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>

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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. 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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. 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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 identified. During mesodermal cell procurement, the integrity and viability of the cells are essential. Cell death will prevent proper RL development. In a similar vein, correct ectoderm manipulation is necessary to guarantee the interaction between mesodermal and ectodermal cells. When ectoderms are stuffed, mesodermal cells must be as close as possible to the distal ectoderm beneath the AER. For both mesodermal and ectodermal procurement, the developmental stage of the donor embryos is also critical. It must be considered that the mesodermal cells will respond differentially according to their developmental stage. However, the developing stage can be freely selected according to the experimental requirements. Still, the 22 HH stage needs to be maintained to make obtaining ectoderms easier, thus maintaining cellular integrity and signaling. Finally, good grafting and fixing are essential for ensuring correct RL integration to the embryo wall and its development.
Edgar Zwilling first reported the RL system in 19646, after which many research groups implemented it to answer several interesting biological questions. The protocol of the RL has been previously described in length by Marian Ros et al. as a standard method to manipulate the developing chick limb bud18, which explains other ways to window the eggs and perform RL with whole wing or leg and to perform RL with reaggregated mesoderm. However, some variations between the Ros. et al. protocol and the present protocol described here can be found. In their protocol, limb buds from embryos as ectoderm donors are incubated with trypsin in ice-cold PBS for ~2 h. After initiating ectoderm incubation, they immediately obtained limb buds from embryos as mesoderm donors, then fragmented the limb buds, digested them, and removed the ectoderm manually to form the mesodermal pellet after incubating for 30 min. Here, first, the limb buds were obtained from embryos to be used as mesodermal donors, after which the whole limb bud is digested by incubating with trypsin and collagenase. Ectoderms are then removed by filtration, and the pellet is incubated between 1-1.5 h. The advantage of this method to obtain the mesodermal pellet is that the treatment with trypsin detaches the intact ectoderms from the mesoderm while the collagenase treatment digests mesodermal cells. Therefore, it is possible to filter the ectodermal tissue and discard it. On the other hand, more pellet incubation time allows it to compact better, which helps when ectoderms fill. Another difference between the two protocols is that Ros et al. peeled off the ectoderms one by one and transferred them to the Petri dish containing the pellet. In contrast, all the ectoderms are dissected and collected in a Petri dish in the present protocol. The pellet is transferred to the Petri dish to fill the ectoderms. By following this method, the ectoderms can be prepared simultaneously with grafting. As with many other protocols, how RLs are performed may vary; however, the steps in both protocols adequately describe the critical stages of the technique to produce a successful manipulation.
Previous work has demonstrated that dissociated polarizing zone cells inhibit morphogenesis when randomly dispersed among mesoderm in RL7,10. Thus, it is optional to eliminate cells from the ZPA before forming the mesodermal pellet. Later, the ZPA cells (or sonic hedgehog embedded beads) can be used to induce the RL to develop A-P polarity8,9,19.
The RL experimental model is adaptable to a variety of scenarios. Recombination can be implemented with limb cells from different developmental or mature (fore- or hindlimb) stages, other positions along the three limb axes, and with dissociated-reaggregated cells or undissociated-fragmented mesoderm15. Interestingly, previous studies have reported using the RL model to study the behavior of different combinations of mutant and wild-type mesoderm and ectoderms13,14,20,21&#160;or using electroporated cells22.
Considering that limb development is an evolutionarily conserved process, the ectoderm sources also can vary from the chicken, quail, duck, mouse, or rat ectoderms can be obtained following the same described protocol. Another possibility is changing the mesodermal -or even other cell types or sources to produce interspecies RL.
In conclusion, RL is a phenomenal model to study morphogenesis, patterning, cell-cell interactions, cell migration, and cell differentiation at the cellular and molecular levels. Because the procedure of RL allows multiple variations, it permits potential applications across numerous biological questions without being restricted to chicken-limb developmental biology.

