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: All these experiments follow the animal care and ethical guidelines of the University of Nottingham.
1. Preparation of Heparin Beads for Grafting
<ol>
	<li>Wash heparin beads thoroughly in PBS before use. Note: Beads can be stored at 4 &#176;C as a slurry in PBS.
	<ol>
		<li>Select beads for grafting by removing them from the stock with a 20 &#181;l pipette into a 1 ml drop of PBS. Then use a micropipette set to 2 &#181;l to transfer beads into a 20 &#181;l drop of PBS in a 3 cm petri dish. Choose beads based on size and, using a stereo dissecting microscope, transfer selected beads with a P2 pipette tip; those around 100 &#181;m diameter are ideal.</li>
	</ol>
	</li>
	<li>Remove the PBS from the beads. Remove most of the liquid with a 20 &#181;l pipette and the residual liquid by capillary action with fine watchmakers forceps. Note: It is important to remove as much as possible so the growth factor is not diluted when it is added to the beads.</li>
	<li>Pipette the growth factor directly onto the beads. For FGF18 add 0.5 &#181;l of recombinant FGF18 reconstituted at 0.5 mg/ml in 0.1% BSA in PBS. Place several drops of water around the edges of the dish to prevent the liquid from evaporating.</li>
	<li>Incubate the beads for 1 hr at RT. Remove the drops of water from the dish with a Pasteur pipette and place on ice ready for grafting.</li>
	<li>If required (for transparent beads) transfer 4-5 beads to 2% phenol red before grafting to help visualize them. Rinse in PBS before transferring to the embryo.</li>
</ol>
2. Preparation of AG 1-X2 Beads for Grafting
<ol>
	<li>Prior to use derivatize beads with 0.2 N formic acid.
	<ol>
		<li>To do this use a spatula to transfer beads to a 1.5 ml microcentrifuge tube. Add 1ml of 0.2 N formic acid and wash on a shaking platform for 1 hr.</li>
		<li>Then remove the formic acid with a p1000 pipette and replace with 1 ml of water. Wash for 1 hr and repeat six times to remove remaining formic acid.</li>
		<li>Remove remaining liquid with a p1000 pipette and store the beads at 4 &#176;C. Note: It is not necessary to remove all the residual water at this stage.</li>
	</ol>
	</li>
	<li>Remove a small pellet of beads of approximately 5-10 &#181;l volume from the stock using a small spatula into a 3 cm petri dish and add 1 ml of DMSO (or whichever solvent the drug is dissolved in). Note: This should provide several hundred beads from which to select those of the right size.</li>
	<li>Then use a P2 pipette set to 2 &#181;l to transfer beads of approximately 100 &#181;m diameter to a 20 &#181;l drop of solvent in another 3 cm petri dish.</li>
	<li>Remove solvent with a 20 &#181;l pipette and any residual solvent with a 2 &#181;l pipette. Replace with 20 &#181;l of the drug to be applied. 
	NOTE: For consistency dissolve the drug at a concentration of 100 &#181;M in DMSO (or which ever solvent is used), aliquot into 20 &#181;l and store at -80 &#176;C as some small molecule inhibitors are unstable and it is best to avoid repeated freeze-thawing. If necessary then dilute the drug to the desired concentration before soaking beads. Effective concentrations may need to be determined empirically for each drug.</li>
	<li>Incubate for 1 hr. Protect from light as many of these molecules are light sensitive.</li>
	<li>Before grafting transfer 4-5 beads with a p2 pipette set to 2 &#181;l into 20 &#181;l of 2% phenol red dissolved in water. Perform this step using a stereo dissecting microscope to visualize the beads. Remove a single bead with fine watchmakers forceps or a wire loop and rinse in PBS before transferring to the embryo. Do not leave beads in 2% phenol red for more than 15 min before use as this can cause loss of activity.</li>
</ol>
3. Preparing Tungsten Needles
<ol>
	<li>Cut fine tungsten wire into 3-4 cm lengths. Insert one piece of wire into the end of a glass Pasteur pipette and melt in a Bunsen burner to fix in position. Prepare up to 10 needles to ensure that there are spares if they are damaged during use.</li>
	<li>Place the end of the wire into a blowtorch flame and hold it there until the tungsten glows white and the tip is sharp (around 2-3 min). Note: During use the needle can be cleaned and re-sharpened in the blowtorch as needed. 
	NOTE: These needles can also be used to make loops for transferring beads. Gently touch the needle to the bench and it will bend to form a loop.</li>
</ol>
4. Preparing Embryos for Bead Grafts
<ol>
	<li>Incubate eggs with the blunt end up until they reach the desired stage (3-5 days for most limb bud manipulations). Using blunt forceps tap on the blunt end to break the shell and then use the forceps to remove the shell and expose the embryo. Ensure the eggshell membrane is also removed. 
	NOTE: 1-2 ml of PBS can be added with a Pasteur pipette to assist with this if the membrane sticks to the yolk. Gently pipette the PBS onto the surface of the yolk, grasp the membrane with the forceps, taking care not to damage the yolk, and gently pull it away from the egg.</li>
	<li>Remove up to 5 ml of albumen from the egg with a 10 ml syringe and a 19 G needle. Take only as much as needed to ensure the embryo does not make contact with the tape used to seal the egg as removal of larger volumes can reduce embryo survival rates.</li>
	<li>To visualize the embryo add a 5-6 drops of artists india ink to 15 ml of PBS containing 100 U penicillin and 0.1 mg streptomycin/ml. Inject 0.5-1 ml directly under the embryo with a 1 ml syringe and a 25 G needle.</li>
	<li>Add 2-3 ml of PBS with 1% fetal calf serum and 100 U penicillin and 0.1 mg streptomycin/ml into the egg to ensure the embryo does not dehydrate. Using sharpened watchmakers forceps remove the vitelline membrane and open the amnion over the developing limb buds. Ensure the embryo does not dry out and, if needed, add more PBS / FCS. 
	NOTE: The limb buds can be clearly seen as paired outgrowths on the flank of the embryo. At these stages the embryo will turn such the right side is normally uppermost. As a result it is normally easier to graft beads into the right limbs and use the left ones as contralateral controls.</li>
	<li>Use a sharpened tungsten wire to make an incision into the limb bud where the bead will be implanted. Avoid cutting all the way through the limb bud where possible although at younger stages this is difficult. 
	NOTE: In older embryos (HH stage 22 and above) it is possible to select specific regions of the limb bud for grafting but at younger stages this is more difficult as the limb bud itself is small. It is also important to remember that the limb bud will grow past the grafted bead so, especially at younger stages, the bead will remain in the proximal limb.</li>
	<li>Pick up a bead from either the growth factor or drug using either extra fine watchmakers forceps or a loop made from a tungsten needle. If the bead has been soaked in either DMSO or phenol red, rinse in PBS before applying to the embryo. Transfer the bead to the embryo with the forceps / wire loop. Then use the sharpened tungsten wire to insert the bead into the incision.</li>
	<li>Add 1-2 ml of PBS/FCS to the egg ensuring that the embryo is kept hydrated but avoid applying directly onto the embryo itself as this can damage the embryos and cause the bead to be dislodged. Seal the egg with tape and return to the incubator.</li>
</ol>

