Rapid functional genetics to study stem cell-niche interactions in the skin

项目摘要 皮肤干细胞受到来自其小生境的信号的严重影响，包括不同的成纤维细胞群体。 虽然在过去的十年里，我们分离和分子分析不同细胞类型的能力已经大大提高， 识别驱动干细胞-小生境相互作用的关键基因的主要障碍是 生成所需的遗传模型（例如，细胞类型特异性Cre或CreER，以及过表达或敲除 小鼠系），以在生理学相关环境中以细胞类型特异性方式测试基因功能。因此，在本发明中， 虽然许多不同的细胞类型已被确定和分子概况，关键基因，驱动许多 发育和再生过程仍不完全清楚。这种巨大的知识差距 这对开发皮肤病的治疗方法提出了重大障碍。 为了解决这一差距并展示快速功能遗传学如何能够在干细胞领域实现新发现- 利基相互作用，我们将首先建立腺相关病毒（AAV）工具包，以扩大该领域的能力， 在小鼠的多种真皮细胞类型中的功能遗传学。这一目标扩大了我们目前在使用AAVs方面的成功 到真皮细胞，目标是建立工具，使所有皮肤研究人员能够修改基因， 在真皮群体如真皮成纤维细胞和DP中快速表达。我们最近开发了 并在皮肤上进行SHARE-seq，这是一种高通量单细胞测序方法， 测量细胞内的染色质可及性（单细胞ATACseq）和基因表达（单细胞RNAseq）。 相同单元SHARE-seq数据使我们能够通过计算推断关键的调控元件（增强子，启动子） 这进一步使得能够构建细胞类型特异性AAV工具。 我们知道所提出的策略是可行的，因为我们已经用它来构建可以操纵基因的工具， 在竖皮利肌（APM）中表达，这是一种目前缺乏特异性Cre/CreER构建体的细胞类型。 APM是毛囊干细胞（HFSC）的一种新兴的小生境细胞类型。然而，分子机制 对杀伤人员地雷如何调节HFSC行为的了解仍然很少。在Aim 2中，我们将使用我们的AAV工具来 发现APM衍生的分泌因子，调节HFSC的激活和维持。总的来说，这些 结果将为皮肤社区提供急需的工具，以加速不同主题的研究， 可能与理解和潜在地治疗广泛的脱发病症有关。由于AAVs 无毒和无免疫原性，并且由于许多关键的组织特异性调节元件保留了它们的特异性， 跨物种，有一个令人兴奋的潜力，联合收割机结合我们的生物学发现与我们的技术进步 在不久的将来开发新的基因治疗策略来治疗这些皮肤病。

Project Summary Skin stem cells are heavily influenced by signals from their niches including different fibroblasts populations. While our ability to isolate and molecularly profile diverse cell types has improved drastically in the past decade, a major roadblock in identifying key genes driving stem cell-niche interactions is the lengthy process of generating the genetic models needed (e.g., cell-type specific Cre or CreER, and overexpression or knockout mouse lines) to test gene functions in a cell-type specific manner in a physiologically relevant context. As such, while many different cell types have been identified and molecularly profiled, the critical genes that drive many developmental and regeneration processes remain incompletely understood. This substantial knowledge gap presents a significant impediment to developing therapies for skin diseases. To address this gap and to showcase how rapid functional genetics can enable new discoveries in stem cell- niche interactions, we will first build adeno-associated viral (AAV) toolkits to expand the field’s capacity for rapid functional genetics in multiple dermal cell types in mice. This aim expands on our current success in using AAVs to transduce dermal cells, with the goal of building tools that allows all skin researchers to modify gene expression rapidly in dermal populations such as the dermal fibroblasts and DP. We have recently developed and conducted SHARE-seq on the skin, a high-throughput single cell sequencing method that simultaneously measures chromatin accessibility (single cell ATACseq) and gene expression (single cell RNAseq) within the same cell. SHARE-seq data allow us to computationally infer key regulatory elements (enhancers, promoters) of signature genes for distinct cell types, which further enables the construction of cell-type specific AAV tools. We know the proposed strategy is feasible, because we have used it to build tools that can manipulate gene expression in the arrector pili muscles (APMs), a cell type that currently lacks specific Cre/CreER constructs. APMs are an emerging niche cell type for hair follicle stem cells (HFSCs). However, the molecular mechanisms by which APMs regulates HFSC behavior remain poorly understood. In Aim2, we will use our AAV tools to discover APM-derived secreted factors that regulate HFSC activation and maintenance. Collectively, these results will provide the skin community with much-needed tools to accelerate research in diverse topics, and may be relevant for understanding and potentially treating a wide range of alopecia conditions. Since AAVs are non-toxic and non-immunogenic, and since many key tissue-specific regulatory elements retain their specificity across species, there is an exciting potential to combine our biological findings with our technical advancements to develop novel gene therapy strategies to treat these skin diseases in the near future.