Diese Arbeit wurde von theNIH grantNS042626 unterstützt. Wir danken Kurt Thorn und UCSF Nikon Imaging Center für die Unterstützung bei Bildgebung.

1. Vorbereitung des Zebrafisch-Embryos <ol><li> Zwei Tage vor der Elektroporation eingerichtet Paarung Käfigen von Wildtyp-Fisch mit Trennwänden zu trennen Männchen von Weibchen. </li><li> Einen Tag vor der Elektroporation, ziehen Sie den Teilern in Paarung Käfigen vom Vortag. </li><li> Sammeln Embryonen und inkubieren 50 befruchteten Embryonen in 30 ml embryonalen Medium mit 0,003% Phenylthioharnstoff (PTU) bei 28,5 ° C. </li><li> Am Tag der Elektroporation, dechorionate die Embryonen von Hand mit feinen Pinzetten (Inox 5, Dumont Electronic, Schweiz), wenn die Embryonen der Entwicklungsphase 22hpf erreichen. Übertragen Sie anschließend die Embryonen bis 10ml Elektroporation Ringer-3 (180 mM NaCl, 5 mM KCl, 1,8 mM CaCl2, 5 mM Hepes pH 7,2) mit 0,003% PTU. </li><li> Add 420 ul Tricaine Lager (4mg/ml), um die 10ml Elektroporation Ringer, die Embryonen zu betäuben. </li></ol> 2. Vorbereitung für die Elektroporation <ol><li> Glass Injektionsnadeln wurden von Kapillaren (1,2 mm OD, 0,9 mm ID, mit Filament) auf einem Flaming-Brown P897 Puller (Sutter Instruments, Novato, CA, USA) gezogen. Die Nadelspitze wurde mit einer Pinzette gebrochen, um ein scharfes Ende bekommen, wie in Abbildung 1D gezeigt. Der Durchmesser der Spitze sollte etwa 10 um und der Kegelwinkel sollte etwa 30 Grad. </li><li> Bereiten Sie 0,8% und 1% niedriger Schmelzpunkt Agarose (Shelton Scientific, Inc. Katalog # IB70050) in Elektroporation Ringerlösung. Aliquot der Agarose-Lösung in 2 ml Reaktionsgefäße. Halten Sie die Aliquots in einem Heizblock bei ~ 37 ° C. </li><li> Die Gal4/UAS System wird verwendet, um die Genexpression in neuronalen Vorläuferzellen elektroporiert Embryonen zu fahren. Zwei Plasmide verwendet: Plasmid EF1α-GFF-PT2KXIG, in denen Gal4FF durch die allgegenwärtige Promoter EF1α angetrieben wird, und Plasmid-UAS-E1B-EGFP-PT2KXIG. </li><li> Bereiten EF1α-GFF-PT2KXIG und UAS-E1B-EGFP-PT2KXIG Plasmid-DNA mit GenEluteTM HP Plasmid Midiprep Kit (Sigma). </li><li> Die Plasmide werden dann von Natriumacetat / Ethanolfällung auf die Endkonzentration von 1-2 ug / ul in 10 mM Tris · Cl (pH 8,5) konzentriert. </li><li> Mischen Sie die beiden Plasmide und verdünnt mit Nuklease-freiem H2O auf eine Endkonzentration von 500 ng / ul für jedes Plasmid. Die Zugabe von Phenolrot (verwässert 1 / 10 Volumen), um die Lösung für die Visualisierung der Lieferung bevorzugt. Halten Sie die DNA-Lösung auf Eis. </li><li> Kurz vor der Elektroporation, tendieren, die Injektionsnadel mit 2 ul DNA-Lösung und stellen Sie das Injektionsvolumen zu 2 nl. </li></ol> 3. Elektroporation <ol><li> Ein stero Dissektionsmikroskop (Zeiss Stemi 2000 mit einer maximalen Vergrößerung von 50x), einem Luftdruck-Injektor (Narishige IM 300 Mikroinjektor), zwei Mikromanipulatoren (WPI M3301R, World Precision Instruments, Sarasota, FL, USA), und einer elektrischen Stimulator (SD9 Rechteckimpuls Stimulator, Grass Tlefactor, West Warwick, Rhode Island, USA) werden wie in Abbildung 1A dargestellt eingestellt. </li><li> Um die Embryonen, tauchen 2 Embryonen in 1% niedrig schmelzenden Agarose montieren. Dann ziehen Sie sofort den Embryonen aus der Agarose mit einer Glaspipette. </li><li> Legen Sie die Embryonen auf eine umgekehrte Kunststoff Petrischalendeckel in einzelne Tropfen aus Agarose. Richten Sie die Embryonen vorsichtig mit einem Faser-Sonde an eine dorsale-up-Position vor dem Agarose verfestigt, so dass das Vorderhirn zugänglich sowohl die Elektroden und die Mikroinjektionsnadel (Abbildung 1B) ist. Embryonen müssen in einzelnen Agarose Tropfen montiert werden. </li><li> Bearbeiten Sie die Elektroden mit der linken Mikromanipulator. Legen Sie die Elektroden in die Agarose, wie in Abbildung 1B gezeigt. Benutzerdefinierte Platin-Iridium parallel