Name: grafting of beads into developing chicken embryo limbs to identify signal transduction pathways affecting gene expression
Uploaded: 2016-01-17T14:00:00.000+00:00
Duration: 11 min 48 s
Description: Watch this Scientific Journal Video about Grafting of Beads into Developing Chicken Embryo Limbs to Identify Signal Transduction Pathways Affecting Gene Expression at JoVE.com

This work was partly funded by a University of Nottingham Early Career award to DS. RM is funded by the Higher Committee for Education Development of Iraq.

Ethics Statement: Alle diese Versuche folgen der Tierpflege und ethischen Richtlinien der University of Nottingham. 1. Herstellung von Heparin-Korne für Pfropfen <ol><li> Waschen Sie Heparin Perlen sorgfältig in PBS vor dem Gebrauch. Hinweis: Die Glasperlen können bei 4 ° C als Aufschlämmung in PBS gelagert werden. <ol><li> Wählen Perlen zum Pfropfen von ihnen aus dem Lager entfernt mit einer 20 ul Pipette in ein 1 ml Tropfen PBS. Dann mit einem Mikropipette bis 2 &amp; mgr; l eingestellt, um die Kügelchen in eine 20 &amp; mgr; l Tropfen PBS in einer 3 cm Petrischale übertragen. Wählen Perlen basierend auf Größe und mit einem Stereo-Binokular, Transfer ausgewählten Perlen mit einem P2 Pipettenspitze; die um 100 &amp; mgr; m Durchmesser sind ideal. </li></ol></li><li> Entfernen Sie das PBS aus den Perlen. Entfernen Sie die meisten der Flüssigkeit mit einer 20 ul Pipette und der Restflüssigkeit durch Kapillarwirkung mit feinen Uhrmacher Pinzette. Anmerkung: Es ist wichtig, so viel wie möglich, damit das Wachstum facto entfernenR nicht verdünnt, wenn es an den Beads zugegeben. </li><li> Pipettieren den Wachstumsfaktor direkt auf die Perlen. Für FGF18 werden 0,5 ul rekombinanter FGF18 bei 0,5 mg / ml in 0,1% BSA in PBS rekonstituiert. Einige Tropfen von Wasser um die Ränder der Schale, um die Flüssigkeit verflüchtigt. </li><li> Inkubieren der Kügelchen für 1 h bei RT. Nehmen Sie die Tropfen von Wasser aus der Schale mit einer Pasteurpipette und auf Eis bereit für die Transplantation. </li><li> Bei Bedarf (für transparente Kügelchen) Transfer 4-5 Kügelchen bis 2% Phenolrot vor dem Pfropfen zu helfen, zu visualisieren. Spülen in PBS vor der Übertragung auf den Embryo. </li></ol> 2. Herstellung der AG 1-X2 Perlen zum Pfropfen <ol><li> Vor derivatisieren Perlen mit 0,2 N Ameisensäure verwenden. <ol><li> Verwenden Sie hierzu einen Spachtel, um die Kügelchen in ein 1,5 ml Mikrozentrifugenröhrchen übertragen. In 1 ml 0,2 N Ameisensäure und waschen auf einem Schütteltisch für 1 Stunde. </li><li> Entfernen Sie dann die Ameisensäure mit einer Pipette p1000 und ersetzen 1ml Wasser. Waschen für 1 Stunde und wiederholen Sie sechs Mal zu entfernen restlichen Ameisensäure. </li><li> Entfernen Sie verbleibende Flüssigkeit mit einer Pipette p1000 und die Perlen lagern bei 4 ° C. Anmerkung: Es ist nicht notwendig, alle das Restwasser in dieser Stufe zu entfernen. </li></ol></li><li> Entfernen Sie ein kleines Pellet von Kügelchen von etwa 