Regulation of Appendage Regeneration in Zebrafish

描述（由申请人提供）：成年硬骨鱼和有尾目两栖动物有能力再生整个截肢附肢。相比之下，哺乳动物肢体的再生愈合仅限于指尖。在有尾目动物的肢体再生和硬骨鱼类动物的鳍再生过程中，再生受到精确的调节，只有适当的结构被替换。附件再生的一个主要的未解释的问题是，受伤区域的成年细胞如何保留或识别完成这一过程所必需的位置信息。在硬骨鱼鳍，一个复杂的器官，包括骨，结缔组织，表皮，血管，神经，和色素细胞，位置信息似乎是部分编码的梯度再生率沿着proximodistal（PD）轴。也就是说，近端结构比远端结构再生得快得多。目前在细胞和分子水平上无法解释潜在调控的差异。然而，根据我们发表的结果和新数据，Fgfs通过Erk途径的信号传导可能是一个重要的贡献者。在这里，我们建议解剖斑马鱼尾鳍再生生长率的调节机制。我们假设，分级分子调控程序的基础上，在解剖学和细胞增殖沿着PD轴再生鳍的分级差异。为了验证这一假设，我们提出了三个具体目标。1)我们将定义PD梯度的再生率，细胞增殖，胚基长度，和FGF信号转导活性。2)我们将定义FGF信号编码再生速率的机制。在这里，我们将研究如何实验操作的FGF和ERK信号传导活性影响再生率，使用新的转基因株系，促进诱导增强，或减少，通过这些途径的信号。3)使用这些转基因系之一，一个菌株，有利于诱导型表达的显性负性FGF受体，我们将进行基因筛选突变，抑制要求FGF信号在鳍再生。我们将对在该筛选中发现的不同突变体进行分类，目的是鉴定被破坏的基因。我们的分子遗传学方法来调节斑马鱼附肢的再生率将为理解再生器官发生过程中的重要调控机制提供基础，也可能促进各种人类退行性疾病的再生治疗。

DESCRIPTION (provided by applicant): Adult teleost fish and urodele amphibians have the capacity to regenerate entire amputated appendages. By contrast, regenerative healing of mammalian limbs is limited to digit tips. During limb regeneration in urodeles and fin regeneration in teleosts, regeneration is precisely regulated such that only the appropriate structures are replaced. One of the major unexplained questions of appendage regeneration is how adult cells in the injured area retain or recognize the positional information necessary to accomplish this. In the teleost fin, a complex organ containing bone, connective tissue, epidermis, blood vessels, nerves, and pigment cells, positional information appears to be encoded in part by a gradient in the rate of regeneration along the proximodistal (PD) axis. That is, proximal structures regenerate much more quickly than distal structures. The differences in underlying regulation are currently unexplained at the cellular and molecular levels. However, based on our published results and new data, it is likely that signaling by Fgfs through the Erk pathway is an important contributor. Here, we propose to dissect regulatory mechanisms of regenerative growth rate in the zebrafish caudal fin. We hypothesize that, graded molecular regulatory programs underlie graded differences in anatomy and cellular proliferation along the PD axis of the regenerating fin. To test this hypothesis, we propose 3 Specific Aims. 1) We will define PD gradients of regenerative rate, cellular proliferation, blastemal length, and Fgf signaling activity. 2) We will define the mechanism by which Fgf signaling encodes the regenerative rate. Here, we will examine how experimental manipulation of Fgf and Erk signaling activity affect regenerative rate, using new transgenic lines that facilitate inducible augmentation, or reduction, of signaling through these pathways. 3) Using one of these transgenic lines, a strain that facilitates inducible expression of a dominant-negative Fgf receptor, we will perform a genetic screen for mutations that suppress the requirement for Fgf signaling during fin regeneration. We will classify the different mutants found in this screen with the goal of identifying the disrupted genes. Our molecular genetic approach to the regulation of regenerative rate in zebrafish appendages will provide a foundation for understanding important regulatory mechanisms active during regenerative organogenesis, and may also facilitate regenerative therapies for a variety of human degenerative diseases.