Regulation of Appendage Regeneration in Zebrafish

描述（由申请人提供）：成年硬骨鱼和尾形两栖动物具有再生整个截肢附肢的能力。相比之下，哺乳动物四肢的再生愈合仅限于趾尖。在尾尾动物的肢体再生和硬骨鱼的鳍再生过程中，再生过程受到精确的调控，只有适当的结构才会被替换。附属物再生的主要未解问题之一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。硬骨鱼鳍是一个包含骨、结缔组织、表皮、血管、神经和色素细胞的复杂器官，其位置信息似乎部分由近端远端（PD）轴上的再生速率梯度编码。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节的差异。然而，根据我们发表的结果和新的数据，Fgfs通过Erk途径发出的信号可能是一个重要的贡献者。为了验证这一假设，我们提出了3个具体目标：1)我们将定义再生速率、细胞增殖、囊胚长度和Fgf信号活性的PD梯度。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Fgf和Erk信号活动的实验操作如何影响再生率，使用新的转基因系，促进通过这些途径诱导增强或减少信号。3)利用其中一种转基因系，一种促进诱导表达显性阴性Fgf受体的菌株，我们将对鳍再生过程中抑制Fgf信号需求的突变进行遗传筛选。我们将对这个屏幕中发现的不同突变进行分类，目的是识别被破坏的基因。我们对斑马鱼附属物再生率调控的分子遗传学方法将为理解在再生器官发生过程中活跃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.