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趾尖再生中的作用，并提出一种促进手指/肢体再生的新策略。哺乳动物的趾尖，包括它的骨头，可以在截肢后再生。趾尖截肢后，趾甲上皮与下面未分化的间充质母细胞一起再生，使趾骨再生。再生只发生在与指甲相关的区域，目前尚不清楚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（NSCs）经历wnt依赖性指甲分化。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，也是趾尖截肢后导致趾骨再生的间充质母细胞生长所必需的。当手指在Wnt活化的甲祖细胞附近截肢时，伤口区域的上皮细胞中不会发生Wnt活化，指甲/指无法再生。然而，在上皮细胞（包括NSCs）中强制激活Wnt可以克服这一限制。我们假设wnt活性指甲上皮具有谱系特异性功能，通过释放旁分泌信号来促进手指再生。目的1：我们发现，截肢后，FGF2在甲上皮细胞中以wnt依赖的方式表达，并且FGF2在体外通过FGFR1信号传导促进母细胞增殖。我们将通过基因删除囊胚细胞中的FGFR1来测试FGFR1信号是否对囊胚生长至关重要。我们还将测试上皮细胞中fgf2的过表达是否可以克服再生的近端限制。这些实验将首次通过分别靶向上皮细胞和间充质细胞来解剖分子途径在指尖再生中的作用。目的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实，但目前尚不清楚wnt活性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。因此，我们将测试非NSC来源的上皮细胞（即指甲上皮近端上皮细胞）中wnt的激活是否与来自NSC谱系的细胞具有类似的促进再生的功能。具有稳定的ss-catenin的非nsc来源的上皮细胞缺乏TCF/LEF1家族成员的表达，这是Wnt诱导转录所必需的转录因子，与显示ss-catenin稳定和表达TCF1的指甲上皮细胞相反。我们计划在非NSC谱系中通过基因表达TCF1与稳定的ss-catenin结合来诱导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NSCs和正常皮肤角质形成细胞，它们组成性地激活伤口表皮下的Wnt信号。我们将测试这种细胞疗法是否能诱导近端手指再生

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.