Die Autoren möchten, um zu bestätigen, dass diese Arbeit wurde von der Natural Sciences and Engineering Research Council of Canada (NSERC) Zuschuss unterstützt.

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Die Autoren haben nichts zu offenbaren.

Dieses Protokoll beschreibt die Verwendung von verschiedenen Arten von Bildmikroskopie Zellteilungen in der Zwischenembryogenese. Insbesondere stellt dieses Protokoll, wie Bild die Aufteilung der Neuroblasten, epidermalen Zellen, die Morphogenese erleichtern können. Zell-Zell-Kommunikation wichtig für die Gewebebildung während metazoan Entwicklung und C. elegans ist ein ausgezeichnetes Modell zur Gewebebildung in vivo zu studieren. Ein Ereignis, das schön schildert das Zusammenspiel von epidermalen...

Zwar gibt es Protokolle, die beschreiben, wie Bild der Zellteilung in der frühen C elegans Embryo, beschreibt dieses Protokoll, wie man Bild der Zellteilung in einem Gewebe während der Embryogenese Mitte. Eine der großen Herausforderungen bei der Abbildung Organismen während der Entwicklung ist ihre Empfindlichkeit gegenüber Phototoxizität. Allerdings hat erhöhte Zugänglichkeit zu drehenden Scheibe konfokalen Mikroskopen oder Wischfeld-Mikroskopen weiter verbreitet Imaging-Anwendungen erlaubt. Beide Systeme verwenden Festkörperlaser und gestreuten Lichts, die Begrenzung der Pegel von UV, das die Organismen ausgesetzt sind. Allerdings kann Weitfeld steht immer noch für die Bildgebung in vivo verwendet werden, insbesondere, wenn sie mit Kameras mit hoher Empfindlichkeit (z. B. EMCCD), Blendensteuerung und Lichtsteuerung (zB LEDs oder verstellbare Quecksilberbirnen) ausgestattet. Dieses Protokoll beschreibt, wie Sie entweder auf der Basis eines konfokalen System oder ein Weitfeld-System, um Bild die Zellteilung in den Entwicklungs C verwenden elegans &lt;/ Em&gt; Embryos. Als wir beispielsweise, wie man Bild Neuroblast Zellteilung zu beschreiben. Neuroblasten kann epidermalen Morphogenese durch chemische oder mechanische Signale zu den darüberliegenden Epidermiszellen zu erleichtern, und liefern ein ausgezeichnetes Beispiel für die Bedeutung der interzellulären Kommunikation bei der Bildung von Geweben. Caenorhabditis elegans ist ein idealer Modellorganismus für die Mikroskopie basierte Studien aufgrund seiner Transparenz und einfache Gewebeorganisation ein. Außerdem C. elegans zugänglich ist genetischen Methoden und RNAi, und da viele der Gene humanen Homologen, kann es verwendet werden, um konservierte Mechanismen zur Gewebebildung 2-5 identifizieren. In C. elegans, die Bildung der Epidermis tritt in der Zwischenembryogenese, wenn der Embryo&gt; 300 Zellen. Epidermalen Morphogenese besteht aus mehreren Hauptphasen, in denen der Embryo wird in eine Schicht aus epidermalen Zellen, eingeschlossen verengen und zu erweitern, um die Transformation embryo aus eiförmigen Form in die längliche Form eines Schnecken 6. Ventrale Gehäuse beschreibt eine der morphogenetischen Ereignisse, als die ventralen epidermalen Zellen wandern in Richtung der ventralen Mittellinie, um die Bauchseite des Embryos (Abbildung 1) zu decken. Zuerst werden zwei Paare von vorderen Führungskante liegt Zellen wandern in Richtung der ventralen Mittellinie, wo sie anhaften und Sicherung mit ihren Nachbarn kontralateralen 6. Dies wird durch die Migration der hinteren Tasche befindet sich Zellen, die keilartige Formen Schaffung einer Bauchtasche 6-7 bilden gefolgt. Der Mechanismus, der die Tasche geschlossen ist nicht gut verstanden. Eine Möglichkeit ist, dass eine supra Aktin-Myosin kontraktile Struktur bindet die Tasche Zellen zusammen in einer Tabaksbeutel wie Mode, ähnlich wie Wundheilungs 8. Interessanterweise wird die Migration einiger der Tasche Zellen durch spezifische Teilmengen von Basisneuroblasten 9 (neuronale Vorläufer, die unter der epidermi gefunden werden vermitteltens, Fig. 1B). Frühere Studien zeigten, dass die Neuroblasten regulieren ventralen epidermalen Zellmigration und