bipolaren Elektroden 125 um im Durchmesser und Abstand 500 pm voneinander entfernt sind für die Elektroporation (FHC, Katalog # PBSA0575) verwendet. </li><li> Legen Sie die Mikroinjektion Nadel in das Vorderhirn Herzkammer und spritzen 4NL DNA-Mix, fließt der rote Flüssigkeit und die Schwellung der Ventrikel beobachtet werden können. </li><li> Sofort wurde eine 28,5 V Puls Dauer von 1 Millisekunde manuell. Dann umpolen und gelten noch 28,5 V Puls Dauer von 1 Millisekunde. Dies wird in bilateralen Mosaik Kennzeichnung des Vorderhirns neuralen Vorläuferzellen führen. </li><li> Nach der Elektroporation von 10 Embryonen, fügen 5ml embryonalen Medium, um die Embryonen Abdeckung und ziehen Sie die Agarose mit einem mikrochirurgischen Messer, um die Embryonen freizugeben. </li><li> Übertragen Sie die Elektroporation Embryonen in ein frisches Gericht aus 30 ml embryonalen Medium mit 0,003% PTU und inkubieren bei 28,5 ° C. </li></ol> 4. Montage-und Zeitraffer-Live-Imaging <ol><li> Rund 4 Stunden nach der Elektroporation wird die EGFP-Signal zu beobachten, wenn sie unter einer epifluoreszenten Präpariermikroskop angesehen. </li><li> Fünf Stunden nach der Elektroporation, wählen Sie die Embryonen mit hoch Mosaik, aber starke EGFP-Expression im Vorderhirn Region. </li><li> Übertragen Sie die ausgewählten Embryonen embryonale 10ml Medium mit 0,003% PTU. </li><li> Add 420 ul Tricaine Lager (4mg/ml), um die 10ml embryonalen mittleren to betäuben die Embryonen. </li><li> Um die Embryonen mount, tauchen ein Embryo in 0,8% Agarose für niedrigen Schmelzpunkt. Dann ziehen Sie sofort den Embryo aus der Agarose mit einer Glaspipette und Ort der Embryo in der Mitte einer 35-mm Glasboden Kulturschale (MatTek Unternehmen, Part # P35GC-1,5-10-C, <a href="http://www.glass-bottom-dishes.com/">www.glass-bottom-dishes.com</a> ) . </li><li> Richten Sie die Embryonen vorsichtig mit einem Faser-Sonde an eine dorsale-up-Position vor dem Agarose erstarrt. </li><li> Die Schale richtig von der Temperatur gesteuert Phase des konfokalen Mikroskops, wie in Abbildung 1C (Wir verwenden eine Nikon C1 spektralen konfokalen Mikroskop mit bis rechts Ziele) dargestellt. Stellen Sie die Temperatur auf 28,5 ° C. </li><li> Add 3ml 28,5 ° C vorgeheizten embryonalen Medium mit 0,003% PTU um den Embryo zu decken. Der Embryo ist nun bereit für die Bildgebung. </li><li> Führen Sie Zeitraffer-Live-Bildgebung mit einem festgelegten Intervall mit einem Wasser-Tauchen Ziel. </li></ol> 5. Repräsentative Ergebnisse In Abbildung 2 sind Bilder eines Zeitraffer-konfokale Bildgebung leben des Embryos von EF1α-GFF + UAS-E1B-EGFP bei 22 hpf elektroporiert ausgewählt. <img src="/files/ftp_upload/2594/2594fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Apparatus Set-up. (A) Die Versammlung der Elektroporation Ausstattung: the Grass SD9 Rechteckimpuls Stimulator (i), der linke Mikromanipulator (ii) die Elektroden (iii), das Zeiss-Binokular (iv), der Injektionsnadel (v), das Recht Mikromanipulator (vi) und die Narishige IM 300 Mikroinjektor (vii). (B) Die relative Position der Elektroden (i), das Vorderhirn Ventrikels (ii) und der Injektionsnadel (iii). Beachten Sie die rot gefärbten DNA-Lösung in das Vorderhirn Herzkammer injiziert wurde. (C) Die Montage von Live-Aufnahmen auf Nikon C1 konfokalen Mikroskop: der Temperaturregler (i), das Wasser getaucht Ziel len (ii) und die Elektroporation Embryo in Agarose für niedrigen Schmelzpunkt in einem Glasboden Kulturschale (iii) (D embedded ) Bild von einem gezogen und geschnitten Nadel für die Mikroinjektion von DNA. <img src="/files/ftp_upload/2594/2594fig2.jpg" alt="Abbildung 2" /> Abbildung 2. Ausgewählter Bilder eines 18-Stunden-Zeitraffer-konfokale Bildgebung leben des Embryos mit EF1α-GFF + UAS-E1B-EGFP bei 22 hpf elektroporiert. Ventrikel Oberfläche ist auf der Unterseite eines jeden Bildrahmen. Scale-Balken repräsentiert 10 pm.

