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Neurotransmitters are involved in functions related to signaling, stress response, and pathological disorder development, and thus, their real-time monitoring at the site of production is important for observing the changes related to these disorders. Here, we demonstrate the first time-dependent quantification of dopamine in the brains of live zebrafish embryos using electrochemically pretreated carbon fiber microelectrodes (CFMEs) utilizing differential pulse voltammetry as the measurement technique. The pretreatment of the CFMEs in 0.1 M NaOH held at a potential of +1.0 V for 600 s improves the sensitivity toward dopamine and allows for reliable measurements in low ionic strength media. We demonstrate the measurement of extracellular dopamine concentrations in the zebrafish brain during late embryogenesis. The extracellular dopamine concentration in the tectum of zebrafish varies between 200 and 400 nM. The conventional pharmacological manipulation of neurotransmitter levels in the brain demonstrates the selective detection of dopamine at the implantation site. Exposure to the dopamine transporter inhibitor nomifensine induces an increase in extracellular dopamine from 201.9 (±34.9) nM to 352.2 (±20.0) nM, while exposure to the norepinephrine transporter inhibitor desipramine does not lead to a significant modulation of the measured signal. Furthermore, we report the quantitative assessment of the catecholamine stress response of embryos to tricaine, an anesthetic frequently used in zebrafish assays. Exposure to tricaine induces a short-lived increase in brain dopamine from 198.6 (±15.7) nM to a maximum of 278.8 (±14.0) nM. Thus, in vivo electrochemistry can detect real-time changes in zebrafish neurochemical physiology resulting from drug exposure.
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BACKGROUND: Development of the vertebrate intestinal epithelial stem cell niche begins during embryogenesis but maturation occurs postembryonic. The intestinal mammalian crypt contains stem cells interspersed by secretory cells that play a role in regulation of proliferation. Epithelial cells are specified as either secretory or enterocytes as they migrate up the villi in mammals or fold in zebrafish. Zebrafish forms a functional intestine by the end of embryogenesis but takes another 4 weeks to develop the adult proliferation pattern. RESULTS: We characterize development of the intestinal epithelial stem cell niche during the postembryonic period. During the first 2-weeks postembryogenesis, different groups of epithelial cells sequentially proceed through one or two cell cycles, appear to become quiescent, and remain at the interfold base. The third week begins asymmetric divisions with proliferative progeny moving up the folds. Apoptotic cells are not observed at the fold tip until the end of the fourth week. Secretory cells intersperse among interfold base proliferative cells, increasing in number during the third and fourth weeks with a coincident change in proliferation pattern. CONCLUSIONS: Zebrafish postembryonic intestinal epithelial development consists of 2 weeks of slow proliferation followed by 2 weeks of metamorphosis to the adult structure. Developmental Dynamics 2019. © 2019 Wiley Periodicals, Inc.
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Mucosa Intestinal/citología , Intestinos/citología , Nicho de Células Madre/fisiología , Pez Cebra/embriología , Animales , Proliferación Celular/fisiología , Regulación del Desarrollo de la Expresión GénicaRESUMEN
The intestinal epithelium has constant turnover throughout the life of the organ, with apoptosis of cells at the tips of folds or villi releasing cells into the lumen. Due to constant turnover, epithelial cells need to be constantly replaced. Epithelial cells are supplied by stem cell niches that form at the base of the interfold space (zebrafish) and crypts (birds and mammals). Within the adult stem cell niche of mammals, secretory cells such as Paneth and goblet cells play a role in modulation of proliferation and stem cell activity, producing asymmetric divisions. Progeny of asymmetric divisions move up the fold or villi, giving rise to all of the epithelial cell types. Although much is known about function and organization of the adult intestinal stem cell niche, less is understood about regulation within the immature stem cell compartment. Following smooth muscle formation, the intestinal epithelium folds and proliferation becomes restricted to the interfold base. Symmetric divisions continue in the developing interfold niche until stem cell progeny begin asymmetric divisions, producing progeny that migrate up the developing folds. Proliferative progeny from the developing stem cell niche begin migrating out of the niche during the third week post-embryogenesis (zebrafish) or during the postnatal period (mammals). Regulation and organization of epithelial proliferation in the immature stem cell niche may be regulated by signals comparable to the adult niche. Here we identify a novel subset of secretory cells associated with the developing stem cell niche that receive Notch signaling (referred to as NRSCs). Inhibition of the embryonic NRSCs between 74 hpf to 120 hpf increases epithelial proliferation as well as EGF and IGF signaling. Inhibition of post-embryonic NRSCs (6 hpf to 12 dpf) also increases epithelial proliferation and expression level of Wnt target genes. We conclude that NRSCs play a role in modulation of epithelial proliferation through repression of signaling pathways that drive proliferation during both embryogenesis and the post embryonic period.
