoplasmáticas/métodosRESUMEN
The renin-angiotensin system (RAS), including angiotensin II (Ang II), plays an important role in the regulation of blood pressure and body fluid balance. Consequently, the RAS has emerged as a key target for treatment of kidney and cardiovascular disease. In a search for bioactive peptides using an antibody against the N-terminal portion of Ang II, we identified and characterized a novel angiotensin-related peptide from human urine as a major molecular form. We named the peptide Big angiotensin-25 (Bang-25) because it consists of 25 amino acids with a glycosyl chain and added cysteine. Bang-25 is rapidly cleaved by chymase to Ang II, but is resistant to cleavage by renin. The peptide is abundant in human urine and is present in a wide range of organs and tissues. In particular, immunostaining of Bang-25 in the kidney is specifically localized to podocytes. Although the physiological function of Bang-25 remains uncertain, our findings suggest it is processed from angiotensinogen and may represent an alternative, renin-independent path for Ang II synthesis in tissue.
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Angiotensina II/metabolismo , Angiotensina II/orina , Secuencia de Aminoácidos , Glicosilación , Humanos , Datos de Secuencia Molecular , Péptidos/metabolismo , Péptidos/orina , Distribución TisularRESUMEN
It has been suggested that proangiotensin-12 (proang-12), a novel angiotensin peptide recently discovered in rat tissues, may function as a component of the tissue renin-angiotensin system (RAS). To investigate the role of proang-12 in the production of angiotensin II (Ang II), we measured its plasma and tissue concentrations in Wistar-Kyoto (WKY) and spontaneously hypertensive (SHR) rats, with and without RAS inhibition. The 15-week-old male WKY and SHR rats were left untreated or were treated for 7 days with 30 mg kg(-1) per day losartan, an angiotensin receptor blocker, or with 20 mg kg(-1) per day imidapril, an angiotensin-converting enzyme (ACE) inhibitor. Both treatments increased renin activity and the concentrations of angiotensin I (Ang I) and Ang II in the plasma of WKY and SHR rats, but neither affected plasma proang-12 levels. In contrast to the comparatively low level of proang-12 seen in plasma, cardiac and renal levels of proang-12 were higher than those of Ang I and Ang II. In addition, despite activation of the RAS in the systemic circulation, tissue concentrations of proang-12 were significantly reduced following treatment with losartan or imidapril. Similar reductions were also observed in the tissue concentrations of Ang II in both strains, without a reduction in Ang I. These results suggest that tissue concentrations of proang-12 and Ang II are regulated independently of the systemic RAS in WKY and SHR rats, which is consistent with the notion that proang-12 is a component of only the tissue RAS.
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Angiotensinógeno/metabolismo , Fragmentos de Péptidos/metabolismo , Sistema Renina-Angiotensina/efectos de los fármacos , Angiotensina I/sangre , Angiotensina I/metabolismo , Angiotensina II/sangre , Angiotensina II/metabolismo , Bloqueadores del Receptor Tipo 1 de Angiotensina II/farmacología , Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Angiotensinógeno/sangre , Animales , Presión Sanguínea/efectos de los fármacos , Peso Corporal/efectos de los fármacos , Imidazolidinas/farmacología , Riñón/metabolismo , Losartán/farmacología , Masculino , Miocardio/metabolismo , Tamaño de los Órganos/efectos de los fármacos , Fragmentos de Péptidos/sangre , Radioinmunoensayo , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas WKY , Renina/sangreRESUMEN
Successful brain development requires tight regulation of sequential symmetric and asymmetric cell division. Although Pax6 is known to exert multiple roles in the developing nervous system, its role in the regulation of cell division is unknown. Here, we demonstrate profound alterations in the orientation and mode of cell division in the cerebral cortex of mice deficient in Pax6 function (Pax6(Sey/Sey)) or after acute induced deletion of Pax6. Live imaging revealed an increase in non-vertical cellular cleavage planes, resulting in an increased number of progenitors with unequal inheritance of the apical membrane domain and adherens junctions in the absence of Pax6 function. This phenotype appears to be mediated by the direct Pax6 target Spag5, a microtubule-associated protein, reduced levels of which result in the replication of the Pax6 phenotype of altered cell division orientation. In addition, lack of Pax6 also results in premature delamination of progenitor cells from the apical surface due to an overall decrease in proteins mediating anchoring at the ventricular surface. Moreover, continuous long-term imaging in vitro revealed that Pax6-deficient progenitors generate daughter cells with asymmetric fates at higher frequencies. These data demonstrate a cell-autonomous role for Pax6 in regulating the mode of cell division independently of apicobasal polarity and cell-cell interactions. Taken together, our work reveals several direct effects that the transcription factor Pax6 has on the machinery that mediates the orientation and mode of cell division.