5-10 &amp; mgr; l Volumen aus dem Bestand mit einem kleinen Spatel in eine 3 cm Petrischale und 1 ml DMSO (oder je nachdem, was Lösungsmittel der Wirkstoff gelöst in). Hinweis: Diese sollten mehrere hundert Perlen, von denen diejenigen, die richtige Größe zu wählen ist. </li><li> Dann mit einem P2 Pipette 2 ul eingestellt, um die Kügelchen von etwa 100 &amp; mgr; m Durchmesser zu einem 20 ul Tropfen Lösungsmittel in einem anderen 3 cm Petrischale übertragen. </li><li> Entfernung des Lösungsmittels mit einem 20 ul-Pipette und einer restlichen Lösungsmittels mit einem 2 &amp; mgr; l-Pipette. Ersetzen mit 20 ul des Arzneimittels angewandt werden. HINWEIS: Konsistenz auflösen das Medikament in einer Konzentration von 100 uM in DMSO (oder die überhaupt Lösungsmittel verwendet wird),Aliquot in 20 &amp; mgr; l und bei -80 ° C nach einigen niedermolekularen Inhibitoren sind instabil und es ist am besten wiederholtes Einfrieren und Auftauen vermeiden. Gegebenenfalls anschließend die verdünnte Medikament auf die gewünschte Konzentration vor dem Einweichen Perlen. Wirksame Konzentrationen müssen am besten empirisch für jedes Arzneimittel bestimmt. </li><li> Inkubation für 1 Std. Vor Licht, da viele dieser Moleküle Licht empfindlich sind. </li><li> Vor der Transplantation Transfer 4-5 Perlen mit einer p2 Pipette 2 ul in 20 ul 2% Phenolrot in Wasser gelöst gesetzt. Führen Sie diesen Schritt mit Hilfe eines Stereo Binokular, um die Perlen zu visualisieren. Entfernen Sie eine einzelne Perle mit feinen Uhrmacher Pinzette oder eine Drahtschleife und spülen in PBS vor der Übertragung auf den Embryo. NOTleave Kügelchen in 2% Phenolrot für mehr als 15 min vor der Verwendung, da dies zu Aktivitätsverlust führen. </li></ol> 3. Vorbereitung Tungsten Nadeln <ol><li> Fein geschnitten Wolframdraht in 3-4 cm Länge. Stecken Sie ein Stück Draht in das Endeein Glas Pasteurpipette und Schmelze in einem Bunsenbrenner in Position zu fixieren. Vorbereitung bis zu 10 Nadeln, um sicherzustellen, dass es Ersatzteile, wenn sie während des Gebrauchs beschädigt werden. </li><li> Legen Sie das Ende des Drahtes in einer Lötlampe Flamme und halten Sie ihn dort, bis die Wolfram leuchtet weiß und die Spitze ist scharf (ca. 2-3 min). Hinweis: Während der Verwendung kann die Nadel gereinigt werden und in der Lötlampe nach Bedarf neu geschärft. HINWEIS: Diese Nadeln können auch verwendet werden, um Schleifen für die Übertragung von Perlen zu machen. Berühren Sie leicht die Nadel auf der Bank, und es wird zu biegen, um eine Schleife zu bilden. </li></ol> 4. Vorbereitung Embryonen für Bead Grafts <ol><li> Eier auszubrüten mit dem stumpfen Ende, bis sie die gewünschte Stufe (3-5 Tage für die meisten Gliedmaßenknospe Manipulationen) zu erreichen. Verwendung stumpfen Pinzette Hahn auf dem stumpfen Ende, um die Schale zu brechen und verwenden Sie dann die Zange, um die Schale zu entfernen und setzen den Embryo. Sicherstellen, dass die Eierschalenmembran wird ebenfalls entfernt. HINWEIS: 1-2 ml