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

摘要 毛囊(HF)是由上皮细胞和真皮细胞组成的，是研究毛囊的经典模型。 控制附肢发育的上皮-间充质相互作用。在HF开发过程中，第一个 在形态上不同的真皮群体是真皮凝集物(DC)，一群密集的特化细胞 成熟为真皮乳头(DP)。由于民主党拥有诱导新的HF增长的令人尊敬的能力， 已经努力将未分化的成纤维细胞编程为分化的DC/DP细胞，但遇到了 疗效有限。主要的挑战是无法评估细胞之间的分子差异。 在它们从形态上看出来之前。因此，我们缺乏过渡态的分子“路线图” 直接的世系承诺和形态发生，可以指导忠实的方法在 体外培养。为了应对这一挑战，我们最近使用了一种无偏扩散图技术来系统化单细胞 小鼠胚胎皮肤rna测序(scrna-seq)数据。使用这项技术，我们确定了一个 分子DC分化轨迹，一种推测的DC细胞转录状态的途径 通过，在HF形态发生之前和期间。在这张图谱的指引下，我们显示了皮肤中的Wnt/-连结蛋白 DC细胞分化的中间阶段需要信号传递，DC细胞 一个分子上不同(Dkk1)、高度增殖的种群的直接静止后代。目前， Dkk1细胞经历的关键过渡步骤和调节它们的信号仍然未知。 结合创新的计算方法和鼠标模型，我们的初步数据显示，Dkk1 祖细胞利用两条分子途径来产生DC细胞，这是DC起始与DC的区别 在形态发生之前的膨胀过程。我们假设DC的形成是一个动态过程，其中 DC启动和DC扩展利用不同的分子途径来产生DC细胞，并且该信号 Dkk1调控从增殖到静止的转化是DC分化的关键 祖先。在这项资助中，我们使用了一种综合的方法来构建DC过渡态的时空地图 来管理华盛顿特区的形成。在目标1中，我们将转录动力学scRNA-seq数据(RNA速度)与 体内谱系追踪，定义导致DC启动和DC扩展的过渡步骤，与LIVE相结合 成像和定量FISH，以在空间上定位关键的过渡步骤。使用同样的方法，我们将 定义局部上皮信号如何调节不同DC路径中的关键过渡状态。在目标2中，我们将 研究局部增殖在DC形成中的作用以及调节转换的信号(例如YAP/TAZ) 利用遗传小鼠模型研究增殖和静止之间的关系。这一互补的方法将 在剖析导致DC细胞命运的早期事件方面克服重大挑战。这个项目的总体目标是 该项目是建立一个高分辨率路线图，描绘DC如何形成，以加快再生努力， 同时也为研究其他皮肤附属物的形成提供了一个实验范例。

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.