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...

<ol>
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J., Izpis&#250;a-Belmonte, J. C., Abud, H., Heath, J. 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A., Hurle, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+TGF+beta+s+and+BMPs+as+signals+controlling+the+position+of+the+digits+and+the+areas+of+interdigital+cell+death+in+the+developing+chick+limb+autopod.">Role of TGF beta s and BMPs as signals controlling the position of the digits and the areas of interdigital cell death in the developing chick limb autopod.</a> Development. 122 (8), 2349-2357 (1996).</li><li>Merino, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphogenesis+of+digits+in+the+avian+limb+is+controlled+by+FGFs,+TGFbetas,+and+noggin+through+BMP+signaling.">Morphogenesis of digits in the avian limb is controlled by FGFs, TGFbetas, and noggin through BMP signaling.</a> Developmental Biology. 200 (1), 35-45 (1998).</li><li>Montero, J. A., Lorda-Diez, C. I., Ga&#241;an, Y., Macias, D., Hurle, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activin/TGFbeta+and+BMP+crosstalk+determines+digit+chondrogenesis.">Activin/TGFbeta and BMP crosstalk determines digit chondrogenesis.</a> Developmental Biology. 321 (2), 343-356 (2008).</li><li>Hamburger, V., Hamilton, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+series+of+normal+stages+in+the+development+of+the+chick+embryo.">A series of normal stages in the development of the chick embryo.</a> Journal of Morphology. 88 (1), 49-92 (1951).</li><li>D&#237;az-Hern&#225;ndez, M. E., Bustamante, M., Galv&#225;n-Hern&#225;ndez, C. 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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 ...

This assay enables the student to look specific molecules in many developmental process, such as cell differentiation and morphogenesis by implanting the beads into distinct embryo regions. This protocol can be applied to any experimental animal models, cell culture, organotypic model, including organoids. Furthermore, it's a helpful educational tool for teaching basic developmental biology to students.Demonstrating the procedure will be Alexandra Gonzales-Gonzales, an undergraduate student from the laboratory. To begin, incubate the fertilized white Leghorn chicken eggs vertically with the pointy side down in a humidified incubator at 38 degrees Celsius and 70%relative humidity. Once they reach the 28th stage, according to Hamburger and Hamilton, remove the eggs from the incubator, swap them with 70%ethanol and allow them to air dry.Then disinfect the working area, microscopes and instruments with 70%ethanol. Next, candle the eggs to identify blood vessels and locate the embryo. Discard eggs that do not have an embryo.Using the end of non toothed forceps, open a window by tapping the blunt end of an egg, and remove a one square centimeter shell section. Then, transfer the egg into a carton or plastic holder and place it under the stereo microscope. Using fine surgical forceps, remove any small piece of egg shell that could contact the embryo and remove the air membrane by puncturing and pulling it out.Next, using fine surgical forceps, tear the amniotic sack slightly, being careful not to damage the vasculature of the Chorioallantoic membrane. Stage the embryos in-ovo to determine whether they are in the desired stage. For Affi-gel heparin bead preparation, cut a square shaped parafilm section to fit into a 45 millimeter Petri dish.After placing the parafilm across the bottom of the Petri dish, using the end of non toothed forceps, push each vertex and fix it to the bottom of the dish. Next, using a pipette or spatula, transfer the beads into a micro centrifuge tube and wash them twice with PBS by allowing them to settle and pipetting the supernatant. Then, using a micro pipette, transfer 40 to 50 beads to the center of the parafilm covered Petri dish.Under a microscope, select 30 Affi-gel or heparin beads that have a 100 micrometer diameter. Size the beads using a third interdigit of a 28 stage embryo as a reference. The bead must be smaller than the interdigit.After carefully removing the excess PBS surrounding the selected beads, soak them in two to five microliters of the treatment solution. Incubate the beads in the solution for 30 minutes at room temperature. During incubation, to prevent the beads from drying out, pipette a few drops of PBS or water around the beads to humidify the local atmosphere and cover the dish with parafilm to slow evaporation.Pour AG1X2 bead preparation. Use the spatula to transfer the beads into a micro centrifuge tube and add 50 microliters of the treatment solution at the desired final concentration. After wrapping the tubes with foil to protect them from light, incubate the beads for 20 minutes at slow shaking.After incubation, using a pipette, remove as much of the treatment solution as possible in stain with 0.2%phenol red dissolved in water for two minutes with mild agitation in a vortex. After staining, remove the dye, then wash the beads twice in PBS to remove any residual dye. Next, using a micro pipette tip, transfer 40 to 50 AG1X2 beads to the center of the paraform covered Petri dish and soak the beads in five microliters of PBS.Then under a microscope, select around 30 beads of 100 micrometer diameter in size. Submerge the selected beads in the experimental solution with a few drops of PBS or water around the beads to humidify the local atmosphere. Cover the dish with parafilm to slow evaporation.Before manipulating the embryos, arrange two stereo microscopes next to each other on a benchtop. Then, using non toothed forceps, create a window in the remaining eggs. Next, under the microscope tear the amniotic membrane near the right hind limb with fine surgical forceps.Using fine forceps, hold the embryo by the amniotic membrane to expose the right hind limb. Then using a fine Tungsten needle, make a hole centered in the distalmost of the third interdigit of the hind limb. Without releasing the embryo and the exposed hind limb, take one treated bead from the other microscope using one of the tips of the forceps.The forceps must be open for one bead to adhere to its tip. Transfer the bead into the chick embryo near the hind limb, positioning it on top of the interdigit hole. apply pressure on the bead by closing the forceps until it enters the hole.Then release the embryo, seal the eggshell window with tape and return the eggs to the incubator until they have reached the required stage. To ensure assay efficacy, the bead must be placed consistently and precisely in the correct location. In this study, the soaked bead was placed in the distalmost of the third interdigit beneath the apical ectodermal ridge.The embryos can be stained with ocean blue and alizarin red to evidence the formation of skeletal elements and evaluate the effects on cell differentiation. This assay is also well-suited for evaluating cell death with neutral red and gene regulation, buying C2 hybridization. The main advantage of this experimental tool is to control the time and location of the soaked beads.Combining the core positioning to precise developmental timing provides enormous possibilities to study cell differentiation processes.

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3.0K Views.  Universidad Nacional Autónoma de México. This assay enables the student to look specific molecules in many developmental process, such as cell differentiation and morphogenesis by implanting the beads into distinct embryo regions. This protocol can be applied to any experimental animal models, cell culture, organotypic model, including organoids. Furthermore, it's a helpful educational tool for teaching basic developmental biology to students.Demonstrating the procedure will be Alexandra Gonzales-Gonzales, an undergraduate st...