PBS mit einer Pasteur-Pipette, um mit Unterstützungs hinzugefügt werdenDies, wenn die Membran klebt an den Dotter. Gently Pipette den PBS auf die Oberfläche des Dotters, fassen Sie die Membran mit der Zange, kümmert sich nicht um das Eigelb beschädigen, und ziehen Sie sie vorsichtig aus dem Ei. </li><li> Entfernen Sie bis zu 5 ml Albumin aus dem Ei mit einer 10-ml-Spritze und einer 19 G-Nadel. Sich nur so weit wie notwendig, um den Embryo nicht Kontakt mit dem Band verwendet wird, um das Ei die Entfernung von größeren Volumina abdichten kann Embryoüberlebensraten zu reduzieren gewährleisten. </li><li> Zur Visualisierung der Embryo fügen Sie ein 5-6 Tropfen Künstler Tusche 15 ml PBS mit 100 U Penicillin und Streptomycin 0,1 mg / ml. Injizieren 0,5-1 ml direkt nach dem Embryo mit einer 1 ml Spritze und einer 25 G-Nadel. </li><li> Hinzuzufügen 2-3 ml PBS mit 1% fötalem Kälberserum und 100 U Penicillin und Streptomycin 0,1 mg / ml in das Ei, um den Embryo nicht austrocknen zu gewährleisten. Verwendung geschärft Uhrmacher Pinzette entfernen Sie die Dotterhaut und öffnen Sie das Amnion über die Entwicklung von Extremitätenknospen. Stellen Sie sicher, der Embryo nicht austrocknet und, Falls erforderlich, fügen Sie mehr PBS / FCS. HINWEIS: Die Extremitätenknospen deutlich als gepaart Auswüchse an der Flanke des Embryos sichtbar. In diesen Stadien der Embryo wird sich solche auf der rechten Seite ist in der Regel nach oben. Als Ergebnis ist es normalerweise einfacher, Perlen in die rechten Schenkel Transplantat mit den Links- als diejenigen kontralateralen Kontrollen. </li><li> Verwenden Sie einen geschärften Wolfram-Draht, um einen Einschnitt in die Gliedmaßenknospe, wo der Wulst wird implantiert werden zu machen. Vermeiden Sie das Schneiden den ganzen Weg durch die Gliedmaßenknospe, wo möglich, obwohl in jüngeren Stadien ist dies schwierig. HINWEIS: Bei älteren Embryonen (HH-Stufe 22 und höher) ist es möglich, bestimmte Regionen der Extremitätenknospe zum Pfropfen aber in jüngeren Stadien ist dies schwieriger, wenn die Extremitätenknospe selbst klein zu wählen. Es ist auch wichtig zu bedenken, dass der Extremitätenknospe wird über die gepfropft Perle wachsen so, besonders bei jüngeren Stadien, die Perle in der proximalen Extremitäten bleiben. </li><li> Pick-up ein Wulst entweder von der Wachstumsfaktor oder Drogen entweder extra fein watchmakers Zange oder eine Schleife aus einer Wolfram Nadel gemacht. Wenn die Raupe hat entweder DMSO oder Phenolrot getränkt gründlich in PBS vor dem Auftragen auf den Embryo. Übertragen Sie die Perle auf den Embryo mit der Zange / Drahtschleife. Dann nutzen Sie die geschärften Wolfram-Draht, um den Wulst in den Einschnitt einzufügen. </li><li> Fügen Sie 1-2 ml PBS / FCS auf das Ei sicher, dass der Embryo hydratisiert gehalten, aber vermeiden, die Anwendung direkt auf den Embryo selbst, da dies die Embryonen schädigen und die Wulst zu verdrängt werden. Dichten Sie das Ei mit Band und zurück in den Inkubator. </li></ol>

