This work was supported by the Direcci&#243;n General de Asuntos del Personal Acad&#233;mico (DGAPA)-Universidad Nacional Aut&#243;noma de M&#233;xico [grant numbers IN211117 and IN213314] and Consejo Nacional de Ciencia y Tecnolog&#237;a (CONACyT) [grant number 1887 CONACyT-Fronteras de la Ciencia] awarded to JC-M. JC M-L received a postdoctoral fellowship from the Consejo Nacional de Ciencia y Tecnolog&#237;a (CONACyT-Fronteras de la Ciencia-1887). The authors appreciate the help of Lic. Lucia&#160;Brito from Instituto de Investigaciones Biom&#233;dicas, UNAM in the preparation references of this manuscript.

This research was reviewed and approved by the Institutional Review Board for the Care and Use of Laboratory Animals of the Instituto de Investigaciones Biom&#233;dicas, Universidad Nacional Aut&#243;noma de M&#233;xico (UNAM, Mexico City, Mexico).
1. Egg incubation and embryo staging
NOTE: Fertilized hen eggs can be obtained from local farms. Fertilized White Leghorn chicken eggs are most commonly used. Store the freshly fertilized chicken eggs at 15 &#176;C for up to 1 week prior to incubation.
<ol>
	<li>Incubate the fertilized chicken eggs vertically with the pointy side down in a humidified incubator at 38 &#176;C and 70% relative humidity until they reach the 28 HH stage (approximately 5.5 days according to Hamilton and Hamburger, 1951)22. Rotate the eggs during the incubation to prevent the embryo from adhering to the shell membrane. 
	NOTE: The choice of the developmental stage is informed by the experimental aims. In this case, the 28 HH stage is optimal for interdigit bead implantation to induce an ectopic digit or promote cell death.</li>
	<li>Remove the eggs from the incubator, swab them with 70% ethanol, and allow them to air dry. Disinfect the working area, microscopes, and instruments with 70% ethanol.</li>
	<li>Candle the egg to identify blood vessels and locate the embryo. Discard eggs that do not have an embryo.</li>
	<li>Using the end of non-toothed forceps, open a window in around five eggs by tapping the blunt end of an egg, and remove a 1-cm2 section of the shell with the forceps.</li>
	<li>Transfer the egg into a carton or plastic holder and place it under the stereomicroscope. Remove the air membrane by puncturing and pulling it out with fine surgical forceps. Remove any small piece of eggshell that could contact the embryo. 
	NOTE: The air membrane is the white, opaque membrane observed immediately after windowing the egg.</li>
	<li>Observe under the microscope while opening the amniotic sac slightly, tearing it using the fine surgical forceps. Be careful not to damage the vasculature of the chorioallantoic membrane. 
	&#8203;NOTE: The amnion is the transparent membrane closely surrounding the embryo that encapsulates it in amniotic fluid.</li>
	<li>Stage the embryos in ovo to determine whether they are in the desired stage. Embryos in earlier stages can be returned to the incubator after sealing the eggshell window with tape.</li>
</ol>
2. Bead preparation
NOTE: Depending on the experimental aim and treatment in question, alternative bead types (e.g., Affi-Gel, AG1-X2, heparin) may be more suitable. Affi-Gel beads are optimal for proteins (e.g., TGF-&#223;1), while heparin beads are ideal for growth factors (e.g., FGFs, WNT) and AG1-X2 beads for chemicals solubilized in organic solvents (e.g., DMSO).
<ol>
	<li>Affi-Gel and heparin bead preparation
	<ol>
		<li>Cut a square of parafilm to fit a 45-mm Petri dish. Place the parafilm across the bottom of the petri dish to cover it and fix it to the bottom of the Petri dish by pushing each vertex using the end of non-toothed forceps. Set aside.</li>
		<li>Using a pipette or spatula, transfer the beads into a microcentrifuge tube and wash them twice in 1x PBS by settling and pipetting.</li>
		<li>Transfer ~40-50 beads with a micropipette to the center of the parafilmed Petri dish from step 1.</li>
		<li>Using the microscope, select ~30 Affi-Gel or heparin beads ~100 &#181;m in diameter for use. Use a microscope eyepiece reticle to size the beads or use the third interdigit of an HH 28 embryo as a reference; the bead must be smaller than the interdigit.</li>
		<li>Carefully remove as much of the excess PBS surrounding the beads as possible and soak them in 2-5 &#181;L of the treatment solution. Assure that the solution completely covers beads.</li>
		<li>In parallel, prepare control beads by soaking in a solution that contains the same amount of vehicle as used in the experimental treatment solution. 