ventralen Gehäuse. VAB-1 (Ephrin-Rezeptor) und VAB-2 (Ephrin-Liganden) sind hoch in der Neuroblasten ausgedrückt und voneinander erleichtert das Sortieren der vorderen und hinteren Neuroblasten und Mutationen in vab-1-oder-2 vab Ursache ventralen Gehäuse Phänotypen 10-13. Jedoch Promotor Rettungsexperimente zeigten, daß VAB-1 wird auch in der darüberliegenden Bauch epidermalen Zellen und Rezeptoren für andere Führungssignale von den Neuroblasten sezerniert erforderlich sind in den ventralen Hautzellen 9 ausgedrückt. Obwohl Mutationen in einem dieser Rezeptoren verursachen Bauch Gehäuse Phänotypen, ist es nicht klar, ob Defekte entstehen durch Probleme in der Neuroblast Positionierung oder durch Ausfall der ventralen epidermalen Zellen zu reagieren Führung 14 Queues. Ändern der Teilung der Neuroblasten wiThout die ihre Fähigkeit, Führungssignale absondern könnte Licht auf die Rolle der Neuroblasten und ihre Fähigkeit, mechanische Eingangs während epidermalen Morphogenese bieten zu vergießen. Kürzlich wurde festgestellt, daß eine Zellteilung Gen ani-1 (Anillin) ist hoch in Neuroblasten (2A) ausgedrückt und seine Erschöpfung verursacht Neuroblasten Teilungsfehler. Interessant ist, dass diese Embryonen anzuzeigen ventralen Gehäuse Phänotypen (Fotopoulos, Wernicke und Piekny, unveröffentlichte Beobachtungen). Anillin ist für die Zellteilung erforderlich ist, und insbesondere für die Cytokinese, die das Verfahren, bei dem eine Mutterzelle physikalisch unterteilt in zwei Tochterzellen beschreibt. Zytokinese wird durch die Bildung eines Aktomyosin Kontraktionsring, der fest in Raum und Zeit gesteuert werden, um sicherzustellen, dass er richtig mit Schwester-Chromatid-Trennung gekoppelt werden muss angetrieben. Die Master-Regulator der Zellteilung ist metazoan RhoA (RHO-1 in C. elegans), eine kleine GTPase, die eine istctive in seiner GTP-gebundenen Form. Die GEF Ect2/ECT-2 RhoA aktiviert, nach der RhoA-GTP interagiert mit Effektoren, die die Kontraktionsring zu bilden und zu vermitteln seine Eindringen 15. Anillin ist ein Multidomänen-Protein, das RhoA über seinen C-Terminus und an Actin und Myosin über seinen N-Terminus bindet. Anillin erforderlich ist, um die Position des kontraktilen Ring in Säugetier-oder Drosophila S2 Zellen 16 zu stabilisieren. Anillin Erschöpfung verursacht Kontraktionsringe, um seitliche Schwingungen zu unterziehen, und Zytokinese schließlich ausfällt Bildung kernigen Zellen 17-19. Interessant, wenn auch C. elegans ani-1-Koordinaten Aktomyosin Kontraktilität im Embryo, ist es nicht wesentlich für die Cytokinese. Jedoch, wie oben beschrieben, ani-1 ist für Neuroblast Zytokinese in der Zwischenembryogenese (Fotopoulos, Wernicke und Piekny, unveröffentlichte Beobachtungen) erforderlich. Cytokinesis Scheitern würde die Anzahl und Position der Neuroblasten verändern und könnte die Lok beeinflussenation von chemischen Führungssignale, oder es könnte die mechanischen Eigenschaften des Gewebes verändern. Beide Modelle unterstreichen die Rolle der nichtautonomen für Neuroblasten ventralen Gehäuse, und die Bedeutung der Gewebe-Gewebe-Kommunikation während der embryonalen Entwicklung. Das Versuchsprotokoll beschreibt, wie Bild Zellteilung während C. elegans Mitte Embryogenese unter Verwendung von Fluoreszenzmikroskopie. Die Mehrzahl der Experimente, die die Mechanismen der Zellteilung in Einzelzellen in Kulturschalen (zB. HeLa-Zellen oder S2) oder in frühen Embryonen mit einer begrenzten Anzahl von Zellen (z. B. C. elegans ein Embryo, Xenopus oder stachelhäuter durch Embryonen). Jedoch ist es wichtig, auch die Zellteilung im Gewebe zu untersuchen, wie es externe Signale, die den Zeitpunkt und die Platzierung der Teilungsebene beeinflussen können. Weiterhin können Zellen, chemische oder mechanische Signale bereitzustellen, um die Entwicklung der benachbarten Gewebe beeinflussens, und es ist wichtig zu verstehen, wie die interzelluläre Kommunikation hilft Geweben während der Entwicklung zu bilden.