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Proliferación Celular/fisiología , Mucosa Intestinal/metabolismo , Nicho de Células Madre/fisiología , Animales , Apoptosis/fisiología , Diferenciación Celular/fisiología , Desarrollo Embrionario/fisiología , Células Epiteliales/metabolismo , Regulación del Desarrollo de la Expresión Génica/genética , Intestinos/embriología , Células de Paneth/metabolismo , Receptores Notch/metabolismo , Transducción de Señal/fisiología , Células Madre/metabolismo , Pez Cebra/embriologíaRESUMEN
Various parameters can influence the toxic response to silver nanoparticles (Ag NPs), including the size and surface properties, as well as the exposure environment and the biological site of action. Herein, we assess the intestinal toxicity of three different sizes (10, 40, and 100â¯nm) of Ag NPs in embryonic zebrafish, and describe the relationship between the properties and behavior of Ag NPs in the exposure medium, and induction of lethal and sublethal effects. We find that the composition of the medium and the size contribute to differential NPs agglomeration, release of Ag ions, and subsequent effects during exposure. The exposure medium causes dramatic reduction in silver dissolution due to the presence of salts and divalent cations, which limits the lethal potential of silver ions. Lethality is observed primarily for embryos exposed to medium sized Ag NPs (40â¯nm), but not to the supernatant originated from particles, which suggests that the exposure to particulate silver is the main cause of mortality. On the other hand, the exposure to 10â¯nm and 100â¯nm NPs, as well as Ag ions, only causes sublethal developmental defects in skeletal muscles and intestine, and induces a nitric oxide imbalance.
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Embrión no Mamífero/efectos de los fármacos , Nanopartículas del Metal/toxicidad , Plata/toxicidad , Pez Cebra/crecimiento & desarrollo , Animales , Relación Dosis-Respuesta a Droga , Intestinos/efectos de los fármacos , Intestinos/embriología , Nanopartículas del Metal/química , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/embriología , Tamaño de la Partícula , Plata/química , Propiedades de Superficie , Análisis de Supervivencia , Pruebas de ToxicidadRESUMEN
Nitric oxide (NO) is an important signaling molecule that has been implicated in a variety of physiological and pathophysiological processes in living organisms. NO plays an important role in embryonic development in vertebrates and has been reported to influence early organ development and plasticity. Quantifying NO in single embryos and their developing organs is challenging because of the small size of the embryos, the low dynamically changing concentration and the short life-time of NO. Here, we measured the distribution of NO in the intestine of live zebrafish (Danio rerio) embryos in physiological conditions and under the influence of therapeutic agents. NO measurements were performed using a miniaturized electrochemical sensor fabricated on a single carbon fiber (CF) which enables quantitative real time in vivo monitoring, and by fluorescence imaging using the 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM-DA) dye. NO production was detected in the middle segment the intestine at a level of 3.78 (±0.64) µM, and at lower levels in the anterior and posterior segments, 1.08 (±0.22) and 1.00 (±0.41) µM respectively. In the presence of resveratrol and rosuvastatin, the intestinal NO concentration decreased by 87% and 84%, demonstrating a downregulating effect. These results indicate the presence of variable micromolar concentrations of NO along the intestine of zebrafish embryos and demonstrate the usefulness of CF microelectrodes to measure quantitatively the NO release at the level of a single organ in individual zebrafish embryos. This work provides a unique approach to study in real time the modulatory role of NO in vivo and contributes to further understanding of the molecular basis of embryonic development for developmental biology and drug screening applications.
