Induction of digit regeneration by Wnt active nail epithelium

描述（由申请人提供）：目前，没有办法再生因受伤或疾病而失去的肢体。我们的长期目标是了解指甲上皮在指尖再生过程中的作用，并提出一种新的策略来促进手指/肢体的再生。哺乳动物的趾尖包括它的骨头可以在截肢后再生。足趾尖截肢后，指甲上皮细胞与下面的未分化间充质胚细胞一起再生，再生足趾骨。再生仅发生在与指甲相关的区域，并且不知道为什么存在这种再生限制。我们最近鉴定并发现指甲上皮干细胞（NSC）经历Wnt依赖的指甲分化。值得注意的是，指甲上皮中的Wnt活化不仅是指甲再生所需的，而且是间充质芽基生长所需的，所述间充质芽基生长导致趾尖截肢后的趾骨再生。当手指在Wnt活性指甲祖细胞附近被截肢时，Wnt活化不会发生在伤口区域的上皮细胞中，并且指甲/手指不能再生。然而，包括NSC在内的上皮细胞中的强制Wnt激活可以克服这一限制。我们假设Wnt活性指甲上皮细胞具有谱系特异性功能，通过发出旁分泌信号促进手指再生。目标1：我们发现FGF 2在截肢后以Wnt依赖的方式在指甲上皮细胞中表达，并且FGF 2在体外通过FGFR 1信号传导促进胚基增殖。我们将测试是否FGFR 1信号是必需的芽基生长在体内，基因删除fgfr 1芽基细胞。我们也将测试fgf 2在上皮细胞中的过度表达是否能克服再生的近端限制。这些实验将是第一个通过分别靶向上皮细胞和间充质细胞来剖析分子途径在指尖再生中的作用。目标2：[虽然Wnt活性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NSC衍生的上皮细胞是否具有独特的谱系依赖性功能以促进再生仍不清楚。因此，我们将测试非NSC衍生的上皮细胞（即指甲上皮近端的上皮细胞）中的Wnt活化是否可以与衍生自NSC谱系的上皮细胞类似地起作用以促进再生。具有稳定的β-连环蛋白的非NSC衍生的上皮细胞缺乏TCF/LEF 1家族成员的表达，TCF/LEF 1家族成员是Wnt诱导的转录所必需的转录因子，与表现出β-连环蛋白稳定性并表达TCF 1的指甲上皮细胞相反。我们计划在NSC龛附近截肢后，通过基因表达TCF 1与稳定的β-连环蛋白组合来诱导非NSC谱系中的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，组成性激活伤口表皮下的Wnt信号。我们将测试这种细胞疗法是否可以诱导手指再生， 截肢。]这些实验将为在再生医学中利用神经干细胞治疗截肢者奠定基础。

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.