<ol>
	<li>Temple, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+of+neural+stem+cells.">The development of neural stem cells.</a> Nature. 414, 112-117 (2001).</li><li>G#246;tz, M., Huttner, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cell+biology+of+neurogenesis.">The cell biology of neurogenesis.</a> Nat. Rev. Mol. Cell Biol. 6, 777-788 (2005).</li><li>Cerda, G. A., Thomas, J. E., Allende, M. L., Karlstrom, R. O., Palma, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroporation+of+DNA,+RNA,+and+morpholinos+into+zebrafish+embryos.">Electroporation of DNA, RNA, and morpholinos into zebrafish embryos.</a> Methods. 39, 207-211 (2006).</li><li>Hendricks, M., Jesuthasan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroporation-based+methods+for+in+vivo,+whole+mount+and+primary+culture+analysis+of+zebrafish+brain+development.">Electroporation-based methods for in vivo, whole mount and primary culture analysis of zebrafish brain development.</a> Neural. Dev. 2, 6-6 (2007).</li><li>Tawk, M., Bianco, I. H., Clarke, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Focal+electroporation+in+zebrafish+embryos+and+larvae.">Focal electroporation in zebrafish embryos and larvae.</a> Methods Mol. Biol. 546, 145-151 (2009).</li><li>Tawk, M., Tuil, D., Torrente, Y., Vriz, S., Paulin, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-efficiency+gene+transfer+into+adult+fish:+a+new+tool+to+study+fin+regeneration.">High-efficiency gene transfer into adult fish: a new tool to study fin regeneration.</a> Genesis. 32, 27-31 (2002).</li></ol>

In diesem Video zeigen wir eine Methode zur Zeitraffer-Live-Darstellung von neuralen Vorläuferzellen in einem klonalen Ebene in den Entwicklungsländern Zebrafisch Vorderhirn. Wir testeten und modifiziert das bestehende Elektroporation Protokolle 4-6, um genetisch und Fluoreszenz-Label einzelnen neuronalen Vorläuferzellen. Eine Linie Baum klonal verbundenen Nachkommen Zellen zusammengesetzt ist etabliert, da nur wenige Zellen waren spärlich mit einer relativ geringen Spannung von Elektroporation gekennzeic...

time-lapse live imaging of clonally related neural progenitor cells in the developing zebrafish forebrain

