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)调节的一个很好的模型 微环境或生态位，因为它涉及周期性的破坏(再生)、休息(休眠)和再生 (生长期)。来自真皮乳头(DP)--一群间充质HF细胞--的信号被认为诱导了 在毛发从端原到生长期的转变过程中，凸起/胚芽干细胞从静止状态切换到激活状态 周而复始。然而，由于缺乏直接的遗传工具来强大的、可诱导的靶向端原DP的基因 消融，DP在SC激活中作用的功能证据在很大程度上是间接的。我们也知道的很少 关于成人DP的长期命运和潜力及其与真皮鞘和皮肤的血统关系 成纤维细胞。最后，成人DP在毛发周期中的精确分子谱系仍然很差。 由于先前研究中DP分离和基因表达分析的局限性，定义了。我们最近 建立胚胎特异性分离和分子鉴定的遗传标记策略 DP前体和DP来自生长的HFs，这使得鉴定它们的分子特征成为可能。然后我们 开发胚胎DP特异性基因打靶工具并探索DP签名基因在心衰过程中的作用 队形。我们现在已经进一步建立了健壮和可诱导的基因靶向的条件 端原DP，从而使探索DP中的细胞周转和克隆谱系关系成为可能 通过对端原中DP指令激活HF的分子机制的研究， 从生长期过渡到生长期。在这些研究中，我们将严格检验端粒酶原DP作为一种 SC-在头发周期中激活利基。我们将使Crabp1成为第一个健壮的、可诱导的基因驱动程序 用于Cre介导的端粒酶原DP的靶向。然后我们将使用Crabp1iCreer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，通过遗传命运图谱和脉冲追逐标记保留实验，获得 洞察DP身份和血统潜力。我们将使用我们的新型驱动器来烧蚀PdGFRα和PdGFRβ 端粒酶原DP，评估SC在隆起/胚芽中的激活状态，对PDGF信号转导缺失的影响 毛发再生，并破译PDGF信号驱动的DP功能的分子机制 调节SC的激活。我们将在关键的毛发周期分离纯DP、凸起/生殖细胞和相关细胞类型 阶段，并系统地定义它们在整个毛发周期中的分子特征，这是前所未有的 基因表达发现的细胞分辨率和敏感性。然后我们将研究它的功能作用 NR3C1和其他新发现的DP特征基因，通过与端原DP中的Crabp1iCreer基因消融。 通过这项工作，我们将极大地扩展我们对这些利基细胞调节SC的知识，这将是 对包括HFs在内的功能性皮肤再生治疗的发展至关重要。

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.