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

总结 毛囊（HF）由上皮细胞和真皮细胞组成，是研究毛囊发育的经典模型。 上皮-间充质相互作用控制附件的发展。在HF开发过程中，第一个 形态上独特的真皮群是真皮凝聚物（DC），一种密集的特化细胞群 成熟为毛乳头（DP）。由于DP具有诱导新HF生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，但是遇到了困难， 有限的功效。主要的挑战是无法评估细胞之间的分子差异 在它们的形态变得明显之前。因此，我们缺乏过渡态的分子“路线图”， 直接的血统承诺和形态发生，可以指导忠实的方法来重演这些事件， 体外为了应对这一挑战，我们最近使用了无偏扩散图技术来系统化单细胞 来自小鼠胚胎皮肤的RNA测序（scRNA-seq）数据。利用这项技术，我们发现了一个 分子DC分化轨迹，DC细胞通过其分化的转录状态的推断途径， 通过，在HF形态发生之前和期间。在此图谱的指导下，我们发现真皮Wnt/β-连环蛋白 需要信号传导来进展到DC细胞分化的中间阶段，并且DC细胞是 分子上不同的（Dkk 1+）高度增殖群体的直接静止后代。目前 Dkk 1+细胞通过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小鼠模型，我们的初步数据显示，Dkk 1 + 祖细胞利用两种分子途径产生区分DC起始与DC的DC细胞 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， DC起始和DC扩增利用不同的分子途径来产生DC细胞， Dkk 1+调节DC从增殖到静止的转变是DC分化所必需的 祖先在本研究中，我们使用一种综合的方法来建立一个DC过渡态的时空图 控制着特区的形成在目标1中，我们将转录动力学scRNA-seq数据（RNA速度）与 体内谱系追踪以定义导致DC起始和DC扩增的转变步骤， 成像和定量FISH来空间定位关键的转变步骤。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通路内的关键过渡状态。在目标2中，我们 检查局部增殖在DC形成中的作用和调节过渡的信号（例如雅普/TAZ） 增殖和静止之间的关系。这一补充办法将 克服解剖导致DC细胞命运的早期事件的主要挑战。这个项目的总体目标是 该项目的目标是建立一个高分辨率的路线图，描绘如何形成DC，以加速再生的努力， 同时也为研究其他皮肤附属物的形成提供了实验范例。

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.