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The authors have nothing to disclose.

Die Verwendung von Transplantaten Wulst unmittelbar entwickelnden Geweben in ovo angelegt ist ein mächtiges Werkzeug, um die Rolle von Wachstumsfaktor-Signalweges während der Entwicklung geben beispiellose Kontrolle über die Entwicklungsstufe, bei der sie angewendet werden, und die Dauer der Exposition zu sezieren. Die Wahl der Wulst für jede Art von Molekülen ist wichtig. Kleinen hydrophoben Molekülen, wie den hier beschriebenen Inhibitoren und Retinsäure, normalerweise gut binden de...

Vogelembryonen haben ein leistungsfähiges Werkzeug für die Untersuchung der Entwicklung seit vielen Jahren 1 vorgesehen. Eines ihrer nützlichen Eigenschaften ist, dass sie relativ einfach zu manipulieren sind. Externe Entwicklung macht es möglich, das Ei zu öffnen, um den Embryo zugreifen und verschiedene Mikromanipulationen mit Beispielen wie dem klassischen Wachtel-Küken Chimärensystem zur Untersuchung des Zellschicksals 2,3, die Injektion von Retroviren für die Überexpression in bestimmten Geweben während der Entwicklung 4,5 und Explantatkultur entwicklungsSignalQuellen 6 zu identifizieren. Jüngerer Chimären zwischen unmarkiertem Hosts mit Transplantaten von einem transgenen Huhn Linie, die GFP generiert hat gezeigt, dass die Kombination von klassischen Pfropfen und genetische Modifikation kann wichtige Einsichten in die Entwicklung 7,8 bereitzustellen. Die Leichtigkeit, mit der der Vogelembryo kann manipuliert werden, ist es ein hervorragendes Modell für die Untersuchung des Körpers gemachtEntwicklungs 9. Anwendung spezifischer Wachstumsfaktoren auf die Entwicklung Gliedmaßen in vivo war maßgeblich an der Identifizierung von Faktoren, die Gliedmaßen Musterung 10,11 verändern und weiterhin Einblicke in diesen Prozess 12 bereitzustellen. Dieser Ansatz ist auch verwendet worden, um die Faktoren, die die Entwicklung der Muskulatur regulieren zu studieren und hat Rollen für zahlreiche Signale wie Wnts 13, 14 und BMPs HGF 15 freigelegt. Kürzlich wurde diese Technik verwendet, um die Signale, die myogene Genexpression in der Extremitätenknospe untersuchen und zu zeigen, dass Interaktionen zwischen FGF18 und Retinsäure kann das Timing MyoD Expression 16 zu steuern. Mit einer Kombination von Wachstumsfaktoren und kleine Moleküle, die auf Kügelchen geladen werden kann und dann direkt in spezifischen Geweben in bestimmten Entwicklungsstadien gepfropft die Möglichkeit gibt, zu fast jeder Zeit und Region bei der Entwicklung einzugreifen. Dies wurde verwendet, um INVEstigate viele Prozesse einschließlich Somiten Musterung 17,18, neuronale Spezifikation 19, Neuralleiste Migration 20 und Achse Verlängerung 21. Hier beschreiben wir ein Verfahren zum Pfropfen von Kügelchen in entweder Wachstumsfaktoren oder Inhibitoren in der Entwicklung Hühnerschenkel eingeweicht. Dies kann genutzt werden, um die Auswirkungen dieser Signale auf myogenesis durch Analysieren muskelspezifische Genexpression in situ-Hybridisierung zu bestimmen. Wir beschreiben Transplantate unter Verwendung von entweder Heparin getränkten Kügelchen, die für Wachstumsfaktoren oder AG 1-X2 Perlen für kleine hydrophobe Moleküle, wie Retinsäure oder Kleinmolekül-Inhibitoren spezifischer Signalwege verwendet. Aber auch andere Perlen sind ebenfalls verfügbar, die verwendet wurden, um sowohl FGFs und Shh 23 22 zu liefern.

<table><tbody><tr><td>Heparin acrylic beads</td><td>Sigma</td><td>H5263</td><td>The acrylic-heparin beads used have been discontinued. However a replacement product is available, Heparin agarose beads, cat no H6508. These are transparent so harder to work with but can be stained with phenol red in the same way as AG 1-X2 beads,</td></tr><tr><td>AG 1-X2 beads</td><td>Bio-Rad</td><td>140-1231</td><td></td></tr><tr><td>Affi Gel blue beads</td><td>Bio-Rad</td><td>153-7301; 153-7302</td><td>These beads have been used with a range of growth factors including Shh and FGFs and can be used to replace heparin beads</td></tr><tr><td>FGF18</td><td>Peprotech</td><td>100-28</td><td>Resuspend in PBS with 0/1% BSA, prepare single use aliquots of 0.5-1ul and store at -80&amp;deg;C. Batches and suppliers can vary so different concentrations should be tested to determine an effective dose.</td></tr><tr><td>U0126</td><td>Cell Signalling</td><td>9903</td><td>Make to 20mM stock in DMSO and store in single use aliquots at -80&amp;deg;C. Protect from light.</td></tr><tr><td>BMS-493</td><td>Tocris Biosciences</td><td>3509</td><td>Resuspend in DMSO and store in single use aliquots at -80&amp;deg;C. Protect from light.</td></tr><tr><td>Black Indian ink</td><td>Windsor and Newton</td><td>5012572003384 (30 ml)</td><td>While alternatives to this product are available care should be taken as some inks are toxic to embryos</td></tr><tr><td>Tungsten wire, 0.1 mm dia. 99.95%</td><td>Alfa Aesar</td><td>10404</td><td></td></tr><tr><td>Penicillin / streptomycin</td><td>Sigma</td><td>P0781</td><td>Dilute 100X in PBS/ink and PBS/FCS</td></tr></tbody></table>

