Induction of digit regeneration by Wnt active nail epithelium

描述(申请人提供)：目前，因受伤或疾病而失去的肢体无法再生。我们的长期目标是了解指甲上皮在指尖再生中的作用，并提出一种新的策略来促进手指/肢体的再生。哺乳动物的指尖，包括它的骨头，在截肢后可以再生。指尖截断后，指甲上皮与未分化的间充质母细胞一起再生，这些细胞再生指骨。再生只发生在与指甲相关的区域，目前尚不清楚为什么会存在这种再生限制。我们最近鉴定并发现指甲上皮干细胞(NSCs)经历了依赖Wnt的指甲分化。值得注意的是，指甲上皮中Wnt的激活不仅是指甲再生所必需的，也是间充质母细胞生长所必需的，指尖截肢后，间质母细胞生长会导致指骨再生。当手指在Wnt活性的指甲祖细胞近端被截断时，伤口区域的上皮细胞不会发生Wnt激活，指甲/手指无法再生。然而，在包括神经干细胞在内的上皮细胞中强制激活Wnt可以克服这一限制。我们假设Wnt活性的指甲上皮具有谱系特异性功能，通过发出旁分泌信号来促进手指再生。目的：我们发现FGF2在截肢后的指甲上皮细胞中以Wnt依赖的方式表达，FGF2在体外通过FGFR1信号促进胚泡的增殖。我们将通过基因删除胚泡细胞中的FGFR1信号来测试FGFR1信号对胚泡生长是否是体内必需的。我们还将测试在CN上皮细胞中过表达FGF2是否克服了再生的近端限制。这些实验将首次通过分别以上皮细胞和间充质细胞为靶点来剖析分子通路在指尖再生中的作用。目的：[虽然Wnt活性的指甲上皮细胞在诱导手指再生中的重要作用已被证明，但Wnt活性的NSC来源的上皮细胞是否具有独特的谱系依赖功能来促进再生尚不清楚。因此，我们将测试非神经干细胞来源的上皮细胞(即指甲上皮近端的上皮细胞)中的Wnt激活是否具有与来自神经干细胞谱系的细胞类似的促进再生的功能。非神经干细胞来源的上皮细胞缺乏TCF/LEF1家族成员的表达，这些转录因子是Wnt诱导转录所必需的，而指甲上皮细胞则同时表现出ç-catenin稳定和TCF1的表达。我们计划在NSC壁龛附近截肢后，通过基因表达TCF1和稳定的ç-catenin来诱导非NSC谱系的Wnt信号激活，并检查这是否足以诱导手指再生。此外，我们将移植神经干细胞和正常皮肤角质形成细胞，它们构成激活伤口表皮下的Wnt信号。我们将测试这种细胞疗法是否能在近端截肢后诱导手指再生。]这些实验将为开发神经干细胞用于治疗截肢者的再生医学奠定基础。

DESCRIPTION (provided by applicant): Currently, there is no way to regenerate limbs that are lost to injuries or diseases. Our long term goal is to understand the role of the nail epithelim during digit tip regeneration and propose a novel strategy to promote regeneration of the digit/limb. The mammalian digit tip including its bone can regenerate upon amputation. Following amputation of the digit tip, the nail epithelium regrows together with the underlying undifferentiated mesenchymal blastema cells that regenerates the digit bone. Regeneration occurs only in areas associated with the nail and it is unknown why this regenerative limitation exists. We recently identified and discovered nail epithelial stem cells (NSCs) undergo Wnt-dependent nail differentiation. Remarkably, Wnt activation in the nail epithelium is required not only for nail regeneration but also for mesenchymal blastema growth that leads to digit bone regeneration upon digit tip amputation. When the digit is amputated proximal to the Wnt-active nail progenitors, Wnt activation does not occur in epithelial cells in the wound area and the nail/digit fails to regenerate. Nevertheless, forced Wnt activation in epithelial cells including NSCs can overcome this limitation. We hypothesize that Wnt-active nail epithelium holds a lineage specific function to promote digit regeneration by emitting paracrine signals. Aim1: We found that FGF2 is expressed in nail epithelial cells in a Wnt-dependent manner after amputation and that FGF2 promotes blastema proliferation through FGFR1 signaling in vitro. We will test whether FGFR1 signaling is essential for blastema growth in vivo, by genetically deleting fgfr1 in blastema cells. We will also test if overexpression of fgf2 in epithelial cells cn overcome the proximal limitation in regeneration. These experiments will be the first to dissect the role of a molecular pathway in digit tip regeneration by separately targeting the epithelial an mesenchymal cells. Aim2: [While the essential role of Wnt-active nail epithelial cells in inducing digit regeneration has been demonstrated, it remains unclear whether Wnt-active NSC-derived epithelial cells hold a unique lineage-dependent function to promote regeneration. We will thus test whether Wnt-activation in non-NSC-derived epithelial cells (i.e. epithelial cells proximal to nail epithelium) can similarly function to promote regeneration as those derived from the NSC lineage. Non-NSC-derived epithelial cells with stabilized ß-catenin lack expression of TCF/LEF1 family members, transcription factors essential for Wnt induced transcription, in contrast to nail epithelial cells that display both ß-catenin stabilization and express TCF1. We plan to induce Wnt signal activation in non- NSC lineage by genetically expressing TCF1 in combination with stabilized ß-catenin following amputations proximal to the NSC niche and examine whether this is sufficient to induce digit regeneration. Additionally, we will transplant NSCs and normal skin keratinocytes that constitutively activate Wnt signaling underneath the wound epidermis. We will test if this cell therapy can induce digit regeneration following proximal amputation.] These experiments will create a foundation to exploit NSCs in regenerative medicine for treating amputees.