tissue studies by culturing reconstituted chicken skin 

A Comprehensive Review of Avian Embryonic Skin Explant Culture Protocols

Avian embryo skin development is an excellent model for studying the mechanisms of morphogenesis because of the distinct patterns and the accessibility to microsurgery and manipulation1,2. However, evaluating cellular and molecular events in intact tissues can be difficult because the presence of extraneous tissues can complicate microscopic observations. Furthermore, the ability to manipulate gene expression to test their role in skin morphogenesis is not always a simple task. We find we can test gene functions using retroviral transduction with a higher success rate using skin explant models. Here we discuss the advantages of three skin explant models that have been developed.
Avian embryonic skin culture is a powerful system to assess cell behavior, gene regulation, and function during skin feather bud development3,4,5,6. It allows for the evaluation of the molecular mechanisms of feather bud development through the global addition of growth factors placed in the culture media or their local release from growth factor-coated beads. Developmental regulatory genes can also be manipulated using viral gene transduction of intact or dominant negative forms for functional studies evaluating their roles in specific morphogenetic events 7,8.
Avian epithelial-mesenchymal recombination culture enables investigators to determine the contributions of each skin component during the early stages of skin morphogenesis. Rawles&#39; use of this approach revealed that interactions between the mesenchyme and epithelium are essential to forming skin appendages9. The mesenchyme can form condensations and the epithelium is needed to induce and maintain mesenchymal condensation formations2. Later, this approach was used to assess why Scaleless chickens fail to form feathers. The defect was discovered to be in mesenchyme10. Dhouailly performed tissue epithelial-mesenchymal recombination studies in embryos from different species. These studies provided developmental and evolutionary insights into epithelial-mesenchymal communications that promote skin morphogenesis3.
This study was used to better understand factors that control feather growth. The method also improves the visualization of cellular and molecular events involved in skin patterning that take place during feather initiation, development, and elongation along the anterior-posterior axis. When the epithelium is separated from the mesenchyme and the two components are then recombined, new interactions re-establish skin patterning. This approach allows us to evaluate mesenchymal inducing signals and epithelial competence molecules that enable the epidermis to respond to the mesenchymal signals11. The subsequent downstream molecular expression that is required for feather bud development and pattern formation can also be examined. These studies have established that the location of buds is controlled by the mesenchyme. Rotation of the epithelium 90o before recombination with the mesenchyme demonstrates that the direction of feather bud elongation is controlled by the epithelium. This method was essential for us to study the molecular mechanism regulating feather bud orientation12.
Avian skin reconstitution culture, in which the skin mesenchyme is dissociated to single cells before plating at high cell density and overlaid with intact epithelium, resets dermal cells to a primordial state. The explant then self-organizes to form a new periodic pattern independent of the previous cues13. This skin reconstitution model can be used to study the initial processes of feather periodic patterning. We used this approach to explore how modulating the ratio of mesenchymal cells to a single piece of epithelium can influence the size or number of feather buds. The number of buds was found to increase but not the size of buds as the ratio of mesenchymal cells increased. Another advantage to this approach is that mesenchymal cell viral transduction shows higher efficiency than in the other two culture conditions and can produce more obvious phenotypes.

Author Spotlight: Insight into the Current Experimental Avian Skin Explant Methodologies

Using Avian Skin Explants to Study Tissue Patterning and Organogenesis

The research objective of my research team is to understand tissue development and the regeneration. This goal is particularly important in the stem cell research today. We hope to understand the principles how a organ form.And this knowledge is not only help us to understand the nature, but also can be applied to help regenerative medicine. The challenge we face is to understand the molecular and the cellular process involved in these morphogenetic processes. So therefore, we need to have a explant culture system to analyze molecular and cellular processes carefully because it's not possible to do this in vivo.To fill in this knowledge gap, scientists have developed different ways of organoid cultures. In the organoid culture, we will be able to take collective cell movement to see how cells interact with each other. We are also able to manipulate the cells so they will express different molecules to test the function of the molecules.The unique approach we use is the chicken skin explant culture. Chicken skin is used because they are easily accessible from the egg and they form distinct feather buds, arranged the in hexagonal pattern as a readout for tissue patterns. There are three ways we do the culture, as you will see.One is to hold the explant culture, allow us to take the movies or deliver molecules to perturb the system. Second, to study the contribution of epicilia or mesenzyme, we can take them apart and put them back. Third, to further challenge the ability of each cells, we dissociate them into the single cells and test how they come back to reorganize into the tissue patterns.Through these approaches, we have been able to gain new understanding toward the tissue patterning.

Tissue Studies by Culturing Reconstituted Chicken Skin 

To start, incubate fertilized chicken eggs in a humidified incubator at 38 degrees Celsius. Now dissect stage 30 to 33 chicken embryo dorsal skins in HBSS. Then incubate the skins in two times calcium and magnesium free saline with 0.25%EDTA for 15 to 20 minutes.Carefully separate the epithelium and the MESENCHYME using watchmaker's forceps and move the separated material to HBSS, placed on ice. Collect the separated mesenchyme into a 15 milliliter centrifuge tube and add two milliliters of 0.1%collagenase trypsin made in PBS. Then incubate the solution in a water bath.Next, use a pasteur pipette to disperse the skin mesenchyme into a single cell suspension. Then add eight milliliters of DMEM containing 10%fetal bovine serum to halt digestion. Centrifuge the cells at 233G for five minutes.Then resuspend the pellet in culture medium. Next place a cell culture insert in a well of a six well dish, drop a 10 microliter droplet of the mesenchymal cells onto the insert. In the well outside of the insert, pipette two milliliters of DMEM with 10%fetal bovine serum.Incubate the plated cells at 37 degrees in an incubator set with 5%carbon dioxide and 95%air for one hour. Using a one milliliter pipette, transfer the intact epithelium on top of the plated mesenchymal droplet. Flatten the intact epithelium over the mesenchyme to form the reconstituted skin explant.Leave a thin layer of medium on the insert to keep the skin explant semi wet. Providing an air/liquid interface incubate the reconstituted skin explant at 37 degrees in an incubator with 5%carbon dioxide and 95%air environment. The ex vivo organ cultures appear homogeneous at first short feather buds formed after two to three days in culture and long feather buds formed after four to five days in culture.

Research

JoVE Journal

Developmental Biology

The developing avian skin during embryogenesis is a unique model that can provide valuable insights into tissue patterning. Here three variations on skin ...