Name: chicken recombinant limbs assay to understand morphogenesis, patterning, and early steps in cell differentiation
Uploaded: 2022-01-12T13:00:00
Description: Watch this Scientific Journal Video about Chicken Recombinant Limbs Assay to Understand Morphogenesis, Patterning, and Early Steps in Cell Differentiation at JoVE.com

We thank to Estefania Garay-Pacheco for images in Figure 2 and to Maria Valeria Chimal-Montes de Oca for artwork. 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 was the recipient of a postdoctoral fellowship from the Consejo Nacional de Ciencia y Tecnolog&#237;a (CONACyT-Fronteras de la Ciencia-1887).

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). A schematic flowchart of the general steps of this protocol is shown in Figure 1A.
1. Embryo incubation and determination of viability
<ol>
	<li>Incubate fertilized chicken eggs at 38 &#176;C and 60% ...

<ol>
	<li>Malashichev, Y., Christ, B., Pr&#246;ls, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Avian+pelvis+originates+from+lateral+plate+mesoderm+and+its+development+requires+signals+from+both+ectoderm+and+paraxial+mesoderm.">Avian pelvis originates from lateral plate mesoderm and its development requires signals from both ectoderm and paraxial mesoderm.</a> Cell and Tissue Research. 331 (3), 595-604 (2008).</li><li>Mahmood, 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=A+role+for+FGF-8+in+the+initiation+and+maintenance+of+vertebrate+limb+bud+outgrowth.">A role for FGF-8 in the initiation and maintenance of vertebrate limb bud outgrowth.</a> Current Biology. 5 (7), 797-806 (1995).</li><li>Yu, K., Ornitz, D. 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M., Fallon, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+proportion+and+distribution+of+polarizing+zone+cells+causing+morphogenetic+inhibition+when+coaggregated+with+anterior+half+wing+mesoderm+in+recombinant+limbs.">The proportion and distribution of polarizing zone cells causing morphogenetic inhibition when coaggregated with anterior half wing mesoderm in recombinant limbs.</a> Development. 67 (1), 13-25 (1982).</li><li>Ros, M. A., Lyons, G. E., Mackem, S., Fallon, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recombinant+limbs+as+a+model+to+study+homeobox+gene+regulation+during+limb+development.">Recombinant limbs as a model to study homeobox gene regulation during limb development.</a> Developmental Biology. 166 (1), 59-72 (1994).</li><li>Piedra, M. E., Rivero, F. B., Fernandez-Teran, M., Ros, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pattern+formation+and+regulation+of+gene+expressions+in+chick+recombinant+limbs.">Pattern formation and regulation of gene expressions in chick recombinant limbs.</a> Mechanisms of Development. 90 (2), 167-179 (2000).</li><li>Crosby, G. M., Fallon, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibitory+effect+on+limb+morphogenesis+by+cells+of+the+polarizing+zone+coaggregated+with+pre-or+postaxial+wing+bud+mesoderm.">Inhibitory effect on limb morphogenesis by cells of the polarizing zone coaggregated with pre-or postaxial wing bud mesoderm.</a> Developmental Biology. 46 (1), 28-39 (1975).</li><li>Fallon, J. F., Simandl, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+between+chick+limb+bud+mesoderm+and+reptile+ectoderm+result+in+limb+outgrowth+in+the+limbless+mutant.">Interactions between chick limb bud mesoderm and reptile ectoderm result in limb outgrowth in the limbless mutant.