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 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.
Breakthroughs in the molecular mechanisms that control gene expression during embryonic development have allowed us to understand how cell fate is determined. Commitment to different cell lineages occurs once cells begin the molecular expression of transcription factors1. This expression pattern is highly coordinated in space and time and thereby directs the shaping, positioning, and patterning of cells, tissues, and organs1,2,3,4,5. Embryonic induction is the process by which cells are committed to specific lineages by establishing hierarchies that restrict cells' potentiality, which even include the generation of the basic body plan as occurs with the Spemann organizer6,7. The blastopore dorsal lip induces a second embryonic axis in a host embryo8,9. Today, with the aid of grafting and other classical experiments combined with molecular approaches, it is known that different transcription factors and growth factors function to direct embryonic induction in the Spemann organizer10. Thus, experimental manipulation is an important tool to understand cell differentiation, morphogenesis, and patterning processes during embryogenesis.
Interestingly, in embryonic systems where tissue transplantation is difficult or when the inducers are already well known, carriers are used to deliver molecules (e.g., proteins, chemicals, toxins, etc.) to regulate cell differentiation, morphogenesis, and even patterning. One such carrier system involves implanting beads soaked in a specific molecule in any experimental model organism at any developmental time point to determine the effect of the said reagent or direct the differentiation of the said model. For example, by implanting retinoic acid (RA)-soaked beads into the chicken wing limb bud, Cheryl Tickle et al. (1985) demonstrated that RA induces the expression of sonic hedgehog in the zone of polarizing activity (ZPA)11,12. The same experimental strategy was used to discover that RA controls the asymmetry of somites and cell death in the limb bud during digit development and in other embryonic limb regions13,14,15. Other factors, mainly proteins (e.g., fibroblast growth factors [FGF], transforming growth factor-beta [TGF-ß]) have been used to induce limbs in early embryos' flanks and new digits in the interdigital region, respectively16,17,18,19,20,21. These experiments evidence the power and utility of this technique for determining the stage of commitment or competence of tissues or groups of cells exposed to the molecules.
In this protocol, the chick limb at the stage of digit formation served as the experimental model to present step-by-step how to prepare and implant the soaked beads. However, this experimental tool is not limited to this application but can be exploited in any experimental animal model and any timepoint in vitro and in vivo to study induction, differentiation, cell death, and patterning.
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).
1. Egg incubation and embryo staging
NOTE: Fertilized hen eggs can be obtained from local farms. Fertilized White Leghorn chicken eggs are most commonly used. Store the freshly fertilized chicken eggs at 15 °C for up to 1 week prior to incubation.
2. Bead preparation
NOTE: Depending on the experimental aim and treatment in question, alternative bead types (e.g., Affi-Gel, AG1-X2, heparin) may be more suitable. Affi-Gel beads are optimal for proteins (e.g., TGF-ß1), while heparin beads are ideal for growth factors (e.g., FGFs, WNT) and AG1-X2 beads for chemicals solubilized in organic solvents (e.g., DMSO).
3. Embryo manipulation and interdigit implantation
Using soaked beads to evaluate cell behavior in the embryonic chick limb
To assure the efficacy of this assay, the bead must be placed consistently and precisely in the correct location; in this case, the distal-most of the third interdigit beneath the apical ectodermal ridge AER (Figure 1A). This positioning permits the molecule in question to spread equally throughout the interdigital tissue. Moreover, the zone beneath the AER contains undifferentiated cells that are readily responsive to treatment. To evaluate the effects on cell differentiation, the embryos can be stained with Alcian blue and Alizarin red to evidence the formation of skeletal elements (Figure 1B). The soaked-bead assay is also well-suited for evaluating cell death with neutral red (Figure 1C) and gene regulation by in situ hybridization (Figure 1D). Scale bar is set at 250 µm.
Figure 1: Bead implantation into the interdigital tissue of the chick limb.(A)The correct location for the soaked bead beneath the AER in a 28 HH hindlimb. (B) Alcian blue and Alizarin red skeletal staining evidence the formation of an ectopic digit induced 4 days after implanting a TGF-ß1-soaked bead. (C) Neutral red staining marks cell death induction (arrowhead) 24 h after implanting a RA-soaked bead into the interdigital tissue at the 28 HH stage. (D) Sox9 in situ hybridization 4 h after a TGF-ß1-soaked bead was implanted. Scale bar is set at 250 µm. The images shown in B and C were taken from Díaz-Hernández et al.23,24. Please click here to view a larger version of this figure.
The main advantage of the experimental tool detailed in this protocol is being able to control the time and location of the exposure to beads soaked in a given experimental molecule. Combining the correct positioning with precise developmental timing provides enormous possibilities to study cell differentiation processes. Performing these experiments in undifferentiated tissue enables investigating the first crucial events in cellular lineage. For example, placing a TGFß-soaked bead in the interdigital tissue of embryonic limbs 28 HH results in the formation of an ectopic digit in which a molecular cascade is triggered that induces the genetic expression of the master gen Sox925. Remarkably, the induced cartilage tissue also organizes into a digit with phalanx formation.
Interestingly, RA triggers cell death in the same interdigital region by regulating the gene expression of bone morphogenetic proteins that direct the cell fate of undifferentiated cells toward cell death10. Hence, cell differentiation, cell death, morphogenesis, and patterning can be concurrently investigated in the same region of an embryo and tailored to any region and genetic pathway of interest8,9.
The elements crucial to the success of this protocol include never letting the soaked beads dry out (i.e., they must always remain wet). Also, selecting the appropriate beads is essential: Affi-Gel and heparin beads are for proteins, whereas AG1-X2 are for chemicals dissolved in organic solvents. Another critical point is the concentration of the molecule contained in the solution used to soak the beads, which is usually 1000-fold more concentrated than would be used for in vitro studies. Nevertheless, an inconvenience of this method is that the final concentration of molecules released from the soaked beads is unknown, as well as the velocity of release. In the protocol is mentioned that beads-100 µm diameter is more convenient for use. Consider this when limbs are manipulated. Most importantly, the diameter of the beads must be selected according to the implantation zone and consistently maintained in each embryo. However, a slight variation in bead size between experiments is not likely to affect the results.
In conclusion, the potential of the soaked-bead assay outlined here depends only on the imagination of the researcher. This protocol can be applied to any experimental animal, cell culture, or organotypic culture model, including organoids. Furthermore, this protocol is a helpful, straightforward educational tool for teaching students basic developmental biology concepts and technical skills by practicing this experimental manipulation in developmental biology classes.
The authors have nothing to disclose.
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 received a postdoctoral fellowship from the Consejo Nacional de Ciencia y Tecnología (CONACyT-Fronteras de la Ciencia-1887). The authors appreciate the help of Lic. Lucia Brito from Instituto de Investigaciones Biomédicas, UNAM in the preparation references of this manuscript.
Affi-Gel Blue Gel beads | Bio-Rad | 153-7302 | |
AG1-X2 beads | Bio-Rad | 1400123 | |
Egg incubator | Incumatic de Mexico | Incumatic 1000 | |
Fine surgical forceps | Fine Science Tools | 9115-10 | |
Heparine Sepharose beads | Abcam | ab193268 | |
Petri dish | Nest | 705001 | |
Stereomicroscope | Zeiss | Stemi DV4 | |
Tape | NA | NA | |
Tungsten needle | GoodFellow | E74-15096/01 |