Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function^1,2. To understand the behavior of neural progenitor cells in the complex in vivo environment, time-lapse live imaging of neural progenitor cells in an intact brain is critically required. In this video, we exploit the unique features of zebrafish embryos to visualize the development of forebrain neural progenitor cells in vivo. We use electroporation to genetically and sparsely label individual neural progenitor cells. Briefly, DNA constructs coding for fluorescent markers were injected into the forebrain ventricle of 22 hours post fertilization (hpf) zebrafish embryos and electric pulses were delivered immediately. Six hours later, the electroporated zebrafish embryos were mounted with low melting point agarose in glass bottom culture dishes. Fluorescently labeled neural progenitor cells were then imaged for 36hours with fixed intervals under a confocal microscope using water dipping objective lens. The present method provides a way to gain insights into the in vivo development of forebrain neural progenitor cells and can be applied to other parts of the central nervous system of the zebrafish embryo.Download video file.^{(49M, mov)}     

Live Imaging of Cell Motility and Actin Cytoskeleton of Individual Neurons and Neural Crest Cells in Zebrafish Embryos

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily accessible at all stages of development. Moreover, their optical clarity allows high resolution imaging of cell and molecular dynamics in the natural environment of the intact embryo. We are using a live imaging approach to analyze cell behaviors during neural crest cell migration and the outgrowth and guidance of neuronal axons.Live imaging is particularly useful for understanding mechanisms that regulate cell motility processes. To visualize details of cell motility, such as protrusive activity and molecular dynamics, it is advantageous to label individual cells. In zebrafish, plasmid DNA injection yields a transient mosaic expression pattern and offers distinct benefits over other cell labeling methods. For example, transgenic lines often label entire cell populations and thus may obscure visualization of the fine protrusions (or changes in molecular distribution) in a single cell. In addition, injection of DNA at the one-cell stage is less invasive and more precise than dye injections at later stages.Here we describe a method for labeling individual developing neurons or neural crest cells and imaging their behavior in vivo. We inject plasmid DNA into 1-cell stage embryos, which results in mosaic transgene expression. The vectors contain cell-specific promoters that drive expression of a gene of interest in a subset of sensory neurons or neural crest cells. We provide examples of cells labeled with membrane targeted GFP or with a biosensor probe that allows visualization of F-actin in living cells¹.Erica Andersen, Namrata Asuri, and Matthew Clay contributed equally to this work.Open in a separate windowClick here to view.^{(58M, flv)}     

Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the developing zebrafish brain (Gutzman et al, 2008). Such analysis affords a new understanding of the underlying cell biology required for development of the 3D structure of the vertebrate brain, and significantly increases our ability to study neural tube morphogenesis. The embryonic zebrafish brain is shaped beginning at 18 hours post fertilization (hpf) as the ventricles within the neuroepithelium inflate. By 24 hpf, the initial steps of neural tube morphogenesis are complete. Using the method described here, embryos at the one cell stage are injected with mRNA encoding membrane-targeted green fluorescent protein (memGFP). After injection and incubation, the embryo, now between 18 and 24 hpf, is mounted, inverted, in agarose and imaged by confocal microscopy. Notably, the zebrafish embryo is transparent making it an ideal system for fluorescent imaging. While our analyses have focused on the midbrain-hindbrain boundary and the hindbrain, this method could be extended for analysis of any region in the zebrafish to a depth of 80-100 μm.Open in a separate windowClick here to view.^{(44M, flv)}     

Applicability of Live Cell Imaging of mRNA Expression in Combination with Calcium Imaging for in vitro Studies of Neural Network Activity

The effectiveness of live cell mRNA detection for neurobiological studies was evaluated. We modified the commercial protocol for the use of RNA detection probes (SmartFlareTM, Merck) for primary hippocampal cultures. It was shown that RNA probes could be used both as an independent evaluation system and in combination with Ca²⁺ imaging. The complex of these methods provided the unique possibility of performing simultaneous studies of the functional calcium homeostasis of neuronal-glial networks and differential analysis of the plasticity of cells with different levels of mRNA expression.     

