Induction of digit regeneration by Wnt active nail epithelium

Wnt活性指甲上皮诱导手指再生

• 批准号: 9542711
• 负责人: Mayumi Ito
• 金额: $ 37.29万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-30 至 2020-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9542711
• 关键词: Address; Amputation; Amputees; Area; Bone Regeneration; Cell Lineage; Cell Therapy; Cells; Cues; Defect; Digit structure; Disease; Distal; Epithelial; Epithelial Cells; Epithelium; FGF2 gene; FGFR1 gene; Family member; Fibroblast Growth Factor; Foundations; Genetic; Genetic Transcription; Goals; Growth; In Vitro; Injury; Leg; Limb structure; Mediating; Mediator of activation protein; Mesenchymal; Molecular; Molecular Profiling; Nail plate; Natural regeneration; Organ; Organ Model; Paracrine Communication; Pathway interactions; Play; Regenerative Medicine; Regenerative response; Role; Signal Transduction; Skin; Stem cells; Testing; Transgenic Mice; Transgenic Organisms; Transplantation; Undifferentiated; WNT Signaling Pathway; arm; base; beta catenin; blastema; bone; digit regeneration; experimental study; in vivo; innovation; keratinocyte; limb regeneration; mouse model; nerve supply; novel strategies; organ regeneration; overexpression; progenitor; public health relevance; regenerative; stem cell niche; transcription factor; wound; wound epidermis

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.

描述（由适用提供）：目前，无法再生因受伤或疾病而丢失的四肢。我们的长期目标是了解指甲上皮在数字尖端再生期间的作用，并提出建议，一种促进数字/肢体再生的新策略。哺乳动物数字尖端包括截肢时的骨头可以再生。截肢后数字尖端后，指甲上皮与潜在的未分化的间充质胚细胞一起再生长，从而再生数字骨。再生仅在与指甲相关的区域发生，这是为什么存在这种再生限制的原因。我们最近在Wnt依赖性指甲分化下鉴定并发现了指甲上皮干细胞（NSC）。值得注意的是，指甲上皮中的Wnt激活不仅需要指甲再生，而且还需要间充质胚芽生长，这在数字尖端截肢时会导致数字骨再生。当数字截肢到靠近Wnt-Active指甲祖细胞时，Wnt激活不会发生在伤口区域的上皮细胞中，并且指甲/数字无法再生。然而，包括NSC在内的上皮细胞中强迫Wnt激活可以克服此限制。我们假设Wnt活性指甲上皮具有特定的谱系功能，可以通过消除旁分泌信号来促进数字再生。 AIM1：我们发现FGF2在截肢后以Wnt依赖性方式在指甲上皮细胞中表达，并且FGF2在体外通过FGFR1信号传导促进Blastema增殖。我们将通过在胚细胞中遗传删除FGFR1来测试FGFR1信号对于体内胚芽生长是否至关重要。我们还将测试上皮细胞中FGF2的过表达CN克服了再生的近端限制。这些实验将是第一个通过分别靶向上皮A和间质细胞来剖析分子途径在数字尖端再生中的作用的。 AIM2：[虽然已经证明了Wnt活性指甲上皮细胞在诱导的数字再生中的基本作用，但尚不清楚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来促进再生。因此，我们将测试非NSC衍生的上皮细胞中的Wnt激活（即，近端与指甲上皮的上皮细胞）是否可以同样发挥作用以促进再生，而从NSC谱系衍生出来。与指甲上皮细胞所必需的TCF/LEF1家族成员的非NSC衍生上皮细胞缺乏TCF/LEF1家族成员的表达，对于Wnt诱导的转录所必需的转录因子，与表现出ß-catenin稳定化和表达TCF1的指甲上皮细胞相反。我们计划通过遗传表达TCF1与稳定的ß-catenin结合在非NSC利基市场之后诱导非NSC谱系中的Wnt信号激活，并检查这是否足以诱导数字再生。此外，我们将移植NSC和正常的皮肤角质形成细胞，这些角质形成细胞在伤口表皮下的下方激活Wnt信号。我们将测试这种细胞疗法在截肢近端后是否可以诱导数字再生。