<table><tbody><tr><td>Agar</td><td>BioShop Canada Inc.</td><td>#AGR001.1</td><td>For making &lt;em&gt;C. elegans&lt;/em&gt; NGM and RNAi plates</td></tr><tr><td>Agar</td><td>Bio Basic Inc.</td><td>#9002-18-0</td><td>For making bacteria LB agar plates</td></tr><tr><td>Agarose</td><td>BioShop Canada Inc.</td><td>#AGA001.500</td><td></td></tr><tr><td>Anti-mouse Alexa Fluor 488 antibody</td><td>Life Technologies Corporation (Invitrogen)</td><td>#A11029</td><td></td></tr><tr><td>Anti-mouse anti-GFP antibody</td><td>Roche Applied Science</td><td>#11814460001</td><td></td></tr><tr><td>Anti-rabbit Alexa Fluor 568 antibody</td><td>Life Technologies Corporation (Invitrogen)</td><td>#A11011</td><td></td></tr><tr><td>Ampicillin</td><td>BioShop Canada Inc.</td><td>#AMP201.5</td><td>Store powder at 4 &amp;deg;C and dissolved ampicillin at -20 &amp;deg;C</td></tr><tr><td>Bactopetone&amp;nbsp; (peptone-A)</td><td>Bio Basic Inc.</td><td>#G213</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt; (calcium chloride)</td><td>BioShop Canada Inc.</td><td>#C302.1</td><td></td></tr><tr><td>Cholesterol</td><td>BioShop Canada Inc.</td><td>#CHL380.25</td><td>Dissolve in ethanol</td></tr><tr><td>DAPI&amp;nbsp;</td><td>Sigma-Aldrich</td><td>#D9542</td><td>Use to stain nucleic acids (DNA)</td></tr><tr><td>Glycerol</td><td>BioShop Canada Inc.</td><td>#GLY001.1</td><td></td></tr><tr><td>IPTG (isopropylthio-&amp;beta;-galactoside)</td><td>Bio Basic Inc.</td><td>#367-93-1</td><td>Store powder and dissolved IPTG at -20 &amp;deg;C</td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt; (potassium phosphate, monobasic)</td><td>BioShop Canada Inc.</td><td>#PPM666.1</td><td></td></tr><tr><td>K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt; (potassium phosphate, dibasic)</td><td>BioShop Canada Inc.</td><td>#PPD303.1</td><td></td></tr><tr><td>L4440&amp;nbsp; (feeding vector)</td><td>Addgene</td><td>#1654</td><td>Keep as glycerol stock at -80 &amp;deg;C</td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;&amp;nbsp;&amp;nbsp; (magnesium sulfate)</td><td>BioShop Canada Inc.</td><td>#MAG511.500</td><td></td></tr><tr><td>NaCl (sodium chloride)</td><td>Bio Basic Inc.</td><td>#7647-14-5</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt; (sodium phosphate, dibasic)</td><td>Bio Basic Inc.</td><td>#7558-79-4</td><td></td></tr><tr><td>Normal Donkey Serum (NDS)</td><td>Wisent Bioproducts</td><td>#035-110</td><td></td></tr><tr><td>n-Propyl-3,4,5-trihydroxybenzoate (propyl gallate)</td><td>Alfa Aesar</td><td>#A10877</td><td></td></tr><tr><td>Poly-L-lysine</td><td>Sigma-Aldrich</td><td>#P8920</td><td>For optimal results coat microscope slides three times&amp;nbsp;</td></tr><tr><td>Streptomycin</td><td>BioShop Canada Inc.</td><td>#STP101.50</td><td>Store powder at 4 &amp;deg;C and dissolved streptomycin at -20 &amp;deg;C</td></tr><tr><td>Tetracyclin</td><td>BioShop Canada Inc.</td><td>#TET701.10</td><td>Store powder at 4 &amp;deg;C and dissolved tetracycline at -20 &amp;deg;C</td></tr><tr><td>Tween-20</td><td>Bio Basic Inc.</td><td>CAS#9005-64-5</td><td></td></tr><tr><td>Tryptone</td><td>BioShop Canada Inc.</td><td>#TRP402.500</td><td></td></tr><tr><td>Yeast Extract</td><td>Bio Basic Inc.</td><td>#8013-01-2</td><td></td></tr></tbody></table>

