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

项目摘要 在毛发周期中，生长中的毛囊（HF）在以祖细胞死亡和毛囊退化为标志的阶段中退化。 重塑（退化期）、休息期（休止期）和新毛发生长（生长期）之前。来自皮肤的信号 乳头（DP）-一个关键的信号中心-调节祖细胞增殖和分化的灯泡生长 HF，并诱导隆突/生殖干细胞（SC）再生完全生长的HF。在退行期， DP需要从生长的HF的底部重新定位到上部HF中的SC储层，穿过皮肤 几百微米。这是如何实现的，几十年来一直不为人知。在第一次融资中 在此期间，我们发现毛囊内衬真皮鞘（DS）是一种平滑肌， 物理地重新定位DP以到达其基本SC相邻位置。分子的动态变化 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 收缩未知。探索我们的DS和其他几个基因特征表明了一个有趣的作用， 内皮素信号传导，一个众所周知的血管收缩调节途径，在控制DS收缩。与 几个已建立和新开发的功能测定和新的遗传DS靶向，我们现在有 建立了探索内皮素配体、受体等途径动态表达的条件 生长和退化期间祖细胞、DS和DP中的成员;用于研究功能作用 祖细胞衍生的内皮素在信号传导和激活收缩;和解剖下游 途径机制。总的来说，我们将严格测试卵泡祖细胞和DS参与的假设， 在调节DS收缩和毛发周期退化的上皮-间充质串扰中至关重要。我们将 通过对内皮素信号通路关键成员的详细研究， 原位杂交和免疫荧光分析。我们将用新的方法进一步纯化DS和祖细胞。 从生长和退化卵泡中分离方法，并分析其转录组， 潜在的信号串扰一般和内皮素途径系统的所有组成部分，特别是。我们 将在分离的DS细胞和显微切割的细胞中激活和阻断内皮素信号传导激活。 卵泡，并确定功能DS收缩在我们最近建立的体外和离体活成像 测定。然后，我们将探讨收缩抑制在体内的后果，并直接可视化收缩 抑制与活体成像。我们将进行定时遗传受体和配体消融，并评估 影响卵泡退化。最后，我们将定义空间和时间的钙信号动力学 使用钙报告小鼠和Ca 2+激活剂和抑制剂的内皮素途径激活下游 通道以及PLC/PKC激活的主要内皮素信号通路分支， 激活DS收缩。通过这项工作将确定一个关键的生理功能的祖细胞-DS 相互作用，这可能对毛发再生方法有用，包括操纵毛囊退化。

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.