EPIDERMAL FACTORS THAT PROMOTE INTERNAL TISSUE PROGENITOR ACTIVATION FOLLOWING AMPUTATION

 描述（由申请人提供）：在美国，肢体缺失是一个主要的健康问题，有近200万患者生活在主要肢体截肢的后果中。预计这一数字将随着关键风险因素的增加而增加，目前还没有设计出解决这一问题的生物疗法。虽然人类在四肢和其他关键结构上的再生能力非常有限，但蝾螈可以在一生中再生整个四肢。蝾螈的四肢在解剖学上与人类的四肢相似，它们的发育机制也相似。深入了解使蝾螈肢体再生的分子机制，可以为再生医学中可能采取的未来方法提供重要见解，这反过来又可以彻底改变为面临截肢的患者提供的治疗方案。这种彻底的机械理解一直回避研究人员的日期，因为缺乏工具可用于实验操纵基因表达的蝾螈。然而，在过去的八年里，我们和其他人已经开发出了强大的分子遗传工具，在蝾螈体内运行。我们建议利用这些发展，重新审视长期存在的重要问题，脊椎动物肢体再生。 在蝾螈中，截肢后最早的事件之一是在残肢上形成专门的伤口表皮。在伤口表皮下，形成了内部组织的祖细胞库，即芽基。芽基对于肢体再生是至关重要的，但它们的产生和生长却知之甚少，并且这两个方面都很有可能受到伤口表皮的控制。由于迄今为止缺乏研究这些问题的工具，伤口表皮及其分子因子的确切作用一直是难以捉摸的。通过大规模的RNA测序工作扩展到单细胞水平，我们已经确定了候选基因，其表达在伤口表皮与芽基细胞和所有其他组织样品中高度富集。在这里，我们建议利用这一数据集以及我们最近开发的逆转录病毒系统感染蝾螈在体内， 回答有关伤口表皮作用的具体问题，并检查五个特定基因。在具体目标1中，我们将使用逆转录病毒标记活化细胞，确定截肢后软骨和肌肉祖细胞的活化是否需要伤口表皮。在具体目标2中，我们将测试五种伤口表皮富集基因在没有伤口表皮的肢体中引起去分化或干细胞活化的充分性。这些实验将使我们能够建立一个系统，使我们能够研究伤口表皮下游的关键细胞事件，并测试使用这种方法来识别伤口表皮关键分子组分的可行性。进行这项研究可以使未来的实验能够更深入地剖析支持伤口表皮功能的分子途径，并将为考虑这些过程在哺乳动物环境中的作用奠定基础。

 DESCRIPTION (provided by applicant): Limb loss is a major health concern in the U.S. with nearly two million patients living with the consequences of a major limb amputation. This number is expected to rise with increases in key risk factors, and no biological therapeutics has been devised to address this problem. While humans have exceedingly limited regenerative abilities in limbs and other key structures, axolotl salamanders can regenerate entire limbs throughout their lives. Axolotl limbs are anatomically similar to human limbs, and they develop by similar mechanisms. Gaining a thorough understanding of the molecular mechanisms that enable axolotl limb regeneration stands to offer critical insights into future approaches that may be taken in regenerative medicine, which could in turn revolutionize the treatment options offered to patients facing amputation. This thorough mechanistic understanding has evaded researchers to date because of a paucity of tools available for experimentally manipulating gene expression in axolotls. However, within the last eight years, we-and others-have developed powerful molecular genetic tools that are operational in vivo in axolotls. We propose to leverage these developments to take a fresh look at the longstanding and important question of vertebrate limb regeneration. In axolotls, one of the earliest events post-amputation is the formation of a specialized wound epidermis across the stump. Beneath this wound epidermis, progenitor cell pool for internal tissues, the blastema, forms. Blastemas are critical for limb regeneration, but their creation and growth are poorly understood, and there is a strong possibility that both aspects are under the control of the wound epidermis. Precise roles for wound epidermis and its molecular factors have been elusive because of a lack of tools for studying these questions to date. Through a massive RNA-sequencing effort extended to single-cell level, we have identified candidate genes whose expression is highly enriched in the wound epidermis versus blastema cells and all other tissues sampled. Here we propose to leverage this data set as well as our recently-developed retrovirus system for infecting axolotls in vivo to answer specific questions about the role of wound epidermis and to examine five specific genes. In Specific Aim 1, we will determine if wound epidermis is required for activation of cartilage and muscle progenitors post-amputation using a retrovirus to mark activated cells. In Specific Aim 2, we will test the sufficiency of five wound-epidermis-enriched genes to cause dedifferentiation or stem cell activation in limbs without wound epidermis. These experiments will allow us to establish a system whereby we can study critical cellular events downstream of the wound epidermis, and it test the feasibility of using this approach to identify key molecular components of wound epidermis. Performing this research enable future experiments aimed at more intensive dissection of the molecular pathways that support the wound epidermis functions, it and will lay the groundwork for considering the role of these processes in the mammalian context.