visualizing neuroblast cytokinesis during c. elegans embryogenesis

Dieses Protokoll beschreibt die Verwendung von Fluoreszenzmikroskopie, um Bildteilenden Zellen in den Entwicklungs Caenorhabditis elegans Embryonen. Insbesondere konzentriert sich dieses Protokoll auf, wie man Bild Division Neuroblasten, die unterhalb der epidermalen Zellen zu finden sind und kann wichtig für den epidermalen Morphogenese sein. Gewebebildung ist entscheidend für die Entwicklung Metazoen und setzt auf externe Signale von benachbarten Geweben. C elegans ist ein ausgezeichneter Modellorganismus zur Morphogenese in vivo aufgrund seiner Transparenz und einfache Organisation zu studieren, was seine Gewebe einfach über Mikroskopie zu untersuchen. Ventralen Gehäuse ist der Prozess, wo die ventrale Oberfläche des Embryos durch eine einzige Schicht von Epithelzellen bedeckt. Dieses Ereignis wird angenommen, durch die zugrunde liegenden Neuroblasten, die chemische Führungssignale bereitzustellen, um die Migration der darüberliegenden Epithelzellen vermitteln erleichtert werden. Jedoch sind die Neuroblasten hochproliferative und kann auch handelnals mechanischer Träger für die ventrale Epidermiszellen. Studien mit diesen experimentellen Protokoll könnte die Bedeutung der interzellulären Kommunikation bei der Gewebebildung zu enthüllen, und könnte verwendet werden, um die Rollen von Genen in die Zellteilung in sich entwickelnden Geweben beteiligt zu offenbaren.

Das Versuchsprotokoll beschreibt, wie Bild der Zellteilung in C. elegans Embryonen während der Embryogenese Mitte. Insbesondere beschreibt sie, wie man Bild Neuroblasten, die epidermalen Morphogenese erleichtern können. Epidermalen Morphogenese tritt aufgrund einer Kombination von epidermalen Zellformänderungen, Migration und Adhäsion, sondern stützt sich auf chemische oder mechanische Signale aus den zugrundeliegenden Neuroblasten (Abbildung 1B). Die Neuroblasten sezerFührungsSignale, di...

Watch this Scientific Journal Video about Visualizing Neuroblast Cytokinesis During C. elegans Embryogenesis at JoVE.com

Visualizing Neuroblast Cytokinesis During C. elegans Embryogenesis

Dieses Protokoll beschreibt, wie Bild teilenden Zellen innerhalb eines Gewebes in Caenorhabditis elegans Embryonen. Während mehrere Protokolle beschreiben, wie Bild der Zellteilung im frühen Embryo, beschreibt dieses Protokoll, wie man Bild der Zellteilung in einem Entwicklungs Gewebe während der Embryogenese Mitte spät.

Visualisierung Neuroblast Cytokinesis Während<em&gt; C. elegans</em&gt; Embryogenese

This protocol describes the use of fluorescence microscopy to image dividing cells within developing Caenorhabditis elegans embryos. In particular, this ...

visualizing-neuroblast-cytokinesis-during-c-elegans-embryogenesis

Research

JoVE Journal

Neuroscience

Visualizing Neuroblast Cytokinesis During C. elegans Embryogenesis

11.8K Aufrufe.  Concordia University.  Dieses Protokoll beschreibt, wie Bild teilenden Zellen innerhalb eines Gewebes in Caenorhabditis elegans Embryonen. Während mehrere Protokolle beschreiben, wie Bild der Zellteilung im frühen Embryo, beschreibt dieses Protokoll, wie man Bild der Zellteilung in einem Entwicklungs Gewebe während der Embryogenese Mitte spät. 

