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% relative humidity for about three and a half days until they reach the 22 HH stage (according to Hamilton and Hamburger, 1951)16. 
	NOTE: Freshly fertilized chicken eggs can be stored at 15 &#176;C for up to one week.
	<ol>
		<li>To incubate the fertilized eggs, place the eggs vertically with the pointy side down into the humidified incubator. Rotate the eggs during incubation as it is necessary to prevent the developing embryo from adhering to the shell membrane. 
		NOTE: Fertilized hen eggs can be obtained from local farms. Fertilized White Leghorn chicken eggs are generally used to avoid pigmentation of the feathers in late embryos. Consider incubating enough eggs separately to obtain three structures: (1) limb mesodermal cells, (2) limb ectoderms, and (3) donor embryos to graft the RL.</li>
	</ol>
	</li>
	<li>After three and a half days of incubation, remove eggs from the incubator, swab with 70% ethanol, and allow to air dry.</li>
	<li>Identify developing embryos candling the egg to observe the blood vessels and locate the embryo. Discard eggs that do not have an embryo. 
	&#8203;NOTE: At this point, distribute the eggs to obtain mesodermal cells, ectoderms, and the hosts for the RL.</li>
</ol>
2. Obtaining limb mesodermal cells to fill ectoderms
NOTE: Before initiating the manipulations, it is highly recommended to disinfect the working area, the microscopes, and all the instrumental by swabbing with 70% ethanol solution.
<ol>
	<li>Tap the blunt end of the eggshell with the end of a blunt forceps to open a window and remove about 1 cm x 1 cm of the shell using the forceps.</li>
	<li>Transfer eggs to a plastic or carton holder and placed them one by one under the stereomicroscope. Identify the air membrane and then remove it by picking a small hole where no vessels are found. Pull this area with the aid of fine surgical forceps. 
	NOTE: The air membrane can be identified as the white and opaque membrane observed immediately after windowing the egg.
	<ol>
		<li>Remove any small piece of eggshell that may come into contact with the embryo. 
		NOTE: Verify that the embryos are in the 22 HH stage before initiating the protocol. Embryos in earlier stages can be returned to the incubator after sealing the eggshell window with tape.</li>
	</ol>
	</li>
	<li>Open the amniotic sac completely by tearing open the amnion using the fine surgical forceps (see Table of Materials). 
	NOTE: Amnion can be identified as a transparent membrane that closely covers the embryo and is filled with amniotic fluid.</li>
	<li>Carefully remove the embryo from the egg using a pair of blunt forceps and then transfer into a sterile Petri dish containing ice-cold phosphate-buffered saline solution (1x PBS). Repeat this step for the remaining eggs. Consider that only ~8-10 embryos are sufficient to obtain the mesodermal cells.</li>
	<li>Wash the embryos once in ice-cold 1x PBS, and with the aid of a stereomicroscope (see Table of Materials), withdraw any remaining membranes. Locate the hindlimb buds. 
	NOTE: Limb buds are rounded structures situated along the anterior-posterior axis of the embryo as protrusions from the flank. The hindlimbs are located more posteriorly than the forelimbs.</li>
	<li>Maintain the embryos in clean 1x PBS, and using a pair of fine surgical forceps, snip each hindlimb bud longitudinally. Cut the bud very close to the flank of the embryo to dissect out the whole hindlimb buds. Repeat this step with the remaining embryos.</li>
	<li>Use a plastic transfer pipette to transfer the limb buds into an empty 1.5 mL microcentrifuge tube.</li>
	<li>Use a pipette tip to remove any excess of 1x PBS and replace PBS with 500 &#181;L of 0.5% trypsin solution (see Table of Materials). Incubate limb buds in a thermoblock for 7 min at 37 &#176;C.</li>
	<li>Remove the trypsin solution and replace it with 500 &#181;L of collagenase type IV at 2 mg/mL in Hanks Balanced Salt Solution (see Table of Materials), then incubate it in a thermoblock at 37 &#176;C for 8 min. 
	NOTE: Trypsin incubation slightly detaches the ectoderms, while collagenase disaggregate mesodermal cells.</li>
	<li>After incubation, remove as much collagenase as possible and replace it with 1 mL of cold Dulbecco&#39;s Modified Eagle Medium-high glucose (DMEM-HG) medium supplemented with 10% fetal bovine serum (FBS) to inactivate the enzymes. Pipette the mixture gently ~10 times.</li>
	<li>Filter the suspension containing the cells with a cell strainer of 70 &#181;m to leave behind the ectoderms. 
	NOTE: Filtration will also remove any undigested limb buds, leaving behind a suspension of single cells.</li>
	<li>After filtering, pipette the suspension again 5-10 times and centrifuge at 200 x g for 5 min at room temperature, then discard the supernatant.</li>
	<li>Use 1 ml of DMEM-HG supplemented with 10% FBS to wash the excess collagenase. Centrifuge the suspension at 200 x g for 5 min at room temperature.</li>
