We have developed an upper-level undergraduate laboratory exercise that enables students to replicate a key experiment in developmental biology. In this exercise, students have the opportunity to observe live chick embryos and stain the apical ectodermal ridge, a key tissue required for development of the vertebrate limb. Impressively, every student who has tried this protocol has been successful, making it a good introduction to the use of the chick model system in studying development. The array of materials about limb development, using chick embryos in teaching laboratories, and the history of this experiment provide a rich background for teachers and students.

One of the fundamental questions in developmental biology is how different tissues are formed at the correct place and in the correct relationship to one another. Development of the vertebrate limb is one of the best-understood examples of how a complex tissue is patterned. The early limb bud consists of an internal layer of mesenchyme surrounded by an outer layer of epidermal cells. At the distal tip of the limb, the epidermal cells form a special structure called the “apical ectodermal ridge” (AER; Gilbert, 2013). This small region plays an essential role in outgrowth of the limb (Saunders, 1948). When the AER of a developing chick wing is surgically removed early in development, almost all sections of the wing (humerus, radius, ulna, and bones of the wrist and hand) are absent (Saunders, 1948). When the AER is removed later in development, only the most distal parts (bones of the wrist and hand) are missing (Saunders, 1948). Because the process of limb development is largely conserved, research such as this in model organisms has given insights into the evolution of the vertebrate limb and into the causes of human limb defects.

In this laboratory exercise, students replicate the staining experiment done by Dr. John Saunders that led to the discovery of the AER (Fallon, 2002). In our course at the University of Minnesota Duluth, this exercise is done in concert with lectures and discussions on limb development in an upper-level undergraduate Developmental Biology course (materials used in the course include Saunders [1948], Ros et al. [2000], Tyler [2000], Hall [2007], Zuniga et al. [2012], and Gilbert [2013]).

Because this exercise requires vertebrate embryos, experience with fine manipulations under a stereomicroscope, and some specialized equipment, it is most suitable for an upper-level undergraduate course or a specialized course for earlier students. It could be combined with other experiments using chick embryos, studies of limb evolution and development, or guided reading of primary literature on limb development and regeneration (Tyler, 2000; Tyler et al., 2006; Gilbert, 2013).

Learning Objectives

  1. Maintain an accurate and complete lab notebook.

  2. Practice experimental design by writing an experimental protocol.

  3. Cement learning of limb development.

  4. Build lab skills.

Materials

  • Dissecting needles (Carolina Scientific no. 627220)

  • Dissection scissors (Carolina Scientific no. 621810)

  • Forceps (Fine Science Tools Student Dumont no. 5)

  • Fiber optic or LED light (Techniquip FOI-250)

  • Fertilized chicken eggs (e.g., Sunnyside Hatchery, Beaver Dam, WI)

  • Chicken-egg incubator

  • Stereomicroscopes

  • 9-inch borosilicate-glass Pasteur pipets

  • Nile blue agar

  • 1× phosphate buffered saline (PBS) solution

  • Bunsen burners

  • Blue food coloring

  • 1-mL syringes

  • Pencils

Safety Concerns

Students should use good hand-washing practices with antibacterial soap throughout the lab to prevent exposure to salmonella. Glass tools, glass waste, and syringes should be disposed of in a sharps container. Students should be trained in safe use of syringes and Bunsen burners. Waste should be disposed of according to local guidelines. Because embryos are euthanized following this exercise, ethical guidelines for use of animals in the classroom should be reviewed to determine whether it is appropriate for a particular course (http://www.nabt.org/websites/institution/index.php?p=97). The local Institutional Animal Care and Use Committee should be contacted to determine whether approval of an animal protocol is required. Chicken embryos <13 days old, such as the ones used in this exercise, are assumed to be unable to feel pain because of their rudimentary nervous system. Embryos should be euthanized by a method that causes hypothermia, such as placement in a –20°C freezer for ≥2 hours (https://www.avma.org/KB/Policies/Documents/euthanasia.pdf).

Methods

In Advance of the Lab (Instructor)

  1. Incubate the eggs. Place the eggs in a chicken-egg incubator at 99–102°F (37–39°C) so that they will be between 72 and 96 hours post-fertilization on the day of the lab. Make sure to have a few more eggs than students, in case some eggs do not contain embryos.

  2. Make Nile blue agar.

    • 2 g agar

    • 100 mL distilled or milliQ water

    Heat in 10-second intervals in a microwave oven in a 250-mL beaker, swirling between rounds of heating to prevent superheating. Add 1 g Nile blue sulfate and mix. While the mixture is still liquid, pour a small amount into a Petri dish to make a 1–10 mm layer. Store the remaining solution at 4°C for later use.