Clock Genes and Behavioral Responses to Light Are Altered in a Mouse Model of Diabetic Retinopathy

There is increasing evidence that melanopsin-expressing ganglion cells (ipRGCs) are altered in retinal pathologies. Using a streptozotocin-induced (STZ) model of diabetes, we investigated the impact of diabetic retinopathy on non-visual functions by analyzing ipRGCs morphology and light-induced c-Fos and Period 1–2 clock genes in the central clock (SCN). The ability of STZ-diabetic mice to entrain to light was challenged by exposure animals to 1) successive light/dark (LD) cycle of decreasing or increasing light intensities during the light phase and 2) 6-h advance of the LD cycle. Our results show that diabetes induces morphological changes of ipRGCs, including soma swelling and dendritic varicosities, with no reduction in their total number, associated with decreased c-Fos and clock genes induction by light in the SCN at 12 weeks post-onset of diabetes. In addition, STZ-diabetic mice exhibited a reduction of overall locomotor activity, a decrease of circadian sensitivity to light at low intensities, and a delay in the time to re-entrain after a phase advance of the LD cycle. These novel findings demonstrate that diabetes alters clock genes and behavioral responses of the circadian timing system to light and suggest that diabetic patients may show an increased propensity for circadian disturbances, in particular when they are exposed to chronobiological challenges.     

Zebrafish as a Model for Human Ciliopathies

Cilia, microtubule-based structures found on the surface of almost all vertebrate cells, play an array of diverse biological functions. Abnormal ciliary axonemal structure and function can result in a class of genetic disorders that are collectively termed ciliopathies. Model organisms,including Chlamydomonas reinhardtii and Caenorhabditis elegans have been widely used to study the complex genetic basis of ciliopathies.Here, we review the advantages of the zebrafish as a vertebrate model for human ciliopathies. We summarize the features of zebrafish cilia, and the major findings and contributions of the zebrafish model in recent studies of human ciliopathies. We also discuss the new genome editing approaches being efficiently used in zebrafish, and the exciting prospects of these approaches in modeling human ciliopathies.     

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of causing systemic disease in both fish and humans. A better understanding of S. iniae disease pathogenesis requires an appropriate model system. The genetic tractability and the optical transparency of the early developmental stages of zebrafish allow for the generation and non-invasive imaging of transgenic lines with fluorescently tagged immune cells. The adaptive immune system is not fully functional until several weeks post fertilization, but zebrafish larvae have a conserved vertebrate innate immune system with both neutrophils and macrophages. Thus, the generation of a larval infection model allows the study of the specific contribution of innate immunity in controlling S. iniae infection.The site of microinjection will determine whether an infection is systemic or initially localized. Here, we present our protocols for otic vesicle injection of zebrafish aged 2-3 days post fertilization as well as our techniques for fluorescent confocal imaging of infection. A localized infection site allows observation of initial microbe invasion, recruitment of host cells and dissemination of infection. Our findings using the zebrafish larval model of S. iniae infection indicate that zebrafish can be used to examine the differing contributions of host neutrophils and macrophages in localized bacterial infections. In addition, we describe how photolabeling of immune cells can be used to track individual host cell fate during the course of infection.     

Design of a Novel Equi-Biaxial Stretcher for Live Cellular and Subcellular Imaging

Cells in the body experience various mechanical stimuli that are often essential to proper cell function. In order to study the effects of mechanical stretch on cell function, several devices have been built to deliver cyclic stretch to cells; however, they are generally not practical for live cell imaging. We introduce a novel device that allows for live cell imaging, using either an upright or inverted microscope, during the delivery of cyclic stretch, which can vary in amplitude and frequency. The device delivers equi-biaxial strain to cells seeded on an elastic membrane via indentation of the membrane. Membrane area strain was calibrated to indenter depth and the device showed repeatable and accurate delivery of strain at the scale of individual cells. At the whole cell level, changes in intracellular calcium were measured at different membrane area strains, and showed an amplitude-dependent response. At the subcellular level, the mitochondrial network was imaged at increasing membrane area strains to demonstrate that stretch can lead to mitochondrial fission in lung fibroblasts. The device is a useful tool for studying transient as well as long-term mechanotransduction as it allows for simultaneous stretching and imaging of live cells in the presence of various chemical stimuli.     