	<li>Discard the medium carefully by pipetting and then replace it with 1 mL of fresh DMEM-HG supplemented with 10% FBS without disturbing the cells in the bottom of the tube.</li>
	<li>Allow the cells to form a compact pellet (reaggregate) after incubating them for 1-1.5 h at 37 &#176;C in a thermoblock. 
	&#8203;NOTE: While mesodermal cells are forming the pellet, it is convenient to obtain the limb ectoderms.</li>
</ol>
3. Obtaining the limb ectoderms
<ol>
	<li>Repeat steps 1-6 from section 2 separately with the other 22 HH chicken embryos chosen to obtain the ectoderms. 
	NOTE: The number of embryos for this purpose is proportional to the final number of the RL desired. A ratio of 2:1 ectoderms-RL is appropriate.</li>
	<li>After obtaining the hindlimb buds, use a plastic pipette to transfer them into an empty microcentrifuge tube. Remove any excess 1x PBS and replace it with 500 &#181;L of 0.5% trypsin in sterile 1x PBS. Incubate the mixture for 30 min at 37 &#176;C in a thermoblock.</li>
	<li>After the enzymatic digestion, transfer the trypsin solution containing the limb buds into a sterile Petri dish.</li>
	<li>Remove the excess trypsin as much as possible using a micropipette; flood the Petri dish with ice-cold 1x PBS supplemented with 10% of FBS until all limb buds are covered. 
	NOTE: FBS can be substituted for horse serum.</li>
	<li>Identify the limb buds under the stereomicroscope; identify the ectoderm as a slightly detached transparent membrane from the limb mesodermal cells (Figure 1B).</li>
	<li>Hold the most proximal end of the limb bud with the aid of fine surgical forceps while carefully detach and separate the ectoderm layer using the other forceps. 
	NOTE: Obtaining the ectoderms could be difficult due to mesodermal cell clumping and attaching to ectoderms, thus causing them to become sticky. It is recommended to constantly discard the mesodermal cells to maintain only the ectoderms in the Petri dish.</li>
	<li>Maintain the ectoderms in ice-cold 1x PBS-10% FBS solution. 
	&#8203;NOTE: Ectoderms can be stored at 4 &#176;C in case the reaggregated mesoderm is not ready. However, it is preferable to coordinate the incubation time of the pellet with the ectodermal obtaining.</li>
</ol>
4. Assembling mesodermal cells inside the ectodermal cover
NOTE: For this, it is necessary to have the empty ectoderms in a Petri dish with sterile 1x PBS-10% FBS solution containing a formed pellet of mesodermal cells.
<ol>
	<li>After the pellet is formed (see section 2), discard ~600 &#181;L of the medium from the tube by pipetting.</li>
	<li>Carefully detach the pellet from the bottom using a pipette tip and without smashing or destroying it.</li>
	<li>When the pellet is completely detached from the bottom of the tube, turn the tube upside down to transfer the pellet into the Petri dish containing the empty ectoderms (Figure 1C).</li>
	<li>Cut a small piece of the pellet with the aid of a pair of fine surgical forceps and place it as close as possible to the ectoderm.</li>
	<li>As if it were a bag, open the ectoderm with the surgical forceps, and place the piece of pellet as tightly as possible into the ectodermal cover. Repeat this step for as many RLs as desired (Figure 1D). 
	NOTE: Cut the pieces of the pellet one by one, and fill the ectoderms to maintain clean the 1x PBS-10% FBS solution. The size of the pellet needs to correspond to each ectoderm.</li>
	<li>Allow the ectoderm and the mesoderm to heal together for ~30 min at room temperature and then graft them into the embryo host. Discard the unused ectoderm and mesoderm.</li>
</ol>
5. Transplantation of the filled ectoderm into a host embryo 
&#160;
NOTE: Before transplanting the ectoderms, arrange two stereomicroscopes next to each other on a benchtop, one for embryo manipulation and RL grafting. The other is for maintaining the filled ectoderms ready to transfer into the embryo.
<ol>
	<li>Select the number of desired eggs to graft the filled ectoderms.</li>
	<li>Using the end of a blunt forceps, tap the eggshell of the 22 HH host embryos to open a window. Identify the air membrane and, using a pair of fine surgical forceps, remove it completely.</li>
	<li>Open the amniotic sac near the forelimb to expose the right flank of the embryo. Open only the amount needed to accomplish the procedure.</li>
	<li>Guided by the forelimb position, perform wound scratching with a tungsten needle (see Table of Materials) to the length of 2-3 somites, slightly damaging the mesoderm until it bleeds.</li>
	<li>Individually transfer a filled ectoderm (RL) into the chick embryo and place the base of the recombinant limb over the somite wound. 
	NOTE: RL transfer can be done with a plastic pipette or with an angled slit knife.</li>
	<li>Fix the RL correctly with two pieces of palladium wire of 0.025 mm diameter and 0.5-1 mm length (see Table of Materials). Align the base of the RL with the wound to ensure that it will be attached to the flank of the host embryo and vascularized (Figure 1E).</li>
	<li>Seal the window with tape, and return the egg to the incubator. Collect the embryos at the desired time points for analysis.</li>
</ol>

