Egg Handling <ol><li> Eier können bei 13 ° C aufbewahrt werden bis zu einer Woche vor der Inkubation ohne signifikanten Verlust der Lebensfähigkeit (eine kleine Wein-Kühler eignet sich ideal für diesen Zweck). </li><li> Vor der Inkubation, damit Eier auf Raumtemperatur, dann in einem befeuchteten Inkubator Huhn auf 37,8 eingestellt warmen ° C (100 ° F). </li><li> Wir in der Regel elektroporieren Embryonen etwa 48 Stunden nach Beginn der Inkubation, wenn der Embryo hat Hamburger und Hamilton Stufe 10 erreicht hat. </li><li> Die Zeit der Inkubation kann je nach gewünschten Hamburger und Hamilton Bühne variieren. Temperatur ist von entscheidender Bedeutung und eine Abweichung von optimalen Inkubationstemperatur verändert die Inkubationszeit und Abnahme Embryo Lebensfähigkeit. </li><li> Während der Inkubation sollten die Eier auf ihren Seiten gehalten werden, damit der Embryo richtig wird für die Elektroporation positioniert werden. Der Embryo wird oben auf dem Dotter schwimmen und es ist hilfreich, um die Spitze des Ei mit einem Bleistift vor Windowing, so dass Sie, wo zu schneiden wissen. </li></ol> Vorbereitung <ol><li> Warm Leibovitz L-15 Medien bis 37 ° C. Pull Glaskapillaren in Nadeln. Position Elektroden und Mikromanipulator und verbinden Elektroden an den Impulsgenerator. </li></ol> Windowing <ol><li> Mit einer Spritze mit großkalibrigen Kanüle, durchbohren die Schale auf dem kleinen Ende des Eies. </li><li> Und dabei die Nadel vorsichtig nach unten, um Störungen des Dotters zu verhindern, etwa 5 ml Albumin zu entfernen. </li><li> Dichten Sie das Loch mit einem kleinen Stück Klebeband. </li><li> Decken Sie die Oberseite des Eies mit einem anderen Stück Klebeband (ca. 4x4 cm). </li><li> Mit einer gebogenen Schere, und kümmert sich nicht um den Embryo zu stören, schnitt ein Loch gerade groß genug, um ein Fenster in die Arbeit. </li></ol> Optional: Zur Visualisierung der Neuralrohrdefekte, injizieren Tinte Lösung mit 26-Gauge-Nadel unter dem Embryo (insert Nadel unter der Embryo außerhalb der Blut-Ring). Tusche sollte 1:5 mit Hanks oder vergleichbarer sterile gepufferte Lösung sein. Injektion und Elektroporation <ol><li> Break the Kapillarspitze auf gewünschten Durchmesser mit einer Pinzette </li><li> Mit dem Mund pipettieren, laden Sie die Kapillare mit Plasmid / Fast Green Farbstoff. </li><li> Position der Embryo mit dem Kopf in Ihre Richtung und führen Sie die Nadel in das Neuralrohr in einem flachen Winkel. </li><li> Spritzen Sie die Plasmid-Lösung in das Lumen des Neuralrohrs bis Farbstoff füllt den gesamten Raum. </li></ol> Hinweis: Achten Sie darauf, nicht bis zur Spitze größer als Neuralrohr Durchmesser haben. In der Regel werden Sie in der Lage sein zu sagen, ob die optimale Größe der Spitze hat, wie leicht oder schwer es ist, Flüssigkeit in die Kapillare ziehen erzielt worden. Wenn es extreme Beständigkeit, ist die Eröffnung wahrscheinlich zu klein und die Spitze sollte wieder höher werden aufgebrochen. Wenn es wenig oder keinen Widerstand, ist die Öffnung zu groß und eine neue Nadel verwendet werden. <ol><li> Legen Sie 1-2 Tropfen steriler L-15 Medien auf den Embryo. </li><li> Anschließend wird der Elektroden auf beiden Seiten des Embryos, parallel zu den Neuralrohrdefekten und Puls 5 mal bei 10-24 Volt für 50 mseconds Abstand von 1 Sekunde. Sie werden Luftblasen in der Nähe der Elektroden zu sehen, ob alles korrekt funktioniert. </li><li> Entfernen Sie vorsichtig Elektroden, legte 5 Tropfen L-15 auf der Oberseite des Embryos und Dichtung das Ei mit Klebeband. Stellen Sie sicher, dass das Ei gut abgedichtet ist, da die Trocknung ein wesentlicher Faktor für Embryo Verlust nach der Elektroporation. </li><li> Die Eier wieder in den Inkubator, Fenster nach oben, und inkubieren, bis sie H &amp; H-Stufe gewünscht zu erreichen. </li></ol>