Fluorescent-Based Methods for Gene Knockdown and Functional Cardiac Imaging in Zebrafish

A notable advantage of zebrafish as a model organism is the ease of gene knockdown using morpholino antisense oligonucleotide (MO). However, zebrafish morphants injected with MO for a target protein often show heterogeneous phenotypes, despite controlling the injection volume of the MO solution in all embryos. We developed a method for estimating the quantity of MO injected into each living morphant, based on the co-injection of a control MO labeled with the fluorophore lissamine. By applying this method for knockdown of cardiac troponin T (tnnt2a) in zebrafish, we could efficiently select the partial tnnt2a-depleted zebrafish with a decreased heart rate and impairment of cardiac contraction. To investigate cardiac impairment of the tnnt2a morphant, we performed fluorescent cardiac imaging using Bodipy-ceramide. Cardiac image analysis showed moderate reduction of tnnt2a impaired diastolic distensibility and decreased contraction and relaxation velocities. To the best of our knowledge, this is the first report to analyze the role of tnnt2a in cardiac function in tnnt2a-depleted living animals. Our combinatorial approach can be applied for analyzing the molecular function of any protein associated with human cardiac diseases.     

Labeling and Imaging Cells in the Zebrafish Hindbrain

Key to understanding the morphogenetic processes that shape the early vertebrate embryo is the ability to image cells at high resolution. In zebrafish embryos, injection of plasmid DNA results in mosaic expression, allowing for the visualization of single cells or small clusters of cells ¹ . We describe how injection of plasmid DNA encoding membrane-targeted Green Fluorescent Protein (mGFP) under the control of a ubiquitous promoter can be used for imaging cells undergoing neurulation. Central to this protocol is the methodology for imaging labeled cells at high resolution in sections and also in real time. This protocol entails the injection of mGFP DNA into young zebrafish embryos. Embryos are then processed for vibratome sectioning, antibody labeling and imaging with a confocal microscope. Alternatively, live embryos expressing mGFP can be imaged using time-lapse confocal microscopy. We have previously used this straightforward approach to analyze the cellular behaviors that drive neural tube formation in the hindbrain region of zebrafish embryos ². The fixed preparations allowed for unprecedented visualization of cell shapes and organization in the neural tube while live imaging complemented this approach enabling a better understanding of the cellular dynamics that take place during neurulation.Download video file.^{(105M, mp4)}     

Zebrafish as a Model for Monocarboxyl Transporter 8-Deficiency

Allan-Herndon-Dudley syndrome (AHDS) is a severe psychomotor retardation characterized by neurological impairment and abnormal thyroid hormone (TH) levels. Mutations in the TH transporter, monocarboxylate transporter 8 (MCT8), are associated with AHDS. MCT8 knock-out mice exhibit impaired TH levels; however, they lack neurological defects. Here, the zebrafish mct8 gene and promoter were isolated, and mct8 promoter-driven transgenic lines were used to show that, similar to humans, mct8 is primarily expressed in the nervous and vascular systems. Morpholino-based knockdown and rescue experiments revealed that MCT8 is strictly required for neural development in the brain and spinal cord. This study shows that MCT8 is a crucial regulator during embryonic development and establishes the first vertebrate model for MCT8 deficiency that exhibits a neurological phenotype.     

Identification of Selected Gamma-Ray Induced Deficiencies in Zebrafish Using Multiplex Polymerase Chain Reaction

The ease with which mutations can be generated in zebrafish makes this vertebrate an important resource for developmental genetics and genome studies. We have developed a PCR-based screening method that allows the efficient identification of gamma-ray induced deficiencies targeted to selected sequences. We describe three mutants characteristic of our findings and show that these mutations include deletions and translocations that can affect as much as 1% of the genome. These deficiencies provide a basis for analyzing the functions of cloned zebrafish genes using noncomplementation screens for point mutations induced by high-efficiency chemical mutagenesis.     