<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. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FGF+signaling+regulates+mesenchymal+differentiation+and+skeletal+patterning+along+the+limb+bud+proximodistal+axis.">FGF signaling regulates mesenchymal differentiation and skeletal patterning along the limb bud proximodistal axis.</a> Development. 135 (3), 483-491 (2008).</li><li>Riddle, R. D., Johnson, R. L., Laufer, E., Tabin, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sonic+hedgehog+mediates+the+polarizing+activity+of+the+ZPA.">Sonic hedgehog mediates the polarizing activity of the ZPA.</a> Cell. 75 (5), 1401-1416 (1993).</li><li>McQueen, C., Towers, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishing+the+pattern+of+the+vertebrate+limb.">Establishing the pattern of the vertebrate limb.</a> Development. 147 (17), (2020).</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=Development+of+fragmented+and+of+dissociated+limb+bud+mesoderm.">Development of fragmented and of dissociated limb bud mesoderm.</a> Developmental biology. 9 (1), 20-37 (1964).</li><li>Frederick, J. 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>

The authors have nothing to disclose.

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 ...

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2.0K Views.  Universidad Nacional Autónoma de México. 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.

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

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

Organogenesis is the study of how organs are specified and then acquire their specific shape and functions during development. The Xenopuslaevis embryo is very useful for studying organogenesis because their large size makes them very suitable for identifying organs at the earliest steps in organogenesis. At this time, the primary method used for identifying a specific organ or primordium is whole mount in situ hybridization with labeled antisense RNA probes specific to a gene that is expressed in the organ of interest. In addition, it is relatively easy to manipulate genes or signaling pathways in Xenopus and in situ hybridization allows one to then assay for changes in the presence or morphology of a target organ. Whole mount in situ hybridization is a multi-day protocol with many steps involved. Here we provide a simplified protocol with reduced numbers of steps and reagents used that works well for routine assays. In situ hybridization robots have greatly facilitated the process and we detail how and when we utilize that technology in the process. Once an in situ hybridization is complete, capturing the best image of the result can be frustrating. We provide advice on how to optimize imaging of in situ hybridization results. Although the protocol describes assessing organogenesis in Xenopus laevis, the same basic protocol can almost certainly be adapted to Xenopus tropicalis and other model systems.

