Kenneth N. Wallace

Differentiation of the zebrafish enteric nervous system and intestinal smooth muscle.

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. genesis 46:484–498, 2008.

Chitosan coated carbon fiber microelectrode for selective in vivo detection of neurotransmitters in live zebrafish embryos

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.

Electrochemical Quantification of Serotonin in the Live Embryonic Zebrafish Intestine

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.

Comparative Evaluation of Intestinal Nitric Oxide in Embryonic Zebrafish Exposed to Metal Oxide Nanoparticles

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.

Effect of cerium oxide nanoparticles on intestinal serotonin in zebrafish

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.

Loss of ascl1a prevents secretory cell differentiation within the zebrafish intestinal epithelium resulting in a loss of distal intestinal motility

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.

Alterations of intestinal serotonin following nanoparticle exposure in embryonic zebrafish

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.

Developmental toxicity of glycine-coated silica nanoparticles in embryonic zebrafish

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.

Mass Spectrometry for Proteomics-Based Investigation Using the Zebrafish Vertebrate Model System

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.

Interaction, transformation and toxicity assessment of particles and additives used in the semiconducting industry

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.