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 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.
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.
This research was reviewed and approved by the Institutional Review Board for the Care and Use of Laboratory Animals of the Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México (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
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.
3. Obtaining the limb ectoderms
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.
5. Transplantation of the filled ectoderm into a host embryo
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.
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).
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'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) Fixing the RL in the host embryo with palladium wires. Please note that the RL was positioned on the embryo's right flank near the forelimb bud; p: pellet. Scale bar = 500 µm. Please click here to view a larger version of this figure.
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 µm. Please click here to view a larger version of this figure.
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 µm. Please click here to view a larger version of this figure.
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 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 authors have nothing to disclose.
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 Dirección General de Asuntos del Personal Académico (DGAPA)-Universidad Nacional Autónoma de México [grant numbers IN211117 and IN213314] and Consejo Nacional de Ciencia y Tecnología (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 Tecnología (CONACyT-Fronteras de la Ciencia-1887).
Alcian Blue 8GX | Sigma | A5268 | |
Angled slit knife | Alcon | 2.75mm DB | |
Blunt forceps | Fine Science Tools | 11052-10 | |
Collagenase type IV | Gibco | 1704-019 | |
DMEM-HG | Sigma | D5796 | |
Egg incubator | Incumatic de Mexico | Incumatic 1000 | |
Fetal Bovine Serum | Gibco | 16000069 | |
Fine surgical forceps | Fine Science Tools | 9115-10 | |
Hanks Balanced Salt Solution | Sigma | H6648 | |
Microcentrifuge | Eppendorf | 5417R | |
Micropipet | NA | NA | |
Palladium wire | GoodFellow | 7440 05-3 | |
Petri dish | Nest | 705001 | |
Pippette | crmglobe | PF1016 | |
Stereomicroscope | Zeiss | Stemi DV4 | |
Tape | NA | NA | |
Trypsin porcine | Merck | 9002 07-7 | |
Tungsten needle | GoodFellow | E74-15096/01 |