Defining gene expression and regulation in lingual taste and non-taste papilla epithelium

定义舌味和非味乳头上皮的基因表达和调控

• 批准号: 10116729
• 负责人: Archana Kumari
• 金额: $ 10.14万
• 依托单位: ROWAN UNIVERSITY SCHOOL/OSTEOPATHIC MED
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-03-01 至 2022-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10116729
• 关键词: Defining; gene; expression; regulation; lingual

Abstract The tongue is a heterogeneous tissue that comprises taste fungiform papillae (FP) and non-taste filiform papillae (FILIF) in the anterior region. Maintenance of FP and FILIF requires tight regulation of basal epithelial cell renewal and subsequent proper differentiation. The basal epithelia of FP and FILIF differentiate into distinct sub-sites of the tongue, suggesting significant differences in gene regulation. Understanding the genes that regulate the maintenance of these distinct taste and non-taste epithelia will provide fundamental insight into the mechanisms that control tongue function. Our data establish that Hedgehog (HH) signaling in epithelial cells is essential for adult FP maintenance. Strikingly, within the epithelium, expression of the HH transcriptional effector and target gene, Gli1, is restricted to the FP, while the related transcription factor and key HH pathway component, Gli2 is expressed in the entire basal lingual epithelium, including FP and FILIF. Although we have demonstrated an essential role for epithelial Gli2 in FP maintenance, there remains a major gap in understanding how GLI2 regulates FP homeostasis. Further, while essential for many oral functions, non-taste FILIF homeostasis remains understudied. We hypothesize that FP and FILIF epithelia have distinct gene profiles that differentially regulate lingual tissue homeostasis. We will use Gli1 reporters to efficiently distinguish between taste and non-taste epithelia. In Aim 1 we will discover differentially expressed genes and their regulation by HH signaling in the FP and FILIF epithelial cell populations employing RNA-seq simultaneously with GLI2 ChIP-seq, using a novel FLAG-tagged knock-in Gli2 allele. Our published data show that inhibition of HH signaling using the cancer drug sonidegib is sufficient to drive taste organ and sensation loss whereas the non-taste FILIF remain intact. Notably, patients who take sonidegib report severe taste dysgeusia and ageusia. Building on studies to understand the mechanisms of taste alteration after pharmacological inhibition of HH pathway, we will determine deregulated genes and their function. Although expression of Gli1 is eliminated after sonidegib treatment, our preliminary data indicate that Gli2 expression is retained in the FP epithelium. In Aim 2, to understand the exact effects of HH pathway blockade and role of active Gli2 in FP and FILIF, we will rely on similar experimental approaches combining RNA-seq and GLI2 ChIP-seq. The data obtained will help to understand and illustrate mechanisms that underlie taste disruptions in patients receiving HH pathway inhibitor drugs and may lead to identification of novel targets for development of therapeutic approaches. Principally, defining gene expression and regulation in the distinct taste (Gli1+;Gli2+) and non-taste (Gli1-;Gli2+) epithelia will confer advanced knowledge about the mechanisms of tongue homeostasis. Overall, this proposal will lay the foundation for an in-depth understanding of the genetic, molecular and functional architecture of the FP and FILIF basal epithelium and HH signaling regulation in these particular taste and non-taste lingual environments.

抽象的 舌头是一种异质组织，由味觉菌状乳头 (FP) 和非味觉丝状乳头组成 乳头 (FILIF) 位于前部区域。 FP和FILIF的维持需要基底上皮的严格调节 细胞更新和随后的适当分化。 FP 和 FILIF 的基底上皮分化为不同的 舌头的亚位点，表明基因调控存在显着差异。了解基因 调节这些独特的味觉和非味觉上皮细胞的维持将为我们提供基本的见解 控制舌头功能的机制。我们的数据表明上皮细胞中的 Hedgehog (HH) 信号传导是 对于成人 FP 维持至关重要。引人注目的是，在上皮细胞内，HH 转录因子的表达 效应子和靶基因Gli1仅限于FP，而相关转录因子和关键HH通路 Gli2 在整个基底舌上皮中表达，包括 FP 和 FILIF。虽然我们有 证明了上皮细胞 Gli2 在 FP 维持中的重要作用，但在 了解 GLI2 如何调节 FP 稳态。此外，虽然对许多口腔功能至关重要，但非味觉 FILIF 体内平衡仍未得到充分研究。我们假设 FP 和 FILIF 上皮细胞具有不同的基因 差异调节舌组织稳态的概况。我们将使用 Gli1 记者来有效地 区分味觉上皮和非味觉上皮。在目标 1 中，我们将发现差异表达的基因 使用 RNA-seq 在 FP 和 FILIF 上皮细胞群中通过 HH 信号传导对其进行调节 与 GLI2 ChIP-seq 同时进行，使用新型 FLAG 标记的敲入 Gli2 等位基因。我们公布的数据显示 使用抗癌药物 Sonidegib 抑制 HH 信号传导足以驱动味觉器官和感觉 损失，而无味道的 FILIF 保持完整。值得注意的是，服用索尼吉布的患者报告有严重的味觉 味觉障碍和味觉障碍。以了解味觉改变机制的研究为基础 HH 途径的药理学抑制，我们将确定失调的基因及其功能。虽然 Sonidegib处理后Gli1的表达被消除，我们的初步数据表明Gli2的表达是 保留在 FP 上皮中。在目标 2 中，了解 HH 通路阻断的确切效果以及 FP 和 FILIF 中的活性 Gli2，我们将依靠类似的实验方法结合 RNA-seq 和 GLI2 ChIP 测序。获得的数据将有助于理解和说明味觉破坏的机制 在接受 HH 通路抑制剂药物的患者中，可能会导致新的开发靶标的确定 的治疗方法。主要是定义不同味道中的基因表达和调控 （Gli1+；Gli2+）和非味觉（Gli1-；Gli2+）上皮细胞将赋予有关机制的高级知识 舌头稳态。总体而言，该提案将为深入了解遗传、 FP 和 FILIF 基底上皮的分子和功能结构以及这些中的 HH 信号传导调节 特殊味觉和非味觉语言环境。