Modulating Lef1 Activation in Fibroblasts During Hair Follicle Regeneration in Skin Wounds

项目总结/摘要 大疤痕是一个严重的健康问题;他们抑制流动性，是痛苦的，是一个来源的心理 外伤在美国，仅烧伤患者的治疗每年就达75亿美元， 用于疤痕治疗的费用估计约为120亿美元。还有几种临床条件与 瘢痕形成，如硬皮病、糖尿病溃疡、牛皮癣和纤维化瘢痕疙瘩。因此，委员会认为， 了解如何再生皮肤有可能影响任何接受手术的人， 患有与瘢痕形成相关的临床病症的个体。成年人的皮肤伤口会有疤痕愈合， 皮肤可以再生而不会留下疤痕。然而，不知道如何复制的再生能力， 胚胎皮肤在成人皮肤中仍然是一个知识空白。因此，我们研究的长期目标是建立 诱导成人皮肤中的成纤维细胞具有与其胚胎皮肤相同的再生潜力的方法 同行本申请的特定目的是诱导具有胚胎转录的成人成纤维细胞 重新编程皮肤以支持再生的因素。胚胎皮肤具有乳头状成纤维细胞（PF）和网状成纤维细胞（网状成纤维细胞）。 成纤维细胞（RF），在皮肤发育过程中建立。PF位于表皮下，可以 分化成支持毛囊形成的成纤维细胞，并表达典型的Wnt转录因子， 左1。相反，RF位于网状真皮中，分泌大量的真皮细胞外基质，分化为 转化为脂肪细胞，并且不表达Lef1。重要的是，RF不能在伤口中分化成PF，以支持 再生有趣的是，皮肤成熟和老化的标志是分子和结构的退化， PFs和Lef1表达缺失。我们的中心假设是Lef1是PF功能的主要调节器 在成纤维细胞中诱导伤口中的无瘢痕皮肤再生，并将恢复老化皮肤中的PF特性。指导 根据我们令人兴奋的初步数据，我们将使用新的转基因小鼠模型和下一代测序， 通过以下具体目标来研究这一假设的技术：在目标1中，我们将测试假设， Lef1是PF功能的主要调节因子，其在RFs中的诱导支持皮肤再生。这里我们将 使用新的转基因模型系统来测试RF中的Lef1激活是否支持伤口的再生， 小室移植测定。在目标2中，我们将定义Lef1调节的下游途径，以指定PF 功能协调发展的为此，我们将使用再生转基因模型进行ChIPSeq和单细胞RNA-Seq 系统.最后，在目标3中，我们将确定Lef1是否可以恢复老化皮肤中的PF身份和功能。我们将 分析具有重新编程的RF的“老”转基因小鼠，并测试它们再生皮肤和抑制“衰老”的能力。 这项建议是创新的，因为我们的新方法操纵胚胎转录因子， 体内成纤维细胞。这项研究具有重要意义，因为我们的发现将影响临床和体外研究。 利用成纤维细胞诱导人毛囊再生以及无疤痕愈合的方法。

PROJECT SUMMARY/ABSTRACT Large scars are a serious health concern; they inhibit mobility, are painful, and are a source of psychological trauma. In the United States, treatment for burn victims alone amounts to $7.5 billion annually, the total market for scar treatment is estimated to be around $12 billion. There are also several clinical conditions associated with scarring, such as, scleroderma, diabetic ulcers, psoriasis and fibrotic keloid scarring. Consequently, understanding how to regenerate skin has the potential to impact anyone who undergoes surgery, but also individuals with clinical conditions associated with scarring. Adult skin wounds heal with scars, but embryonic skin can regenerate without scarring. However, not knowing how to reproduce the regeneration abilities of embryonic skin in adult skin remains a gap in knowledge. Thus, the long term goal of our research is to establish methods to induce fibroblasts in adult skin to have the same regenerative potential as their embryonic skin counterparts. The particular objective of this application is to induce adult fibroblasts with embryonic transcription factors to reprogram skin to support regeneration. Embryonic skin has Papillary Fibroblasts (PFs) and Reticular Fibroblasts (RFs), which are established during skin development. PFs reside beneath the epidermis, can differentiate into fibroblasts that support hair follicle formation, and express the canonical Wnt transcription factor, Lef1. In contrast, RFs reside in the reticular dermis, secrete the bulk of dermal extracellular matrix, differentiate into adipocytes, and do not express Lef1. Importantly, RFs cannot differentiate into PFs in wounds to support regeneration. Interestingly, a hallmark of skin maturation and aging is the molecular and structural degradation of PFs and the loss of Lef1 expression. Our central hypothesis is that Lef1 is the master regulator of PF function in fibroblasts that induces scar-less skin regeneration in wounds and will restore PF identity in aged skin. Guided by our exciting preliminary data we will use novel transgenic mouse models and next generation sequencing technologies to investigate this hypothesis by the following specific aims: In aim 1 we will test the hypothesis that Lef1 is the master regulator of PF function and that its induction in RFs supports skin regeneration. Here we will use novel transgenic model systems to test if Lef1 activation in RFs supports regeneration in wounds and chamber grafting assays. In aim 2, we will define the downstream pathways that Lef1 regulates to specify PF functions. To do this we will perform ChIPSeq and Single-Cell-RNA-Seq with our regenerative transgenic model systems. Finally, in aim 3 we will determine if Lef1 can restore PF identity and function in aged skin. We will analyze “old” transgenic mice with reprogrammed RFs and test their ability to regenerate skin and inhibit “aging”. This proposal is innovative because of our novel approach of manipulating embryonic transcription factors in fibroblasts in vivo. The proposed research is significant because our findings will influence clinical and in vitro approaches utilizing fibroblasts to induce human hair follicle regeneration as well as scar-less healing.