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可以再生整个被截肢的附肢。相比之下，成年哺乳动物肢体的再生修复仅限于指尖。发育生物学的关键挑战之一是了解组织再生是如何发生的以及为什么发生的。肢体或鳍再生的标志是形成胚泡，这是一种含有新骨骼元素前体细胞的间充质结构。随着再生的进行，胚泡细胞的增殖和图案化得到调节，以取代丢失的大小和形状正确的组织，这一现象被称为位置记忆。胚泡中的骨骼成骨细胞以及最终再生的鳍射线，来自先前存在的成骨细胞的增殖，而另一种来源(S，如果主要的成骨细胞来源被禁用，则存在)。激活和抑制因子影响再生生长，而在鳍细胞整个生命过程中特定区域维持模式转录因子是位置记忆的一个组成部分。尽管我们对附肢再生的了解最近取得了进展，但该领域仍然存在严重的不足。首先，还没有发表过对附件再生过程中细胞增殖的体内动态进行成像和量化的尝试，这些分析将阐明复杂的模式是如何在损伤后恢复的。其次，对一组有限的传统发育信号因子在再生过程中的功能进行了评估。这项提案的总体目标是定义当细胞的大小和模式恢复到被切断的附件时，调节细胞增殖的机制。1)我们将应用新技术来监测转基因斑马鱼体内的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分泌因子瘦素是否以及如何影响斑马鱼鳍的再生。3)我们将确定突变筛选中涉及的两个基因--细胞外基质成分层粘连蛋白β1a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，我们将检验胚泡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规律的理解，并为理解甚至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.