Exploring How Dermal Papilla Precursors Regulate Hair Follicle Formation

描述（由申请人提供）：为了诱导组织干细胞（SC）朝向用于替代疗法的特定细胞命运，我们需要了解SC如何被调节以增殖和分化成其后代谱系。这些SC命运决定被认为是由来自其直接微环境或小生境中的专门细胞的信号协调的。毛囊（HF）的形态发生是一个很好的模型系统SC调控的利基信号，因为发展的步骤和时间是明确的。然而，与大多数其他器官中的SC小生境一样，由于长期缺乏分离、表征和靶向小生境细胞的遗传工具，调节皮肤SC命运的特定小生境信号在很大程度上是未知的。 特化的毛乳头（DP）前体细胞被认为是作为小生境细胞与早期毛基板中的SC相互作用以形成HF。然而，到目前为止，没有直接的证据表明DP前体是HF形成所必需的，我们也不知道难以捉摸的命运指定信号的性质，这主要是由于缺乏现代遗传学方法来靶向这些细胞进行分离和基因/细胞消融。在我们正在进行的努力和新的初步数据，我们已经开发出急需的遗传策略，以分离，表征和靶向胚胎DP龛细胞的基因/细胞消融在HF形成的最早阶段。在这个提议中，我们将利用DP基因Tbx 18和Sox 2的遗传驱动来测试DP细胞为HF干细胞建立生态位的假设。我们将利用我们新建立的Tbx 18驱动的诱导型Cre系作为DP前体细胞毒性细胞消融的第一个遗传驱动因素，并确定其对HF形成的绝对要求。相反，我们将从Tbx 18和Sox 2驱动的GFP细胞系中分离DP前体，并在毛发诱导试验中直接确定与非特化成纤维细胞相比，这些细胞是否足以诱导HF形成。然后，我们将采用可诱导的铬介导的单个DP前体细胞的单细胞命运映射，以确定其与成熟DP和真皮鞘的谱系关系，并调查DP内的潜在层次结构和异质性。我们将进一步确定特定的作用，Wnt信号在胚胎生态位的连接蛋白基因消融。最后，我们将系统地确定新的DP生态位信号和功能研究它们的作用，在体内的DP受体特异性基因缺失。这项工作将确定胚胎DP龛中的必需基因及其在HF形成过程中激活SC的信号。这些研究的应用在于其改善再生疗法的潜力，该再生疗法旨在恢复烧伤受害者或患有衰弱性皮肤病的患者的全功能皮肤，包括HF，由于我们对基本支持利基信号的理解有限，目前缺乏这种技术。这些发现也将对其他器官系统的再生治疗具有全球意义，其中SC壁龛用于维持组织稳态。

DESCRIPTION (provided by applicant): To coax tissue stem cells (SCs) towards specific cell fates for replacement therapies, we need to understand how SCs are regulated to proliferate and differentiate into their progeny lineages. These SC fate decisions are thought to be coordinated by signals from specialized cells in their immediate microenvironment, or niche. Hair follicle (HF) morphogenesis is an excellent model system for SC regulation by niche signals, since the developmental steps and timing are well-defined. However, as with SC niches in most other organs, the specific niche signals that regulate skin SC fates are largely unknown, due to the long-standing lack of genetic tools to isolate, characterize and target the niche cells. Specialized dermal papilla (DP) precursor cells are thought to interact as niche cells with SCs in incipient hair placodes to form HFs. To date, however, no direct proof exists that DP precursors are required for HF formation nor do we know the nature of the elusive fate-specifying signals, largely due to the lack of modern genetic methods for targeting these cells for isolation and gene/cell ablation. In our ongoing effort and new preliminary data, we have developed much-needed genetic strategies to isolate, characterize and target embryonic DP niche cells for gene/cell ablation during the earliest steps of HF formation. In this proposal, we will utilize genetic drivers of DP genes Tbx18 and Sox2 to test the hypothesis that DP cells establish a niche for HF stem cells. We will utilize our newly established Tbx18-driven inducible Cre line as the first genetic driver for cytotoxic cell ablation of DP precursors and determine their absolute requirement for HF formation. Conversely, we will isolate DP precursors from Tbx18- and Sox2-driven GFP lines and directly determine in hair induction assays whether these cells are sufficient to induce HF formation in comparison to unspecialized fibroblasts. We will then employ inducible Cre-mediated single-cell fate mapping of individual DP precursor cells to define their lineage relationships with mature DPs and dermal sheaths and to investigate the potential hierarchy and heterogeneity within DPs. We will further determine the specific role of Wnt signaling in the embryonic niche by ¿-catenin gene ablation. We will finally systematically identify novel DP niche signals and functionally investigate their role in vivo by DP precursor-specific gene deletion. This work will identify essential genes in the embryonic DP niche and its signal(s) that activate SCs during HF formation. The application of these studies lies in their potential to improve regenerative therapies meant to restore fully functional skin including HFs for burn victims or patients with debilitating skin diseases, a technology which currently is lacking due to our limited understanding of essential supporting niche signals. These findings will also have global relevance for regenerative therapies in other organ systems, where SC niches operate to maintain tissue homeostasis.