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Técnicas Electroquímicas , Intestinos/química , Intestinos/embriología , Óxido Nítrico/análisis , Pez Cebra/embriología , Animales , Electrodos , Óxido Nítrico/metabolismo , Factores de TiempoRESUMEN
Chemical mechanical planarization (CMP) is a widely used technique for the manufacturing of integrated circuit chips in the semiconductor industry. The process generates large amounts of waste containing engineered particles, chemical additives, and chemo-mechanically removed compounds. The environmental and health effects associated with the release of CMP materials are largely unknown and have recently become of significant concern. Using a zebrafish embryo assay, we established toxicity profiles of individual CMP particle abrasives (SiO2 and CeO2), chemical additives (hydrogen peroxide, proline, glycine, nicotinic acid, and benzotriazole), as well as three model representative slurries and their resulting waste. These materials were characterized before and after use in a typical CMP process in order to assess changes that may affect their toxicological profile and alter their surface chemistry due to polishing. Toxicity outcome in zebrafish is discussed in relation with the physicochemical characteristics of the abrasive particles and with the type and concentration profile of the slurry components pre and post-polishing, as well as the interactions between particle abrasives and additives. This work provides toxicological information of realistic CMP slurries and their polishing waste, and can be used as a guideline to predict the impact of these materials in the environment.
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Cerio/toxicidad , Embrión no Mamífero/efectos de los fármacos , Residuos Industriales/efectos adversos , Semiconductores , Dióxido de Silicio/toxicidad , Pez Cebra/embriología , Animales , Cerio/química , Exposición a Riesgos Ambientales/efectos adversos , Peróxido de Hidrógeno/química , Peróxido de Hidrógeno/toxicidad , Dióxido de Silicio/químicaRESUMEN
Nanoparticle (NP) surface coatings are known to influence the toxicity of engineered nanomaterials. This work examines the effect of glycine functionalization on silica NPs and investigates changes in viability and developmental defects in the organs of zebrafish embryos upon exposure. Silica NPs and glycine-functionalized silica NPs are synthesized and characterized. Exposure of zebrafish embryos to glycine-silica NPs affects the mortality percentage in a similar manner to soluble glycine. Developmental defects are observed in embryos exposed to soluble glycine, glycine-silica NPs, or silica NPs in comparison with the unexposed embryos. The damage is localized in the brain, heart, and liver of zebrafish embryos. These observations suggest a complex mechanism of toxicity, with glycine maintaining its toxic activity even when covalently bound on silica surface. Our results illustrate that surface modification of non-lethal particles can create different toxicity outcomes in the organs of exposed zebrafish embryos.
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Embrión no Mamífero/efectos de los fármacos , Glicina/toxicidad , Nanopartículas/toxicidad , Dióxido de Silicio/toxicidad , Contaminantes Químicos del Agua/toxicidad , Pez Cebra/embriología , Animales , Tamaño de la Partícula , Pruebas de ToxicidadRESUMEN
The zebrafish (Danio rerio) is frequently being used to investigate the genetics of human diseases as well as resulting pathologies. Ease of both forward and reverse genetic manipulation along with conservation of vertebrate organ systems and disease causing genes has made this system a popular model. Many techniques have been developed to manipulate the genome of zebrafish producing mutants in a vast array of genes. While genetic manipulation of zebrafish has progressed, proteomics have been under-utilized. This review highlights studies that have already been performed using proteomic techniques and as well as our initial proteomic work comparing changes to the proteome between the ascl1a-/- and WT intestine.
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Genoma/fisiología , Mucosa Intestinal/metabolismo , Espectrometría de Masas/métodos , Proteómica/métodos , Proteínas de Pez Cebra/metabolismo , Pez Cebra/metabolismo , Animales , Humanos , Mutación , Pez Cebra/genética , Proteínas de Pez Cebra/genéticaRESUMEN
The increased use of engineered nanoparticles (NPs) in manufacturing and consumer products raises concerns about the potential environmental and health implications on the ecosystem and living organisms. Organs initially and more heavily affected by environmental NPs exposure in whole organisms are the skin and digestive system. We investigate the toxic effect of two types of NPs, nickel (Ni) and copper oxide (CuO), on the physiology of the intestine of a living aquatic system, zebrafish embryos. Embryos were exposed to a range of Ni and CuO NP concentrations at different stages of embryonic development. We use changes in the physiological serotonin (5HT) concentrations, determined electrochemically with carbon fiber microelectrodes inserted in the live embryo, to assess this organ dysfunction due to NP exposure. We find that exposure to both Ni and CuO NPs induces changes in the physiological 5HT concentration that varies with the type, exposure period and concentration of NPs, as well as with the developmental stage during which the embryo is exposed. These data suggest that exposure to NPs might alter development and physiological processes in living organisms and provide evidence of the effect of NPs on the physiology of the intestine.