grafting of beads into developing chicken embryo limbs to identify signal transduction pathways affecting gene expression

Mit Hühnerembryonen es möglich ist, zur direkten Prüfung der Wirkung von entweder Wachstumsfaktoren oder spezifische Inhibitoren von Signalwegen auf die Gen-Expression und Aktivierung von Signaltransduktionswegen. Diese Technik ermöglicht die Abgabe von Signalmolekülen an genau definierten Entwicklungsstadien für bestimmte Zeiten. Nach dieser Embryonen können geerntet werden und Genexpression untersucht, beispielsweise durch in situ Hybridisierung oder Aktivierung des Signals mit der Immunfärbung beobachtet Übertragungswegen. In diesem Video-Heparin-Perlen in FGF18 getränkt oder AG 1-X2 Perlen in U0126, ein MEK-Inhibitor eingeweicht, in die Gliedmaßenknospe in ovo gepfropft. Dies zeigt, dass FGF18 induziert die Expression von MyoD und ERK Phosphorylierung und sowohl endogene als auch FGF18 induziert MyoD Expression durch U0126 gehemmt. Perlen in einer Retinsäure Antagonisten getränkt können vorzeitige MyoD Induktion durch FGF18 potenzieren. Dieser Ansatz kann unsed mit einer Vielzahl von verschiedenen Wachstumsfaktoren und Inhibitoren und wird leicht auf andere Gewebe in den sich entwickelnden Embryo angepasst.

HH Stufe 21 MyoD ist nicht in sich entwickelnden Gliedmaßen Myoblasten obwohl Färbung kann myotome der Entwicklungs Somiten (1A) zu sehen ist, ausgedrückt. 1B zeigt in situ Hybridisierung für MyoD 6 h nach einer FGF18 Wulst Transplantats. MyoD in Myoblasten dicht an dem Wulst induzierten während es keinen Ausdruck in der kontralateralen Extremität. Co-Pfropfen eines Wulstes in U0126 getränkt, einen spezifischen Inhibitor von MEK, Blöcke FGF18 Induktion von MyoD ...

Watch this Scientific Journal Video about Grafting of Beads into Developing Chicken Embryo Limbs to Identify Signal Transduction Pathways Affecting Gene Expression at JoVE.com

Grafting of Beads into Developing Chicken Embryo Limbs to Identify Signal Transduction Pathways Affecting Gene Expression

By grafting beads soaked in growth factors or specific inhibitors of signaling pathways into developing embryos it is possible to directly test their effects in vivo. In this protocol beads are grafted into the limb bud to determine the effects of these molecules on gene expression and signal transduction.

Pfropfen von Beads in die Entwicklung von Hühnerembryo-Glieder zu identifizieren Signaltransduktionswegen Beeinflussung der Genexpression

Using chicken embryos it is possible to test directly the effects of either growth factors or specific inhibitors of signaling pathways on gene expression ...

grafting-beads-into-developing-chicken-embryo-limbs-to-identify

Universidad San Sebastian

Research

JoVE Journal

Developmental Biology

8.8K Aufrufe.  University of Nottingham. By grafting beads soaked in growth factors or specific inhibitors of signaling pathways into developing embryos it is possible to directly test their effects in vivo. In this protocol beads are grafted into the limb bud to determine the effects of these molecules on gene expression and signal transduction.