A Mathematical Model of Lymphangiogenesis in a Zebrafish Embryo

The lymphatic system of a vertebrate is important in health and diseases. We propose a novel mathematical model to elucidate the lymphangiogenic processes in zebrafish embryos. Specifically, we are interested in the period when lymphatic endothelial cells (LECs) exit the posterior cardinal vein and migrate to the horizontal myoseptum of a zebrafish embryo. We wonder whether vascular endothelial growth factor C (VEGFC) is a morphogen and a chemotactic factor for these LECs. The model considers the interstitial flow driving convection, the reactive transport of VEGFC, and the changing dynamics of the extracellular matrix in the embryo. Simulations of the model illustrate that VEGFC behaves very differently in diffusion and convection-dominant scenarios. In the former case, it must bind to the matrix to establish a functional morphogen gradient. In the latter case, the opposite is true and the pressure field is the key determinant of what VEGFC may do to the LECs. Degradation of collagen I, a matrix component, by matrix metallopeptidase 2 controls the spatiotemporal dynamics of VEGFC. It controls whether diffusion or convection is dominant in the embryo; it can create channels of abundant VEGFC and scarce collagen I to facilitate lymphangiogenesis; when collagen I is insufficient, VEGFC cannot influence the LECs at all. We predict that VEGFC is a morphogen for the migrating LECs, but it is not a chemotactic factor for them.     

Live Imaging of Innate Immune and Preneoplastic Cell Interactions Using an Inducible Gal4/UAS Expression System in Larval Zebrafish Skin

Here we describe a method to conditionally induce epithelial cell transformation by the use of the 4-Hydroxytamoxifen (4-OHT) inducible KalTA4-ER^T2/UAS expression system¹ in zebrafish larvae, and the subsequent live imaging of innate immune cell interaction with HRAS^G12V expressing skin cells. The KalTA4-ER^T2/UAS system is both inducible and reversible which allows us to induce cell transformation with precise temporal/spatial resolution in vivo. This provides us with a unique opportunity to live image how individual preneoplastic cells interact with host tissues as soon as they emerge, then follow their progression as well as regression. Recent studies in zebrafish larvae have shown a trophic function of innate immunity in the earliest stages of tumorigenesis^2,3. Our inducible system would allow us to live image the onset of cellular transformation and the subsequent host response, which may lead to important insights on the underlying mechanisms for the regulation of oncogenic trophic inflammatory responses. We also discuss how one might adapt our protocol to achieve temporal and spatial control of ectopic gene expression in any tissue of interest.     

Optimization of Whole-Body Zebrafish Sectioning Methods for Mass Spectrometry Imaging

Mass spectrometry imaging (MSI) methods and protocols have become widely adapted to a variety of tissues and species. However, the MSI literature contains minimal information on whole-body cryosection preparation for the zebrafish (ZF; Danio rerio), a model organism routinely used in developmental, toxicity, and carcinogenicity studies. The optimal medium for embedding and cryosectioning a whole organism or soft-tissue specimen for histological examination is a synthetic polymer mixture that is incompatible with MSI as a result of ion suppression. We describe the optimal methods and results for embedding and cryosectioning whole-body ZF for MALDI-MSI. We evaluated 13 distinct embedding media formulations and found a supportive hydrogel with the consistency of cartilage to be the optimal embedding medium. The hydrogel medium does not interfere with MSI data collection, aids in tissue stability, is readily available for purchase, and is easy to prepare and handle during cryosectioning. Additionally, we decreased the matrix cluster interference commonly caused by α-cyano-4-hydroxycinnamic acid by adding ammonium phosphate to the solvent spray solution. The optimized methods developed in our laboratory produced high-quality cryosections, as well as high-quality mass spectral images of sectioned ZF.     