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Cerium oxide nanoparticles or nanoceria are emerging as a new and promising class of nanoparticle technology for biomedical applications. The safe implementation of these particles in clinical applications requires evaluation of their redox properties and reactivity that might cause neurotoxic effects by interacting with redox components of the physiological environment. We report in vitro and in vivo studies to evaluate the impact of nanoceria exposure on serotonin (5-HT), an important neurotransmitter that plays a critical role in various physiological processes including motility and secretion in the digestive system. In vitro studies of 5-HT in the presence of nanoceria using spectroscopic, electrochemical and surface characterization methods demonstrate that nanoceria interacts with 5-HT and forms a surface adsorbed 5-HT-nanoceria complex. Further in vivo studies in live zebrafish embryos indicate depletion of the 5-HT level in the intestine for exposure periods longer than three days. Intestinal 5-HT was assessed quantitatively in live embryos using implantable carbon fiber microelectrodes and the results were compared to immunohistochemistry of the dissected intestine. 20 and 50 ppm nanoparticle exposure decreased the 5-HT level to 20.5 (±1.3) and 5.3 (±1.5) nM respectively as compared to 30.8 (±3.4) nM for unexposed control embryos. The results suggest that internalized nanoceria particles can concentrate 5-HT at the nanoparticle accumulation site depleting it from the surrounding tissue. This finding might have long term implications in the neurophysiology and functional development of organisms exposed to these particles through intended or unintended exposure.
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Nanoparticle (NP) exposure may induce oxidative stress through generation of reactive oxygen and nitrogen species, which can lead to cellular and tissue damage. The digestive system is one of the initial organs affected by NP exposure. Here, it is demonstrated that exposure to metal oxide NPs induces differential changes in zebrafish intestinal NO concentrations. Intestinal NO concentrations are quantified electrochemically with a carbon fiber microelectrode inserted in the intestine of live embryos. Specificity of the electrochemical signals is demonstrated by NO-specific pharmacological manipulations and the results are correlated with the 4,5-diaminofluorescein-diacetate (DAF-FM-DA). NPs are demonstrated to either induce or reduce physiological NO levels depending on their redox reactivity, type and dose. NO level is altered following exposure of zebrafish embryos to CuO and CeO2 NPs at various stages and concentrations. CuO NPs increase NO concentration, suggesting an intestinal oxidative damage. In contrast, low CeO2 NP concentration exposure significantly reduces NO levels, suggesting NO scavenging activity. However, high concentration exposure results in increased NO. Alterations in NO concentration suggest changes in intestinal physiology and oxidative stress, which will ultimately correspond to NPs toxicity. This work also demonstrates the use of electrochemistry to monitor in vivo changes of NO within zebrafish organs.
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Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Intestinos/enzimología , Nanopartículas del Metal/química , Óxido Nítrico/química , Óxidos/química , Animales , Apoptosis , Electroquímica , Electrodos , Fluoresceína/química , Intestinos/efectos de los fármacos , Metales/química , Microscopía Electrónica de Transmisión , Nanotecnología , Oxidación-Reducción , Estrés Oxidativo , Oxígeno/química , Especies de Nitrógeno Reactivo , Especies Reactivas de Oxígeno , Pez Cebra/embriologíaRESUMEN
The vertebrate intestinal epithelium is renewed continuously from stem cells at the base of the crypt in mammals or base of the fold in fish over the life of the organism. As stem cells divide, newly formed epithelial cells make an initial choice between a secretory or enterocyte fate. This choice has previously been demonstrated to involve Notch signaling as well as Atonal and Her transcription factors in both embryogenesis and adults. Here, we demonstrate that in contrast to the atoh1 in mammals, ascl1a is responsible for formation of secretory cells in zebrafish. ascl1a-/- embryos lack all intestinal epithelial secretory cells and instead differentiate into enterocytes. ascl1a-/- embryos also fail to induce intestinal epithelial expression of deltaD suggesting that ascl1a plays a role in initiation of Notch signaling. Inhibition of Notch signaling increases the number of ascl1a and deltaD expressing intestinal epithelial cells as well as the number of developing secretory cells during two specific time periods: between 30 and 34hpf and again between 64 and 74hpf. Loss of enteroendocrine products results in loss of anterograde motility in ascl1a-/- embryos. 