Dermal Papilla Regulation and Function for Stem Cell Activation in the Hair Cycle

项目摘要 毛囊（HF）周期是研究成体干细胞（SC）调控的一个很好的模型， 微环境或生态位，因为它涉及周期性的破坏（退化期），休息（休止期）和再生 （anagen）。来自毛乳头（DP）--间充质HF细胞簇--的信号被认为诱导了一种新的免疫反应。 在毛发的休止期到生长期的转变期间，在凸起/胚芽SC中从静止状态转换到活化状态 周期然而，由于缺乏直接的遗传工具来稳健地、可诱导地靶向基因的休止期DP， 消融术后，DP在SC激活中的作用的功能证据在很大程度上是间接的。我们也不知道 关于成年DP的长期命运和潜力及其与真皮鞘的谱系关系， 成纤维细胞最后，成年DP在毛发周期中的精确分子库仍然很差 由于以前研究中DP分离和基因表达分析的局限性，我们最近 建立了胚胎特异性分离和分子鉴定的遗传标记策略， DP前体和DP从生长的HF，使其有可能确定其分子签名。然后我们 开发了胚胎DP特异性基因靶向工具，并探索了DP标签基因在HF中的作用。 阵我们现在已经进一步建立了用于稳定的和可诱导的基因靶向的条件， 休止期DP，从而使探索细胞周转和克隆谱系关系的DP沿着 随着对DP指令HF激活的分子机制的研究， 到生长期的过渡在这些研究中，我们将严格检验这一假设，即休止期DP作为一个 在毛发周期中激活SC的小生境。我们将把Crapp 1作为第一个强大的、可诱导的遗传驱动因子， 用于Cre介导的调聚体DP的靶向。然后，我们将使用Crabp 1 iCreER小鼠来确定，空间和 时间精度，DP的细胞周转及其与邻近真皮鞘的谱系关系 和皮肤间充质，通过遗传命运作图和脉冲追踪标记保留实验， 深入了解DP身份和谱系潜力。我们将使用我们的新型驱动器消融PDGFRα和PDGFRβ， 休止期DP，评估隆突/胚芽中的SC活化状态，PDGF信号传导的损失对 毛发再生，并破译PDGF信号驱动DP功能的分子机制 调节SC活化。我们将在关键的毛发周期中分离纯DP、隆突/生殖SC和相关细胞类型 阶段，并在整个头发周期中系统地定义其分子特征， 细胞分辨率和基因表达发现的灵敏度。然后，我们将研究 Nr 3c 1和其他新鉴定的DP标签基因，通过在休止期DP中用Crabp 1 iCreER进行基因切除。 通过这项工作，我们将大大扩展我们对这些小生境细胞的SC调节的知识，这将是 对于开发涉及功能性皮肤（包括HF）再生的治疗至关重要。

Project Summary The hair follicle (HF) cycle is an excellent model for studying adult stem cell (SC) regulation by the microenvironment or niche, as it involves cyclical bouts of destruction (catagen), rest (telogen) and regrowth (anagen). Signals from the dermal papilla (DP) – a cluster of mesenchymal HF cells – are thought to induce a switch from a resting to an activated state in bulge/germ SCs during the telogen-to-anagen transition of the hair cycle. However, due to the lack of direct genetic tools for robust, inducible targeting of the telogen DP for gene ablation, the functional evidence for a role of the DP in SC activation is largely indirect. We also know little about the long-term fate and potential of the adult DP and its lineage relationships to the dermal sheath and fibroblasts. Finally, the precise molecular repertoire of the adult DP during the hair cycle has remained poorly defined, due to the limitations of DP isolation and gene expression analysis in previous studies. We recently established genetic labeling strategies for the specific isolation and molecular characterization of embryonic DP precursors and the DP from growing HFs, making it possible to identify their molecular signatures. We then developed embryonic DP-specific gene targeting tools and explored the role of DP signature genes during HF formation. We have now further established the conditions for the robust and inducible genetic targeting of the telogen DP, thus enabling exploration of the cellular turnover and clonal lineage relationships in the DP along with investigation into the molecular mechanisms underlying DP instruction of HF activation during the telogen- to-anagen transition. In these studies, we will rigorously test the hypothesis that the telogen DP serves as a SC-activating niche during the hair cycle. We will establish Crabp1 as the first robust, inducible genetic driver for Cre-mediated targeting of the telogen DP. We will then use Crabp1iCreER mice to determine, with spatial and temporal precision, the cellular turnover of the DP and its lineage relationship to the neighboring dermal sheath and the skin mesenchyme, through genetic fate mapping and pulse-chase label retention experiments, gaining insight into DP identity and lineage potential. We will use our novel driver to ablate PDGFRα and PDGFRβ in the telogen DP, assess SC activation status in the bulge/germ, the impact of the loss of PDGF signaling on hair regeneration, and decipher the molecular mechanisms underlying the PDGF signaling-driven DP functions regulating SC activation. We will isolate pure DP, bulge/germ SCs and related cell types at key hair cycle stages, and systematically define their molecular signatures throughout the hair cycle, with unprecedented cellular resolution and sensitivity of gene expression discovery. We will then investigate the functional role of Nr3c1 and other newly identified DP signature genes, by gene ablation with Crabp1iCreER in the telogen DP. With this work, we will greatly expand our knowledge of SC regulation by these niche cells, which will be essential for the development of treatments involving the regeneration of functional skin, including HFs.