EPIDERMAL FACTORS THAT PROMOTE INTERNAL TISSUE PROGENITOR ACTIVATION FOLLOWING AMPUTATION

截肢后促进内组织祖细胞激活的表皮因素

• 批准号: 9253350
• 负责人: JESSICA L. WHITED
• 金额: $ 8.85万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-05-01 至 2018-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9253350
• 关键词: Address; Ambystoma; Amputation; Anatomy; Animals; Biological Response Modifier Therapy; Candidate Disease Gene; Cartilage; Cell Cycle; Cells; Data Set; Development; Differentiation Antigens; Dissection; Epidermis; Event; Future; Gene Expression; Genes; Growth; Health; Human; Injury; Label; Lead; Limb Development; Limb structure; Mammals; Methodology; Mitotic; Molecular; Molecular Genetics; Muscle; Natural regeneration; Organism; Pathway interactions; Patients; Process; Regenerative Medicine; Research; Research Personnel; Resources; Retroviridae; Risk Factors; Role; Salamander; Stem cells; Structure; Surgical sutures; System; Testing; Thick; Tissue Sample; Tissues; Wound Healing; base; blastema; cartilage cell; design; experimental study; in vivo; insight; interest; limb amputation; limb regeneration; molecular marker; novel; progenitor; programs; public health relevance; regenerative; satellite cell; tool; transcriptome; transcriptome sequencing; wound epidermis

 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.

 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200万患者生活在重大肢体截肢的后果中。这一数字预计会随着关键风险因素的增加而上升，而且还没有设计出生物疗法来解决这个问题。虽然人类四肢和其他关键结构的再生能力极其有限，但Axolotl火蜥蜴可以在一生中再生整个肢体。Axolotl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。彻底了解使Axolotl肢体再生的分子机制将为再生医学未来可能采取的方法提供关键的见解，这反过来可能会彻底改变为面临截肢的患者提供的治疗选择。到目前为止，由于缺乏可用于实验操作Axolotl基因表达的工具，这种彻底的机制理解一直让研究人员望而却步。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，可以在活体内对Axolotls进行操作。我们建议利用这些发展来重新审视脊椎动物肢体再生这一长期而重要的问题。在Aaxolotls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。在伤口的表皮下，形成了内部组织的祖细胞池，即胚泡。胚泡对肢体再生至关重要，但对其产生和生长知之甚少，很有可能这两个方面都在创面表皮的控制之下。由于迄今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难以捉摸。通过扩展到单细胞水平的大规模RNA测序工作，我们已经确定了在伤口表皮与胚泡细胞和所有其他组织样本中高度丰富表达的候选基因。在这里，我们建议利用这个数据集以及我们最近开发的逆转录病毒系统在体内感染Axolotl，以 回答关于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激活的细胞来确定截肢后软骨和肌肉祖细胞的激活是否需要伤口表皮。在具体目标2中，我们将测试五个富含创面表皮的基因是否足以在没有创面表皮的肢体中引起去分化或干细胞激活。这些实验将使我们能够建立一个系统，在这个系统中，我们可以研究伤口表皮下游的关键细胞事件，并测试使用这种方法识别伤口表皮关键分子成分的可行性。进行这项研究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通路，并将为考虑这些过程在哺乳动物环境中的作用奠定基础。