<ol>
	<li>Itasaki, N., Bel-Vialar, S., Krumlauf, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=&apos;Shocking&apos;+developments+in+chick+embryology:+electroporation+and+in+ovo+gene+expression.">&apos;Shocking&apos; developments in chick embryology: electroporation and in ovo gene expression.</a> Nat Cell Biol. 1, 203-207 (1999).</li></ol>

The authors have nothing to disclose.

Dieses Protokoll bietet eine Schritt-für Schritt-Anleitung zum Neuralrohr Elektroporation von HH10 Hühnerembryonen. Zusätzlich zu zeigen, die Technik wird Richtlinien für die Lagerung und Handhabung von Eiern zur Verfügung gestellt. Diese Technik hat eine breite Palette von Anwendungen für die genetische Analyse und die Leichtigkeit und geringen Kosten von Experimenten macht sie möglich für viele Labors.

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		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Chicken egg incubator</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>humidified, set to 37.80&deg;C (100F). </td>
</tr>
<tr>
<td>Pulse generator </td>
<td>&nbsp;</td>
<td>Genetronics </td>
<td>BTX T820 </td>
<td>Square wave generator or comparable</td>
</tr>
<tr>
<td>Electrodes</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>our electrodes were made from 0.25 mm diameter platinum wire and were 5 mm long and spaced 1 mm apart</td>
</tr>
<tr>
<td>Connecter cables</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Micromanipulator</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Dissecting scope </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>with large working distance</td>
</tr>
<tr>
<td>Glass capillaries </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>and capillary puller or commercial glass needles</td>
</tr>
<tr>
<td>Leibovitz&#x2019;s L-15 media</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>chicken eggs</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>fertilized</td>
</tr>
<tr>
<td>Curved scissors</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>10 ml syringe</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Large bore needle </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>16-18G</td>
</tr>
<tr>
<td>Mouth pipette</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>India ink </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Insulin syringe/syringe </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Plasmid DNA </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&#x2265; 1&#181;g/&#181;l in sterile TE or 1X PBS containing 0.05% Fast Green dye</td>
</tr>		</tbody>
		</table>
		</div>
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in ovo electroporations of hh stage 10 chicken embryos

Größe und externe Entwicklung des Hühnerembryo schon lange gemacht, ein wertvolles Werkzeug in der Studie der Entwicklungsbiologie. Mit dem Aufkommen von molekularbiologischen Techniken, hat das Küken zu einem nützlichen System, in dem Gen-Regulation und Funktion zu studieren. Durch Elektroporation DNA-oder RNA-Konstrukte in den Entwicklungsländern Hühnerembryo können Gene exprimiert werden können oder nach unten, um in vivo Funktion von Genen zu analysieren klopfte. Ebenso kann Reporter-Konstrukte für das Schicksal Mapping verwendet werden oder zur vermeintlichen Gen regulatorische Elemente zu untersuchen. Im Vergleich zu ähnlichen Versuchen in der Maus hat chick Elektroporation den Vorteil, dass schnell, einfach und kostengünstig. Dieses Video zeigt zunächst, wie man ein Fenster in die Eischale zu machen, um den Embryo zu manipulieren. Anschließend wird der Embryo sichtbar gemacht mit einer verdünnten Lösung von Tusche unter den Embryo injiziert. Ein Glas Nadel und Pipette werden verwendet, um DNA und Fast Green Farbstoff in die Entwicklung des Neuralrohrs, dann Platin-Elektroden sind parallel zu den Embryo gelegt und kurze elektrische Impulse werden mit einem Impulsgeber verabreicht zu injizieren. Schließlich wird das Ei mit Klebeband verschlossen und wieder in einen Inkubator für die weitere Entwicklung. Zusätzlich zeigt das Video richtigen Ei Lagerung und Handhabung und diskutiert mögliche Ursachen des Embryos Verlust nach der Elektroporation.