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><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 contrary, whether either cell viability and/or the graft failed, the RL was detached from the mesodermal wall or presented a rough morphology (Figure 2B).
Morphological and patterning examination of recombinant limbs 
For morphological examination, RL was stained with Alcian blue17 to observe the formation of skeletal elements and their patterning. It is recommended to stain the whole trunk of the donor embryo to avoid missing the RL during the procedure (Figure 3A). Alternatively, before clearing the RL, images in ethanol solution were obtained to observe the morphology of the RL or perform quantitative measurements (Figure 3B). Stained or unstained RL was sliced to observe tissue structure or identify cell type (Figure 3C).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63183/63183fig01.jpg" /> 
Figure 1: Schematic representation of the experimental design of recombinant limbs (RL). (A) RL was performed by assembling limb bud mesoderm from a donor 22 HH chick embryo inside an ectodermal cover obtained from another 22 HH chick embryo donor. Ectoderms were tightly stuffed with mesodermal cells. After assembly, stuffed ectoderms were transferred and fixed with palladium wires on top of a previous somite&#39;s wound. (B) A limb bud with its ectoderm detached after trypsin treatment. (C) The pellet was obtained after its formation near to the empty ectoderms, ready to be filled. (D) An ectodermal cover filled with mesodermal cells is shown. (E)&#160;Fixing the RL in the host embryo with palladium wires. Please note that the RL was positioned on the embryo&#39;s right flank near the forelimb bud; p: pellet. Scale bar = 500 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63183/63183fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63183/63183fig02.jpg" /> 
Figure 2: Freshly obtained chicken-chicken recombinant limbs. (A) A 24 h RL attached to the mesoderm wall is shown. (B) An unsuccessful 24 h RL is shown. Please note that the palladium wire is not fixing the RL; consequently, the RL detached from the mesodermal wall and presented a rough morphology. Scale bar = 500 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63183/63183fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63183/63183fig03.jpg" /> 
Figure 3: Morphological analysis of recombinant limbs. (A) Alcian blue staining to demonstrate skeletal elements in a 6-day chicken-chicken RL. (B) The same RL was shown in (A) before clearing the Alcian blue. (C) Sagittal slice of an RL stained with Alcian blue staining and with hematoxylin and eosin. Scale bar = 100 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63183/63183fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

chicken-recombinant-limbs-assay-to-understand-morphogenesis

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

2.1K 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

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 ...

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

The chick embryo provides a superb vertebrate model that can be used to dissect developmental questions in a direct way. Its accessibility and robustness following surgical intervention are key experimental strengths. Mica plates were the first barriers used to prevent chick limb bud initiation1. Protocols that use aluminum foil as an impermeable barrier to wing bud or leg bud induction and or initiation are described. We combine this technique with bead placement lateral to the barrier to exogenously supply candidate endogenous factors that have been blocked by the barrier. The results are analyzed using in situ hybridization of subsequent gene expression. Our main focus is on the role of retinoic acid signaling in the induction and later initiation of the chick embryo fore and hindlimb. We use BMS 493 (an inverse agonist of retinoic acid receptors (RAR)) soaked beads implanted in the lateral plate mesoderm (LPM) to mimic the effect of a barrier placed between the somites (a source of retinoic acid (RA)) and the LPM from which limb buds grow. Modified versions of these protocols could also be used to address other questions on the origin and timing of inductive cues. Provided the region of the chick embryo is accessible at the relevant developmental stage, a barrier could be placed between the two tissues and consequent changes in development studied. Examples may be found in the developing brain, axis extension and in organ development, such as liver or kidney induction.

Protocols using impermeable barriers to block induction events between tissues of the main body axis and flank in the chick embryo required for limb formation are described. Beads soaked in candidate inductive signals are used to overcome the effect of barrier placement and analysis of gene expression confirms this.

The chick embryo provides a superb vertebrate model that can be used to dissect developmental questions in a direct way. Its accessibility and robustness ...

Assay for Neural Induction in the Chick Embryo

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying the formation and initial patterning of the nervous system. This video demonstrates how to graft organizer tissue into a host, a method by which Hensen s node (the organizer in the chick embryo) is grafted to a host competent ectoderm. The organizer graft instructs overlying na ve tissue to adopt a neural fate via neural inducing signals. This mechanism is referred to as neural induction, and constitutes the initial step in the formation of brain and spinal cord in amniotes. This method is essentially used for the characterization of putative neural inducing molecules in chick. This video demonstrates the different steps in the assay for neural induction; First, the donnor embryo is explanted and pinned on a dish. Then, the host embryo is prepared for New culture. The graft is excised and transplanted to the host area pellucida margin. The host is cultured for 18-22 hrs. The assembly is fixed and processed for further applications (e.g. in situ hybridization). This method was originally devised by Waddington 1,2 and Gallera 3,4.

