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可以再生整个被截肢的附肢。相比之下，成年哺乳动物四肢的再生愈合仅限于手指的尖端。发育生物学的关键挑战之一是理解组织再生是如何以及为什么发生的。肢体或鳍再生的标志是胚质的形成，这是一种间充质结构，含有用于新骨骼元素的祖细胞。随着再生的进行，胚细胞的增殖和形成模式受到调节，这样失去的正确大小和形状的组织就会被替换，这种现象被称为位置记忆。母细胞中的骨骼成骨细胞以及最终再生的鳍，都来自于先前存在的成骨细胞的增殖，而如果原成骨细胞来源被禁用，则存在替代来源。激活因子和抑制因子影响再生生长，鳍细胞一生中模式转录因子的区域特异性维持是位置记忆的一个组成部分。尽管我们对附属物再生的理解最近取得了进展，但在该领域仍存在关键缺陷。首先，目前还没有发表过对附属物再生过程中细胞增殖的体内动态进行成像和量化的尝试，这些分析将阐明损伤后如何恢复复杂的模式。其次，一组有限的传统发育信号因子已经被评估在再生过程中的功能。本建议的总体目标是确定调节细胞增殖的机制，因为大小和模式被恢复到截肢的附属物。1)应用新技术监测转基因斑马鱼体内细胞周期进程和生长指标，建立鱼鳍再生过程中细胞增殖的时空图谱。2)我们将使用新的试剂来确定分泌因子瘦素的信号传导是否以及如何影响斑马鱼鳍的再生。3)我们将定义突变筛选中涉及的两个基因（细胞外基质成分层粘连蛋白β 1a和电压门控钙通道钙稳态调节剂1）影响鳍再生的机制。通过这些方法，我们将验证胚细胞增殖受激活和抑制影响的复杂微环境控制的假设。这项工作将增加对脊椎动物组织再生过程中发育调控的理解，并为理解甚至可能改变人类组织再生能力的现有局限性提供重要视角。

DESCRIPTION (provided by applicant): Adult teleost fish and urodele amphibians can regenerate entire amputated appendages. By contrast, regenerative healing of adult mammalian limbs is limited to the very tips of digits. One of the key challenges in developmental biology is to understand how and why tissue regeneration occurs. The hallmark of limb or fin regeneration is formation of a blastema, a mesenchymal structure that contains progenitor cells for new skeletal elements. As regeneration proceeds, blastemal cell proliferation and patterning are regulated such that lost tissues of correct size and shape are replaced, a phenomenon called positional memory. Skeletal osteoblasts in the blastema and, ultimately, the regenerated fin rays, derive from the proliferation of pre- existing osteoblasts, while an alternative source(s exists if the primary osteoblast source is disabled. Activating and inhibitory factors influence regenerative growth, and region-specific maintenance of patterning transcription factors in fin cells throughout life is a component of positional memory. Despite recent advances in our understanding of appendage regeneration, there remain key deficiencies in the field. First, there have been no published attempts to image and quantify the in vivo dynamics of cell proliferation during appendage regeneration, analyses that would illuminate how complex pattern is restored after injury. Second, a limited set of traditional developmental signaling factors has been assessed for function during regeneration. The overall goal of this proposal is to define mechanisms that regulate cell proliferation as size and pattern are restored to an amputated appendage. 1) We will apply new technologies to monitor in vivo cell cycle progression and growth indicators in transgenic zebrafish, to create a spatiotemporal map of cell proliferation during fin regeneration. 2) We will use new reagents to define whether and how signaling by the secreted factor Leptin influences zebrafish fin regeneration. 3) We will define mechanisms by which two genes implicated from a mutagenesis screen, the extracellular matrix component laminin beta 1a and the voltage-gated calcium channel calcium homeostasis modulator 1, influence fin regeneration. With these approaches, we will test the hypothesis that blastemal cell proliferation is controlled by a complex microenvironment of activating and inhibitory influences. This work will increase understanding of developmental regulation during vertebrate tissue regeneration, and provide important perspective for comprehending, and perhaps changing, the existing limitations in regenerative capacity of human tissues.