Watch this Scientific Journal Video about In Ovo Electroporations of HH Stage 10 Chicken Embryos at JoVE.com

In Ovo Electroporations of HH Stage 10 Chicken Embryos

Küken in ovo Elektroporation ist eine Technik, die genetische Manipulation der Vogelembryo ermöglicht. Typische Anwendungen für diese Technik sind die funktionelle Analyse von Genen und vermeintlichen Enhancer-Elemente. Dieses Video zeigt Neuralrohr Elektroporation in HH 10 Hühnerembryonen. Injektionstechnik und ordnungsgemäße Eibehandlung werden diskutiert.

In Ovo Electroporations von HH Stage 10 Hühnerembryonen

Large size and external development of the chicken embryo have long made it a valuable tool in the study of developmental biology.  With the advent of ...

in-ovo-electroporations-of-hh-stage-10-chicken-embryos

Research

JoVE Journal

Biology

18.2K Aufrufe.  University of Chicago. Küken in ovo Elektroporation ist eine Technik, die genetische Manipulation der Vogelembryo ermöglicht. Typische Anwendungen für diese Technik sind die funktionelle Analyse von Genen und vermeintlichen Enhancer-Elemente. Dieses Video zeigt Neuralrohr Elektroporation in HH 10 Hühnerembryonen. Injektionstechnik und ordnungsgemäße Eibehandlung werden diskutiert.

Serie: In Ovo Electroporations of HH Stage 10 Chicken Embryos

Preparation of Neuronal Cultures from Midgastrula Stage Drosophila Embryos

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos and their dechorionation using bleach are demonstrated. Using a glass pipet attached to a mouth suction tube, we illustrate the removal of all cells from single embryos. The method for dispersing cells from each embyro into a small (5 l) drop of medium on an uncoated glass coverslip is demonstrated. A view through the microscope at 1 hour after plating illustrates the preferred cell density. Most of the cells that survive when grown in defined medium are neuroblasts that divide one or more times in culture before extending neuritic processes by 12-24 hours. A view through the microscope illustrates the level of neurite outgrowth and branching expected in a healthy culture at 2 days in vitro. The cultures are grown in a simple bicarbonate based defined medium, in a 5% CO2 incubator at 22-24&deg;C. Neuritic processes continue to elaborate over the first week in culture and when they make contact with neurites from neighboring cells they often form functional synaptic connections. Neurons in these cultures express voltage-gated sodium, calcium, and potassium channels and are electrically excitable. This culture system is useful for studying molecular genetic and environmental factors that regulate neuronal differentiation, excitability, and synapse formation/function.

This video demonstrates the preparation of primary neuronal cultures from midgastrula stage Drosophila embryos. Views of live cultures show cells 1 hour after plating and differentiated neurons after 2 days of growth in a bicarbonate-based defined medium. The neurons are electrically excitable and form synaptic connections.

Vorbereitung der neuronalen Kulturen aus Midgastrula Bühne Drosophila Embryonen

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos ...

Forschung

Biologie

Windowing Chicken Eggs for Developmental Studies

The study of development has been greatly aided by the use of the chick embryo as an experimental model.  The ease of accessibility of the embryo has allowed for experiments to map cell fates using several approaches, including chick quail chimeras and focal dye labeling.  In addition, it allows for molecular perturbations of several types, including placement of protein-coated beads and introduction of plasmid DNA using in ovo electroporation.  These experiments have yielded important data on the development of the central and peripheral nervous systems.  For many of these studies, it is necessary to open the eggshell and reclose it without perturbing the embryo's growth.  The embryo can be examined at successive developmental stages by re-opening the eggshell.  While there are several excellent methods for opening chicken eggs, in this article we demonstrate one method that has been optimized for long survival times.  In this method, the egg rests on its side and a small window is cut in the shell.  After the experimental procedure, the shell is used to cover the egg for the duration of its development.  Clear plastic tape overlying the eggshell protects the embryo and helps retain hydration during the remainder of the incubation period.  This method has been used beginning at two days of incubation and has allowed survival through mature embryonic ages.

