Hyde David R, Godwin Alan R, Thummel Ryan
Department of Biological Sciences, Center for Zebrafish Research, University of Notre Dame, USA.
J Vis Exp. 2012 Mar 29(61):3632. doi: 10.3791/3632.
Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration of adult tissues, such as the heart, retina, spinal cord, optic nerve, sensory hair cells, and fins. The zebrafish fin is a relatively simple appendage that is easily manipulated to study multiple stages in epimorphic regeneration. Classically, fin regeneration was characterized by three distinct stages: wound healing, blastema formation, and fin outgrowth. After amputating part of the fin, the surrounding epithelium proliferates and migrates over the wound. At 33 °C, this process occurs within six hours post-amputation (hpa, Figure 1B). Next, underlying cells from different lineages (ex. bone, blood, glia, fibroblast) re-enter the cell cycle to form a proliferative blastema, while the overlying epidermis continues to proliferate (Figure 1D). Outgrowth occurs as cells proximal to the blastema re-differentiate into their respective lineages to form new tissue (Figure 1E). Depending on the level of the amputation, full regeneration is completed in a week to a month. The expression of a large number of gene families, including wnt, hox, fgf, msx, retinoic acid, shh, notch, bmp, and activin-betaA genes, is up-regulated during specific stages of fin regeneration. However, the roles of these genes and their encoded proteins during regeneration have been difficult to assess, unless a specific inhibitor for the protein exists, a temperature-sensitive mutant exists or a transgenic animal (either overexpressing the wild-type protein or a dominant-negative protein) was generated. We developed a reverse genetic technique to quickly and easily test the function of any gene during fin regeneration. Morpholino oligonucleotides are widely used to study loss of specific proteins during zebrafish, Xenopus, chick, and mouse development. Morpholinos basepair with a complementary RNA sequence to either block pre-mRNA splicing or mRNA translation. We describe a method to efficiently introduce fluorescein-tagged antisense morpholinos into regenerating zebrafish fins to knockdown expression of the target protein. The morpholino is micro-injected into each blastema of the regenerating zebrafish tail fin and electroporated into the surrounding cells. Fluorescein provides the charge to electroporate the morpholino and to visualize the morpholino in the fin tissue. This protocol permits conditional protein knockdown to examine the role of specific proteins during regenerative fin outgrowth. In the Discussion, we describe how this approach can be adapted to study the role of specific proteins during wound healing or blastema formation, as well as a potential marker of cell migration during blastema formation.
某些蝾螈和硬骨鱼物种能够再生其组织。斑马鱼已成为广泛用于研究成年组织自发再生的模型，如心脏、视网膜、脊髓、视神经、感觉毛细胞和鳍。斑马鱼鳍是一种相对简单的附属物，易于操作以研究形态发生再生的多个阶段。传统上，鳍再生的特征在于三个不同阶段：伤口愈合、芽基形成和鳍生长。切除部分鳍后，周围上皮细胞增殖并迁移覆盖伤口。在33℃时，此过程在截肢后6小时内发生（hpa，图1B）。接下来，来自不同谱系（如骨、血液、神经胶质、成纤维细胞）的底层细胞重新进入细胞周期以形成增殖性芽基，而覆盖的表皮继续增殖（图1D）。随着芽基近端的细胞重新分化为各自的谱系以形成新组织，生长阶段发生（图1E）。根据截肢水平，一周到一个月内完成完全再生。在鳍再生的特定阶段，包括wnt、hox、fgf、msx、视黄酸、shh、notch、bmp和激活素-βA基因在内的大量基因家族的表达上调。然而，除非存在该蛋白质的特异性抑制剂、温度敏感突变体或产生转基因动物（过表达野生型蛋白质或显性负性蛋白质），否则这些基因及其编码蛋白质在再生过程中的作用很难评估。我们开发了一种反向遗传技术，可快速轻松地测试任何基因在鳍再生过程中的功能。吗啉代寡核苷酸广泛用于研究斑马鱼、非洲爪蟾、鸡和小鼠发育过程中特定蛋白质的缺失。吗啉代与互补RNA序列碱基配对，以阻断前体mRNA剪接或mRNA翻译。我们描述了一种将荧光素标记的反义吗啉代有效导入再生斑马鱼鳍中以敲低靶蛋白表达的方法。将吗啉代显微注射到再生斑马鱼尾鳍的每个芽基中，并通过电穿孔导入周围细胞。荧光素提供电荷以使吗啉代电穿孔并在鳍组织中可视化吗啉代。该方案允许条件性蛋白质敲低，以检查特定蛋白质在再生鳍生长过程中的作用。在讨论部分，我们描述了如何调整这种方法来研究特定蛋白质在伤口愈合或芽基形成过程中的作用，以及芽基形成过程中细胞迁移的潜在标志物。