Serie: Visualizing Neuroblast Cytokinesis During C. elegans Embryogenesis

Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

The Drosophila embryo is an attractive model system for investigating the cellular and molecular basis of neuronal development. Here we describe the procedure for the visualization of Drosophila embryonic nervous system using antibodies to neuronal proteins. Since the entire embryonic peripheral nervous and central nervous systems are well characterized at the level of individual cells (Dambly-Chaudi&egrave;re et al., 1986; Bodmer et al., 1987; Bodmer et al., 1989), any aberrations to these systems can be easily identified using antibodies to different neuronal proteins. The developing embryos are collected at certain times to ensure that the embryos are in the proper developmental stages for visualization. After collection, the outer layers of the embryo, the chorion membrane and the vitelline envelope that surrounds the embryo, are removed before fixation. Embryos are then incubated with neuronal antibodies and visualized using fluorescently labeled secondary antibodies. Embryos at stages 12-17 are visualized to access the embryonic nervous system. At stage 12 the CNS germ band starts shortening and by stage 15 the definitive pattern of the commissure has been achieved. By stage 17 the CNS contracts and the PNS is fully developed (Campos-Ortega et al. 1985). Thus changes in the pattern of the PNS and CNS can be easily observed during these developmental stages.

Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

We describe the procedure to prepare staged Drosophila embryos for the visualization of the embryonic nervous system during embryogenesis.

Visualisierung des embryonalen Nervensystems in Whole-mount Drosophila Embryos

The Drosophila embryo is an attractive model system for investigating the cellular and molecular basis of neuronal development. Here we describe the ...

Forschung

Neurowissenschaften

Isotropic Light-Sheet Microscopy and Automated Cell Lineage Analyses to Catalogue Caenorhabditis elegans Embryogenesis with Subcellular Resolution

Caenorhabditis elegans (C. elegans) stands out as the only organism in which the challenge of understanding the cellular origins of an entire nervous system can be observed, with single cell resolution, in vivo. Here, we present an integrated protocol for the examination of neurodevelopment in C. elegans embryos. Our protocol combines imaging, lineaging and neuroanatomical tracing of single cells in developing embryos. We achieve long-term, four-dimensional (4D) imaging of living C. elegans&#160;embryos with nearly isotropic spatial resolution through the use of Dual-view Inverted Selective Plane Illumination Microscopy (diSPIM). Nuclei and neuronal structures in the nematode embryos are imaged and isotropically fused to yield images with resolution of ~330 nm in all three dimensions. These minute-by-minute high-resolution 4D data sets are then analyzed to correlate definitive cell-lineage identities with gene expression and morphological dynamics at single-cell and subcellular levels of detail. Our protocol is structured to enable modular implementation of each of the described steps and enhance studies on embryogenesis, gene expression, or neurodevelopment.

Here, we present a combinatorial approach using high-resolution microscopy, computational tools, and single-cell labeling in living C. elegans embryos to understand single cell dynamics during neurodevelopment.

Isotrope Licht-Sheet-Mikroskopie und automatisierte Zelllineage-Analysen zu Katalog-Caenorhabditis elegans Embryogenese mit subzellulärer Auflösung

Caenorhabditis elegans (C. elegans) stands out as the only organism in which the challenge of understanding the cellular origins of an entire nervous ...

Entwicklungsbiologie

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-&#945;-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment.