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

The Xenopus laevis embryo continues to be exceptionally useful in the study of early development due to its large size and ease of manipulation. A simplified protocol for whole mount in situ hybridization protocol is provided that can be used in the identification of specific organs in this model system.

Organogenesis is the study of how organs are specified and then acquire their specific shape and functions during development.  The Xenopuslaevis embryo ...

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

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 ...

Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo

Understanding the genetic programs underlying neural development is an important goal of developmental and stem cell biology. In the amphibian blastula, cells from the roof of the blastocoel are pluripotent. These cells can be isolated, and programmed to generate various tissues through manipulation of genes expression or induction by morphogens. In this manuscript protocols are described for the use of Xenopus laevis blastocoel roof explants as an assay system to investigate key in vivo and in vitro features of early neural development. These protocols allow the investigation of fate acquisition, cell migration behaviors, and cell autonomous and non-autonomous properties. The blastocoel roof explants can be cultured in a serum-free defined medium and grafted into host embryos. This transplantation into an embryo allows the investigation of the long-term lineage commitment, the inductive properties, and the behavior of transplanted cells in vivo. These assays can be exploited to investigate molecular mechanisms, cellular processes and gene regulatory networks underlying neural development. In the context of regenerative medicine, these assays provide a means to generate neural-derived cell types in vitro that could be used in drug screening.

Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo

In Xenopus embryos, cells from the roof of the blastocoel are pluripotent and can be programmed to generate various tissues. Here, we describe protocols to use amphibian blastocoel roof explants as an assay system to investigate key in vivo and in vitro features of early neural development.

Understanding the genetic programs underlying neural development is an important goal of developmental and stem cell biology. In the amphibian blastula, ...

Initiating Differentiation in Immortalized Multipotent Otic Progenitor Cells

Use of human induced pluripotent stem cells (iPSC) or embryonic stem cells (ESC) for cell replacement therapies holds great promise. Several limitations including low yields and heterogeneous populations of differentiated cells hinder the progress of stem cell therapies. A fate restricted immortalized multipotent otic progenitor (iMOP) cell line was generated to facilitate efficient differentiation of large numbers of functional hair cells and spiral ganglion neurons (SGN) for inner ear cell replacement therapies. Starting from dissociated cultures of single iMOP cells, protocols that promote cell cycle exit and differentiation by basic fibroblast growth factor (bFGF) withdrawal were described. A significant decrease in proliferating cells after bFGF withdrawal was confirmed using an EdU cell proliferation assay. Concomitant with a decrease in proliferation, successful differentiation resulted in expression of molecular markers and morphological changes. Immunostaining of Cdkn1b (p27KIP) and Cdh1 (E-cadherin) in iMOP-derived otospheres was used as an indicator for differentiation into inner ear sensory epithelia while immunostaining of Cdkn1b and Tubb3 (neuronal &#946;-tubulin) was used to identify iMOP-derived neurons. Use of iMOP cells provides an important tool for understanding cell fate decisions made by inner ear neurosensory progenitors and will help develop protocols for generating large numbers of iPSC or ESC-derived hair cells and SGNs. These methods will accelerate efforts for generating otic cells for replacement therapies.

The current protocols to maintain immortalized multipotent otic progenitor (iMOP) cells and otic differentiation are described. Culture conditions and molecular markers that indicate differentiation into sensory epithelia and spiral ganglion neurons (SGN) are highlighted.

Use of human induced pluripotent stem cells (iPSC) or embryonic stem cells (ESC) for cell replacement therapies holds great promise. Several limitations ...

