An in vitro model of acute horizontal basal cell activation reveals dynamic gene regulatory networks underlying the acute activation phase

Unlike most of the adult nervous system, the olfactory epithelium (OE) can regenerate neurons lost during normal homeostasis or after injury. This life-long regeneration is propagated by two populations of stem cells, the actively proliferating globose basal cells (GBCs) and the dormant horizontal basal cells (HBCs). HBCs only activate and contribute to epithelial regeneration in the context of severe injury, and this activation is mediated by the loss of the transcription factor Tp63. While the HBC differentiation trajectories that occur after activation have been described with lineage tracing and single-cell RNA seq experiments, the immediate consequences of injury on HBC gene expression and fate commitment have not been explored. We present an in vitro model of the acute activation process for HBCs in response to treatment with phorbol 12-myristate 13-acetate (PMA) to explore the molecular underpinnings of these early activation events. We find that treating HBCs with PMA induces the rapid degradation of TP63, and we find that this effect is partially reversed when cells are allowed to recover in maintenance media. Using bulk RNA sequencing we found that PMA-treated HBCs pass through various stages of acute activation identifiable by specific gene regulatory signatures. These transcriptomic phases are associated with varying degrees of plasticity with regards to activated HBCs ability to engraft in transplant models.与成体神经系统的多数组织不同，嗅觉上皮（olfactory epithelium, OE）可再生正常稳态过程中或损伤后丢失的神经元。这种终生性的再生过程由两类干细胞群体驱动：活跃增殖的球形基细胞（globose basal cells, GBCs）与静息态的水平基细胞（horizontal basal cells, HBCs）。水平基细胞仅在严重损伤的情境下才会被激活并参与上皮再生，而该激活过程由转录因子Tp63的缺失所介导。尽管已有研究通过谱系示踪（lineage tracing）与单细胞RNA测序（single-cell RNA seq）实验描述了激活后水平基细胞的分化轨迹，但损伤对水平基细胞基因表达与命运决定的即时影响尚未得到探索。本研究构建了一套体外模型，用于模拟水平基细胞经佛波醇12-肉豆蔻酸酯13-乙酸酯（phorbol 12-myristate 13-acetate, PMA）处理后的急性激活过程，以探究这些早期激活事件的分子基础。我们发现，用PMA处理水平基细胞可诱导TP63快速降解，且当细胞在维持培养基中恢复培养时，该效应可被部分逆转。通过批量RNA测序（bulk RNA sequencing）分析，我们发现经PMA处理的水平基细胞会经历多个急性激活阶段，这些阶段可通过特定的基因调控特征加以区分。这些转录组学阶段与激活后的水平基细胞在移植模型中的定植能力所对应的不同可塑性程度相关。