Live-Imaging der Mitose in der Entwicklung von embryonalen Maus-Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

A Semi-high-throughput Imaging Method and Data Visualization Toolkit to Analyze C. elegans Embryonic Development

C. elegans is the premier system for the systematic analysis of cell fate specification and morphogenetic events during embryonic development. One challenge is that embryogenesis dynamically unfolds over a period of about 13 h; this half day-long timescale has constrained the scope of experiments by limiting the number of embryos that can be imaged. Here, we describe a semi-high-throughput protocol that allows for the simultaneous 3D time-lapse imaging of development in 80&#8211;100 embryos at moderate time resolution, from up to 14 different conditions, in a single overnight run. The protocol is straightforward and can be implemented by any laboratory with access to a microscope with point visiting capacity. The utility of this protocol is demonstrated by using it to image two custom-built strains expressing fluorescent markers optimized to visualize key aspects of germ-layer specification and morphogenesis. To analyze the data, a custom program that crops individual embryos out of a broader field of view in all channels, z-steps, and timepoints and saves the sequences for each embryo into a separate tiff stack was built. The program, which includes a user-friendly graphical user interface (GUI), streamlines data processing by isolating, pre-processing, and uniformly orienting individual embryos in preparation for visualization or automated analysis. Also supplied is an ImageJ macro that compiles individual embryo data into a multi-panel file that displays maximum intensity fluorescence projection and brightfield images for each embryo at each time point. The protocols and tools described herein were validated by using them to characterize embryonic development following knock-down of 40 previously described developmental genes; this analysis visualized previously annotated developmental phenotypes and revealed new ones. In summary, this work details a semi-high-throughput imaging method coupled with a cropping program and ImageJ visualization tool that, when combined with strains expressing informative fluorescent markers, greatly accelerates experiments to analyze embryonic development.

A Semi-high-throughput Imaging Method and Data Visualization Toolkit to Analyze C. elegans Embryonic Development

This work describes a semi-high-throughput protocol that allows simultaneous 3D time-lapse imaging of embryogenesis in 80&#8211;100 C. elegans embryos in a single overnight run. Additionally, image processing and visualization tools are included to streamline data analysis. The combination of these methods with custom reporter strains enables detailed monitoring of embryogenesis.

Ein Instrument zur Analyse der C. elegans Embryonal development

C. elegans is the premier system for the systematic analysis of cell fate specification and morphogenetic events during embryonic development. One ...

4D Microscopy: Unraveling Caenorhabditis elegans Embryonic Development Using Nomarski Microscopy

4D microscopy is an invaluable tool for unraveling the embryonic developmental process in different animals. Over the last decades, Caenorhabditis elegans has emerged as one of the best models for studying development. From an optical point of view, its size and transparent body make this nematode an ideal specimen for DIC (Differential Interference Contrast or Nomarski) microscopy. This article illustrates a protocol for growing C. elegans nematodes, preparing and mounting their embryos, performing 4D microscopy and cell lineage tracing. The method is based on multifocal time-lapse records of Nomarski images and analysis with specific software. This technique reveals embryonic developmental dynamics at the cellular level. Any embryonic defect in mutants, such as problems in spindle orientation, cell migration, apoptosis or cell fate specification, can be efficiently detected and scored. Virtually every single cell of the embryo can be followed up to the moment the embryo begins to move. Tracing the complete cell lineage of a C. elegans embryo by 4D DIC microscopy is laborious, but the use of specific software greatly facilitates this task. In addition, this technique is easy to implement in the lab. 4D microscopy is a versatile tool and opens the possibility of performing an unparalleled analysis of embryonic development.

4D Microscopy: Unraveling Caenorhabditis elegans Embryonic Development Using Nomarski Microscopy

Here, we present a protocol for preparing and mounting Caenorhabditis elegans embryos, recording development under a 4D microscope and tracing cell lineage.

4D-Mikroskopie: Entschlüsselung der Caenorhabditis elegans Embryonalentwicklung mittels Nomarski-Mikroskopie

4D microscopy is an invaluable tool for unraveling the embryonic developmental process in different animals. Over the last decades, Caenorhabditis elegans ...

Visualizing Cytoskeleton-Dependent Trafficking of Lipid-Containing Organelles in Drosophila Embryos

Early Drosophila embryos are large cells containing a vast array of conventional and embryo-specific organelles. During the first three hours of embryogenesis, these organelles undergo dramatic movements powered by actin-based cytoplasmic streaming and motor-driven trafficking along microtubules. The development of a multitude of small, organelle-specific fluorescent probes (FPs) makes it possible to visualize a wide range of different lipid-containing structures in any genotype, allowing live imaging without requiring a genetically encoded fluorophore. This protocol shows how to inject vital dyes and molecular probes into Drosophila embryos to monitor the trafficking of specific organelles by live imaging. This approach is demonstrated by labeling lipid droplets (LDs) and following their bulk movement by particle image velocimetry (PIV). This protocol provides a strategy amenable to the study of other organelles, including lysosomes, mitochondria, yolk vesicles, and the ER, and for tracking the motion of individual LDs along microtubules. Using commercially available dyes brings the benefits of separation into the violet/blue and far-red regions of the spectrum. By multiplex co-labeling of organelles and/or cytoskeletal elements via microinjection, all the genetic resources in Drosophila are available for trafficking studies without the need to introduce fluorescently tagged proteins. Unlike genetically encoded fluorophores, which have low quantum yields and bleach easily, many of the available dyes allow for rapid and simultaneous capture of several channels with high photon yields.