Single-Molecule and Superresolution Imaging in Live Bacteria Cells

Single-molecule imaging enables biophysical measurements devoid of ensemble averaging, gives enhanced spatial resolution beyond the diffraction limit, and permits superresolution reconstructions. Here, single-molecule and superresolution imaging are applied to the study of proteins in live Caulobacter crescentus cells to illustrate the power of these methods in bacterial imaging. Based on these techniques, the diffusion coefficient and dynamics of the histidine protein kinase PleC, the localization behavior of the polar protein PopZ, and the treadmilling behavior and protein superstructure of the structural protein MreB are investigated with sub-40-nm spatial resolution, all in live cells.Since its advent 20 years ago, single-molecule fluorescence imaging has given rise to a host of exciting experiments (Ambrose and Moerner 1991). Beyond enabling fundamental investigations of the physics of emissive molecules, one main advantage of this technique is its use in biologically relevant, live-cell experiments. Optical fluorescence microscopy is an important instrument for cell biology, as light can be used to noninvasively probe a sample with relatively small perturbation of the specimen, enabling dynamical observation of the motions of internal structures in living cells. Single-molecule epifluorescence microscopy extends these capabilities by achieving nanometer-scale resolution, taking advantage of the fact that one can precisely characterize the point spread function (PSF) of a microscope, allowing the center of a distribution, and thus the exact position of an emitter, to be localized with accuracy much better than the diffraction limit itself. This localization accuracy improves beyond the diffraction limit roughly as one over the square root of the number of detected photons (Thompson et al. 2002). Detecting 100 photons from a single, isolated molecule can therefore improve the resolution of an optical measurement from the ∼250-nm diffraction limit down to 25 nm.Single-molecule imaging has been used in the investigation of a number of live-cell samples. In 2000, the lateral heterogeneity of the plasma membrane was investigated by tracing the motion of single dye-labeled lipids in native human airway smooth muscle (HASM) cells (Schütz et al. 2000), and epidermal growth factor (EGF) receptor signaling was explored with a fluorescent protein fusion and a labeled ligand (Sako et al. 2000). Single fluorophore-labeled molecules have subsequently been used in many ways (Moerner 2003), for instance to investigate the effect of varying cholesterol concentration on the mobility of proteins in the plasma membrane of Chinese hamster ovary (CHO) cells (Vrljic et al. 2002; Vrljic et al. 2005) and to explore the real-time dynamic behavior of cell-penetrating-peptide (CPP) molecular transporters on the plasma membrane of CHO cells (Lee et al. 2008). Furthermore, in 2001, Harms et al. characterized the emission of fluorescent proteins in biocompatible environments and noted that the yellow fluorescent protein EYFP was well-suited to single-molecule imaging in cells (Harms et al. 2001). Such fluorescent proteins can be genetically encoded as tags for native proteins in cells; these fusions have been used in many live-cell single-molecule experiments.More recently, single-molecule epifluorescence microscopy has been used to probe the inner workings of live bacteria. The small size of prokaryotic cells makes the optical diffraction limit particularly noticeable, which has stimulated the push toward superlocalization and superresolution to overcome this obstacle. As a result, the nascent field of bacterial structural biology has benefited greatly from single-molecule investigations of proteins in live cells. The overall shapes of such cells can be seen in a standard light microscope, but those interested in probing subcellular details, such as protein structure and localization, have typically had to resort to in vitro characterization combined with extrapolation to the cellular environment, as well as to indirect methods such as biochemical assays. Although cryo-electron microscopy can provide extremely high spatial resolution, fixation or plunge-freezing is essential, and methods for identifying specific proteins out of many are still lacking. As a consequence, bacterial cell biology is an area of study ripe for investigation with direct, noninvasive optical methods of probing position, coupling and structure, with resolution below the standard diffraction limit.Several groups have extended single-molecule imaging techniques to live bacterial samples. In 2004, single PleC proteins were visualized in Caulobacter crescentus cells (Deich et al. 2004), and the behavior of this system is described in more detail later. More recently, Xie and coauthors have used single-molecule fluorescence techniques to study DNA-binding proteins, mRNA, and membrane proteins to provide much insight into the mechanisms of bacterial gene expression; these efforts have been documented in a recent review (Xie et al. 2008). As well, Conley et al. used covalently linked Cy3-Cy5-thiol switchable fluorophores to illuminate the stalks of C. crescentus cells with high resolution (Conley et al. 2008). In this article, we focus on the application of single-molecule imaging and single-molecule-based superresolution imaging to investigate the localization, movement, and structure of three important proteins, PleC, PopZ, and MreB, in live C. crescentus cells.