Präzise Muster der Teilung, Migration und Differenzierung neuronaler Vorläuferzellen sind entscheidend für die richtige Entwicklung des Gehirns und die Funktion 1,2. Um zu verstehen, das Verhalten des neuronalen Vorläuferzellen in der komplexen in vivo-Umgebung ist Zeitraffer-Live-Darstellung von neuralen Vorläuferzellen in einem intakten Gehirn kritisch erforderlich. In diesem Video, nutzen wir die einzigartigen Eigenschaften von Zebrafisch-Embryonen zur Entwicklung des Vorderhirns neuralen Vorläuferzellen in vivo sichtbar zu machen. Wir verwenden Elektroporation, genetisch und dünn Label einzelnen neuronalen Vorläuferzellen. Kurz gesagt, Konstrukte kodierenden DNA Fluoreszenzmarker wurden in das Vorderhirn Ventrikel von 22 Stunden nach der Befruchtung (HPF) Zebrafischembryonen und elektrische Impulse wurden sofort injiziert. Sechs Stunden später wurden die Elektroporation Zebrafischembryonen mit niedrigem Schmelzpunkt Agarose in Glasboden Kulturschalen montiert. Fluoreszenzmarkierten neuralen Vorläuferzellen wurden dann für 36 Stunden mit festen Abständen unter einem konfokalen Mikroskop mit Wasser getaucht Objektiv abgebildet. Das vorliegende Verfahren bietet eine Möglichkeit, Einblicke in die in vivo Entwicklung von Vorderhirn neuralen Vorläuferzellen zu gewinnen und kann auch auf andere Teile des zentralen Nervensystems des Zebrafisch-Embryos angewendet werden.

Watch this Scientific Journal Video about Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain at JoVE.com

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

Die vorliegende Video zeigt eine Methode, die nutzt die Kombination von Elektroporation und konfokale Mikroskopie, um Live-Aufnahmen zu den einzelnen neuronalen Vorläuferzellen im sich entwickelnden Zebrafisch Vorderhirn führen.<em&gt; In-vivo-</em&gt; Analyse der Entwicklung des Vorderhirns neuralen Vorläuferzellen in einem klonalen Ebene kann auf diese Weise erreicht werden.

Zeitraffer-Live-Imaging von klonal verbundenen neuronalen Vorläuferzellen in der Entwicklung von Zebrafisch Vorderhirnneuronen

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To ...

time-lapse-live-imaging-clonally-related-neural-progenitor-cells

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

10.2K Aufrufe.  University of California San Francisco. Die vorliegende Video zeigt eine Methode, die nutzt die Kombination von Elektroporation und konfokale Mikroskopie, um Live-Aufnahmen zu den einzelnen neuronalen Vorläuferzellen im sich entwickelnden Zebrafisch Vorderhirn führen. In-vivo- Analyse der Entwicklung des Vorderhirns neuralen Vorläuferzellen in einem klonalen Ebene kann auf diese Weise erreicht werden.

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

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.

Key to understanding the morphogenetic processes that shape the early embryo is the ability to image cells at high resolution. We describe here a technique for labeling single cells or small clusters of cells in whole zebrafish embryos with membrane-targeted Green Fluorescent Protein.

Labeling und Imaging Cells in der Zebrafisch Hinterhirn

Key to understanding the morphogenetic processes that shape the early vertebrate embryo is the ability to image cells at high resolution. In zebrafish ...

Forschung

Neurowissenschaften

Multi-Photon Time Lapse Imaging to Visualize Development in Real-time: Visualization of Migrating Neural Crest Cells in Zebrafish Embryos

Congenital eye and craniofacial anomalies reflect disruptions in the neural crest, a transient population of migratory stem cells that give rise to numerous cell types throughout the body. Understanding the biology of the neural crest has been limited, reflecting a lack of genetically tractable models that can be studied in vivo and in real-time. Zebrafish is a particularly important developmental model for studying migratory cell populations, such as the neural crest. To examine neural crest migration into the developing eye, a combination of the advanced optical techniques of laser scanning microscopy with long wavelength multi-photon fluorescence excitation was implemented to capture high-resolution, three-dimensional, real-time videos of the developing eye in transgenic zebrafish embryos, namely Tg(sox10:EGFP) and Tg(foxd3:GFP), as sox10 and foxd3 have been shown in numerous animal models to regulate early neural crest differentiation and likely represent markers for neural crest cells. Multi-photon time-lapse imaging was used to discern the behavior and migratory patterns of two neural crest cell populations contributing to early eye development. This protocol provides information for generating time-lapse videos during zebrafish neural crest migration, as an example, and can be further applied to visualize the early development of many structures in the zebrafish and other model organisms.