Visualizing Cytoskeleton-Dependent Trafficking of Lipid-Containing Organelles in Drosophila Embryos

In the early Drosophila embryo, many organelles are motile. In principle, they can be imaged live via specific fluorescent probes, but the eggshell prevents direct application to the embryo. This protocol describes how to introduce such probes via microinjection, and then analyze bulk organelle motion via particle image velocimetry.

Visualisierung des zytoskelettabhängigen Handels von lipidhaltigen Organellen in Drosophila-Embryonen

Early Drosophila embryos are large cells containing a vast array of conventional and embryo-specific organelles. During the first three hours of ...

Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

Cellular processes, such as chromosome assembly, segregation and cytokinesis,are inherently dynamic. Time-lapse imaging of living cells, using fluorescent-labeled reporter proteins or differential interference contrast (DIC) microscopy, allows for the examination of the temporal progression of these dynamic events which is otherwise inferred from analysis of fixed samples1,2. Moreover, the study of the developmental regulations of cellular processes necessitates conducting time-lapse experiments on an intact organism during development. The Caenorhabiditis elegans embryo is light-transparent and has a rapid, invariant developmental program with a known cell lineage3, thus providing an ideal experiment model for studying questions in cell biology4,5and development6-9. C. elegans is amendable to genetic manipulation by forward genetics (based on random mutagenesis10,11) and reverse genetics to target specific genes (based on RNAi-mediated interference and targeted mutagenesis12-15). In addition, transgenic animals can be readily created to express fluorescently tagged proteins or reporters16,17. These traits combine to make it easy to identify the genetic pathways regulating fundamental cellular and developmental processes in vivo18-21. In this protocol we present methods for live imaging of C. elegans embryos using DIC optics or GFP fluorescence on a compound epifluorescent microscope. We demonstrate the ease with which readily available microscopes, typically used for fixed sample imaging, can also be applied for time-lapse analysis using open-source software to automate the imaging process.

Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

The C. elegans embryo is a powerful system for studying cell biology and development. We present a protocol for live imaging of C. elegans embryos utilizing DIC optics or fluorescence using readily available epifluorescent microscopes and open-source software.

Imaging C. elegans Embryos mit einem Epifluoreszenz-Mikroskop und Open Source Software

Cellular processes, such as chromosome assembly, segregation and cytokinesis,are inherently dynamic. Time-lapse imaging of living cells, using ...

Biologie

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes. The transparency of the 
embryos, the genetic resources, and the relative ease of transformation are qualities that make C. elegans an excellent model for early embryogenesis. Laser-based 
confocal microscopy and fluorescently labeled tags allow researchers to follow specific cellular structures and proteins in the developing embryo. For example, 
one can follow specific organelles, such as lysosomes or mitochondria, using fluorescently labeled dyes. These dyes can be delivered to the early embryo by means 
of microinjection into the adult gonad. Also, the localization of specific proteins can be followed using fluorescent protein tags. Examples are presented here 
demonstrating the use of a fluorescent lysosomal dye as well as fluorescently tagged histone and ubiquitin proteins. The labeled histone is used to visualize 
the DNA and thus identify the stage of the cell cycle. GFP-tagged ubiquitin reveals the dynamics of ubiquitinated vesicles in the early embryo. Observations 
of labeled lysosomes and GFP:: ubiquitin can be used to determine if there is colocalization between ubiquitinated vesicles and lysosomes. A technique for 
the microinjection of the lysosomal dye is presented. Techniques for generating transgenenic strains are presented elsewhere (1, 2). For imaging, embryos 
are cut out of adult hermaphrodite nematodes and mounted onto 2% agarose pads followed by time-lapse microscopy on a standard laser scanning confocal 
microscope or a spinning disk confocal microscope. This methodology provides for the high resolution visualization of early embryogenesis.