5HT produced by enterochromaffin cells is critical in motility and secretion within the intestine. We find that addition of exogenous 5HT to ascl1a-/- embryos at near physiological levels (measured by differential pulse voltammetry) induce anterograde motility at similar levels to wild type velocity, distance, and frequency. Removal or doubling the concentration of 5HT in WT embryos does not significantly affect anterograde motility, suggesting that the loss of additional enteroendocrine products in ascl1a-/- embryos also contributes to intestinal motility. Thus, zebrafish intestinal epithelial cells appear to have a common secretory progenitor from which all subtypes form. Loss of enteroendocrine cells reveals the critical need for enteroendocrine products in maintenance of normal intestinal motility.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/fisiología , Células Epiteliales/citología , Intestinos/embriología , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/fisiología , Animales , Diferenciación Celular , Linaje de la Célula , Células Enterocromafines/citología , Enterocitos/metabolismo , Motilidad Gastrointestinal , Regulación del Desarrollo de la Expresión Génica , Células Caliciformes/citología , Modelos Biológicos , Modelos Genéticos , Mutación , Transducción de Señal , Factores de Transcripción , Pez CebraRESUMEN
We report the development of a chitosan modified carbon fiber microelectrode for in vivo detection of serotonin. We find that chitosan has the ability to reject physiological levels of ascorbic acid interferences and facilitate selective and sensitive detection of in vivo levels of serotonin, a common catecholamine neurotransmitter. Presence of chitosan on the microelectrode surface was investigated using scanning electron microscopy (SEM) and cyclic voltammetry (CV). The electrode was characterized using differential pulse voltammetry (DPV). A detection limit of 1.6 nM serotonin with a sensitivity of 5.12 nA/µM, a linear range from 2 to 100 nM and a reproducibility of 6.5% for n=6 electrodes were obtained. Chitosan modified microelectrodes selectively measure serotonin in presence of physiological levels of ascorbic acid. In vivo measurements were performed to measure concentration of serotonin in the live embryonic zebrafish intestine. The sensor quantifies in vivo intestinal levels of serotonin while successfully rejecting ascorbic acid interferences. We demonstrate that chitosan can be used as an effective coating to reject ascorbic acid interferences at carbon fiber microelectrodes, as an alternative to Nafion, and that chitosan modified microelectrodes are reliable tools for in vivo monitoring of changes in neurotransmitter levels.
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Carbono/química , Quitosano/química , Embrión no Mamífero/química , Neurotransmisores/análisis , Serotonina/análisis , Pez Cebra/embriología , Animales , Fibra de Carbono , Electroquímica , MicroelectrodosRESUMEN
We monitored real-time in vivo levels of serotonin release in the digestive system of intact zebrafish embryos during early development (5 days postfertilization, dpf) using differential pulse voltammetry with implanted carbon fiber microelectrodes modified with carbon nanotubes dispersed in nafion. A detection limit of 1 nM, a linear range between 5 and 200 nM, and a sensitivity of 83.65 nA x microM(-1) were recorded. The microelectrodes were implanted at various locations in the intestine of zebrafish embryos. Serotonin levels of up to 29.9 (+/-1.13) nM were measured in vivo in normal physiological conditions. Measurements were performed in intact live embryos without additional perturbation beyond electrode insertion. The sensor was able to quantify pharmacological alterations in serotonin release and provide the longitudinal distribution of this neurotransmitter along the intestine with high spatial resolution. In the presence of fluvoxamine, a selective serotonin reuptake inhibitor (SSRI), concentrations of 54.1 (+/-1.05) nM were recorded while in the presence of p-chloro-phenylalanine (PCPA), a tryptophan hydroxylase inhibitor, the serotonin levels decreased to 7.2 (+/-0.45) nM. The variation of serotonin levels was correlated with immunohistochemical analysis. We have demonstrated the first use of electrochemical microsensors for in vivo monitoring of intestinal serotonin levels in intact zebrafish embryos.