The ease of accessibility of the embryo has allowed for experiments to map cell fates using several approaches, including chick quail chimeras and focal dye labeling. In this article we demonstrate one egg preparation method that has been optimized for long survival times.

Windowing Chicken Eggs für Developmental Studies

The study of development has been greatly aided by the use of the chick embryo as an experimental model.  The ease of accessibility of the embryo has ...

Placing Growth Factor-Coated Beads on Early Stage Chicken Embryos

The neural tube expresses many proteins in specific spatiotemporal patterns during development. These proteins have been shown to be critical for cell fate determination, cell migration, and formation of neural circuits. Neuronal induction and patterning involve bone morphogenetic protein (BMP), sonic hedgehog (SHH), fibroblast growth factor (FGF), among others. In particular, the expression pattern of Fgf8 is in close proximity to regions expressing BMP4 and SHH. This expression pattern is consistent with developmental interactions that facilitate patterning in the telencephalon.

Here we provide a visual demonstration of a method in which an in ovo preparation can be used to test the effects of Fgfs in the formation of the forebrain. Beads are coated with protein and placed in the developing neural tube to provide sustained exposure. Because the procedure uses small, carefully placed beads, it is minimally invasive and allows several beads to be placed within a single neural tube. Moreover, the method allows for continued development so that embryos can be analyzed at a more mature stage to detect changes in anatomy and in neural patterning. This simple but useful protocol allows for real time imaging. It provides a means to make spatially and temporally limited changes to endogenous protein levels.

A variety of growth factors and proteins interact to induce cells to take on different cell fates during development. Here we demonstrate the use of an in ovo preparation to address possible interactions between different proteins in development by placing beads on E2.5 chick embryos.

Platzieren Growth Factor-Coated Perlen auf Early Stage Hühnerembryonen

The neural tube expresses many proteins in specific spatiotemporal patterns during development.   These proteins have been shown to be critical for cell ...

Chick ex ovo Culture and ex ovo CAM Assay: How it Really Works

Chicken eggs in the early phase of breeding are between in vitro and in vivo systems and provide a vascular test environment not only to study angiogenesis but also to study tumorigenesis. After the chick chorioallantoic membrane (CAM) has developed, its blood vessel network can be easily accessed, manipulated and observed and therefore provides an optimal setting for angiogenesis assays. Since the lymphoid system is not fully developed until late stages of incubation, the chick embryo serves as a naturally immunodeficient host capable of sustaining grafted tissues and cells without species-specific restrictions. In addition to nurturing developing allo- and xenografts, the CAM blood vessel network provides a uniquely supportive environment for tumor cell intravasation, dissemination, and vascular arrest and a repository where arrested cells extravasate to form micro metastatic foci.
For experimental purposes, in most of the recent studies the CAM was exposed by cutting a window through the egg shell and experiments were carried out in ovo, resulting in significant limitations in the accessibility of the CAM and possibilities for observation and photo documentation of effects. When shell-less cultures of the chick embryo were used1-4, no experimental details were provided and, if published at all, the survival rates of these cultures were low. We refined the method of ex ovo culture of chick embryos significantly by introducing a rationally controlled extrusion of the egg content. These ex ovo cultures enhance the accessibility of the CAM and chick embryo, enabling easy in vivo documentation of effects and facilitating experimental manipulation of the embryo. This allows the successful application to a large number of scientific questions: (1) As an improved angiogenesis assay5,6, (2) an experimental set up for facilitated injections in the vitreous of the chick embryo eye7-9, (3) as a test environment for dissemination and intravasation of dispersed tumor cells from established cell lines inoculated on the CAM10-12, (4) as an improved sustaining system for successful transplantation and culture of limb buds of chicken and mice13 as well as (5) for grafting, propagation, and re-grafting of solid primary tumor tissue obtained from biopsies on the surface of the CAM14.
In this video article we describe the establishment of a refined chick ex ovo culture and CAM assay with survival rates over 50%. Besides we provide a step by step demonstration of the successful application of the ex ovo culture for a large number of scientific applications. 
Daniel S. Dohle, Susanne D. Pasa, and Sebastian Gustmann contributed equally to this study.

