chicken skin explant culture for organogenesis study

鳥類胚性皮膚外植片培養プロトコルの包括的なレビュー

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.

著者スポットライト:現在の実験的鳥類皮膚外植片方法論への洞察

鳥類の皮膚外植片を用いた組織パターン形成と器官形成の研究

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.

器官形成研究のための鶏皮外植片培養

まず、受精した鶏の卵を摂氏38度の加湿インキュベーターでインキュベートします。次に、ステージ28〜32の鶏の胚をHBSSで満たされた60ミリメートルのシャーレに入れます。背側胚の皮膚と頭部を解剖した後、手足のつぼみをそっと引っ張って、体の腹側を下向きに再配置します。次に、胚体の両側に沿って、前方から後方に切開します。一対の鉗子で手足のつぼみを縦方向につかみ、体を安定させます。時計職人の鉗子を使って、首から尾の部分まで丁寧に皮膚をはがします。鋭利なハサミを使って、剥がした皮膚にまだ付着している皮下組織を繊細に取り除き、皮膚のエッジを滑らかにします。次に、2ミリリットルのサプリメントDMEMを6ウェルディッシュの各ウェルに加えます。ウェルに抗生物質を添加した後、ウェル内に滅菌組織培養インサートを配置し、培地が培養インサートの外側を囲むようにします。次に、HBSSに浸した状態で皮膚をヘラに移し、しわや折り目を防ぎます。完了したら、切除した皮膚をHBSSを含む皿に移します。ヘラから皮膚を折りたたむことなくゆっくりと培養インサートにスライドさせます。200マイクロリットルのピペットを使用して、インサートから余分なHBSSを取り除きます。最後に、皮膚外植片培養物を摂氏37度でインキュベートし、5%の二酸化炭素と95%の空気をブレンドします。2日ごとにミディアムを交換してください。羽の原基は、培養で2日後に短い羽芽に発達しました。そして長い羽芽は培養で4日後に発達しました。真皮のプラコードは、側面よりも正中線でより成熟していました。

Research

JoVE Journal

Developmental Biology

Chicken Skin Explant Culture for Organogenesis Study

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 ...