A combination of the advanced optical techniques of laser scanning microscopy with long wavelength multi-photon fluorescence excitation was implemented to capture high-resolution, three-dimensional, real-time imaging of neural crest migration in Tg(sox10:EGFP) and Tg(foxd3:GFP) zebrafish embryos.

Multi-Photonen-Time Lapse Imaging Entwicklung in Echtzeit zu visualisieren: Visualisierung der Migration Neuralleisten-Zellen in den Zebrafish Embryos

Congenital eye and craniofacial anomalies reflect disruptions in the neural crest, a transient population of migratory stem cells that give rise to ...

Entwicklungsbiologie

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 &mu;m.

In this video, we demonstrate a method by which to analyze the developing vertebrate brain in live zebrafish embryos at single cell resolution by confocal microscopy. This includes the method by which we inject the single-cell zebrafish embryo and subsequently mount and image the developing brain.

Live-Imaging der Zebrafisch embryonalen Gehirn durch konfokale Mikroskopie

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 ...

Biologie

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.

This protocol describes imaging of individual neurons or neural crest cells in living zebrafish embryos. This method is used to examine cellular behaviors and actin localization using fluorescence confocal time-lapse microscopy.

Live-Imaging von Zellbewegung und Aktin-Zytoskelett von einzelnen Neuronen und Zellen der Neuralleiste in Zebrafisch-Embryos

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily ...

Visualizing the Developing Brain in Living Zebrafish using Brainbow and Time-lapse Confocal Imaging

Development of the vertebrate nervous system requires a precise coordination of complex cellular behaviors and interactions. The use of high resolution in vivo imaging techniques can provide a clear window into these processes in the living organism. For example, dividing cells and their progeny can be followed in real time as the nervous system forms. In recent years, technical advances in multicolor techniques have expanded the types of questions that can be investigated. The multicolor Brainbow approach can be used to not only distinguish among like cells, but also to color-code multiple different clones of related cells that each derive from one progenitor cell. This allows for a multiplex lineage analysis of many different clones and their behaviors simultaneously during development. Here we describe a technique for using time-lapse confocal microscopy to visualize large numbers of multicolor Brainbow-labeled cells over real time within the developing zebrafish nervous system. This is particularly useful for following cellular interactions among like cells, which are difficult to label differentially using traditional promoter-driven colors. Our approach can be used for tracking lineage relationships among multiple different clones simultaneously. The large datasets generated using this technique provide rich information that can be compared quantitatively across genetic or pharmacological manipulations. Ultimately the results generated can help to answer systematic questions about how the nervous system develops.

In vivo imaging is a powerful tool that can be used to investigate the cellular mechanisms underlying nervous system development. Here we describe a technique for using time-lapse confocal microscopy to visualize large numbers of multicolor Brainbow-labeled cells in real time within the developing zebrafish nervous system.

Visualisierung des sich entwickelnden Gehirns bei lebenden Zebrafischen mit Brainbow und Zeitraffer-Konfokalbildnis

Development of the vertebrate nervous system requires a precise coordination of complex cellular behaviors and interactions. The use of high resolution in ...

Multicolor Time-lapse Imaging of Transgenic Zebrafish: Visualizing Retinal Stem Cells Activated by Targeted Neuronal Cell Ablation

High-resolution time-lapse imaging of living zebrafish larvae can be utilized to visualize how biological processes unfold (for review see 1). Compound transgenic fish which express different fluorescent reporters in neighboring cell types provide a means of following cellular interactions 2 and/or tissue-level responses to experimental manipulations over time. In this video, we demonstrate methods that can be used for imaging multiple transgenically labeled cell types serially in individual fish over time courses that can span from minutes to several days. The techniques described are applicable to any study seeking to correlate the "behavior" of neighboring cells types over time, including: 1) serial 'catch and release' methods for imaging a large number of fish over successive days, 2) simplified approaches for separating fluorophores with overlapping excitation/emission profiles (e.g., GFP and YFP), 3) use of hypopigmented mutant lines to extend the time window available for high-resolution imaging into late larval stages of development, 4) use of membrane targeted fluorescent reporters to reveal fine morphological detail of individual cells as well as cellular details in larger populations of cells, and 5) a previously described method for chemically-induced ablation of transgenically targeted cell types; i.e., nitroreductase (NTR) mediated conversion of prodrug substrates, such as metronidazole (MTZ), to cytotoxic derivatives 3,5.
As an example of these approaches, we will visualize the ablation and regeneration of a subtype of retinal bipolar neuron within individual fish over several days. Simultaneously we will monitor several other retinal cell types, including neighboring non-targeted bipolar cells and potential degeneration-stimulated retinal stem cells (i.e., M&#971;ller glia). This strategy is being applied in our lab to characterize cell- and tissue-level (e.g., stem cell niche) responses to the selective loss and regeneration of targeted neuronal cell types.