Serie: Grafting of Beads into Developing Chicken Embryo Limbs to Identify Signal Transduction Pathways Affecting Gene Expression

Optimized Ex-ovo Culturing of Chick Embryos to Advanced Stages of Development

Research in anatomy, embryology, and developmental biology has largely relied on the use of model organisms. In order to study development in live embryos model organisms, such as the chicken, are often used. The chicken is an excellent model organism due to its low cost and minimal maintenance, however they present observational challenges because they are enclosed in an opaque eggshell. In order to properly view the embryo as it develops, the shell must be windowed or removed. Both windowing and ex ovo techniques have been developed to assist researchers in the study of embryonic development. However, each of the methods has limitations and challenges. Here, we present a simple, optimized ex ovo culture technique for chicken embryos that enables the observation of embryonic development from stage HH 19 into late stages of development (HH 40), when many organs have developed. This technique is easy to adopt in both undergraduate classes and more advanced research laboratories where embryo manipulations are conducted.

Optimized Ex-ovo Culturing of Chick Embryos to Advanced Stages of Development

Viewing and accessing the chicken embryo during development can be challenging. We have developed an ex ovo method that is simple, cost effective, and can easily be used in a classroom or research setting. This method provides access to the embryo into late stages of embryonic development (HH 40).

Optimierte<em&gt; Ex-ovo</em&gt; Züchtung von Hühnerembryonen zu fortgeschrittenen Entwicklungsstadium

Research in anatomy, embryology, and developmental biology has largely relied on the use of model organisms. In order to study development in live embryos ...

Forschung

Entwicklungsbiologie

Dual Labeling of Neural Crest Cells and Blood Vessels Within Chicken Embryos Using ChickGFP Neural Tube Grafting and Carbocyanine Dye DiI Injection

All developing organs need to be connected to both the nervous system (for sensory and motor control) as well as the vascular system (for gas exchange, fluid and nutrient supply). Consequently both the nervous and vascular systems develop alongside each other and share striking similarities in their branching architecture. Here we report embryonic manipulations that allow us to study the simultaneous development of neural crest-derived nervous tissue (in this case the enteric nervous system), and the vascular system. This is achieved by generating chicken chimeras via transplantation of discrete segments of the neural tube, and associated neural crest, combined with vascular DiI injection in the same embryo. Our method uses transgenic chickGFP embryos for intraspecies grafting, making the transplant technique more powerful than the classical quail-chick interspecies grafting protocol used with great effect since the 1970s. ChickGFP-chick intraspecies grafting facilitates imaging of transplanted cells and their projections in intact tissues, and eliminates any potential bias in cell development linked to species differences. This method takes full advantage of the ease of access of the avian embryo (compared with other vertebrate embryos) to study the co-development of the enteric nervous system and the vascular system.

Dual Labeling of Neural Crest Cells and Blood Vessels Within Chicken Embryos Using ChickGFP Neural Tube Grafting and Carbocyanine Dye DiI Injection

Here we report dual labeling of neural crest cells and blood vessels using chickGFP neural tube intraspecies grafting combined with intra-vascular DiI injection. This experimental technique allows us to simultaneously visualize and study development of the NCC-derived (enteric) nervous system and the vascular system, during organogenesis.

Dual-Kennzeichnung von neuralen Kammzellen und Blutgefäßen innerhalb von Hühnerembryonen mit ChickGFP Neural Tube Grafting und Carbocyanin Dye DiI Injektion

All developing organs need to be connected to both the nervous system (for sensory and motor control) as well as the vascular system (for gas exchange, ...

Microbead Implantation in the Zebrafish Embryo

The zebrafish has emerged as a valuable genetic model system for the study of developmental biology and disease. Zebrafish share a high degree of genomic conservation, as well as similarities in cellular, molecular, and physiological processes, with other vertebrates including humans. During early ontogeny, zebrafish embryos are optically transparent, allowing researchers to visualize the dynamics of organogenesis using a simple stereomicroscope. Microbead implantation is a method that enables tissue manipulation through the alteration of factors in local environments. This allows researchers to assay the effects of any number of signaling molecules of interest, such as secreted peptides, at specific spatial and temporal points within the developing embryo. Here, we detail a protocol for how to manipulate and implant beads during early zebrafish development.