Neural induction is the first step in the formation of the brain. It is a mechanism by which Hensen's node (organizer), instructs adjacent tissue to adopt a neural fate, i.e. to give rise to the nervous system. This video demonstrates an assay for neural induction in chick embryo.

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Biology

Chicken Embryo Spinal Cord Slice Culture Protocol

Slice cultures can facilitate the manipulation of embryo development both pharmacologically and through gene manipulations. In this reduced system, potential lethal side effects due to systemic drug applications can be overcome. However, culture conditions must ensure that normal development proceeds within the reduced environment of the slice. We have focused on the development of the spinal cord, particularly that of spinal motor neurons. We systematically varied culture conditions of chicken embryo slices from the point at which most spinal motor neurons had been born. We assayed the number and type of motor neurons that survived during the culture period and the position of those motor neurons compared to that in vivo. We found that serum type and neurotrophic factors were required during the culture period and were able to keep motor neurons alive for at least 24 hr and allow those motor neurons to migrate to appropriate positions in the spinal cord. We present these culture conditions and the methodology of preparing the embryo slice cultures using eviscerated chicken embryos embedded in agarose and sliced using a vibratome.

Slice cultures facilitate the manipulation of embryo development by gene and pharmacological perturbations. However, culture conditions must ensure that normal development can proceed within the reduced environment of the slice. We illustrate a protocol that facilitates normal spinal cord development to proceed for at least 24 hr.

Slice cultures can facilitate the manipulation of  embryo development both pharmacologically and through gene manipulations. In  this reduced system, ...

Neuroscience

A Label-free Xenograft Model for Investigating the Behavior of Human Stem Cell Spheroids in Chick Embryos

Xenografts are valuable methods to investigate the behavior of human cells in vivo. In particular, the embryonic environment provides cues for cell migration, differentiation, and morphogenesis, with unique instructive signals and germ layer identity that are often absent from adult xenograft models. In addition, embryonic models cannot discriminate self versus non-self tissues, eliminating the risk of rejection of the graft and the need for immune suppression of the host. This paper presents a methodology for transplantation of spheroids of human cells into chicken embryos, which are accessible, amenable to manipulation, and develop at 37 &#176;C. 
Spheroids allow the selection of a specific region of the embryo for transplantation. After being grafted, the cells become integrated into the host tissue, allowing the follow-up of their migration, growth, and differentiation. This model is flexible enough to allow the utilization of different adherent populations, including heterogeneous primary cell populations and cancer cells. To circumvent the need for prior cell labeling, a protocol for the identification of donor cells through hybridization of human-specific Alu probes is also described, which is particularly important when investigating heterogeneous cell populations. Furthermore, DNA probes can be easily adapted to identify other donor species. This protocol will describe the general methods for preparing spheroids, grafting into chicken embryos, fixing and processing tissue for paraffin sectioning, and finally identifying the human cells using DNA in situ hybridization. Suggested controls, examples of interpretation of results and various cell behaviors that can be assayed will be discussed in addition to the limitations of this method.

Here, we describe a method to transplant and identify human cell spheroids into chick embryos. This xenograft model uses the embryonic microenvironment as a source of instructive signals to assay cell migration, differentiation, and tropism and is especially suited for the study of primary and/or heterogeneous cell populations.

Xenografts are valuable methods to investigate the behavior of human cells in vivo. In particular, the embryonic environment provides cues for cell ...

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

A multitude of genetic programs is activated during embryonic development that orchestrates cell differentiation to generate an astounding diversity of somatic cells, tissues, and organs. The precise activation of these genetic programs is regulated by morphogens, diffusible molecules that direct cell fate at different thresholds. Understanding how genetic activation coordinates morphogenesis requires the study of local interactions triggered by morphogens during development. The use of beads soaked in proteins or drugs implanted into distinct regions of the embryo enables studying the role of specific molecules in the establishment of digits and other developmental processes. This experimental technique provides information on the control of cell induction, cell fate, and pattern formation. Thus, this soaked bead assay is an extremely powerful and valuable experimental tool applicable to other embryonic models.

