Characterization of progenitor populations in adult taste epithelium

项目摘要 味觉感受器细胞（TRC）不断地从成体干/祖细胞中被替换，并且这种替换的保真度（fidelity）是不稳定的。 这个过程是我们味觉相对恒定的基础。然而，许多癌症治疗方法 我们认为这是由于味觉细胞更新的扰动。Wnt/β-catenin和Hedgehog通路 与许多癌症有关，许多药物已经并将继续开发针对这些癌症的药物。 这些药物总是会导致患者的味觉功能障碍。味觉干细胞亚群 表达Wnt靶基因Lgr 5和Hedgehog靶基因Gli 1，以及Wnt和Hedgehog途径 已经显示在体内调节味觉细胞更新。因此，从长远来看， Wnt和刺猬敏感干细胞在味觉稳态中的关系，如这里所提出的，将揭示 这些祖细胞如何被化疗药物破坏，导致味觉功能障碍， 制定减轻味觉障碍的战略。在我们的应用中，我们提出测试明确的假设， LGR 5+和GLI 1+干细胞在小鼠轮廓味觉乳头中的功能关系。 假设1：表达高水平LGR 5的祖细胞是缓慢循环的多能干细胞， 快速增殖的GLI 1 +/LGR 5低/阴性祖细胞直接产生TRCs。 假设2：在LGR 5+细胞消融后，GLI 1+祖细胞扩展其重建轮廓蛋白的潜力 上皮并产生新的LGR 5+干细胞。 为了测试这些想法，我们结合联合收割机体内分子遗传学，体外生产舌类器官， 细胞转录组分析-所有的方法，我们已经成为熟练。在目标1中，我们测试 LGR 5与GLI 1祖细胞产生味细胞丰富的类器官的能力，并进一步评估 每种祖细胞类型的谱系产生依赖于Wnt信号传导的程度。在目标2中， 探索GLI 1+祖细胞在遗传后再生味觉细胞和LGR 5+干细胞的能力。 LGR 5+细胞的消融。在目标3中，我们将联合收割机时间细粒度谱系追踪与单细胞RNA相结合 测序以转录组学地定义连续产生每种功能性味道的细胞谱系 细胞类型，即，神经胶质样细胞和甜、苦、鲜味、盐和酸TRCs。总括而言，我们建议的研究将 导致我们对维持我们的细胞和分子机制的理解取得重大进展。 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.