Live Zebrafish Larvae Research Articles

Real-time observation of listeria monocytogenes-phagocyte interactions in living zebrafish larvae.

The zebrafish, Danio rerio, has become a popular vertebrate model for the study of infections, mainly because of its excellent optical accessibility at the embryonic and larval stages, when the innate immune system is already effective. We have thus tested the susceptibility of zebrafish larvae to the human pathogen Listeria monocytogenes, a gram-positive, facultative, intracellular bacterium that is known to survive and multiply in professional phagocytes and that causes fatal meningitis and abortions. Intravenous injection of early zebrafish larvae resulted in a progressive and ultimately fatal infection. Blood-borne L. monocytogenes bacteria were quickly trapped and engulfed by macrophages, an event that, for the first time, could be captured in vivo and in real time. Granulocytes also participated in the innate immune response. As in mammals, bacteria could escape the macrophage phagosome in a listeriolysin-dependent manner and accessed the cytosol; this event was critical for bacterial virulence, as listeriolysin-deficient bacteria were completely avirulent. Actin comet tails and protrusions were observed, suggesting cell-to-cell spread; these phenomena also played a role in virulence in zebrafish larvae, as actA-deficient bacteria were attenuated. These results demonstrate the relevance of the genetically tractable and optically accessible zebrafish model for the study of L. monocytogenes pathogenesis and particularly for the dissection of its interactions with phagocytes in vivo, a key factor of L. monocytogenes virulence.

Zebrafish Embryology and Cartilage Staining Protocols for High School Students

The Life Sciences-Howard Hughes Medical Institute Outreach Program at Harvard University supports high school science education by offering an on-campus program for students and their teachers to participate in investigative, hands-on laboratory sessions. The outreach program has recently designed and launched a successful zebrafish embryology protocol that we present here. The main objectives of this protocol are to introduce students to zebrafish as a model research organism and to provide students with direct experience with current techniques used in embryological research. The content of the lab is designed to generate discussions on embryology, genetics, fertilization, natural selection, and animal adaptation. The protocol produces reliable results in a time-efficient manner using a minimum of reagents. The protocol presented here consists of three sections: observations of live zebrafish larvae at different developmental stages, cartilage staining of zebrafish larvae, and a mutant hunt involving identification of two zebrafish mutants (nacre and chokh). Here, we describe the protocol, show the results obtained for each section, and suggest possible alternatives for different lab settings.

A reverse genetic screen in the zebrafish identifies crb2b as a regulator of the glomerular filtration barrier

The glomerular filtration barrier is necessary for the selective passage of low molecular weight waste products and the retention of blood plasma proteins. Damage to the filter results in proteinuria. The filtration barrier is the major pathogenic site in almost all glomerular diseases and its study is therefore of clinical significance. We have taken advantage of the zebrafish pronephros as a system for studying glomerular filtration. In order to identify new regulators of filtration barrier assembly, we have performed a reverse genetic screen in the zebrafish testing a group of genes which are enriched in their expression within the mammalian glomerulus. In this novel screen, we have coupled gene knockdown using morpholinos with a physiological glomerular dye filtration assay to test for selective glomerular permeability in living zebrafish larvae. Screening 20 genes resulted in the identification of ralgps1, rapgef2, rabgef1, and crb2b. The crumbs (crb) genes encode a family of evolutionarily conserved proteins important for apical-basal polarity within epithelia. The crb2b gene is expressed in zebrafish podocytes. Electron microscopic analysis of crb2b morphants reveals a gross disorganization of podocyte foot process architecture and loss of slit diaphragms while overall polarity is maintained. Nephrin, a major component of the slit diaphragm, is apically mis-localized in podocytes from crb2b morphants suggesting that crb2b is required for the proper protein trafficking of Nephrin. This report is the first to show a role for crb function in podocyte differentiation. Furthermore, these results suggest a novel link between epithelial polarization and the maintenance of a functional filtration barrier.

Optical Lock-in Detection (OLID) and OLID-FRET Imaging Microscopy

The success of imaging fluorescent proteins within living cells is limited by the presence of background signals - while some improvements to image contrast can be achieved through background subtraction, these benefits diminish when the background signal varies in time and space. Further this approach does not allow imaging below the background signal, a regime of interest for single molecule biology. We have developed an optical lock-in detection (OLID) approach and associated signal analysis that involve modulating the fluorescence emission of the probe through deterministic, optical control of its fluorescent and non-fluorescent states, and subsequently applying a lock-in detection method to isolate the modulated signal of interest from non-modulated background signals. The lock-in detection method involves pixel-by-pixel based cross-correlation analysis that maps the correlation between the total fluorescence emission within single pixels of an image detected over several cycles of optical switching, and a reference waveform that represents the defined response of the switch probe to the switching cycle. The fundamentally new approach to imaging, which is similar in principle to radar, allows for the selective detection of emission from optical switch probes even in the presence of a larger population of conventional fluorescent probes. We have developed new synthetic and genetically-encoded optical switches for OLID and OLID-FRET imaging in living cells and tissues including nitrospirobenzopyran and naphthozaxine based probes as well as fusion proteins of Dronpa and a new optically switchable mcherry. OLID imaging using these probes is remarkably efficient at imaging specific structures and proteins in high and time varying background signal environments such as in living cells, in culture, in brain slices and in live Xenopus embryos and zebrafish larvae.

