chicken skin explant culture for organogenesis study

조류 배아 피부 이식 배양 프로토콜에 대한 종합적인 검토

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.

저자 스포트라이트: 현재 실험적인 조류 피부 이식 방법론에 대한 통찰력

조직 패터닝 및 유기 형성을 연구하기 위해 조류 피부 이식물을 사용합니다.

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.

유기 형성 연구를 위한 닭 피부 이식 배양

시작하려면 섭씨 38도의 가습 인큐베이터에서 수정된 닭고기 알을 부화시킵니다. 그런 다음 HBSS로 채워진 60mm 페트리 접시에 28-32 단계 닭 배아를 놓습니다. 등쪽 배아 피부와 머리를 절개한 후 팔다리 싹을 부드럽게 당겨 몸의 복부 쪽을 아래쪽으로 재배치합니다.이제 배아체의 양쪽을 따라 전방에서 후방 절개를 만듭니다. 한 쌍의 집게로 팔다리 싹을 세로로 잡고 몸을 안정시킵니다. 워치메이커의 집게를 사용하여 목에서 꼬리 부분까지 피부를 조심스럽게 벗겨냅니다.날카로운 가위를 사용하여 벗겨진 피부에 아직 붙어 있는 남아 있는 피하 조직을 섬세하게 제거하고 피부 가장자리를 매끄럽게 합니다. 다음으로, 6웰 접시의 각 웰에 2ml의 보충제 DMEM을 추가합니다. 웰에 항생제를 추가한 후 멸균 조직 배양 삽입물을 웰 내부에 넣고 배지가 배양 삽입물의 외부를 둘러싸도록 합니다.그런 다음 HBSS에 담근 상태에서 피부를 주걱으로 옮겨 주름이 생기거나 접히는 것을 방지합니다. 완료되면 절제된 피부를 HBSS가 함유된 접시에 옮깁니다. 주걱의 피부를 접지 않고 배양 삽입물에 천천히 밀어 넣습니다.200마이크로리터 피펫을 사용하여 인서트에서 과도한 HBSS를 제거합니다. 마지막으로 섭씨 37도에서 5%의 이산화탄소와 95%의 공기를 혼합하여 피부 이식 배양액을 배양합니다. 이틀에 한 번씩 배지를 교체하십시오.깃털 primordia는 배양 된 지 이틀 후에 짧은 깃털 싹으로 발달했습니다. 그리고 긴 깃털 싹은 배양 4일 후에 발달했습니다. 피부 플라코드는 측면보다 정중선에서 더 성숙했습니다.

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 ...