The zebrafish is an excellent model system for genetic and developmental studies. Bead implantation is a valuable tissue manipulation technique that can be used to interrogate developmental mechanisms by introducing alterations in local cellular environments. This protocol describes how to perform microbead implantation in the zebrafish embryo.

Microbead Implantation im Zebrafischembryo

The zebrafish has emerged as a valuable genetic model system for the study of developmental biology and disease. Zebrafish share a high degree of genomic ...

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 ...

Biologie

In Ovo Electroporations of HH Stage 10 Chicken Embryos

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 molecular biological techniques, the chick has become a useful system in which to study gene regulation and function.  By electroporating DNA or RNA constructs into the developing chicken embryo, genes can be expressed or knocked down in order to analyze in vivo gene function.  Similarly, reporter constructs can be used for fate mapping or to examine putative gene regulatory elements.  Compared to similar experiments in mouse, chick electroporation has the advantages of being quick, easy and inexpensive.  This video demonstrates first how to make a window in the eggshell to manipulate the embryo.  Next, the embryo is visualized with a dilute solution of India ink injected below the embryo.  A glass needle and pipette are used to inject DNA and Fast Green dye into the developing neural tube, then platinum electrodes are placed parallel to the embryo and short electrical pulses are administered with a pulse generator.  Finally, the egg is sealed with tape and placed back into an incubator for further development.  Additionally, the video shows proper egg storage and handling and discusses possible causes of embryo loss following electroporation.

Chick in ovo electroporation is a technique which allows genetic manipulation of the avian embryo. Common applications of this technique include functional analysis of genes and putative enhancer elements. This video demonstrates neural tube electroporation in HH 10 chick embryos. Injection technique and proper egg handling are discussed.

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 ...

Application of Impermeable Barriers Combined with Candidate Factor Soaked Beads to Study Inductive Signals in the Chick

The chick embryo provides a superb vertebrate model that can be used to dissect developmental questions in a direct way. Its accessibility and robustness following surgical intervention are key experimental strengths. Mica plates were the first barriers used to prevent chick limb bud initiation1. Protocols that use aluminum foil as an impermeable barrier to wing bud or leg bud induction and or initiation are described. We combine this technique with bead placement lateral to the barrier to exogenously supply candidate endogenous factors that have been blocked by the barrier. The results are analyzed using in situ hybridization of subsequent gene expression. Our main focus is on the role of retinoic acid signaling in the induction and later initiation of the chick embryo fore and hindlimb. We use BMS 493 (an inverse agonist of retinoic acid receptors (RAR)) soaked beads implanted in the lateral plate mesoderm (LPM) to mimic the effect of a barrier placed between the somites (a source of retinoic acid (RA)) and the LPM from which limb buds grow. Modified versions of these protocols could also be used to address other questions on the origin and timing of inductive cues. Provided the region of the chick embryo is accessible at the relevant developmental stage, a barrier could be placed between the two tissues and consequent changes in development studied. Examples may be found in the developing brain, axis extension and in organ development, such as liver or kidney induction.

Protocols using impermeable barriers to block induction events between tissues of the main body axis and flank in the chick embryo required for limb formation are described. Beads soaked in candidate inductive signals are used to overcome the effect of barrier placement and analysis of gene expression confirms this.

Die Anwendung der undurchlässige Barrieren In Kombination mit Candidate Factor Soaked Beads zu studieren Induktive Signale im Küken

The chick embryo provides a superb vertebrate model that can be used to dissect developmental questions in a direct way. Its accessibility and robustness ...

