Investigating the role of Lef1 in fibroblast lineages to induce skin regeneration and reverse aging

项目总结/摘要 了解如何诱导皮肤再生而不是疤痕将具有广泛的临床意义， 这是因为它们抑制了移动性，是痛苦的，并且是心理创伤的来源。在联合 在美国，仅烧伤患者的治疗每年就达75亿美元，疤痕治疗的总市场是 估计在120亿美元左右。对成纤维细胞异质性的作用缺乏了解抑制了进展 在开发用于与瘢痕形成相关的临床病症（例如硬皮病、糖尿病性 溃疡、牛皮癣和纤维化瘢痕疙瘩。因此，了解如何再生皮肤有 可能会影响任何接受手术的人，但也可能影响患有与以下疾病相关的临床疾病的人： 疤痕成人皮肤伤口愈合后会留下疤痕，但胚胎皮肤可以再生而不会留下疤痕。但并非 知道如何安全地将胚胎皮肤的再生能力转移到成人皮肤， 知识因此，我们研究的长期目标是建立诱导成人皮肤成纤维细胞的方法， 具有与胚胎皮肤相同的再生潜力，而不会改变发育， 体内平衡本申请的目的是用胚胎转录因子诱导成人成纤维细胞 重新编程皮肤以支持再生和逆转衰老。胚胎和新生儿皮肤有一个短暂的 乳头状成纤维细胞（PF）在皮肤成熟和老化过程中丢失。新生儿肺纤维化位于 表皮，支持皮肤再生，并表达经典的Wnt转录因子Lef 1。的标志 皮肤老化的主要原因是PFs的降解和Lef 1表达的缺失。我们的中心假设是Lef 1是 新生儿PF功能的主要调节剂，可诱导无疤痕皮肤再生，并恢复PF特性 皮肤老化。在我们令人兴奋的初步数据的指导下，我们将使用新的转基因小鼠模型， 第二代测序技术通过以下具体目标来研究这一假设：在目标1中，我们将 通过成人组织特异性诱导来检验Lef 1是新生儿PF功能的主要调节因子这一假设 皮肤在这里，我们将使用创伤实验和室移植试验，以测试是否成人瘢痕成纤维细胞 可以被转化为可再生的。在目标2中，我们将定义Lef 1调节的下游途径， 指定新生儿PF功能。为此，我们将使用我们的 再生转基因模型系统。最后，在目标3中，我们将确定Lef 1是否可以安全地恢复新生儿 老年皮肤中PF的身份和功能。我们将分析具有重新编程皮肤的“老”转基因小鼠， 它们具有再生皮肤和抑制“衰老”的能力。这项建议是创新的，因为我们的新方法， 在成纤维细胞中操纵胚胎转录因子。这项研究意义重大，因为 了解如何安全地调节成纤维细胞谱系将导致新的分子和细胞靶点， 再生器官，同时还为其他相关临床疾病提供重要的知识基础 与功能障碍的成纤维细胞，如牛皮癣、硬皮病和关节炎。

PROJECT SUMMARY/ABSTRACT Understanding how to induce skin regeneration instead of scarring will have broad implications clinically and cosmetically because; 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. A lack of understanding of the role of fibroblast heterogeneity inhibits progress in developing novel treatments for 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 safely transfer the regeneration abilities of embryonic skin to 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, without altering development and homeostasis. The objective of this application is to induce adult fibroblasts with embryonic transcription factors to reprogram skin to support regeneration and reverse aging. Embryonic and Neonatal skin have a transient Papillary Fibroblasts (PFs) that are lost during skin maturation and aging. Neonatal PFs reside beneath the epidermis, support skin regeneration, and express the canonical Wnt transcription factor, Lef1. A hallmark of skin aging is the degradation of PFs and the loss of Lef1 expression. Our central hypothesis is that Lef1 is the master regulator of Neonatal PF function, which induces scar-less skin regeneration 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 Neonatal PF function by tissue specific induction in adult skin. Here we will use wounding experiments and chamber grafting assays to test if adult scarring fibroblasts can be transformed to be regenerative. In aim 2, we will define the downstream pathways that Lef1 regulates to specify Neonatal 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 safely restore Neonatal PF identity and function in aged skin. We will analyze “old” transgenic mice with reprogrammed skin 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 understanding how to safely regulate fibroblast lineages will lead to new molecular and cellular targets to regenerate organs, while also providing an important knowledge base for other clinical conditions associated with dysfunctional fibroblasts such as; psoriasis, scleroderma, and arthritis.