Specification of Dermal Papilla Cell Fate in the Hair Follicle Stem Cell Niche

描述(由申请人提供)：干细胞(SC)激活和自我更新的调节对于成功的组织发育和动态平衡是必要的。在许多组织中，这是由微环境中的支持细胞发出的信号指挥的，这种微环境被称为干细胞小生境。彻底了解哺乳动物干细胞龛细胞在发育过程中如何编程和功能以调节干细胞是未来组织特异性再生治疗的关键要求。虽然对无脊椎动物的研究已经获得了很多知识，但由于组织的复杂性和缺乏分离和表征细胞的方法，对于哺乳动物干细胞龛中的干细胞如何获得专门化状态的分子理解尚不清楚。我们最近开发了新的遗传工具来研究毛囊SC壁龛中的毛乳头(DP)细胞，这些细胞被认为决定了毛囊SC的命运。我们已经纯化了这些细胞，确定了它们的分子身份，并建立了研究它们命运规范的方法。这建立了一个亟需的哺乳动物模型，可以在其中研究SC生态位的形成和功能。我们的长期目标是以毛囊形态发生和再生为模型系统，了解支配SC生态位命运的分子机制。我们的工作已经确定了DP细胞的分子特征，包括一系列信号和转录因子(TF)。在这个建议中，我们将检验这样的假设，即DP签名TFS包含调节SC激活的必要且充分的活性，毛发诱导DP生态位命运。我们将在体外和体内系统地操纵DP细胞中标志性转铁蛋白的表达，以确定指定DP利基细胞的分子特性和功能活性的转录调控机制。我们将进一步建立一种新的可诱导的DP特异性体内基因打靶小鼠模型，这是目前该领域所缺乏的。最后，我们将使用这一新的体内基因消融模型，并与我们建立的体外/体内杂交基因敲除试验并行，使用可用于条件性基因消融的现有小鼠，测试用于DP利基命运规范的候选DP签名TF的必要性。我们的工作将确定毛囊DP细胞SC生态位命运的核心转录调控。在分离的DP细胞和正常的成纤维细胞中激活转录程序有望扩大全功能细胞，用于皮肤重建的治疗。这些发现将对其他再生组织具有全球意义，在这些组织中，SC利基的作用是维持组织的动态平衡，并可能导致3D组织再生疗法的开发。 公共卫生相关性：彻底了解通过支持利基细胞来调节干细胞(SC)的激活和自我更新是未来组织特异性再生疗法的关键要求。哺乳动物干细胞壁龛中的壁龛细胞如何获得其专门化地位在很大程度上是未知的。我们将定义毛囊SC生态位中毛乳头细胞生态位命运的转录调控，建立一个急需的哺乳动物模型，在此模型中可以研究SC生态位的形成和功能。我们的工作将提高我们为未来的治疗创造更正常行为的皮肤组织的能力，并将对其他再生组织具有全球相关性，在这些组织中，SC利基的运作是为了维持组织的动态平衡。

DESCRIPTION (provided by applicant): Regulation of stem cell (SC) activation and self-renewal is necessary for successful tissue development and homeostasis. In many tissues this is orchestrated by signals from supporting cells within the microenvironment called the SC niche. A thorough understanding of how mammalian SC niche cells become programmed and function during development to regulate SCs is a crucial requirement for future tissue-specific regenerative therapies. While much knowledge has been gained from studies in invertebrates, a molecular understanding of how niche cells in mammalian SC niches, such as the bone marrow, brain, intestine and hair follicles, acquire their specialized status is unknown due to the complexity of the tissues and the lack of methods to isolate and characterize the cells. We have recently developed novel genetic tools to study dermal papilla (DP) cells in the hair follicle SC niche, which are thought to direct follicular SC fate. We have purified these cells, defined their molecular identity and established assays to study their fate specification. This establishes a much needed mammalian model where formation and function of the SC niche can be studied. Our long- term goal is to understand the molecular mechanisms that govern SC niche fate specification, using hair follicle morphogenesis and regeneration as a model system. Our work has identified the molecular signature of DP cells, including a battery of signaling and transcription factors (TFs). In this proposal, we will test the hypothesis that the DP signature TFs contains the activity necessary and sufficient to regulate the SC activating, hair inducing DP niche fate. We will systematically manipulate signature TF expression in DP cells in vitro and in vivo to define the transcriptional control mechanisms that specify the molecular identity and functional activity of DP niche cells. We will further establish a novel inducible DP-specific in vivo gene targeting mouse model that is currently missing in the field. Finally, we will test with available mice that are targeted for conditional gene ablation, the necessity of candidate DP signature TFs for DP niche fate specification, using this novel in vivo gene ablation model, and in parallel with our established in vitro/in vivo hybrid knockout assay. Our work will identify the core transcriptional regulation of the SC niche fate of hair follicle DP cells. Activating the transcriptional program in isolated DP cells and in regular fibroblasts bears the promise to expand fully functional cells for therapeutic use in skin reconstructive efforts. These findings will have global relevance for other regenerative tissues, where SC niches operate to maintain tissue homeostasis, and potentially will lead to development of 3D-tissue regenerative therapies. PUBLIC HEALTH RELEVANCE: A thorough understanding of the regulation of stem cell (SC) activation and self-renewal by supporting niche cells is a crucial requirement for future tissue-specific regenerative therapies. How the niche cells in mammalian SC niches acquire their specialized status is largely unknown. We will define the transcriptional control of niche fate specification of dermal papilla cells in the hair follicle SC niche, establishing a much needed mammalian model, where formation and function of the SC niche can be studied. Our work will improve our ability to create a more normal behaving skin tissue for future therapies and will also have global relevance for other regenerative tissues, where SC niches operate to maintain tissue homeostasis.