Zebrafish development and regeneration: new tools for biomedical research

Basic research in pattern formation is concerned with the generation of phenotypes and tissues. It can therefore lead to new tools for medical research. These include phenotypic screening assays, applications in tissue engineering, as well as general advances in biomedical knowledge. Our aim here is to discuss this emerging field with special reference to tools based on zebrafish developmental biology. We describe phenotypic screening assays being developed in our own and other labs. Our assays involve: (i) systemic or local administration of a test compound or drug to zebrafish in vivo; (ii) the subsequent detection or "readout" of a defined phenotypic change. A positive readout may result from binding of the test compound to a molecular target involved in a developmental pathway. We present preliminary data on assays for compounds that modulate skeletal patterning, bone turnover, immune responses, inflammation and early-life stress. The assays use live zebrafish embryos and larvae as well as adult fish undergoing caudal fin regeneration. We describe proof-of-concept studies on the localised targeting of compounds into regeneration blastemas using microcarriers. Zebrafish are cheaper to maintain than rodents, produce large numbers of transparent eggs, and some zebrafish assays could be scaled-up into medium and high throughput screens. However, advances in automation and imaging are required. Zebrafish cannot replace mammalian models in the drug development pipeline. Nevertheless, they can provide a cost-effective bridge between cell-based assays and mammalian whole-organism models.

In vivo imaging of zebrafish digestive organ function using multiple quenched fluorescent reporters

Optical clarity of larvae makes the zebrafish ideal for real-time analyses of vertebrate organ function through the use of fluorescent reporters of enzymatic activities. A key function of digestive organs is to couple the generation of enzymes with mechanical processes that enable nutrient availability and absorption. However, it has been extremely difficult, and in many cases not possible, to directly observe digestive processes in a live vertebrate. Here we describe a new method to visualize intestinal protein and lipid processing simultaneously in live zebrafish larvae using a quenched fluorescent protein (EnzChek) and phospholipid (PED6). By employing these reagents, we found that wild-type larvae exhibit significant variation in intestinal phospholipase and protease activities within a group but display a strong correlation between the activities within individuals. Furthermore, we found that pancreas function is essential for larval digestive protease activity but not for larval intestinal phospholipase activity. Although fat-free (ffr) mutant larvae were previously described to exhibit impaired lipid processes, we found they also had significantly reduced protease activity. Finally, we selected and evaluated compounds that were previously suggested to have altered phospholipase activity and are known or suspected to have inflammatory effects in the intestinal tract including nonsteroidal anti-inflammatory drugs, and identified a compound that significantly increases intestinal phospholipid processing. Thus the multiple fluorescent reporter-based methodology facilitates the rapid analysis of digestive organ function in live zebrafish larvae.

A high throughput live transparent animal bioassay to identify non-toxic small molecules or genes that regulate vertebrate fat metabolism for obesity drug development

BackgroundThe alarming rise in the obesity epidemic and growing concern for the pathologic consequences of the metabolic syndrome warrant great need for development of obesity-related pharmacotherapeutics. The search for such therapeutics is severely limited by the slow throughput of animal models of obesity. Amenable to placement into a 96 well plate, zebrafish larvae have emerged as one of the highest throughput vertebrate model organisms for performing small molecule screens. A method for visually identifying non-toxic molecular effectors of fat metabolism using a live transparent vertebrate was developed. Given that increased levels of nicotinamide adenine dinucleotide (NAD) via deletion of CD38 have been shown to prevent high fat diet induced obesity in mice in a SIRT-1 dependent fashion we explored the possibility of directly applying NAD to zebrafish.MethodsZebrafish larvae were incubated with daily refreshing of nile red containing media starting from a developmental stage of equivalent fat content among siblings (3 days post-fertilization, dpf) and continuing with daily refreshing until 7 dpf.ResultsPPAR activators, beta-adrenergic agonists, SIRT-1 activators, and nicotinic acid treatment all caused predicted changes in fat, cholesterol, and gene expression consistent with a high degree of evolutionary conservation of fat metabolism signal transduction extending from man to zebrafish larvae. All changes in fat content were visually quantifiable in a relative fashion using live zebrafish larvae nile red fluorescence microscopy. Resveratrol treatment caused the greatest and most consistent loss of fat content. The resveratrol tetramer Vaticanol B caused loss of fat equivalent in potency to resveratrol alone. Significantly, the direct administration of NAD decreased fat content in zebrafish. Results from knockdown of a zebrafish G-PCR ortholog previously determined to decrease fat content in C. elegans support that future GPR142 antagonists may be effective non-toxic anti-obesity therapeutics.ConclusionOwing to the apparently high level of evolutionary conservation of signal transduction pathways regulating lipid metabolism, the zebrafish can be useful for identifying non-toxic small molecules or pharmacological target gene products for developing molecular therapeutics for treating clinical obesity. Our results support the promising potential in applying NAD or resveratrol where the underlying target protein likely involves Sirtuin family member proteins. Furthermore data supports future studies focused on determining whether there is a high concentration window for resveratrol that is effective and non-toxic in high fat obesity murine models.