		NOTE: Concentrations need to be calculated for each treatment according to the experiment. Use the appropriate personal protective equipment when handling potentially harmful reagents.</li>
		<li>Incubate the beads in the solution for 30 min at room temperature. To prevent the beads from drying out during the incubation, pipette a few drops of 1x PBS or water around the beads to humidify the local atmosphere and cover the dish with parafilm to slow evaporation.</li>
		<li>Place the Petri dish on ice and implant the beads within the same day.</li>
	</ol>
	</li>
	<li>AG 1-X2 bead preparation
	<ol>
		<li>Cut a square of parafilm to fit a 45-mm Petri dish. Cover the bottom of the Petri dish with parafilm and affix by pushing each vertex using the end of non-toothed forceps. Set aside.</li>
		<li>Use a spatula to transfer the AG 1-X2 beads into a microcentrifuge tube. Add 30-50 &#181;L of the treatment solution at the desired final concentration.</li>
		<li>In parallel, incubate the control beads in a solution with the vehicle alone, prepared without the experimental chemical or protein of interest.</li>
		<li>Incubate the beads for 20 min while slowly shaking at room temperature. Wrap the microcentrifuge tubes with foil to protect from light during all incubation, given that many of these molecules are light-sensitive</li>
		<li>Remove as much of the solution as possible using a pipette and stain with 2% phenol red dissolved in water at room temperature for 2 min with mild agitation in a vortex. 
		NOTE: Dyeing the transparent AG 1-X2 beads facilitates their implantation in the embryo.</li>
		<li>Remove the 2% phenol red and wash the beads twice in 1x PBS to remove any excess dye.</li>
		<li>With a micropipette tip, transfer ~40-50 AG 1-X2 beads to the center of the parafilm-covered Petri dish prepared in step 1 and soak these in 5 &#181;L of 1x PBS. Under the microscope, select around 30 beads that are ~100 &#181;m diameter in size. Discard the unused beads.</li>
		<li>Beads are ready to implant. Ensure the beads are submerged in the PBS throughout the implantation by pipetting a few drops of 1x PBS or water around the beads to humidify the local atmosphere and/or cover the dish with parafilm to slow evaporation.</li>
	</ol>
	</li>
</ol>
3. Embryo manipulation and interdigit implantation
<ol>
	<li>Before manipulating the embryos, arrange two stereomicroscopes next to each other on a benchtop. One is for embryo manipulation and bead implantation, and the other is for maintaining the treated beads ready to implant into the embryo.</li>
	<li>Using non-toothed forceps, create a window in the remaining eggs as described in the egg incubation and embryo staging section.</li>
	<li>Under the microscope, open the amniotic sac by tearing the amniotic membrane with fine surgical forceps near the right hindlimb only by the amount needed to accomplish the procedure (i.e., as little as possible).</li>
	<li>Using fine forceps, hold the embryo by the amniotic membrane to expose the right hindlimb. Using a fine tungsten needle, make a hole centered in the distal-most of the third interdigit of the hindlimb.</li>
	<li>Without releasing the embryo and the exposed hindlimb, take one treated bead from the other microscope using one of the tips of the forceps. The forceps must be in the open position for one bead to adhere to the forceps tip.</li>
	<li>Transfer the bead into the chick embryo near the hindlimb and position it on top of the interdigit hole.</li>
	<li>Close the forceps to apply pressure to the bead until it enters the hole.</li>
	<li>Release the embryo and seal the eggshell window with tape.</li>
	<li>Return the eggs to the incubator until they have reached the required stage. Repeat the procedure until the required number of manipulated embryos is reached. It is imperative to ensure that the beads do not dry out at any point. 
	NOTE: To observe a complete ectopic finger, incubate for ~72 h after bead implantation. In contrast, early differentiation genes (e.g., Sox9) are expressed ~30 min after the implantation of a TGF-&#223;1-soaked bead.</li>
</ol>

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The authors have nothing to disclose.

The main advantage of the experimental tool detailed in this protocol is being able to control the time and location of the exposure to beads soaked in a given experimental molecule. Combining the correct positioning with precise developmental timing provides enormous possibilities to study cell differentiation processes. Performing these experiments in undifferentiated tissue enables investigating the first crucial events in cellular lineage. For example, placing a TGF&#223;-soaked bead in the interdigital tissue of emb...