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

This article describes a technique for the visualization of the early events of embryogenesis in the nematode Caenorhabditis elegans.

Zeitraffer-Mikroskopie der frühen Embryogenese in Caenorhabditis elegans

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes.  The transparency of the 
embryos, the genetic ...

Tracking Calcium in the Early Developmental Phases of C. elegans

In Vivo Visualization of Calcium Transients during Fertilization and Early Development in C. elegans

Calcium is an important signaling molecule during the oocyte-to-embryo transition (OET) and early embryogenesis. The hermaphroditic nematode Caenorhabditis elegans provides several unique advantages for the study of the OET as it is transparent and has an ordered gonad that produces one mature oocyte every ~23 min at 20 &#176;C. We have modified the genetically encoded calcium indicator jGCaMP7s to fluorescently indicate the moment of fertilization within a living organism. We have termed this reporter &#34;CaFE&#34; for Calcium during Fertilization in C. elegans. The CaFE reporter was&#160;engineered into a safe harbor locus in single copy, has no significant impact on physiology or fecundity, and produces a robust signal upon fertilization. Here, a series of protocols is presented for utilizing the CaFE reporter as an in vivo tool for dissecting the OET and embryogenesis. We include methods to synchronize worms, examine the effects of RNAi knockdown, mount worms for imaging, and to visualize calcium in oocytes and embryos. Additionally, we present the generation of additional worm strains to aid in this type of analysis. Demonstrating the utility of the CaFE reporter to visualize the timing of fertilization, we report that double ovulation occurs when ipp-5 is targeted by RNAi and that only the first oocyte undergoes immediate fertilization. Furthermore, the discovery of single-cell calcium transients during early embryogenesis is reported here, demonstrating that the CaFE reporter persists into early development. Importantly, the CaFE reporter in worms is simple enough to use for incorporation into course-based undergraduate research (CURE) laboratory classes. The CaFE reporter, coupled with the ordered gonad and ease of RNAi in worms, facilitates inquiry into the cell-cell dynamics required to regulate internal fertilization and early embryogenesis.

Here, we present protocols and tools for visualizing calcium during fertilization and early embryogenesis using a genetically encoded calcium reporter expressed in the germline of the model nematode C. elegans.

Calcium is an important signaling molecule during the oocyte-to-embryo transition (OET) and early embryogenesis. The hermaphroditic nematode ...

Visualization of the Axonal Projection Pattern of Embryonic Motor Neurons in Drosophila

The establishment of functional neuromuscular circuits relies on precise connections between developing motor axons and target muscles. Motor neurons extend growth cones to navigate along specific pathways by responding to a large number of axon guidance cues that emanate from the surrounding extracellular environment. Growth cone target recognition also plays a critical role in neuromuscular specificity. This work presents a standard immunohistochemistry protocol to visualize motor neuron projections of late stage-16 Drosophila melanogaster embryos. This protocol includes a few key steps, including a genotyping procedure, to sort the desired mutant embryos; an immunostaining procedure, to tag embryos with fasciclin II (FasII) antibody; and a dissection procedure, to generate filleted preparations from fixed embryos. Motor axon projections and muscle patterns in the periphery are much better visualized in flat preparations of filleted embryos than in whole-mount embryos. Therefore, the filleted preparation of fixed embryos stained with FasII antibody provides a powerful tool to characterize the genes required for motor axon pathfinding and target recognition, and it can also be applied to both loss-of-function and gain-of-function genetic screens.

Visualization of the Axonal Projection Pattern of Embryonic Motor Neurons in Drosophila

This work details a standard immunohistochemistry method to visualize motor neuron projections of late stage-16 Drosophila melanogaster embryos. The filleted preparation of fixed embryos stained with FasII antibody provides a powerful tool to characterize the genes required for motor axon pathfinding and target recognition during neural development.

Visualisierung des axonalen Projektionsmusters der embryonalen motorischen Neuronen in<em&gt; Drosophila</em

The establishment of functional neuromuscular circuits relies on precise connections between developing motor axons and target muscles. Motor neurons ...

Visualisierung Neuroblast Cytokinesis Während

Zusammenfassung

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