Delineating spatiotemporal dynamics of hair follicle dermal niche specification at the single-cell level

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SUMMARY The hair follicle (HF) is composed of an epithelial and dermal population and is a classic model to study the epithelial-mesenchymal interactions governing appendage development. During HF development, the first morphologically distinct dermal population is the dermal condensate (DC), a dense cluster of specialized cells that matures into the dermal papilla (DP). As the DP holds the revered capacity to induce new HF growth, large efforts have been made to program undifferentiated fibroblasts into differentiated DC/DP cells but met with limited efficacy. The principal challenge has been the inability to assess molecular differences between cells before they are morphologically apparent. As such, we lack a molecular “roadmap” of the transition states that direct lineage commitment and morphogenesis that could guide faithful methods to recapitulate these events in vitro. To meet this challenge, we recently used an unbiased diffusion map technique to systemize single-cell RNA sequencing (scRNA-seq) data from mouse embryonic skin. Using this technique, we identified a molecular DC differentiation trajectory, an inferred pathway of transcriptional states through which DC cells pass, before and during HF morphogenesis. Guided by this map, we showed that dermal Wnt/-catenin signaling is required to progress to an intermediate phase of DC cell differentiation and that DC cells are immediate quiescent progeny of a molecularly distinct (Dkk1+), highly proliferative population. Currently, the critical transition steps that Dkk1+ cells pass through and the signals that regulate them remain unknown. Combining innovative computational methods and mouse models, our preliminary data reveal that Dkk1+ progenitors utilize two molecular pathways to generate DC cells that distinguishes DC initiation from DC expansion processes prior to morphogenesis. We hypothesize that DC formation is a dynamic process wherein DC initiation and DC expansion utilize distinct molecular pathways to generate DC cells and that signals that regulate the transition from proliferation to quiescence are essential for DC differentiation by Dkk1+ progenitors. In this grant, we use an integrative approach to build a temporospatial map of DC transition states that govern DC formation. In Aim 1, we will couple transcriptional kinetic scRNA-seq data (RNA velocity) with in vivo lineage tracing to define transition steps that lead to DC initiation and DC expansion, coupled with live imaging and quantitative FISH to spatially locate critical transition steps. Using this same approach, we will define how local epithelial signals regulate key transition states within distinct DC paths. In Aim 2, we will examine the role of local proliferation in DC formation and signals (e.g. YAP/TAZ) that regulate the transition between proliferation and quiescence using genetic mouse models. This complementary approach will overcome major challenges in dissecting the early events that lead to DC cell fate. The overall goal of this project is to build a high-resolution roadmap that delineates how DCs form to accelerate regenerative efforts, while also providing an experimental paradigm to study the formation of other cutaneous appendages.