In this video, techniques for multicolor confocal time-lapse imaging and targeted cell ablation are provided. Time-lapse imaging is used to monitor the behavior of multiple cell types of interest in vivo. Targeted cell ablation facilitates the study neural circuit function and cell-specific neuronal regeneration paradigms.

Multicolor Zeitraffer-Imaging transgener Zebrafische: Visualisierung von Retinal Stammzellen durch gezielte Nervenzellen Ablation Activated

High-resolution time-lapse imaging of living zebrafish larvae can be utilized to visualize how biological processes unfold (for review see 1). Compound ...

Live Imaging Followed by Single Cell Tracking to Monitor Cell Biology and the Lineage Progression of Multiple Neural Populations

Understanding the mechanisms that control critical biological events of neural cell populations, such as proliferation, differentiation, or cell fate decisions, will be crucial to design therapeutic strategies for many diseases affecting the nervous system. Current methods to track cell populations rely on their final outcomes in still images and they generally fail to provide sufficient temporal resolution to identify behavioral features in single cells. Moreover, variations in cell death, behavioral heterogeneity within a cell population, dilution, spreading, or the low efficiency of the markers used to analyze cells are all important handicaps that will lead to incomplete or incorrect read-outs of the results. Conversely, performing live imaging and single cell tracking under appropriate conditions represents a powerful tool to monitor each of these events. Here, a time-lapse video-microscopy protocol, followed by post-processing, is described to track neural populations with single cell resolution, employing specific software. The methods described enable researchers to address essential questions regarding the cell biology and lineage progression of distinct neural populations.

A robust protocol to monitor neural populations by time-lapse video-microscopy followed by software-based post-processing is described. This method represents a powerful tool to identify biological events in a selected population during live imaging experiments.

Leben Sie Imaging, gefolgt von einzelligen Tracking Monitor Zellbiologie und die Linie Fortschreiten der mehrere neuronalen Populationen

Understanding the mechanisms that control critical biological events of neural cell populations, such as proliferation, differentiation, or cell fate ...

Analyzing Craniofacial Morphogenesis in Zebrafish Using 4D Confocal Microscopy

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical clarity and amenability to genetic manipulation, the zebrafish embryo has become a popular model organism with which to perform time-lapse analysis of morphogenesis in living embryos. Confocal imaging of a live zebrafish embryo requires that a tissue of interest is persistently labeled with a fluorescent marker, such as a transgene or injected dye. The process demands that the embryo is anesthetized and held in place in such a way that healthy development proceeds normally. Parameters for imaging must be set to account for three-dimensional growth and to balance the demands of resolving individual cells while getting quick snapshots of development. Our results demonstrate the ability to perform long-term in vivo imaging of fluorescence-labeled zebrafish embryos and to detect varied tissue behaviors in the cranial neural crest that cause craniofacial abnormalities. Developmental delays caused by anesthesia and mounting are minimal, and embryos are unharmed by the process. Time-lapse imaged embryos can be returned to liquid medium and subsequently imaged or fixed at later points in development. With an increasing abundance of transgenic zebrafish lines and well-characterized fate mapping and transplantation techniques, imaging any desired tissue is possible. As such, time-lapse in vivo imaging combines powerfully with zebrafish genetic methods, including analyses of mutant and microinjected embryos.

Time-lapse confocal imaging is a powerful technique useful for characterizing embryonic development. Here, we describe the methodology and characterize craniofacial morphogenesis in wild-type, as well as pdgfra, smad5, and smo mutant embryos.

Analyse Craniofacial Morphogenese in Zebrafisch Mit 4D konfokale Mikroskopie

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical ...