Chick ex ovo Culture and ex ovo CAM Assay: How it Really Works

The chick chorioallantoic membrane (CAM) is a unique, naturally immunodeficient supportive culture environment to study angiogenesis and tumorigenesis. This video article demonstrates the different steps in chick ex ovo culture, application of potentially angiogenic substances and successful inoculation of tumor cells and tissues on the surface of the CAM.

Küken Ex ovo Kultur und Ex ovo CAM-Assay: Wie es wirklich funktioniert

Chicken eggs in the early phase of breeding are between in vitro and in vivo systems and provide a vascular test environment not only to study ...

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

The morphogenesis of the Drosophila embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell shape changes during embryonic development. One of the challenges faced in studying this structure is that the lumen of the heart tube, as well as the membrane features that are crucial to heart tube formation, are difficult to visualize in whole mount embryos, due to the small size of the heart tube and intra-lumenal space relative to the embryo. The use of transmission electron microscopy allows for higher magnification of these structures and gives the advantage of examining the embryos in cross section, which easily reveals the size and shape of the lumen. In this video, we detail the process for reliable fixation, embedding, and sectioning of late stage Drosophila embryos in order to visualize the heart tube lumen as well as important cellular structures including cell-cell junctions and the basement membrane.

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

We describe a process for fixation, embedding, sectioning, and imaging of late stage Drosophila embryos for Trasmission Electron Microscopy of the embryonic heart tube. This technique allows for the visualization of the heart tube lumen as well as the basement membrane, which lines the lumen of the heart.

Vorbereitung der Embryonen für Elektronenmikroskopie der Drosophila Embryonale Herz Rohr

The morphogenesis of the Drosophila  embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell ...

In-vivo Centrifugation of Drosophila Embryos

A major strategy for purifying and isolating different types of intracellular organelles is to separate them from each other based on differences in buoyant density. However, when cells are disrupted prior to centrifugation, proteins and organelles in this non-native environment often inappropriately stick to each other. Here we describe a method to separate organelles by density in intact, living Drosophila embryos. Early embryos before cellularization are harvested from population cages, and their outer egg shells are removed by treatment with 50% bleach. Embryos are then transferred to a small agar plate and inserted, posterior end first, into small vertical holes in the agar. The plates containing embedded embryos are centrifuged for 30 min at 3000g. The agar supports the embryos and keeps them in a defined orientation. Afterwards, the embryos are dug out of the agar with a blunt needle. Centrifugation separates major organelles into distinct layers, a stratification easily visible by bright-field microscopy. A number of fluorescent markers are available to confirm successful stratification in living embryos. Proteins associated with certain organelles will be enriched in a particular layer, demonstrating colocalization. Individual layers can be recovered for biochemical analysis or transplantation into donor eggs. This technique is applicable for organelle separation in other large cells, including the eggs and oocytes of diverse species.

In-vivo Centrifugation of Drosophila Embryos

We describe a method to separate organelles by density in living Drosophila embryos. Embryos are embedded in agar and centrifuged. This technique yields reproducible separation of major organelles along the anterior-posterior embryo axis. This method facilitates colocalization experiments and yields organelle fractions for biochemical analysis and transplantation experiments.

In-vivo-Zentrifugation von Drosophila Embryonen

A major strategy for purifying and isolating different types of intracellular organelles is to separate them from each other based on differences in ...