Early functional development of interneurons in the zebrafish olfactory bulb

In the adult olfactory bulb (OB) of vertebrates, local GABAergic interneurons (INs) mediate recurrent and lateral inhibition between the principal neurons of the OB, the mitral cells (MCs), and play pivotal roles in the processing of odor-evoked activity patterns. The properties and functions of INs in the developing OB are, however, not well understood. We studied the functional development of INs in the OB of living zebrafish larvae 3-6 days postfertilization using anatomical techniques and in-vivo two-photon Ca2+ imaging. We identified MCs and INs by cell-type-specific expression of transgenic fluorescent markers and found that the IN:MC ratio was lower than in the adult fish. Moreover, the fraction of INs responding with Ca2+ signals to a set of natural odors was substantially lower than in adults. Odors of different chemical classes evoked overlapping patterns of Ca2+ signals that were concentrated in the center of the IN layer. The GABA(A) receptor agonists GABA and muscimol strongly suppressed odor responses, whereas a GABA(A) receptor antagonist enhanced responses and altered the spatial distribution of odor-evoked activity. These results indicate that IN odor responses at early developmental stages are sparse and exhibit no obvious chemotopic organization. Nevertheless, GABAergic signaling is already inhibitory at early stages of OB development and strongly influences odor-evoked activity patterns. Hence, INs already participate in the processing of odor information at very early stages of OB development even though the majority of INs emerge only at later stages.

A GFP-based genetic screen reveals mutations that disrupt the architecture of the zebrafish retinotectal projection

The retinotectal projection is a premier model system for the investigation of molecular mechanisms that underlie axon pathfinding and map formation. Other important features, such as the laminar targeting of retinal axons, the control of axon fasciculation and the intrinsic organization of the tectal neuropil, have been less accessible to investigation. In order to visualize these processes in vivo, we generated a transgenic zebrafish line expressing membrane-targeted GFP under control of the brn3c promoter/enhancer. The GFP reporter labels a distinct subset of retinal ganglion cells (RGCs), which project mainly into one of the four retinorecipient layers of the tectum and into a small subset of the extratectal arborization fields. In this transgenic line, we carried out an ENU-mutagenesis screen by scoring live zebrafish larvae for anatomical phenotypes. Thirteen recessive mutations in 12 genes were discovered. In one mutant, ddl, the majority of RGCs fail to differentiate. Three of the mutations, vrt, late and tard, delay the orderly ingrowth of retinal axons into the tectum. Two alleles of drg disrupt the layer-specific targeting of retinal axons. Three genes, fuzz, beyo and brek, are required for confinement of the tectal neuropil. Fasciculation within the optic tract and adhesion within the tectal neuropil are regulated by vrt, coma, bluk, clew and blin. The mutated genes are predicted to encode molecules essential for building the intricate neural architecture of the visual system.

In vivo imaging of synapse formation on a growing dendritic arbor

The form of a neuron's dendritic arbor determines the set of axons with which it may form synaptic contacts, thus establishing connectivity within neural circuits. However, the dynamic relationship between dendrite growth and synaptogenesis is not well understood. To observe both processes simultaneously, we performed long-term imaging of non-spiny dendritic arbors expressing a fluorescent postsynaptic marker protein as they arborized within the optic tectum of live zebrafish larvae. Our results indicate that almost all synapses form initially on newly extended dendritic filopodia. A fraction of these nascent synapses are maintained, which in turn stabilizes the subset of filopodia on which they form. Stabilized filopodia mature into dendritic branches, and successive iterations of this process result in growth and branching of the arbor. These findings support a 'synaptotropic model' in which synapse formation can direct dendrite arborization.