Breakthroughs in the molecular mechanisms that control gene expression during embryonic development have allowed us to understand how cell fate is determined. Commitment to different cell lineages occurs once cells begin the molecular expression of transcription factors1. This expression pattern is highly coordinated in space and time and thereby directs the shaping, positioning, and patterning of cells, tissues, and organs1,2,3,4,5. Embryonic induction is the process by which cells are committed to specific lineages by establishing hierarchies that restrict cells&#39; potentiality, which even include the generation of the basic body plan as occurs with the Spemann organizer6,7. The blastopore dorsal lip induces a second embryonic axis in a host embryo8,9. Today, with the aid of grafting and other classical experiments combined with molecular approaches, it is known that different transcription factors and growth factors function to direct embryonic induction in the Spemann organizer10. Thus, experimental manipulation is an important tool to understand cell differentiation, morphogenesis, and patterning processes during embryogenesis.
Interestingly, in embryonic systems where tissue transplantation is difficult or when the inducers are already well known, carriers are used to deliver molecules (e.g., proteins, chemicals, toxins, etc.) to regulate cell differentiation, morphogenesis, and even patterning. One such carrier system involves implanting beads soaked in a specific molecule in any experimental model organism at any developmental time point to determine the effect of the said reagent or direct the differentiation of the said model. For example, by implanting retinoic acid (RA)-soaked beads into the chicken wing limb bud, Cheryl Tickle et al. (1985) demonstrated that RA induces the expression of sonic hedgehog in the zone of polarizing activity (ZPA)11,12. The same experimental strategy was used to discover that RA controls the asymmetry of somites and cell death in the limb bud during digit development and in other embryonic limb regions13,14,15. Other factors, mainly proteins (e.g., fibroblast growth factors [FGF], transforming growth factor-beta [TGF-&#223;]) have been used to induce limbs in early embryos&#39; flanks and new digits in the interdigital region, respectively16,17,18,19,20,21. These experiments evidence the power and utility of this technique for determining the stage of commitment or competence of tissues or groups of cells exposed to the molecules.
In this protocol, the chick limb at the stage of digit formation served as the experimental model to present step-by-step how to prepare and implant the soaked beads. However, this experimental tool is not limited to this application but can be exploited in any experimental animal model and any timepoint in vitro and in vivo to study induction, differentiation, cell death, and patterning.

<table><tbody><tr><td>Affi-Gel Blue Gel beads</td><td>Bio-Rad</td><td>153-7302</td><td></td></tr><tr><td>AG1-X2 beads</td><td>Bio-Rad</td><td>1400123</td><td></td></tr><tr><td>Egg incubator</td><td>Incumatic de Mexico</td><td>Incumatic 1000</td><td></td></tr><tr><td>Fine surgical forceps</td><td>Fine Science Tools</td><td>9115-10</td><td></td></tr><tr><td>Heparine Sepharose beads</td><td>Abcam</td><td>ab193268</td><td></td></tr><tr><td>Petri dish</td><td>Nest</td><td>705001</td><td></td></tr><tr><td>Stereomicroscope</td><td>Zeiss</td><td>Stemi DV4</td><td></td></tr><tr><td>Tape</td><td>NA</td><td>NA</td><td></td></tr><tr><td>Tungsten needle</td><td>GoodFellow</td><td>E74-15096/01</td><td></td></tr></tbody></table>

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.

Using soaked beads to evaluate cell behavior in the embryonic chick limb 
To assure the efficacy of this assay, the bead must be placed consistently and precisely in the correct location; in this case, the distal-most of the third interdigit beneath the apical ectodermal ridge AER (Figure 1A). This positioning permits the molecule in question to spread equally throughout the interdigital tissue. Moreover, the zone beneath the AER contains undifferentiated cells that are...

Watch this Scientific Journal Video about Analysis of Cell Differentiation, Morphogenesis, and Patterning During Chicken Embryogenesis Using the Soaked-Bead Assay at JoVE.com

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

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 ...

analysis-cell-differentiation-morphogenesis-patterning-during-chicken

Universidad San Sebastian

Research

JoVE Journal

Developmental Biology

3.1K Views.  Universidad Nacional Autónoma de México. 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.

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

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 ...

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.

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 ...

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.

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 ...

Biology

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 ...

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 ...