Loss- and Gain-of-function Approach to Investigate Early Cell Fate Determinants in Preimplantation Mouse Embryos

Gene silencing and overexpression techniques are instrumental for the identification of genes involved in embryonic development. Direct target gene modification in preimplantation embryos provides a means to study the underlying mechanisms of genes implicated in, for instance, cellular differentiation into the trophectoderm (TE) and the inner cell mass (ICM). Here, we describe a protocol that examines the role of neogenin as an authentic receptor for initial cell fate determination in preimplantation mouse embryos. First, we discuss the experimental manipulations that were used to produce gain and loss of neogenin function by microinjecting neogenin cDNA and shRNA; the effectiveness of this approach was confirmed by a strong correlation between the pair-wise expression levels of either red fluorescent protein (RFP) or green fluorescent protein (GFP) and the immunocytochemical quantification of neogenin expression. Secondly, overexpression of neogenin in preimplantation mouse embryos leads to normal ICM development while neogenin knockdown causes the ICM to develop abnormally, implying that neogenin could be a receptor that relays extracellular cues to drive blastomeres to early cell fates. Given the success of this detailed protocol in investigating the function of a novel embryonic developmental stage-specific receptor, we propose that it has the potential to aid in exploration and identification of other stage-specific genes during embryogenesis.

The goal of this protocol is to describe a loss- and gain-of function method that is applicable to identify neogenin as a stage-specific receptor that leads to trophectoderm and inner cell mass differentiation in preimplantation mouse embryos.

 Gene silencing and overexpression techniques are instrumental for the identification of genes involved in embryonic development. Direct target gene ...

A Novel Ex Ovo Banding Technique to Alter Intracardiac Hemodynamics in an Embryonic Chicken System

The new model presented here can be used to understand the influence of hemodynamics on specific cardiac developmental processes, at the cellular and molecular level. To alter intracardiac hemodynamics, fertilized chicken eggs are incubated in a humidified chamber to obtain embryos of the desired stage (HH17). Once this developmental stage is achieved, the embryo is maintained ex ovo and hemodynamics in the embryonic heart are altered by partially constricting the outflow tract (OFT) with a surgical suture at the junction of the OFT and ventricle (OVJ). Control embryos are also cultured ex ovo but are not subjected to the surgical intervention. Banded and control embryos are then incubated in a humidified incubator for the desired period of time, after which 2D ultrasound is employed to analyze the change in blood flow velocity at the OVJ as a result of OFT banding. Once embryos are maintained ex ovo, it is important to ensure adequate hydration in the incubation chamber so as to prevent drying and eventually embryo death. Using this new banded model, it is now possible to perform analyses of changes in the expression of key players involved in valve development and to understand the role of hemodynamics on cellular responses in vivo, which could not be achieved previously.

A Novel Ex Ovo Banding Technique to Alter Intracardiac Hemodynamics in an Embryonic Chicken System

For the first time we present here a reproducible banding procedure to alter hemodynamics in the developing heart ex ovo. This is achieved by partially constricting the outflow tract (OFT).

The new model presented here can be used to understand the influence of hemodynamics on specific cardiac developmental processes, at the cellular and ...

In Ovo Electroporation in the Chicken Auditory Brainstem

Electroporation is a method that introduces genes of interest into biologically relevant organisms like the chicken embryo. It is long established that the chicken embryo is an effective research model for studying basic biological functions of auditory system development. More recently, the chicken embryo has become particularly valuable in studying gene expression, regulation and function associated with hearing. In ovo electroporation can be used to target auditory brainstem regions responsible for highly specialized auditory functions. These regions include the chicken nucleus magnocellularis (NM) and nucleus laminaris (NL). NM and NL neurons arise from distinct precursors of rhombomeres 5 and 6 (R5/R6). Here, we present in ovo electroporation of plasmid-encoded genes to study gene-related properties in these regions. We show a method for spatial and temporal control of gene expression that promote either gain or loss of functional phenotypes. By targeting auditory neural progenitor regions associated with R5/R6, we show plasmid transfection in NM and NL. Temporal regulation of gene expression can be achieved by adopting a tet-on vector system. This is a drug inducible procedure that expresses the genes of interest in the presence of doxycycline (Dox). The in ovo electroporation technique &#8211; together with either biochemical, pharmacological, and or in vivo functional assays &#8211; provides an innovative approach to study auditory neuron development and associated pathophysiological phenomena.

