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可以再生整个截肢附肢。相比之下，成年哺乳动物肢体的再生愈合仅限于脚趾尖。发育生物学的关键挑战之一是了解组织再生如何以及为什么发生。肢或鳍再生的标志是胚基的形成，胚基是一种含有新骨骼成分的祖细胞的间充质结构。随着再生的进行，胚基细胞的增殖和图案化受到调节，使得丢失的正确大小和形状的组织被替换，这种现象称为位置记忆。胚基中的成骨细胞和最终再生的鳍条来源于预先存在的成骨细胞的增殖，而如果初级成骨细胞来源被禁用，则存在替代来源。激活和抑制因子影响再生生长，并且在整个生命过程中鳍细胞中图案化转录因子的区域特异性维持是位置记忆的组成部分。尽管最近我们对附肢再生的理解取得了进展，但该领域仍存在关键缺陷。首先，还没有发表的尝试，图像和量化的体内动态的细胞增殖过程中的附件再生，分析，将阐明如何复杂的模式是恢复损伤后。第二，一组有限的传统发育信号因子已被评估再生过程中的功能。这项建议的总体目标是确定机制，调节细胞增殖的大小和模式恢复到截肢的附件。1)我们将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，以创建鳍再生过程中细胞增殖的时空图。2)我们将使用新的试剂来确定是否以及如何通过分泌因子瘦素信号影响斑马鱼鳍再生。3)我们将定义两个基因的诱变筛选，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，我们将检验胚基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，并为理解和改变人类组织再生能力的现有限制提供重要的视角。

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.