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The authors have nothing to disclose.

The use of bead grafts applied directly to developing tissues in ovo is a powerful tool to dissect the role of growth factor signaling during development giving unparalleled control over the developmental stage at which they are applied and the duration of exposure.
The choice of bead for each type of molecule is important. Small hydrophobic molecules, such as the inhibitors described here and retinoic acid, usually bind well to derivatized AG 1-X2 beads although it is necessary to test the ef...

Avian embryos have provided a powerful tool for the study of development for many years1. One of their most useful characteristics is that they are relatively easy to manipulate. External development makes it possible to open the egg to access the embryo and perform various micromanipulations including examples such as the classic quail-chick chimera system for studying cell fate2,3, the injection of retroviruses for overexpression in specific tissues during development4,5 and explant culture to identify developmental signaling sources6. More recently chimeras generated between unlabeled hosts with grafts from a transgenic chicken line expressing GFP has shown that the combination of classical grafting and genetic modification can provide important insights into development7,8.
The ease with which the avian embryo can be manipulated has made it an excellent model for studying limb development9. Application of specific growth factors to developing limbs in vivo has been instrumental in identifying factors that alter limb patterning10,11 and continues to provide insights into this process12. This approach has also been used to study the factors that regulate muscle development and has uncovered roles for numerous signals such as Wnts13, BMPs14 and HGF15.
Recently this technique has been used to investigate the signals controlling myogenic gene expression in the limb bud and has shown that interactions between FGF18 and retinoic acid can control the timing of MyoD expression16. Using a combination of growth factors and small molecules that can be loaded onto beads and then grafted directly into specific tissues at defined developmental stages gives the opportunity to intervene at almost any time and region during development. This has been used to investigate many processes including somite patterning17,18, neural specification19, neural crest migration20 and axis extension21.
Here we describe a method for grafting beads soaked in either growth factors or inhibitors into developing chicken limbs. This has been used to determine the effects of these signals on myogenesis by analyzing muscle specific gene expression with in situ hybridization. We describe grafts using either heparin soaked beads, which are used for growth factors, or AG 1-X2 beads for small hydrophobic molecules such as retinoic acid or small molecule inhibitors of specific signaling pathways. However other beads are also available which have been used to deliver both FGFs22 and Shh23.

<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

Using chicken embryos it is possible to test directly the effects of either growth factors or specific inhibitors of signaling pathways on gene expression and activation of signal transduction pathways. This technique allows the delivery of signaling molecules at precisely defined developmental stages for specific times. After this embryos can be harvested and gene expression examined, for example by in situ hybridization, or activation of signal transduction pathways observed with immunostaining.
In this video heparin beads soaked in FGF18 or AG 1-X2 beads soaked in U0126, a MEK inhibitor, are grafted into the limb bud in ovo. This shows that FGF18 induces expression of MyoD and ERK phosphorylation and both endogenous and FGF18 induced MyoD expression is inhibited by U0126. Beads soaked in a retinoic acid antagonist can potentiate premature MyoD induction by FGF18.
This approach can be used with a wide range of different growth factors and inhibitors and is easily adapted to other tissues in the developing embryo.

At HH stage 21 MyoD is not expressed in developing limb myoblasts although staining can be seen in the myotome of the developing somites (Figure 1A). Figure 1B shows in situ hybridization for MyoD 6 hr following an FGF18 bead graft. MyoD is induced in myoblasts close to the bead while there is no expression in the contralateral limb. Co-grafting a bead soaked in U0126, a specific inhibitor of MEK, blocks FGF18 induction of MyoD (Figure 1C). Similarly grafting a ...

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.

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 de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

8.8K Views.  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.

Video: 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).

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

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.

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.

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.

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

Biology

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.

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.

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.

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.

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.

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.

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

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

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Chapters in this video

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

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

Microbead Implantation in the Zebrafish Embryo

Placing Growth Factor-Coated Beads on Early Stage Chicken Embryos

In Ovo Electroporations of HH Stage 10 Chicken Embryos

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

Assay for Neural Induction in the Chick Embryo

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

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

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