</a> Anatomical Record. 208, 53-54 (1984).</li><li>Kuhlman, J., Niswander, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Limb+deformity+proteins:+role+in+mesodermal+induction+of+the+apical+ectodermal+ridge.">Limb deformity proteins: role in mesodermal induction of the apical ectodermal ridge.</a> Development. 124 (1), 133-139 (1997).</li><li>Goetinck, P. F., Abbott, U. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+limb+morphogenesis.+I.+Experiments+with+the+polydactylous+mutant,+talpid.">Studies on limb morphogenesis. I. Experiments with the polydactylous mutant, talpid.</a> Journal of Experimental Zoology. 155, 161-170 (1964).</li><li>Carrington, J. L., Fallon, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initial+limb+budding+is+independent+of+apical+ectodermal+ridge+activity;+evidence+from+a+limbless+mutant.">Initial limb budding is independent of apical ectodermal ridge activity; evidence from a limbless mutant.</a> Development. 104 (3), 361-367 (1988).</li><li>Fernandez-Teran, M., Piedra, M. E., Ros, M. A., Fallon, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+recombinant+limb+as+a+model+for+the+study+of+limb+patterning,+and+its+application+to+muscle+development.">The recombinant limb as a model for the study of limb patterning, and its application to muscle development.</a> Cell and Tissue Research. 296 (1), 121-129 (1999).</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>Ganan, Y., Macias, D., Duterque-Coquillaud, M., Ros, M. 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>Ros, M. A., Simandl, B. K., Clark, A. W., Fallon, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+manipulating+the+chick+limb+bud+to+study+gene+expression,+tissue+interactions,+and+patterning.">Methods for manipulating the chick limb bud to study gene expression, tissue interactions, and patterning.</a> Developmental Biology Protocols. 137, 245-266 (2000).</li><li>MacCabe, J. A., Saunders, J. W., Pickett, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+control+of+the+anteroposterior+and+dorsoventral+axes+in+embryonic+chick+limbs+constructed+of+dissociated+and+reaggregated+limb-bud+mesoderm.">The control of the anteroposterior and dorsoventral axes in embryonic chick limbs constructed of dissociated and reaggregated limb-bud mesoderm.</a> Developmental Biology. 31 (2), 323-335 (1973).</li><li>Zwilling, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+contact+between+mutant+(wingless)+limb+buds+and+those+of+genetically+normal+chick+embryos:+confirmation+of+a+hypothesis.">Effects of contact between mutant (wingless) limb buds and those of genetically normal chick embryos: confirmation of a hypothesis.</a> Developmental Biology. 39 (1), 37-48 (1974).</li><li>Prahlad, K. V., Skala, G., Jones, D. G., Briles, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Limbless:+A+new+genetic+mutant+in+the+chick.">Limbless: A new genetic mutant in the chick.</a> Journal of Experimental Zoology. 209 (3), 427-434 (1979).</li><li>Marin Llera, J. C., Lorda-Diez, C. I., Hurle, J., Chimal-Monroy, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SCA-1/Ly6A+mesodermal+skeletal+progenitor+subpopulations+reveal+differential+commitment+of+early+limb+bud+cells.">SCA-1/Ly6A mesodermal skeletal progenitor subpopulations reveal differential commitment of early limb bud cells.</a> Frontiers in Cell and Developmental Biology. 9, 656999 (2021).</li></ol>