An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

Understanding the relationships between genetic and microenvironmental factors that drive normal and malformed embryonic development is fundamental for discovering new therapeutic strategies. Advancements in imaging technology have enabled quantitative investigation of the organization and maturing of the body plan, but later stage embryonic morphogenesis is less clear. Chicken embryos are an attractive vertebrate animal model system for this application because of its ease of culture and surgical manipulation. Early embryos can be cultured for a short time on filter paper rings, which enables complete optical access for cell patterning and fate studies1,2. Studying advanced developmental processes such as cardiac morphogenesis are traditionally performed through a window of the eggshell3-5, but this technique limits optical access due to window size. We previously developed a simple method to culture whole embryos ex-ovo on hexagonal weigh boats for up to 10 days, which enabled high resolution imaging via ultrasonography6,7. These cultures were difficult to transport, limiting the types of imaging tools available for live experiments. We here present an improved shell-less culture system with a cost-effective, portable environmental chamber. Eggs were cracked onto a hammock created by a polyurethane membrane (cling wrap) affixed circumferentially to a plastic cup partially filled with sterile water. The dimensions of the circumference and depth of the hammock were both critical to maintain surface tension, while the mechanics of the hammock and water beneath helped dampen vibrations induced by transportation. A small footprint circulating water bath was also developed to enable continuous temperature control during experimentation. We demonstrate the ability to culture embryos in this way for at least 14 days without morphogenic defect or delay and employ this system in several microsurgical and imaging applications.

An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

In this article, we present a simple methodology to enable long-term ex-ovo avian embryo culture. This technique is ideal for longitudinal experimentation requiring complete optical accessibility and/or sterile transportation in avian embryos.

Ein Ex-ovo Chicken Embryo Culture-System Geeignet für Imaging und Mikrochirurgie Anwendungen

Understanding the relationships between genetic and microenvironmental factors that drive normal and malformed embryonic development is fundamental for ...

Imaging Cell Shape Change in Living Drosophila Embryos

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging. Cell shape changes in the fly are analogous to those in higher organisms, and they drive tissue morphogenesis. So, in many cases, their study has direct implications for understanding human disease (Table 1)1-5. On the sub-cellular scale, these cell shape changes are the product of activities ranging from gene expression to signal transduction, cell polarity, cytoskeletal remodeling and membrane trafficking. Thus, the Drosophila embryo provides not only the context to evaluate cell shape changes as they relate to tissue morphogenesis, but also offers a completely physiological environment to study the sub-cellular activities that shape cells.
The protocol described here is designed to image a specific cell shape change called cellularization. Cellularization is a process of dramatic plasma membrane growth, and it ultimately converts the syncytial embryo into the cellular blastoderm. That is, at interphase of mitotic cycle 14, the plasma membrane simultaneously invaginates around each of ~6000 cortically anchored nuclei to generate a sheet of primary epithelial cells. Counter to previous suggestions, cellularization is not driven by Myosin-2 contractility6, but is instead fueled largely by exocytosis of membrane from internal stores7. Thus, cellularization is an excellent system for studying membrane trafficking during cell shape changes that require plasma membrane invagination or expansion, such as cytokinesis or transverse-tubule (T-tubule) morphogenesis in muscle.
Note that this protocol is easily applied to the imaging of other cell shape changes in the fly embryo, and only requires slight adaptations such as changing the stage of embryo collection, or using "embryo glue" to mount the embryo in a specific orientation (Table 1)8-19. In all cases, the workflow is basically the same (Figure 1). Standard methods for cloning and Drosophila transgenesis are used to prepare stable fly stocks that express a protein of interest, fused to Green Fluorescent Protein (GFP) or its variants, and these flies provide a renewable source of embryos. Alternatively, fluorescent proteins/probes are directly introduced into fly embryos via straightforward micro-injection techniques9-10. Then, depending on the developmental event and cell shape change to be imaged, embryos are collected and staged by morphology on a dissecting microscope, and finally positioned and mounted for time-lapse imaging on a confocal microscope.

Imaging Cell Shape Change in Living Drosophila Embryos

Early development of the fruit fly, Drosophila melanogaster, is characterized by a number of cell shape changes that are well suited for imaging approaches. This article will describe basic tools and methods required for live confocal imaging of Drosophila embryos, and will focus on a cell shape change called cellularization.

Imaging Zellgröße Veränderung der Lebens- Drosophila Embryos

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging.  Cell shape ...

