Tripotent Lgr5 stem cells in the posterior tongue generate non-taste lingual, taste and salivary gland cells [scRNA-seq]

The circumvallate papillae (CVP) and foliate papillae (FoP) of the posterior tongue contain taste buds in close proximity to specialized salivary glands, known as von Ebner and minor salivary glands, respectively. The developmental relationship between taste buds and these salivary glands remains largely unexplored. Lineage tracing studies in mice have revealed that Lgr5 marks taste bud stem cells. Here, we report single-cell RNA sequencing of the entire CVP and FoP of mice, yielding transcriptional profiles of cells from tongue surface epithelium, taste buds and the associated salivary glands. We unveiled a developmental trajectory in which taste buds, the associated salivary glands and the non-taste tongue surface epithelium originate from a common Lgr5+ cell. We describe long-term organoid culture conditions for these cells and confirm their tripotency at the clonal level in vitro. CVP and FoP harbor chemosensory units consisting of taste bud and salivary gland cells derived from the same parental Lgr5+ stem cell. Overall design: Circumvallate papillae (n=20) and foliate papillae (n=40) were send for single-cell RNA seq. Lineage trajectory analysis revealed a potential bi/tripotent Lgr5+ stem cell in the posterior tongue. Subsequent scRNA-seq analysis of clonal cultures derived from a single Lgr5+ stem cell presented its tripotent nature. Next, in-vivo mulitcolour lineage tracing confirmed this phenomenon. Studying the cell fate decisions resulted in a putative role for Foxe1 in the lineage trajectory. Bulk RNA-sequencing of knock-out of Foxe1 in the posterior tongue organoids revealed an ablation of mature saliva markers and a more progenitor-like phenotype.

舌后部的轮廓乳头（circumvallate papillae, CVP）与叶状乳头（foliate papillae, FoP）内分布有味蕾，且这些味蕾分别紧邻特化唾液腺——冯埃布纳腺（von Ebner）与小唾液腺。目前学界对味蕾与这类唾液腺之间的发育关联仍知之甚少。小鼠体内的谱系示踪研究已证实，Lgr5可标记味蕾干细胞。本研究对小鼠完整的轮廓乳头与叶状乳头开展单细胞RNA测序（single-cell RNA sequencing, scRNA-seq），获取了舌表面上皮细胞、味蕾及相关唾液腺细胞的转录组图谱。本研究揭示了一条发育轨迹：味蕾、相关唾液腺与非味觉舌表面上皮细胞均起源于同一种Lgr5阳性（Lgr5+）细胞。本研究建立了这类细胞的长期类器官（organoid）培养体系，并在体外克隆水平证实了其三向分化潜能。轮廓乳头与叶状乳头包含化学感受单元，该单元由源自同一亲本Lgr5+干细胞的味蕾细胞与唾液腺细胞构成。整体实验设计：我们共收集20个轮廓乳头与40个叶状乳头用于单细胞RNA测序。谱系轨迹分析显示，舌后部存在一种兼具双向/三向分化潜能的Lgr5+干细胞。后续针对单个Lgr5+干细胞来源的克隆培养物开展的单细胞RNA测序分析，证实了其三向分化潜能。随后，体内多色谱系示踪实验验证了这一现象。通过对细胞命运决定的研究，我们推测Foxe1在该发育轨迹中发挥调控作用。对舌后部类器官中Foxe1敲除样本的批量RNA测序结果显示，成熟唾液标志物的表达被完全消除，且细胞呈现出更接近祖细胞的表型。