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

摘要 舌是一种异质组织，包括味觉菌状乳头（FP）和非味觉丝状乳头（FP）。 乳头（FILIF）在前区。FP和FILIF的维持需要基底上皮细胞的紧密调节， 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细胞， 亚位点的舌头，表明基因调控的显着差异。了解那些 调节这些不同的味觉和非味觉上皮细胞的维持将提供基本的洞察力， 控制舌头功能的机制。我们的数据证实上皮细胞中的Hedgehog（HH）信号是 对成年人的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 效应基因和靶基因Gli 1仅限于FP，而相关的转录因子和关键HH途径 作为组成部分，Gli 2在整个基底舌上皮中表达，包括FP和FILIF。虽然我们 虽然已经证明上皮Gli 2在FP维持中的重要作用，但在 了解GLI 2如何调节FP稳态。此外，虽然对于许多口腔功能是必不可少的，但非味觉功能是不可或缺的。 FILIF体内平衡仍然研究不足。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特征。我们将使用Gli 1报告基因， 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， 采用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GLI 2 ChIP-seq同时，使用新的FLAG标记的敲入Gli 2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 而非味觉的FILIF保持完整。值得注意的是，服用sonidegib的患者报告说， 味觉障碍和味觉丧失。建立在研究的基础上，以了解味觉改变的机制， 通过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治疗后Gli 1的表达被消除，我们的初步数据表明，Gli 2的表达是 保留在FP上皮中。在目的2中，为了了解HH通路阻断的确切效果和 在FP和FILIF中激活Gli 2，我们将依靠类似的实验方法结合RNA-seq和GLI 2 ChIP-seq.获得的数据将有助于理解和说明味觉中断的机制 在接受HH通路抑制剂药物的患者中，可能导致识别新的开发靶点 的治疗方法。原则上，定义不同味觉中的基因表达和调控 (Gli1+; Gli 2+）和非味觉（Gli 1-; Gli 2+）上皮细胞将提供关于 舌稳态总的来说，这一建议将为深入了解遗传， FP和FILIF基底上皮的分子和功能结构以及这些细胞中的HH信号调节 特殊的味觉和非味觉语言环境。

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.