Specification, Molecular Control and Niche Functions of the Hair Follicle Mesenchyme

项目摘要 在毛发周期中，生长的毛囊(HF)在以祖细胞死亡和毛囊为标志的阶段倒退 重塑(生发)、休止期前(生发)和新发生长(生发)。来自皮肤的信号 乳头(DP)--一个关键的信号中心--调节生长鳞茎中的前体细胞的增殖和分化 HFS，并诱导隆起/生殖细胞(SC)再生完全生长的HFs。在退行性变过程中， DP需要从生长HFs的底部重新定位到上层HF的Sc水库，穿过皮肤 几百微米。几十年来，人们一直不知道这是如何实现的。在第一笔资金中 期间，我们发现毛囊衬里真皮鞘(DS)是一种收缩到 物理上重新定位DP，以达到其基本的SC相邻位置。发生的动态分子变化 在相邻的祖细胞和DS中，以及它们之间的上皮-间充质串扰是否调节 收缩是未知的。探索我们的DS和其他几个基因签名表明了一个有趣的角色 内皮素信号，众所周知的血管收缩调节途径，在控制DS收缩中的作用。使用 一些已建立的和新开发的功能分析和新的遗传DS靶向，我们现在 为探索内皮素配体、受体等途径的动态表达奠定了条件 祖细胞中的成员，生长和退化过程中的DS和DP；用于研究其功能作用(S) 祖细胞衍生的内皮素在信号和激活收缩中的作用；以及对下游的解剖 途径机制(S)。总体而言，我们将严格测试卵泡祖细胞和DS交配的假设 在上皮-间充质串扰中，对调节DS收缩和毛发周期倒退至关重要。我们会 通过详细的研究，精确地定位和定位关键的内皮素信号通路成员的表达时序 原位杂交和免疫荧光分析。我们将用新奇的方式进一步净化DS和祖先 生长和退化卵泡的多色分离方法及其转录本分析 潜在的信号串扰，特别是内皮素途径系统的所有组件。我们 会在药物上激活和阻断分离的DS细胞和显微解剖中的内皮素信号激活 卵泡，并在我们最近建立的体外和体外活体成像中确定功能性DS收缩 化验。然后，我们将探索体内收缩抑制的后果，并直接将收缩可视化 通过活体成像进行抑制。我们将进行定时的遗传受体和配体消融，并评估 对卵泡退化的影响。最后，我们将定义空间和时间钙信号动力学 利用钙报告鼠和钙激活剂和抑制剂激活内皮素途径下游 以及PLC/PKC的激活途径来解剖主要的内皮素信号通路分支 操作以激活DS收缩。通过这项工作将确定先祖细胞的一个关键生理功能-DS 相互作用，这可能是有用的头发再生方法，包括操纵毛囊倒退。

Project Summary During the hair cycle, growing hair follicles (HF) regress in a phase marked by progenitor death and follicle remodeling (catagen), before a rest period (telogen) and new hair growth (anagen). Signals from the dermal papilla (DP) – a key signaling center – regulate progenitor proliferation and differentiation in the bulb of growing HFs, and induce bulge/germ stem cells (SC) to regenerate fully growing HFs. During catagen regression, the DP needs to relocate from the base of growing HFs to the SC reservoir in the upper HF, traversing the skin several hundred micrometers. How this is accomplished has been unknown for decades. In the first funding period, we have uncovered that the follicle-lining dermal sheath (DS) is a smooth muscle that contracts to physically relocate the DP to reach its essential SC-adjacent position. The dynamic molecular changes that occur in adjacent progenitors and the DS and whether epithelial-mesenchymal crosstalk between them regulates contraction is unknown. Exploring our DS and several other gene signatures suggested an intriguing role of endothelin signaling, a well-known vasoconstriction regulatory pathway, in controlling DS contraction. With several established and newly developed functional assays and novel genetic DS targeting, we have now established the conditions for exploring the dynamic expression of endothelin ligand, receptor and other pathway members in progenitors, DS and DP during both growth and regression; for investigating the functional role(s) of progenitor-derived endothelin in signaling and activating contraction; and for dissecting the downstream pathway mechanism(s). Overall, we will rigorously test the hypothesis that follicle progenitors and the DS engage in epithelial-mesenchymal crosstalk crucial for regulating DS contraction and hair cycle regression. We will precisely map expression localization and timing of key endothelin signaling pathway members through detailed in situ hybridization and immunofluorescence analyses. We will further purify DS and progenitors with novel multicolor isolation methods from growing and regressing follicles and analyze their transcriptomes to define potential signaling crosstalk in general and all components of the endothelin pathway system in particular. We will pharmacologically activate and block endothelin signaling activation in isolated DS cells and microdissected follicles, and determine functional DS contraction in our recently established in vitro and ex vivo live imaging assays. We will then explore the consequences of contraction inhibition in vivo and directly visualize contraction inhibition with intravital imaging. We will perform timed genetic receptor and ligand ablations and assess the impact on follicle regression. Finally, we will define the spatial and temporal calcium signaling dynamics downstream of endothelin pathway activation using calcium reporter mice and activators and inhibitors of Ca2+ channels as well as of the PLC/PKC activation to dissect the major endothelin signaling pathway branches that operate to activate DS contraction. With this work will define a key physiological function of progenitor-DS interaction, which may be useful for hair regeneration approaches, including manipulating follicle regression.