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Electroquímica/métodos , Intestinos/química , Serotonina/análisis , Pez Cebra/embriología , Animales , Intestinos/embriologíaRESUMEN
Metallic nanoparticles such as nickel are used in catalytic sensing, and electronic applications, but health and environmental affects have not been fully investigated. While some metal nanoparticles result in toxicity, it is also important to determine whether nanoparticles of the same metal but of different size and shape changes toxicity. Three different size nickel nanoparticle (Ni NPs) of 30, 60, and 100 nm and larger particle clusters of aggregated 60 nm entities with a dendritic structure were synthesized and exposed to zebrafish embryos assessing mortality and developmental defects. Ni NPs exposure was compared to soluble nickel salts. All three 30, 60, and 100 nm Ni NPs are equal to or less toxic than soluble nickel while dendritic clusters were more toxic. With each Ni NP exposure, thinning of the intestinal epithelium first occurs around the LD10 continuing into the LD50. LD50 exposure also results in skeletal muscle fiber separation. Exposure to soluble nickel does not cause intestinal defects while skeletal muscle separation occurs at concentrations well over LD50. These results suggest that configuration of nanoparticles may affect toxicity more than size and defects from Ni NPs exposure occur by different biological mechanisms than soluble nickel.
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Embrión no Mamífero/anomalías , Embrión no Mamífero/efectos de los fármacos , Nanopartículas/toxicidad , Níquel/toxicidad , Tamaño de la Partícula , Pruebas de Toxicidad , Pez Cebra/embriología , Animales , Tipificación del Cuerpo/efectos de los fármacos , Tracto Gastrointestinal/efectos de los fármacos , Tracto Gastrointestinal/metabolismo , Concentración de Iones de Hidrógeno/efectos de los fármacos , Maxilares/efectos de los fármacos , Maxilares/embriología , Anomalías Maxilomandibulares/embriología , Nanopartículas/ultraestructura , Níquel/química , Solubilidad , Difracción de Rayos XRESUMEN
An advantage in zebrafish is that we can identify spatial and temporal patterns of protein expression using whole-mount immunohistochemistry. To allow primary antibodies to interact with their targets, most tissues must undergo some type of antigen retrieval. Many retrieval techniques have utilized protein-digesting enzymes to access antigens. Here we investigate the use of phospholipase A(2) (PLA(2)) as the sole enzyme for antigen retrieval as well as in combination with low concentrations of proteinase K. Concentrations of proteinase K used with PLA(2) are unable to expose the antigen when used as the sole enzyme. We demonstrate that PLA(2) is useful for both nuclear and cytoplasmic antigens but not for extracellular matrix components.
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Antígenos/análisis , Antígenos/metabolismo , Inmunohistoquímica/métodos , Fosfolipasas A2 Secretoras/metabolismo , Pez Cebra/inmunología , Pez Cebra/metabolismo , Animales , Antígenos/inmunología , Núcleo Celular/química , Núcleo Celular/inmunología , Núcleo Celular/metabolismo , Citoplasma/química , Citoplasma/inmunología , Citoplasma/metabolismo , Endopeptidasa K/metabolismo , Matriz Extracelular/química , Matriz Extracelular/inmunología , Matriz Extracelular/metabolismoRESUMEN
Development of the enteric nervous system is critical for normal functioning of the digestive system. In vertebrates, enteric precursors originate from the neural crest and migrate into the digestive system. Enteric neurons enable the digestive system to sense and respond to local conditions without the need for central nervous system input. Here we describe major steps in differentiation of the zebrafish enteric nervous system. During migration and neural differentiation of enteric precursors, we identify regions of the enteric nervous system in different phases of differentiation. Early in migration, a small group of anterior enteric neurons are first to form. This is followed by an anterior to posterior wave of enteric neural differentiation later in the migratory phase. Enteric precursors continue proliferating and differentiating into the third day of embryogenesis. nNOS neurons form early while serotonin neurons form late toward the end of enteric neural differentiation. Numbers of enteric neurons increase gradually except during periods of circular and longitudinal intestinal smooth muscle differentiation.
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Sistema Nervioso Entérico/citología , Sistema Nervioso Entérico/embriología , Intestinos/citología , Intestinos/embriología , Músculo Liso/citología , Músculo Liso/embriología , Pez Cebra/embriología , Animales , Diferenciación Celular , Movimiento Celular , Proliferación Celular , Embrión no Mamífero/citología , Embrión no Mamífero/embriologíaRESUMEN
Zebrafish meltdown (mlt) mutants develop cystic expansion of the posterior intestine as a result of stromal invasion of nontransformed epithelial cells. Positional cloning identified zebrafish smooth muscle myosin heavy chain (myh11) as the responsible gene. The mlt mutation constitutively activates the Myh11 ATPase, which disrupts smooth muscle cells surrounding the posterior intestine. Adjacent epithelial cells ectopically express metalloproteinases, integrins, and other genes implicated in human cancer cell invasion. Knockdown and pharmacological inhibition of these genes restores intestinal structure in mlt mutants despite persistent smooth muscle defects. These data identify an essential role for smooth muscle signaling in the maintenance of epithelial architecture and support gene expression analyses and other studies that identify a role for stromal genes in cancer cell invasion. Furthermore, they suggest that high-throughput screens to identify regulators of cancer cell invasion may be feasible in zebrafish.
