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

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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.