Characterization of progenitor populations in adult taste epithelium

项目总结 味觉感受器细胞(TrCs)不断地从成体干细胞/祖细胞中被取代，而这一点的保真度 过程是我们味觉相对恒定的基础。然而，许多癌症治疗方法令人不安。 味觉，我们假设这是由于味觉细胞更新的扰动。Wnt/？-catenin和Hedgehog通路 与数十种癌症有关，许多药物已经并将继续被开发来针对这些 肿瘤中的通路；这些药物总是会导致患者的味觉障碍。味觉干细胞亚群 表达Wnt靶基因Lgr5和Hedgehog靶基因Gli1，以及Wnt和Hedgehog途径 已经被证明在体内调节味觉细胞的更新。因此，从长远来看，理解功能性 Wnt和Hedgehog敏感干细胞在味觉动态平衡中的关系，如本文所述，将有助于揭示 关于这些祖细胞是如何被导致味觉障碍的化疗药物干扰的，并允许 制定缓解地貌障碍的战略。在我们的应用程序中，我们建议测试显式假设 LGR5和GLI1干细胞在小鼠环状味觉乳头中的功能关系 假设1：表达高水平LGR5的祖细胞是循环缓慢的多能干细胞，能产生 快速增殖的GLI1/LGR5low/neg祖细胞，可直接分化为TRCs。 假设2：在LGR5细胞消融后，GLI1祖细胞扩大其重建环状结构的潜力 并产生新的LGR5干细胞。 为了测试这些想法，我们结合了体内分子遗传学、体外生产舌质器官和单一的 细胞转录组分析--我们已经熟练使用的所有方法。在目标1中，我们测试 LGR5与GLI1前体细胞产生味觉细胞丰富的有机化合物的能力，并进一步评估 每种祖细胞类型的谱系生产在多大程度上依赖于Wnt信号。在目标2中，我们 探讨GLI1前体细胞在遗传后再生味觉细胞和LGR5干细胞的能力 消融LGR5细胞。在目标3中，我们将时间上的细粒度谱系追踪与单细胞rna相结合。 对持续产生每一种功能性味道的细胞谱系进行转录定义的测序 细胞类型，即胶质样细胞和甜、苦、鲜味、盐和酸的TRC。总括而言，我们建议的研究将会 导致我们对细胞和分子机制的理解取得重大进展，这些机制维持我们的 味觉。

PROJECT SUMMARY Taste receptor cells (TRCs) are continually replaced from adult stem/progenitor cells, and the fidelity of this process underlies the relative constancy of our sense of taste. However, a host of cancer therapies perturb taste and we posit this is due to perturbation of taste cell renewal. The Wnt/ß-catenin and Hedgehog pathways are implicated in scores of cancers, and many drugs have been and continue to be developed to target these pathways in tumors; these drugs invariably cause taste dysfunction for patients. Subsets of taste stem cells express the Wnt target gene Lgr5 and the Hedgehog target gene Gli1, and both Wnt and Hedgehog pathways have been shown to regulate taste cell renewal in vivo. Thus, in the long term, understanding the functional relationship of Wnt- and Hedgehog-sensitive stem cells in taste homeostasis, as proposed here, will shed light on how these progenitors are disrupted by chemotherapeutics that cause taste dysfunction, and allow development of strategies to mitigate dysgeusia. In our application, we propose to test explicit hypotheses of the functional relationship of LGR5+ and GLI1+ stem cells in the circumvallate taste papillae of mice. Hypothesis 1: Progenitors expressing high levels of LGR5 are slow cycling, multipotent stem cells that produce rapidly proliferating GLI1+/LGR5low/neg progenitors that give rise directly to TRCs. Hypothesis 2: Upon LGR5+ cell ablation, GLI1+ progenitors expand their potential to reconstitute circumvallate epithelium and give rise to new LGR5+ stem cells. To test these ideas, we combine in vivo molecular genetics, in vitro production of lingual organoids, and single cell transcriptome profiling – all approaches with which we have become skilled. In Aim 1, we test the competency of LGR5 vs GLI1 progenitors to produce taste cell-replete organoids, and further assess the degree to which lineage production by each progenitor type is dependent upon Wnt signaling. In Aim 2, we explore the capacity of GLI1+ progenitors to regenerate both taste cells and LGR5+ stem cells following genetic ablation of LGR5+ cells. In Aim 3, we combine temporally fine-grained lineage tracing with single cell RNA sequencing to transcriptomically define the cell lineages that continually produce each of the functional taste cell types, i.e., glial-like cells and sweet, bitter, umami, salt and sour TRCs. In sum, our proposed studies will lead to significant advances in our understanding of the cellular and molecular mechanisms that maintain our sense of taste.