In general, the RL protocol can be divided into five steps: (1) embryo incubation, (2) obtaining limb mesodermal cells to fill the ectoderms, (3) obtaining the ectoderms, (4) assembling mesodermal cells inside the ectodermal covers, and (5) transplantation of the filled ectoderms into the host embryos. The major limitation of the RL technique is the long, detailed protocol, which has many critical points that require patience to perform appropriately. To successfully complete the protocol, critical moments need to be ide...

The vertebrate limb is a formidable model for studying cell differentiation, cell proliferation, cell death, pattern formation, and morphogenesis1,2. During development, limbs emerge as bulges from the cells derived from lateral plate mesoderm1. Limb buds consist of a central core of mesodermal cells covered by an ectoderm. From this early structure, a whole and well-formed limb emerge. After the limb bud arises, three axes are recognized: (1) the proximo-distal axis ([PD] shoulder to fingers), (2) the dorso-ventral axis ([DV] from the back of the hand to palm), and (3) the anterior-posterior ([AP] thumb to finger). The proximal-distal axis depends on the apical ectodermal ridge (AER), specialized ectoderm located at the distal tip of the limb bud. The AER is required for outgrowth, survival maintenance, proliferation, and the undifferentiated state of cells receiving signals2,3. On the other hand, the zone of polarizing activity (ZPA) controls anteroposterior patterning4, while the dorsal and ectoderm controls dorsoventral patterning7,8. Integration of three-dimensional patterning implies complex crosstalk between these three axes5. Despite understanding the molecular pathway during limb development, open questions about the mechanisms that control patterning and proper outgrowth to form a whole limb remain unanswered.
Edgar Zwilling developed the recombinant limb (RL) system in 1964 to study the interactions between limb mesenchymal cells and the ectoderm in developing limbs6. The RL system assembles the dissociated-reaggregated limb bud mesoderm into the embryonic limb ectoderm to graft it into the dorsal part of a donor chick embryo. The signals provided by the ectoderm induce the expression of differentiation genes and patterning genes in a spatio-temporal manner, thus inducing the formation of a limb-like structure that can recapitulate the cell programs that occur during limb development7,8,9.
The RL model is valuable for understanding the properties of limb components and the interaction between mesodermal and ectodermal cells6. An RL can be defined as a limb-like structure created by the experimentally assembling or recombining limb bud mesodermal cells inside an ectodermal cover6. The morphogenesis of the RL depends on the characteristics of the mesodermal cells (or other types) that will respond to the ectodermal patterning signals. One of the advantages of this experimental system is its versatility. This characteristic permits the creation of multiple combinations by varying the source of mesodermal cells, such as cells from different developmental stages, from different positions along the limb, or whole (undissociated) or reaggregated cells7,8,9,10. Another example is the capability of obtaining the embryonic ectoderm from species other than chicken, for example, turtle11, quail, or mouse12.
In this sense, the RL technique helps study limb development and the interactions between limb mesenchymal and ectodermal cells from an evolutionary point of view. This technique also has great potential for analyzing the capability of different sources of progenitor cells to differentiate into a limb-like structure by taking advantage of the signals provided by the embryonic ectoderm12,13,14. In contrast to in vitro cultures, the RL permits evaluating the differentiation and morphogenetic potential of a cell population by interpreting embryonic signals from a developing limb9,15.
In this protocol, a step-by-step guide to performing successful RL with reaggregated mesodermal limb bud cells is provided, thus opening the possibility of adapting this protocol with different sources of reaggregated cells or even different ectoderm sources.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Alcian Blue 8GX</td>
			<td>Sigma</td>
			<td>A5268</td>
			<td></td>
		</tr>
		<tr>
			<td>Angled slit knife</td>
			<td>Alcon</td>
			<td>2.75mm DB</td>
			<td></td>
		</tr>
		<tr>
			<td>Blunt forceps</td>
			<td>Fine Science Tools</td>
			<td>11052-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase type IV</td>
			<td>Gibco</td>
			<td>1704-019</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM-HG</td>
			<td>Sigma</td>
			<td>D5796</td>
			<td></td>
		</tr>
		<tr>
			<td>Egg incubator</td>
			<td>Incumatic de Mexico</td>
			<td>Incumatic 1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Gibco</td>
			<td>16000069</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine surgical forceps</td>
			<td>Fine Science Tools</td>
			<td>9115-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Hanks Balanced Salt Solution</td>
			<td>Sigma</td>
			<td>H6648</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td>Eppendorf</td>
			<td>5417R</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipet</td>
			<td>NA</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Palladium wire</td>
			<td>GoodFellow</td>
			<td>7440 05-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>Nest</td>
			<td>705001</td>
			<td></td>
		</tr>
		<tr>
			<td>Pippette</td>
			<td>crmglobe</td>
			<td>PF1016</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>Trypsin porcine</td>
			<td>Merck</td>
			<td>9002 07-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Tungsten needle</td>
			<td>GoodFellow</td>
			<td>E74-15096/01</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Recognizing a well-performed recombinant limb 
After grafting, the manipulated embryos were returned to the incubator to allow the RL to develop. The incubation time correlated with the requirements of the experiment. Nevertheless, the RL can be easily distinguished after 12 h of implantation. To determine whether the implantation was adequate, the RL was observed as a protuberance that was securely attached to the mesodermal wall of the donor embryo (Figure 2A). On the...

Watch this Scientific Journal Video about Chicken Recombinant Limbs Assay to Understand Morphogenesis, Patterning, and Early Steps in Cell Differentiation at JoVE.com

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

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 ...