Assay for Neural Induction in the Chick Embryo

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying the formation and initial patterning of the nervous system. This video demonstrates how to graft organizer tissue into a host, a method by which Hensen s node (the organizer in the chick embryo) is grafted to a host competent ectoderm. The organizer graft instructs overlying na ve tissue to adopt a neural fate via neural inducing signals. This mechanism is referred to as neural induction, and constitutes the initial step in the formation of brain and spinal cord in amniotes. This method is essentially used for the characterization of putative neural inducing molecules in chick. This video demonstrates the different steps in the assay for neural induction; First, the donnor embryo is explanted and pinned on a dish. Then, the host embryo is prepared for New culture. The graft is excised and transplanted to the host area pellucida margin. The host is cultured for 18-22 hrs. The assembly is fixed and processed for further applications (e.g. in situ hybridization). This method was originally devised by Waddington 1,2 and Gallera 3,4.

Neural induction is the first step in the formation of the brain. It is a mechanism by which Hensen's node (organizer), instructs adjacent tissue to adopt a neural fate, i.e. to give rise to the nervous system. This video demonstrates an assay for neural induction in chick embryo.

Assay für Neural Induction im Hühnerembryo

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

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 ...

Chicken Recombinant Limbs Assay to Understand Morphogenesis, Patterning, and Early Steps in Cell Differentiation

Cell differentiation is the fine-tuned process of cell commitment leading to the formation of different specialized cell types during the establishment of developing tissues and organs. This process is actively maintained in adulthood. Cell differentiation is an ongoing process during the development and homeostasis of organs. Understanding the early steps of cell differentiation is essential to know other complex processes such as morphogenesis. Thus, recombinant chicken limbs are an experimental model that allows the study of cell differentiation and pattern generation under embryonic patterning signals. This experimental model imitates an in vivo environment; it assembles reaggregated cells into an ectodermal cover obtained from an early limb bud. Later, ectoderms are transferred and implanted in a chick embryo receptor to allow its development. This assay was mainly used to evaluate mesodermal limb bud cells; however, it can be applied to other stem or progenitor cells from other organisms.

Recombinant limbs are a powerful experimental model that allows for studying the process of cell differentiation and the generation of patterns under the influence of embryonic signals. This protocol presents a detailed method for generating recombinant limbs with chicken limb-mesodermal cells, adaptable to other cell types obtained from different organisms.

Chicken Recombinant Limbs Assay, um Morphogenese, Musterung und frühe Schritte in der Zelldifferenzierung zu verstehen

Cell differentiation is the fine-tuned process of cell commitment leading to the formation of different specialized cell types during the establishment of ...

Analysis of Cell Differentiation, Morphogenesis, and Patterning During Chicken Embryogenesis Using the Soaked-Bead Assay

A multitude of genetic programs is activated during embryonic development that orchestrates cell differentiation to generate an astounding diversity of somatic cells, tissues, and organs. The precise activation of these genetic programs is regulated by morphogens, diffusible molecules that direct cell fate at different thresholds. Understanding how genetic activation coordinates morphogenesis requires the study of local interactions triggered by morphogens during development. The use of beads soaked in proteins or drugs implanted into distinct regions of the embryo enables studying the role of specific molecules in the establishment of digits and other developmental processes. This experimental technique provides information on the control of cell induction, cell fate, and pattern formation. Thus, this soaked bead assay is an extremely powerful and valuable experimental tool applicable to other embryonic models.

The soaked bead assay involves targeted delivery of test reagent at any developmental time point to study the regulation of cell differentiation and morphogenesis. A detailed protocol, applicable to any experimental animal model, for preparing three different types of soaked beads and implanting these in the interdigit of a chicken embryo is presented.

Analyse der Zelldifferenzierung, Morphogenese und Musterung während der Hühnerembryogenese mit dem Soaked-Bead-Assay

A multitude of genetic programs is activated during embryonic development that orchestrates cell differentiation to generate an astounding diversity of ...

Pfropfen von Beads in die Entwicklung von Hühnerembryo-Glieder zu identifizieren Signaltransduktionswegen Beeinflussung der Genexpression

Zusammenfassung

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Dual-Kennzeichnung von neuralen Kammzellen und Blutgefäßen innerhalb von Hühnerembryonen mit Chick^GFPNeural Tube Grafting und Carbocyanin Dye DiI Injektion