Chromatin Immunoprecipitation Assay for Tissue-specific Genes using Early-stage Mouse Embryos

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This technique has been widely exploited in tissue culture cells, and to a lesser extent, in primary tissue. The application of ChIP to rodent embryonic tissue, especially at early times of development, is complicated by the limited amount of tissue and the heterogeneity of cell and tissue types in the embryo. Here we present a method to perform ChIP using a dissociated embryonic day 8.5 (E8.5) embryo. Sheared chromatin from a single E8.5 embryo can be divided into up to five aliquots, which allows the investigator sufficient material for controls and for investigation of specific protein:chromatin interactions.
We have utilized this technique to begin to document protein:chromatin interactions during the specification of tissue-specific gene expression programs. The heterogeneity of cell types in an embryo necessarily restricts the application of this technique because the result is the detection of protein:chromatin interactions without distinguishing whether the interactions occur in all, a subset of, or a single cell type(s). However, examination of tissue-specific genes during or following the onset of tissue-specific gene expression is feasible for two reasons. First, immunoprecipitation of tissue specific factors necessarily isolates chromatin from the cell type where the factor is expressed. Second, immunoprecipitation of coactivators and histones containing post-translational modifications that are associated with gene activation should only be found at genes and gene regulatory sequences in the cell type where the gene is being or has been activated. The technique should be applicable to the study of most tissue-specific gene activation events.
In the example described below, we utilized E8.5 and E9.5 mouse embryos to examine factor binding at a skeletal muscle specific gene promoter. Somites, which are the precursor tissues from which the skeletal muscles of the trunk and limbs will form, are present at E8.5-9.54,5. Myogenin is a regulatory factor required for skeletal muscle differentiation 6-9. The data demonstrate that myogenin is associated with its own promoter in E8.5 and E9.5 embryos. Because myogenin is only expressed in somites at this stage of development 6,10, the data indicate that myogenin interactions with its own promoter have already occurred in skeletal muscle precursor cells in E8.5 embryos.

We demonstrate a chromatin immunoprecipitation (ChIP) method to identify factor interactions at tissue-specific genes during or after the onset of tissue-specific gene expression in mouse embryonic tissue. This protocol should be widely applicable for the study of tissue-specific gene activation as it occurs during normal embryonic development.

Chromatin Immunopräzipitation-Assay für Tissue-spezifische Gene mit Frühzeitige muriner Embryonen

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This ...

In Ovo Electroporation in Embryonic Chick Retina

Chicken embryonic retina is an excellent tool to study retinal development in higher vertebrates. Because of large size and external development, it is comparatively very easy to manipulate the chick embryonic retina using recombinant DNA/RNA technology. Electroporation of DNA/RNA constructs into the embryonic retina have a great advantage to study gene regulation in retinal stem/progenitor cells during retinal development. Different type of assays such as reporter gene assay, gene over-expression, gene knock down (shRNA) etc. can be performed using the electroporation technique. This video demonstrates targeted retinal injection and in ovo electroporation into the embryonic chick retina at the Hamburger and Hamilton stage 22-23, which is about embryonic day 4 (E4). Here we show a rapid and convenient in ovo electroporation technique whereby a plasmid DNA that expresses green fluorescent protein (GFP) as a marker is directly delivered into the chick embryonic subretinal space and followed by electric pulses to facilitate DNA uptake by retinal stem/progenitor cells. The new method of retinal injection and electroporation at E4 allows the visualization of all retinal cell types, including the late-born neurons1, which has been difficult with the conventional method of injection and electroporation at E1.52.

In Ovo Electroporation in Embryonic Chick Retina

The overall goal of this video is to show how to perform targeted retinal injection and in ovo electroporation of DNA/RNA constructs into the chick embryonic retina at the Hamburger and Hamilton stage 22-23, which is about embryonic day 4 (E4). This technique is very useful to study gene expression, gene regulation, and morphological change in developing chick retina.

In ovo Elektroporation in embryonalen Hühnchenretina

Chicken embryonic retina is an excellent tool to study retinal development in higher vertebrates. Because of large size and external development, it is ...