The soaked bead assay involves targeted delivery of test reagent at any developmental time point to study the regulation of cell differentiation and morphogenesis. A detailed protocol, applicable to any experimental animal model, for preparing three different types of soaked beads and implanting these in the interdigit of a chicken embryo is presented.

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

Assessing Signaling Properties of Ectodermal Epithelia During Craniofacial Development

The accessibility of avian embryos has helped experimental embryologists understand the fates of cells during development and the role of tissue interactions that regulate patterning and morphogenesis of vertebrates (e.g., 1, 2, 3, 4). Here, we illustrate a method that exploits this accessibility to test the signaling and patterning properties of ectodermal tissues during facial development. In these experiments, we create quail-chick 5 or mouse-chick 6 chimeras by transplanting the surface cephalic ectoderm that covers the upper jaw from quail or mouse onto either the same region or an ectopic region of chick embryos. The use of quail as donor tissue for transplantation into chicks was developed to take advantage of a nucleolar marker present in quail but not chick cells, thus allowing investigators to distinguish host and donor tissues 7. Similarly, a repetitive element is present in the mouse genome and is expressed ubiquitously, which allows us to distinguish host and donor tissues in mouse-chick chimeras 8. The use of mouse ectoderm as donor tissue will greatly extend our understanding of these tissue interactions, because this will allow us to test the signaling properties of ectoderm derived from various mutant embryos.

This article describes a tissue transplantation technique that was designed to test the signaling and patterning properties of surface cephalic ectoderm during craniofacial development.

The accessibility of avian embryos has helped experimental embryologists understand the fates of cells during development and the role of tissue ...

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. ...

A Rapid In Vivo Bioassay for Developmentally Active Enhancers

A potential regulatory sequence (PRS) can be identified by several approaches including conservation analysis and transcription factor-specific chromatin immunoprecipitation followed by next generation sequencing (TF ChIP-seq). TF ChIP-seq generates a large data set (TF-regulatome) that includes PRSs and background interactions. These approaches require secondary low throughput validation in model systems that replicate the temporal and spatial specificity of the PRS. 
In this report, a protocol is described to rapidly validate activity of PRSs in their developmental context. Following selection of candidate PRSs, the associated sequences are isolated, cloned into a GFP reporter construct, and then transfected into chick embryos. For PRSs that are anticipated to be functional during early development, from gastrulation through early organogenesis, whole embryo electroporation (EP) is recommended. With this technique transfection, incubation and monitoring occurs ex ovo during early gastrulation. The entire embryo is transfected and as structures form, they can be monitored live for PRS activity. This ex ovo approach supports development up to early limb bud outgrowth. 
If the functional activity of a PRS is expected to be later in development or if the investigation is to ascertain its activity during the development of a specific organ, targeted regional electroporation (TREP) is used. This approach requires injection of construct DNA directly into the developing organ, precursor or adjacent cavity prior to electroporation. An approach to transfect the presumptive limb bud using TREP is also outlined and illustrated. TREP supports a rapid organ-specific validation of a PRS' activity. Both techniques can also be used to investigate regulatory domains by site-directed mutagenesis of the PRS-reporter construct. 
The chick as a bioassay offers a rapid effective tool for PRS validation, functional domain determination, and further characterization of the regulatory events responsible for development.

With the increased use of transcription factor-specific ChIP-seq technology, techniques to validate potential regulatory regions are critical. The authors describe a rapid chick bioassay to validate the activity of regulatory sequences functioning during development.

A potential regulatory sequence (PRS) can be identified by several approaches including conservation analysis and transcription factor-specific chromatin ...

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

Summary

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Chapters in this video

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

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

Assay for Neural Induction in the Chick Embryo

Chicken Embryo Spinal Cord Slice Culture Protocol

A Label-free Xenograft Model for Investigating the Behavior of Human Stem Cell Spheroids in Chick Embryos

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

Assessing Signaling Properties of Ectodermal Epithelia During Craniofacial Development

Using Avian Skin Explants to Study Tissue Patterning and Organogenesis

Tracking Morphogenetic Tissue Deformations in the Early Chick Embryo

A Rapid In Vivo Bioassay for Developmentally Active Enhancers