  3. Make 10 L 1× phosphate buffered saline (PBS).

    • 8 L distilled water

    • 8 g NaCl

    • 0.2 g KCl

    • 1.44 g Na2HPO4

    • 0.24 g KH2PO4

    Adjust the pH to 7.4 with HCl, and then bring to a total volume of 10 L with distilled water. Premade 1× PBS can also be purchased from many suppliers.

In Advance of the Lab (Students)

  1. Developing a protocol. Before the day of the lab, develop a step-by-step protocol based on a methods paper by John Fallon and colleagues (Ros et al., 2000).

Day of the Lab (Students)

  1. Keeping a lab notebook. Keep a complete record of your experiment in your lab notebook.

  2. Making the staining needles. Make staining needles from 9-inch Pasteur pipets. Hold one end of the Pasteur pipet with each hand, with the middle over the flame of a Bunsen burner. When the glass starts to turn red, gently pull the two ends away from one another until the glass separates into two pieces. Place the pulled end into the flame to melt it into a round shape.

  3. Candling the egg. Use a fiber optic light to shine bright light through the intact fertilized egg (Figure 1A). The position of the embryo will have extensive vasculature visible through the shell on the blunt side of the egg. Draw a 1-inch-diameter circle around the site of the embryo.

  4. Windowing the egg. Using a sharp tool such as a dissecting needle, poke a small hole in the center of the 1-inch circle, and then cut out the circle using dissecting scissors (Figure 1B, C).

  5. Identification of limb buds. Using a syringe, add a small amount of blue food coloring just underneath the embryo. Identify the limb buds under a dissecting microscope with light coming from above (Figure 1D, E).

  6. Transferring the embryo. Older embryos typically start to sink into the yolk after the shell is opened. If this happens, use a spatula or forceps to lift the embryo and transfer it to a Petri dish containing 1× PBS (Figure 1F).

  7. Dissection of overlying membranes. Remove the membranes covering the limb bud under a stereomicroscope using a pair of fine forceps or dissecting needles (Figure 1G).

  8. Staining the limb buds. After removing the membranes, load the staining needle by running the round end through a thin layer of Nile blue agar. Touch the stained area of the needle to the apex of the limb bud (Figure 1H).

  9. Observing the staining process. Initially, the whole region around where the needle touched will be equally blue (Figure 2A). After 15–20 minutes, the staining will become darker in the AER than in the surrounding tissues (Figure 2B–D). There is an almost invisible membrane covering the limb bud. If you get diffuse instead of localized staining, it is likely that this membrane is still present. Remove this membrane and repeat the staining procedure.

Figure 1.

Steps in the AER staining protocol. (A) Candling the egg. (B) Poking a small hole in the eggshell with a dissecting needle. (C) Using dissecting scissors to make a hole in the egg. (D, E) Images of embryos and the surrounding vasculature taken through the hole in the shell. Developing limb buds are marked with white arrows. (F) Transferring an older embryo (≥96 hours post-fertilization) into a Petri dish using a spatula. (G) Removing the thin membranes covering the limb bud. (H) Staining the apical region of the limb bud.

Figure 1.

Steps in the AER staining protocol. (A) Candling the egg. (B) Poking a small hole in the eggshell with a dissecting needle. (C) Using dissecting scissors to make a hole in the egg. (D, E) Images of embryos and the surrounding vasculature taken through the hole in the shell. Developing limb buds are marked with white arrows. (F) Transferring an older embryo (≥96 hours post-fertilization) into a Petri dish using a spatula. (G) Removing the thin membranes covering the limb bud. (H) Staining the apical region of the limb bud.

Figure 2.

Examples of AER staining. (A) Whole chick embryo just after staining with Nile blue (white arrow; anterior to the top and dorsal to the right). (B) A stained limb 15–30 minutes after staining, with darker staining in the AER (white arrowhead; anterior to the top and distal to the left). (C) Embryo 15–30 minutes after staining, with the stained limb bud indicated (white arrow; anterior to the top and dorsal to the right). (D) Close-up view of the stained AER (white arrowhead; anterior to the top and distal to the left) in the same embryos as in panel C. Images in panels C and D were taken by undergraduate student Jeffrey Finlon.

Figure 2.

Examples of AER staining. (A) Whole chick embryo just after staining with Nile blue (white arrow; anterior to the top and dorsal to the right). (B) A stained limb 15–30 minutes after staining, with darker staining in the AER (white arrowhead; anterior to the top and distal to the left). (C) Embryo 15–30 minutes after staining, with the stained limb bud indicated (white arrow; anterior to the top and dorsal to the right). (D) Close-up view of the stained AER (white arrowhead; anterior to the top and distal to the left) in the same embryos as in panel C. Images in panels C and D were taken by undergraduate student Jeffrey Finlon.