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Intestinos/crecimiento & desarrollo , Cadenas Pesadas de Miosina/genética , Cadenas Pesadas de Miosina/metabolismo , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , ADN/genética , Epitelio/crecimiento & desarrollo , Humanos , Hibridación in Situ , Datos de Secuencia Molecular , Músculo Liso/crecimiento & desarrollo , Músculo Liso/metabolismo , Mutación , Fenotipo , Homología de Secuencia de Aminoácido , Transducción de Señal , Pez Cebra/genética , Pez Cebra/crecimiento & desarrollo , Pez Cebra/metabolismoRESUMEN
Intestinal development in amniotes is driven by interactions between progenitor cells derived from the three primary germ layers. Genetic analyses and gene targeting experiments in zebrafish offer a novel approach to dissect such interactions at a molecular level. Here we show that intestinal anatomy and architecture in zebrafish closely resembles the anatomy and architecture of the mammalian small intestine. The zebrafish intestine is regionalized and the various segments can be identified by epithelial markers whose expression is already segregated at the onset of intestinal differentiation. Differentiation of cells derived from the three primary germ layers begins more or less contemporaneously, and is preceded by a stage in which there is rapid cell proliferation and maturation of epithelial cell polarization. Analysis of zebrafish mutants with altered epithelial survival reveals that seemingly related single gene defects have different effects on epithelial differentiation and smooth muscle and enteric nervous system development.
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Regulación del Desarrollo de la Expresión Génica , Intestinos/embriología , Intestinos/crecimiento & desarrollo , Animales , Antimetabolitos/farmacología , Tipificación del Cuerpo , Bromodesoxiuridina/farmacología , Diferenciación Celular , Proliferación Celular , Sistema Nervioso Entérico/embriología , Sistema Nervioso Entérico/crecimiento & desarrollo , Células Epiteliales/citología , Epitelio/embriología , Epitelio/crecimiento & desarrollo , Femenino , Peroxidasa de Rábano Silvestre/farmacología , Inmunohistoquímica , Hibridación in Situ , Mucosa Intestinal/metabolismo , Masculino , Modelos Biológicos , Músculo Liso/citología , Músculo Liso/metabolismo , Mutación , Neuronas/metabolismo , Fenotipo , ARN/metabolismo , Factores de Tiempo , Pez CebraRESUMEN
Although the development of the digestive system of humans and vertebrate model organisms has been well characterized, relatively little is known about how the zebrafish digestive system forms. We define developmental milestones during organogenesis of the zebrafish digestive tract, liver, and pancreas and identify important differences in the way the digestive endoderm of zebrafish and amniotes is organized. Such differences account for the finding that the zebrafish digestive system is assembled from individual organ anlagen, whereas the digestive anlagen of amniotes arise from a primitive gut tube. Despite differences of organ morphogenesis, conserved molecular programs regulate pharynx, esophagus, liver, and pancreas development in teleosts and mammals. Specifically, we show that zebrafish faust/gata-5 is a functional ortholog of gata-4, a gene that is essential for the formation of the mammalian and avian foregut. Further, extraembryonic gata activity is required for this function in zebrafish as has been shown in other vertebrates. We also show that a loss-of-function mutation that perturbs sonic hedgehog causes defects in the development of the esophagus that parallel those associated with targeted disruption of this gene in mammals. Perturbation of sonic hedgehog also affects zebrafish liver and pancreas development, and these effects occur in a reciprocal fashion, as has been described during mammalian liver and ventral pancreas development. Together, these data define aspects of digestive system development necessary for the characterization of zebrafish mutants. Given the similarities of teleost and mammalian digestive physiology and anatomy, these findings have implications for developmental and evolutionary studies as well as research of human diseases, such as diabetes, liver cirrhosis, and cancer.