A Submerged Filter Paper Sandwich for Long-term Ex Ovo Time-lapse Imaging of Early Chick Embryos

Due to its availability, low cost, flat geometry, and transparency, the ex ovo chick embryo has become a major vertebrate animal model for the study of morphogenetic events, such as gastrulation2, neurulation3-5, somitogenesis6, heart bending7,8, and brain formation9-13, during early embryogenesis. Key to understanding morphogenetic processes is to follow them dynamically by time-lapse imaging. The acquisition of time-lapse movies of chick embryogenesis ex ovo has been limited either to short time windows or to the need for an incubator to control temperature and humidity around the embryo14. Here, we present a new technique to culture chick embryos ex ovo for high-resolution time-lapse imaging using transmitted light microscopy. The submerged filter paper sandwich is a variant of the well-established filter paper carrier technique (EC-culture)1 and allows for the culturing of chick embryos without the need for a climate chamber. The embryo is sandwiched between two identical filter paper carriers and is kept fully submerged in a simple, temperature-controlled medium covered by a layer of light mineral oil. Starting from the primitive streak stage (Hamburger-Hamilton stage 5, HH5)15 up to at least the 28-somite stage (HH16)15, embryos can be cultured with either their ventral or dorsal side up. This allows the acquisition of time-lapse movies covering about 30 hr of embryonic development. Representative time-lapse frames and movies are shown. Embryos are compared morphologically to an embryo cultured in the standard EC-culture.
The submerged filter paper sandwich provides a stable environment to study early dorsal and ventral morphogenetic processes. It also allows for live fluorescence imaging and micromanipulations, such as microsurgery, bead implantation, microinjection, gene silencing, and electroporation, and has a strong potential to be combined with immersion objectives for laser-based imaging (including light-sheet microscopy).

A Submerged Filter Paper Sandwich for Long-term Ex Ovo Time-lapse Imaging of Early Chick Embryos

Here, we present a new technique to culture chick embryos ex ovo for time-lapse imaging using transmitted light and fluorescence microscopy. The submerged filter paper sandwich is a variant of the well-established filter paper carrier technique1 that allows for the culturing of chick embryos fully-submerged in a liquid culture medium.

Due to its availability, low cost, flat geometry, and transparency, the ex ovo chick embryo has become a major vertebrate animal model for the study of ...

Analysis of Neural Crest Migration and Differentiation by Cross-species Transplantation

Avian embryos provide a unique platform for studying many vertebrate developmental processes, due to the easy access of the embryos within the egg. Chimeric avian embryos, in which quail donor tissue is transplanted into a chick embryo in ovo, combine the power of indelible genetic labeling of cell populations with the ease of manipulation presented by the avian embryo.
Quail-chick chimeras are a classical tool for tracing migratory neural crest cells (NCCs)1-3. NCCs are a transient migratory population of cells in the embryo, which originate in the dorsal region of the developing neural tube4. They undergo an epithelial to mesenchymal transition and subsequently migrate to other regions of the embryo, where they differentiate into various cell types including cartilage5-13, melanocytes11,14-20, neurons and glia21-32. NCCs are multipotent, and their ultimate fate is influenced by 1) the region of the neural tube in which they originate along the rostro-caudal axis of the embryo11,33-37, 2) signals from neighboring cells as they migrate38-44, and 3) the microenvironment of their ultimate destination within the embryo45,46. Tracing these cells from their point of origin at the neural tube, to their final position and fate within the embryo, provides important insight into the developmental processes that regulate patterning and organogenesis.
Transplantation of complementary regions of donor neural tube (homotopic grafting) or different regions of donor neural tube (heterotopic grafting) can reveal differences in pre-specification of NCCs along the rostro-caudal axis2,47. This technique can be further adapted to transplant a unilateral compartment of the neural tube, such that one side is derived from donor tissue, and the contralateral side remains unperturbed in the host embryo, yielding an internal control within the same sample2,47. It can also be adapted for transplantation of brain segments in later embryos, after HH10, when the anterior neural tube has closed47.
Here we report techniques for generating quail-chick chimeras via neural tube transplantation, which allow for tracing of migratory NCCs derived from a discrete segment of the neural tube. Species-specific labeling of the donor-derived cells with the quail-specific QCPN antibody48-56 allows the researcher to distinguish donor and host cells at the experimental end point. This technique is straightforward, inexpensive, and has many applications, including fate-mapping, cell lineage tracing, and identifying pre-patterning events along the rostro-caudal axis45. Because of the ease of access to the avian embryo, the quail-chick graft technique may be combined with other manipulations, including but not limited to lens ablation40, injection of inhibitory molecules57,58, or genetic manipulation via electroporation of expression plasmids59-61, to identify the response of particular migratory streams of NCCs to perturbations in the embryo's developmental program. Furthermore, this grafting technique may also be used to generate other interspecific chimeric embryos such as quail-duck chimeras to study NCC contribution to craniofacial morphogenesis, or mouse-chick chimeras to combine the power of mouse genetics with the ease of manipulation of the avian embryo.62

An approach for analyzing migration and eventual fate of avian neural crest cells in quail-chick chimeric embryos is described. This method is a simple and straightforward technique for tracing neural crest cells during migration and differentiation that are otherwise difficult to distinguish within an unmanipulated chick embryo.

