Regulation of Appendage Regeneration in Zebrafish

描述(申请人提供)：成年硬骨鱼和尾鱼两栖动物具有再生整个截肢附肢的能力。相比之下，哺乳动物肢体的再生修复仅限于指尖。在尾龙的肢体再生和硬骨鱼的鳍再生过程中，再生受到精确的调控，只有合适的结构才能被替换。附件再生的一个尚未解释的主要问题是，损伤区域的成体细胞如何保留或识别完成这一任务所需的位置信息。硬骨鳍是一种复杂的器官，包括骨、结缔组织、表皮、血管、神经和色素细胞，位置信息似乎部分由近端远端(PD)轴再生速度的梯度编码。也就是说，近端结构比远端结构再生快得多。潜在调控的差异目前还不能在细胞和分子水平上解释。然而，根据我们已发表的结果和新的数据，FGFs通过ERK通路发出的信号很可能是一个重要的贡献者。在这里，我们建议剖析斑马鱼尾鳍再生生长速度的调节机制。我们假设，分级的分子调控程序是沿着再生鳍的PD轴在解剖和细胞增殖方面的分级差异的基础。为了验证这一假设，我们提出了三个具体目标。1)我们将定义再生率、细胞增殖、胚泡长度和成纤维细胞生长因子信号活性的PD梯度。2)我们将定义成纤维细胞生长因子信号编码再生速率的机制。在这里，我们将使用新的转基因株系来研究对成纤维细胞生长因子和ERK信号活性的实验性操作如何影响再生率，这些转基因系通过这些途径促进信号的可诱导增强或减少。3)使用这些转基因品系中的一个，一种能够促进显性负向成纤维细胞生长因子受体的可诱导表达的菌株，我们将对在鳍再生过程中抑制成纤维细胞生长因子信号需求的突变进行遗传筛选。我们将对在这一筛选中发现的不同突变进行分类，目的是识别被破坏的基因。我们对斑马鱼附肢再生率调控的分子遗传学方法将为理解再生器官发生过程中活跃的重要调控机制提供基础，也可能为各种人类退行性疾病的再生治疗提供便利。

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.