Lrig1-Expressing Colonic Stem Cells in Homeostasis and Repair

描述（由申请人提供）：这份NIDDK指导研究科学家发展奖申请描述了一个为期三年的培训计划，旨在让我获得额外的技能和知识，以便我能够过渡到指导我自己的独立和富有成效的研究项目。在进行拟议的研究中，通过在大学研究环境中遵循精心指导的职业发展计划，我将通过获得肠上皮损伤模型以及活细胞和超分辨率显微镜方面的专业知识来增加我的科学技能。利用这些新获得的专业知识，我将建立一个科学利基，这将使我有别于我的导师，并为一个强大的，外部资助的研究项目铺平道路。本应用程序的研究将集中在了解结肠干细胞在上皮损伤后修复中的功能。众所周知，Egfr信号在损伤后被激活，这有助于修复；然而，这种激活AD随后的生长和增殖，必须严格调节，以避免异常过度生长。我发现一种Egfr抑制剂Lrig1，标记了一个很大程度上静止的结肠干细胞群，而不像由Lgr5标记的特征明确的增殖性干细胞群。我的建议旨在阐明Lrig1+干细胞的作用，以及Lrig1作为Egfr的负调节因子在结肠组织干细胞稳态和伤口愈合中的作用。干细胞群在损伤后修复中的需求尚未得到测试；这对我们理解结肠上皮隐窝生态位对促进适当再生的要求至关重要。我的具体目标是为了验证我的总体假设，即Lrig1在再生祖细胞中表达，以响应活跃的生长因子信号，如激活的Egfr信号。在Aim 1中，我将在伤口愈合前后同时清除表达Lrig1-和lgr5的干细胞，以测试它们在损伤修复中的需求。我将计算初始值

DESCRIPTION (provided by applicant): This NIDDK Mentored Research Scientist Development Award application describes a three-year training plan designed to allow me to acquire additional skill and knowledge so that I can transition into directing my own independent and productive research program. In carrying out the proposed research, and by following a carefully mentored career development plan in a collegial research environment, I will add to my scientific repertoire by acquiring expertise in intestinal epithelial injury models and as well as live-cell and super-resolution microscopy. Using this newly acquired expertise, I will establish a scientific niche that will set me apart from my mentors and pave the way to a robust, extramurally funded research program. The research proposed in this application will focus on understanding how colonic stem cells function in repair after epithelial injury. It is known that Egfr signaling is activated after injury and this contributes to repair; however, this activation ad subsequent growth and proliferation, must be tightly regulated to avoid aberrant overgrowth. I have found that an Egfr inhibitor, Lrig1, marks a population of largely quiescent colonic stem cells, unlike the well-characterized, proliferative stem cell population marked by Lgr5. My proposal seeks to clarify the role of both Lrig1+ stem cells, and the role of Lrig1 as a negative regulator of Egfr, in colonic tissue stem cell homeostasis and wound healing. The requirement of stem cell populations in repair after injury has not yet been tested; this is critical to our understanding of the colonic epithelial crypt niche requirements for promoting proper regeneration. My Specific Aims are designed to test my over-arching hypothesis that Lrig1 is expressed in regenerative progenitor cells, in response to an active growth factor signal, such as activated Egfr signaling. In Aim 1, I will eliminate both Lrig1- and Lgr5-expressing stem cells before and after wound healing, to test their requirement in injury repair. I will evaluate initial injury and end-stage wound-repair by histological, molecular, and super-resolution analysis. In Aim 2, to examine the influence of Lrig1 on Egfr in wound healing, I will examine the effects of the absence of Lrig1 in wound healing, using Lrig1 null mice, and use advanced microscopy to examine the dynamics of Egfr activation. With the evolution of tools to manipulate stem cell populations in vivo, only now are the studies proposed here possible. Moreover, Vanderbilt's super-resolution microscopic capabilities will enable me to examine colon cell dynamics in tissue repair after injury at a resolution not previously possible. By coupling sophisticated mouse modeling with super-resolution imaging, the studies proposed here directly test the relationship between Lrig1- and Lgr5-expressing stem cells in injury repair, as well as examine the role of Lrig1 as a negative regulator of Egfr activity in this wound-healing context. The goal of this Mentored Research Scientist Development Award is to develop the expertise that will allow me to run an independent, funded research program that will contribute to improving human health and disease.