Avian embryos provide a unique platform for studying many vertebrate developmental processes, due to the easy access of the embryos within the egg. ...

Neuroscience

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 ...

A Comprehensive Review of Avian Embryonic Skin Explant Culture Protocols

Using Avian Skin Explants to Study Tissue Patterning and Organogenesis

The developing avian skin during embryogenesis is a unique model that can provide valuable insights into tissue patterning. Here three variations on skin explant cultures to examine different aspects of skin development are described. First, ex vivo organ cultures and manipulations offer researchers opportunities to observe and study the development of feather buds directly. Skin explant culture can grow for 7 days enabling direct analysis of cellular behavior and 4D imaging at intervals during this growth period. This also allows for physical and molecular manipulations of culture conditions to visualize tissue response. For example, growth factor-coated beads can be applied locally to induce changes in feather patterning in a limited area. Alternatively, viral transduction can be delivered globally in the culture media to up or downregulate gene expression. Second, the skin recombination protocol allows researchers to investigate tissue interactions between the epidermis and mesenchyme that are derived from different skin regions, different life stages, or different species. This affords an opportunity to test the time window in which the epithelium is competent to respond to signals and its ability to form different skin appendages in response to signals from different mesenchymal sources. Third, skin reconstitution using dissociated dermal cells overlaid with intact epithelium resets skin development and enables the study of the initial processes of periodic patterning. This approach also enhances our ability to manipulate gene expression among the dissociated cells before creating the reconstituted skin explant. This paper provides the three culture protocols and exemplary experiments to demonstrate their utility.

Author Spotlight: Insight into the Current Experimental Avian Skin Explant Methodologies

Here we describe protocols for three types of avian embryonic skin explant cultures that can be used to examine tissue interactions, 4D imaging timelapse movie (3D plus time), global or local perturbation of molecular function, and systems biology characterization.

The developing avian skin during embryogenesis is a unique model that can provide valuable insights into tissue patterning. Here three variations on skin ...

Tracking Morphogenetic Tissue Deformations in the Early Chick Embryo 

Embryonic epithelia undergo complex deformations (e.g. bending, twisting, folding, and stretching) to form the primitive organs of the early embryo. Tracking fiducial markers on the surfaces of these cellular sheets is a well-established method for estimating morphogenetic quantities such as growth, contraction, and shear. However, not all surface labeling techniques are readily adaptable to conventional imaging modalities and possess different advantages and limitations. Here, we describe two labeling methods and illustrate the utility of each technique. In the first method, hundreds of fluorescent labels are applied simultaneously to the embryo using magnetic iron particles. These labels are then used to quantity 2-D tissue deformations during morphogenesis. In the second method, polystyrene microspheres are used as contrast agents in non-invasive optical coherence tomography (OCT) imaging to track 3-D tissue deformations. These techniques have been successfully implemented in our lab to studythe physical mechanisms of early head fold, heart, and brain development, and should be adaptable to a wide range morphogenetic processes.

Tracking Morphogenetic Tissue Deformations in the Early Chick Embryo

This article describes surface labeling and ex ovo tissue culture in the early chick embryo. Techniques amenable to time-lapse bright field, fluorescence, and optical coherence tomography imaging are presented. Tracking surface labels with high spatiotemporal resolution enables kinematic quantities such as morphogenetic strains (deformations) to be calculated in both two and three dimensions.

Embryonic epithelia undergo complex deformations (e.g. bending, twisting, folding, and stretching) to form the primitive organs of the early embryo. ...

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

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

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

Windowing Chicken Eggs for Developmental Studies

Placing Growth Factor-Coated Beads on Early Stage Chicken Embryos

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

A Submerged Filter Paper Sandwich for Long-term Ex Ovo Time-lapse Imaging of Early Chick Embryos

Analysis of Neural Crest Migration and Differentiation by Cross-species Transplantation

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

Using Avian Skin Explants to Study Tissue Patterning and Organogenesis

Tracking Morphogenetic Tissue Deformations in the Early Chick Embryo