In Ovo Electroporation in the Chicken Auditory Brainstem

Auditory brainstem neurons of avians and mammals are specialized for fast neural encoding, a fundamental process for normal hearing functions. These neurons arise from distinct precursors of embryonic hindbrain. We present techniques utilizing electroporation to express genes in the hindbrain of chicken embryos to study gene function during auditory development.

Electroporation is a method that introduces genes of interest into biologically relevant organisms like the chicken embryo. It is long established that ...

In Vitro Ovule Cultivation for Live-cell Imaging of Zygote Polarization and Embryo Patterning in Arabidopsis thaliana

In most flowering plants, the zygote and embryo are hidden deep in the mother tissue, and thus it has long been a mystery of how they develop dynamically; for example, how the zygote polarizes to establish the body axis and how the embryo specifies various cell fates during organ formation. This manuscript describes an in vitro ovule culture method to perform live-cell imaging of developing zygotes and embryos of Arabidopsis thaliana. The optimized cultivation medium allows zygotes or early embryos to grow into fertile plants. By combining it with a poly(dimethylsiloxane) (PDMS) micropillar array device, the ovule is held in the liquid medium in the same position. This fixation is crucial to observe the same ovule under a microscope for several days from the zygotic division to the late embryo stage. The resulting live-cell imaging can be used to monitor the real-time dynamics of zygote polarization, such as nuclear migration and cytoskeleton rearrangement, and also the cell division timing and cell fate specification during embryo patterning. Furthermore, this ovule cultivation system can be combined with inhibitor treatments to analyze the effects of various factors on embryo development, and with optical manipulations such as laser disruption to examine the role of cell-cell communication.

In Vitro Ovule Cultivation for Live-cell Imaging of Zygote Polarization and Embryo Patterning in Arabidopsis thaliana

This manuscript describes an in vitro ovule cultivation method that enables live-cell imaging of Arabidopsis zygotes and embryos. This method is utilized to visualize the intracellular dynamics during zygote polarization and the cell fate specification in developing embryos.

In most flowering plants, the zygote and embryo are hidden deep in the mother tissue, and thus it has long been a mystery of how they develop dynamically; ...

Using In Vivo and Tissue and Cell Explant Approaches to Study the Morphogenesis and Pathogenesis of the Embryonic and Perinatal Aorta

The aorta is the largest artery in the body. The aortic wall is composed of an inner layer of endothelial cells, a middle layer of alternating elastic lamellae and smooth muscle cells (SMCs), and an outer layer of fibroblasts and extracellular matrix. In contrast to the widespread study of pathological models (e.g., atherosclerosis) in the adult aorta, much less is known about the embryonic and perinatal aorta. Here, we focus on SMCs and provide protocols for the analysis of the morphogenesis and pathogenesis of embryonic and perinatal aortic SMCs in normal development and disease. Specifically, the four protocols included are: i) in vivo embryonic fate mapping and clonal analysis; ii) explant embryonic aorta culture; iii) SMC isolation from the perinatal aorta; and iv) subcutaneous osmotic mini-pump placement in pregnant (or non-pregnant) mice. Thus, these approaches facilitate the investigation of the origin(s), fate, and clonal architecture of SMCs in the aorta in vivo. They allow for modulating embryonic aorta morphogenesis in utero by continuous exposure to pharmacological agents. In addition, isolated aortic tissue explants or aortic SMCs can be used to gain insights into the role of specific gene targets during fundamental processes such as muscularization, proliferation, and migration. These hypothesis-generating experiments on isolated SMCs and the explanted aorta can then be assessed in the in vivo context through pharmacological and genetic approaches.

Using In Vivo and Tissue and Cell Explant Approaches to Study the Morphogenesis and Pathogenesis of the Embryonic and Perinatal Aorta

Protocols for studying the embryonic and perinatal murine aorta using in vivo clonal analysis and fate mapping, aortic explants, and isolated smooth muscle cells are detailed here. These diverse approaches facilitate the investigation of the morphogenesis of the embryonic and perinatal aorta in normal development and the pathogenesis in disease.

The aorta is the largest artery in the body. The aortic wall is composed of an inner layer of endothelial cells, a middle layer of alternating elastic ...