Results & Discussion

This lab had an extremely high rate of success. Every student (n = 63 of 63) in two semesters was able to carry out the complete protocol and effectively stain the AER. Students found a wide variety of activities in this lab valuable for their learning, with the highest number of students finding observations of chick embryos and AER staining the most valuable (Table 1 and Figure 3).

Table 1.

Student assessment of the AER staining laboratory.

Which part of the chick development laboratory contributed the
most to your understanding of development?aleast to your understanding of development?a
Reading the introductory material for the laboratory 4% 5% 
Doing the homework to generate a protocol for AER staining 2% 4% 
Writing a laboratory notebook entry 2% 10% 
Carrying out the steps needed to observe chick embryos 8% 1% 
Observing live chick embryos 10% 0% 
Watching the interview with Nicole LeDouarinb (neural crest development) 5% 3% 
Watching the interview with John Saunders b (limb development) 5% 2% 
Staining the AER 10% 0% 
Discussing the laboratory with other students 2% 2% 
Discussing the laboratory as a class 0% 1% 
Outside reading of primary literature 1% 6% 
Other 1% 1% 
Which part of the chick development laboratory contributed the
most to your understanding of development?aleast to your understanding of development?a
Reading the introductory material for the laboratory 4% 5% 
Doing the homework to generate a protocol for AER staining 2% 4% 
Writing a laboratory notebook entry 2% 10% 
Carrying out the steps needed to observe chick embryos 8% 1% 
Observing live chick embryos 10% 0% 
Watching the interview with Nicole LeDouarinb (neural crest development) 5% 3% 
Watching the interview with John Saunders b (limb development) 5% 2% 
Staining the AER 10% 0% 
Discussing the laboratory with other students 2% 2% 
Discussing the laboratory as a class 0% 1% 
Outside reading of primary literature 1% 6% 
Other 1% 1% 

aNumber of students out of 21 who replied with each answer.

bThese interviews were from the CD-ROM Differential Expressions (Sinauer and Associates, Sunderland, MA).

Figure 3.

Students’ evaluations of the value of this laboratory for their learning. This figure is based on evaluations by 21 students in the 2011 class of Developmental Biology at the University of Minnesota Duluth.

Figure 3.

Students’ evaluations of the value of this laboratory for their learning. This figure is based on evaluations by 21 students in the 2011 class of Developmental Biology at the University of Minnesota Duluth.

The other main advantage of this lab exercise is the variety of materials that are available to support student learning. Developmental Biology: A Guide for Experimental Study (Tyler, 2000) provides guidance on how to work with chicken embryos. Many developmental biology textbooks have extensive sections on limb development, and historical perspective comes from interviews with John Saunders and from a review by Saunders on recent advancements in the field (Saunders, 1998; Fallon, 2002; Tyler et al., 2006; Gilbert, 2010; Wolpert & Tickle, 2010).

Acknowledgments

We thank Tonya Connor for guidance in student assessment, Nicholas Lamon for excellent lab support, Nadejda Bozadjieva and Frank Liu for their work as graduate teaching assistants, and the undergraduate students in Developmental Biology for their help in establishing this protocol and their valuable feedback.

References

References
Fallon, J.F. (2002). How serendipity shaped a life; an interview with John W. Saunders. International Journal of Developmental Biology, 46, 853–861.
Gilbert, S.F. (2010). Developmental Biology, 9th Ed. Sunderland, MA: Sinauer Associates.
Gilbert, S.F. (2013). Developmental Biology, 10th Ed. Sunderland, MA: Sinauer Associates.
Hall, B.K., Ed. (2007). Fins into Limbs: Evolution, Development, and Transformation. Chicago, IL: University of Chicago Press.
Ros, M.A., Simandl, B.K., Clark, A.W. & Fallon, J.F. (2000). Methods for manipulating the chick limb bud to study gene expression, tissue interactions, and patterning. Methods in Molecular Biology, 137, 245–266.
Saunders, J.W., Jr. (1948). The proximo-distal sequence of origin of the parts of the chick wing and the role of the ectoderm. Journal of Experimental Zoology, 108, 363–403.
Saunders, J.W., Jr. (1998). Apical ectodermal ridge in retrospect. Journal of Experimental Zoology, 282, 669–676.
Tyler, M.S. (2000). Developmental Biology: A Guide for Experimental Study, 2nd Ed. Sunderland, MA: Sinauer Associates.
Tyler, M.S., Kozlowski, R.N. & Gilbert, S.F. (2006). Differential Expressions2: Key Experiments in Developmental Biology. Sunderland, MA: Sinauer Associates.
Wolpert, L. & Tickle, C. (2010). Principles of Development, 4th Ed. Oxford, UK: Oxford University Press.
Zuniga, A., Zeller, R. & Probst, S. (2012). The molecular basis of human congenital limb malformations. Wiley Interdisciplinary Reviews: Developmental Biology, 1, 803–822.