4-Dimensional Imaging of Zebrafish Optic Cup Morphogenesis

Visual system function requires the establishment of precise tissue and organ structures. In the vertebrate eye, structural defects are a common cause of visual impairment, yet mechanisms of eye morphogenesis are still poorly understood. The basic organization of the embryonic eye is conserved throughout vertebrates, thus live imaging of zebrafish embryos has become a powerful approach to directly observe eye development at real time under normal and pathological conditions. Dynamic cell processes including movements, morphologies, interactions, division, and death can be visualized in the embryo. We have developed methods for uniform labeling of subcellular structures and timelapse confocal microscopy of early eye development in zebrafish. This protocol outlines the method of generating capped mRNA for injection into the 1-cell zebrafish embryo, mounting embryos at optic vesicle stage (~12 hours post fertilization, hpf), and performing multi-dimensional timelapse imaging of optic cup morphogenesis on a laser scanning confocal microscope, such that multiple datasets are acquired sequentially in the same imaging session. Such an approach yields data that can be used for a variety of purposes, including cell tracking, volume measurements, three-dimensional (3D) rendering, and visualization. Our approaches allow us to pinpoint the cellular and molecular mechanisms driving optic cup development, in both wild type and genetic mutant conditions. These methods can be employed directly by other groups or adapted to visualize many additional aspects of zebrafish eye development.

This protocol describes an approach for in toto labeling and multidimensional imaging of zebrafish early eye development. We describe labeling, embedding, and four dimensional (4D) imaging using laser scanning confocal microscopy, and considerations for optimizing acquisition of datasets for dissecting mechanisms of optic cup morphogenesis.

4-dimensionale Bildgebung der Zebrafisch-Optikbecher-Morphogenese

Visual system function requires the establishment of precise tissue and organ structures. In the vertebrate eye, structural defects are a common cause of ...

In vivo Imaging of Fully Active Brain Tissue in Awake Zebrafish Larvae and Juveniles by Skull and Skin Removal

Understanding the ephemeral changes that occur during brain development and maturation requires detailed high-resolution imaging in space and time at cellular and subcellular resolution. Advances in molecular and imaging technologies have allowed us to gain numerous detailed insights into cellular and molecular mechanisms of brain development in the transparent zebrafish embryo. Recently, processes of refinement of neuronal connectivity that occur at later larval stages several weeks after fertilization, which are for example control of social behavior, decision making or motivation-driven behavior, have moved into focus of research. At these stages, pigmentation of the zebrafish skin interferes with light penetration into brain tissue, and solutions for embryonic stages, e.g., pharmacological inhibition of pigmentation, are not feasible anymore.
Therefore, a minimally invasive surgical solution for microscopy access to the brain of awake zebrafish is provided that is derived from electrophysiological&#160;approaches. In teleosts, skin and soft skull cartilage can be carefully removed by micro-peeling these layers, exposing underlying neurons and axonal tracts without damage. This allows for recording neuronal morphology, including synaptic structures and their molecular contents, and the observation of physiological changes such as Ca2+ transients or intracellular transport events. In addition, interrogation of these processes by means of pharmacological inhibition or optogenetic manipulation is feasible. This brain exposure approach provides information about structural and physiological changes in neurons as well as the correlation and interdependence of these events in live brain tissue in the range of minutes or hours. The technique is suitable for in vivo brain imaging of zebrafish larvae up to 30 days post fertilization, the latest developmental stage tested so far. It, thus, provides access to such important questions as synaptic refinement and scaling, axonal and dendritic transport, synaptic targeting of cytoskeletal cargo or local activity-dependent expression. Therefore, a broad use for this mounting and imaging approach can be anticipated.

Here we present a method to image the zebrafish embryonic brain in vivo upto larval and juvenile stages. This microinvasive procedure, adapted from electrophysiological approaches, provides access to cellular and subcellular details of mature neuron and can be combined with optogenetics and neuropharmacological studies for characterizing brain function and drug intervention.

In vivo Bildgebung von vollaktivem Hirngewebe bei wachen Zebrafischlarven und Jungtieren durch Schädel- und Hautentfernung

Understanding the ephemeral changes that occur during brain development and maturation requires detailed high-resolution imaging in space and time at ...