Recombinant limbs are a powerful model for a study in the process of cell differentiation and power formation under the influence of embryonic signals in an in vivo environment. This assay allows multiple variations permitting potential applications without being restricted to chicken limb developmental biology. It can be applied to other stem or progenitor from other organisms.To begin, remove eggs from the incubator after three and a half days, swab with 70%ethanol, and leave them to air dry. Next, identify and locate the developing embryos by candling the egg and observing the blood vessels. Discard eggs that do not have an embryo.Tap the blunt end of the egg shell with the end of a pair of blunt forceps to open a window and remove about one square centimeter of the shell using the forceps. Then transfer the eggs to a plastic or carton holder. Place the eggs one by one under the stereomicroscope.Identify the air membrane and remove it by picking a small hole in the area without any vessels. Pull this area out with the aid of fine surgical forceps and remove pieces of eggshell in contact with the embryo. Tear open the amniotic sac with fine surgical forceps.Carefully remove the embryo from the egg with blunt forceps and transfer it into a sterile Petri dish containing ice-cold 1x PBS. Wash the embryos once in ice-cold 1x PBS and withdraw any remaining membranes under the stereomicroscope. Then locate the hindlimb buds.Snip each hindlimb bud longitudinally, cutting very close to the embryo flank with fine surgical forceps. Maintaining the embryos in 1x PBS. Then transfer the limb buds into a 1.5 milliliter micro centrifuge tube with a plastic transfer pipette.Next, replace the PBS with 500 microliters of 0.5%trypsin solution. Incubate the limb buds in a thermoblock for seven minutes at 37 degrees Celsius. Then replace the trypsin solution with 500 microliters of two milligram per milliliter collagenase type four in HBSS and incubate it in the thermoblock for another eight minutes.Remove as much collagenase as possible and replace it with one milliliter of cold high glucose DMEM with 10%FBS to inactivate the enzymes. Pipette the mixture gently around 10 times. Filter the suspension containing the cells with a 70 micrometer cell strainer to leave behind the ectoderms.Pipette the suspension again around five to 10 times and centrifuge at 200 times g for five minutes at room temperature. Then discard the supernatant. Wash the excess collagenase with one milliliter of media, then centrifuge the suspension and discard the medium carefully.Next, add 1.5 milliliters of media without disturbing the cells and incubate them for one to 1.5 hours at 37 degrees Celsius in a thermoblock, allowing the cells to form a compact pellet. After obtaining the hind limb buds, transfer them into an empty micro centrifuge tube with a plastic pipette. Remove any excess 1x PBS and replace it with 500 microliters of 0.5%trypsin in sterile 1x PBS.Incubate the mixture for 30 minutes at 37 degrees Celsius in a thermoblock. Transfer the trypsin solution with the limb buds into a sterile Petri dish. Then remove as much of the excess trypsin as possible and flood the Petri dish with ice-cold 1x PBS with 10%FBS until all limb buds are covered.Identify the limb buds under the stereomicroscope. Next, identify the ectoderm as a slightly detached transparent membrane from the limb mesodermal cells. Detach and separate the ectoderm with forceps holding the most proximal end of the limb bud with fine surgical forceps.Take the mesodermal pellet from the previous steps and discard around 600 microliters of the medium. Then carefully detach the pellet with a pipette tip without smashing it. Turn the tube upside down to transfer the pellet into the Petri dish containing the empty ectoderms.Cut a small piece of the pellet with a pair of fine surgical forceps and place it as close as possible to the ectoderm. Open the ectoderm with the surgical forceps and place the pellet as tightly as possible into the ectodermal hall. Open the amniotic sac near the forelimb to expose the right flank of the embryo.Guided by the forelimb position, perform wound scratching with a tungsten needle to the length of two to three somites, slightly damaging the mesoderm until it bleeds. Individually transfer the recombinant limbs into the chick embryo, placing its base over a somite wound. Fix the recombinant limb correctly with two pieces of palladium wire.Align the base of the recombinant limb with the wound. Seal the window with tape and return the egg to the incubator. In successful implantation, the recombinant limb was observed as a protuberance that was securely attached to the mesodermal wall of the donor embryo.On the contrary, the recombinant limb was detached from the mesodermal wall or presented a rough morphology when either cell viability or the graft failed. Alcian blue staining demonstrated skeletal elements in a six day chicken, chicken recombinant limb. Before clearing the Alcian blue, images in ethanol were obtained for morphological and quantitative measurements.H&E stained or unstained limbs were sliced to observe tissues and cells. This technique can develop limb organoids in vivo and study cell differentiation or morphogenetic process by using the stem cells from different sources or species.

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