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División Celular/fisiología , Polaridad Celular/fisiología , Corteza Cerebral/citología , Corteza Cerebral/embriología , Proteínas del Ojo/metabolismo , Proteínas de Homeodominio/metabolismo , Factores de Transcripción Paired Box/metabolismo , Proteínas Represoras/metabolismo , Células Madre/fisiología , Análisis de Varianza , Animales , Proteínas de Ciclo Celular , Inmunoprecipitación de Cromatina , Cartilla de ADN/genética , Electroporación , Femenino , Inmunohistoquímica , Luciferasas , Ratones , Factor de Transcripción PAX6 , Reacción en Cadena de la Polimerasa/métodos , EmbarazoRESUMEN
The regeneration of lens tissue from the iris of newts has become a classical model of developmental plasticity, although little is known about the corresponding plasticity of the mammalian iris. We here demonstrate and characterize multipotent cells within the iris pigment epithelium (IPE) of postnatal and adult rodents. Acutely-isolated IPE cells were morphologically homogeneous and highly pigmented, but some produced neurospheres which expressed markers characteristic of neural stem/progenitor cells. Stem/progenitor cell markers were also expressed in the IPE in vivo both neonatally and into adulthood. Inner and outer IPE layers differentially expressed Nestin (Nes) in a manner suggesting that they respectively shared origins with neural retina (NR) and pigmented epithelial (RPE) layers. Transgenic marking enabled the enrichment of Nes-expressing IPE cells ex vivo, revealing a pronounced capacity to form neurospheres and differentiate into photoreceptor cells. IPE cells that did not express Nes were less able to form neurospheres, but a subset initiated the expression of pan-neural markers in primary adherent culture. These data collectively suggest that discrete populations of highly-pigmented cells with heterogeneous developmental potencies exist postnatally within the IPE, and that some of them are able to differentiate into multiple neuronal cell types.
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Diferenciación Celular/fisiología , Iris/citología , Células Madre Multipotentes/citología , Epitelio Pigmentado Ocular/citología , Animales , Bromodesoxiuridina , Cartilla de ADN , Inmunohistoquímica , Proteínas de Filamentos Intermediarios/metabolismo , Iris/metabolismo , Ratones , Ratones Endogámicos C57BL , Proteínas del Tejido Nervioso/metabolismo , Nestina , Epitelio Pigmentado Ocular/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The remarkable capability of planarian regeneration is mediated by a group of adult stem cells referred to as neoblasts. Although these cells possess many unique cytological characteristics (e.g. they are X-ray sensitive and contain chromatoid bodies), it has been difficult to isolate them after cell dissociation. This is one of the major reasons why planarian regenerative mechanisms have remained elusive for a long time. Here, we describe a new method to isolate the planarian adult stem cells as X-ray-sensitive cell populations by fluorescence-activated cell sorting (FACS). Dissociated cells from whole planarians were labeled with fluorescent dyes prior to fractionation by FACS. We compared the FACS profiles from X-ray-irradiated and non-irradiated planarians, and thereby found two cell fractions which contained X-ray-sensitive cells. These fractions, designated X1 and X2, were subjected to electron microscopic morphological analysis. We concluded that X-ray-sensitive cells in both fractions possessed typical stem cell morphology: an ovoid shape with a large nucleus and scant cytoplasm, and chromatoid bodies in the cytoplasm. This method of isolating X-ray-sensitive cells using FACS may provide a key tool for advancing our understanding of the stem cell system in planarians.