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

摘要 舌头是一种异质组织，由味觉真菌状乳头(FP)和非味觉丝状组织组成 前部乳头(FILIF)。FP和FILIF的维持需要对基底上皮细胞的严格调控 细胞更新和随后的适当分化。FP和FILIF的基底上皮细胞分化为不同的 舌子的不同部位，表明在基因调控方面存在显著差异。了解这些基因 调节这些独特的味觉和非味觉上皮的维持将提供基本的洞察 控制舌头功能的机制。我们的数据表明，上皮细胞中的Hedgehog(HH)信号是 对成人FP维护至关重要。引人注目的是，在上皮内，HH转录的表达 效应器和靶基因Gli1仅限于FP，而相关的转录因子和关键的HH途径 Gli2在包括FP和FILIF在内的整个舌基底部上皮细胞中均有表达。尽管我们有 证明了上皮Gli2在FP维持中的重要作用，在以下方面仍然存在重大差距 了解GLI2如何调节FP动态平衡。此外，虽然对许多口腔功能是必不可少的，但无味 FILIF动态平衡仍未得到充分研究。我们假设FP和FILIF上皮细胞有不同的基因 差异调节舌组织动态平衡的概况。我们将利用Gli1记者高效地 区分味觉和非味觉上皮。在目标1中，我们将发现差异表达的基因和 利用RNA-seq技术在FP和FILIF上皮细胞群中HH信号对它们的调节 与GLI2 CHIP-SEQ同步，使用一种新的标志标记的敲入Gli2等位基因。我们公布的数据显示 使用抗癌药物sonidegib抑制HH信号足以驱动味觉器官和感觉 损失，而无味道的滤纸则保持完好。值得注意的是，服用索尼吉布的患者报告味道很重。 感觉障碍和失语。建立在研究基础上，以了解口感变化的机制 药物抑制HH途径，我们将确定解除调控的基因及其功能。虽然 Sonidegib治疗后Gli1的表达被消除，我们的初步数据表明Gli2的表达是 滞留在FP上皮中。在目标2中，了解HH通路阻断的确切效果和 在FP和FILIF中激活Gli2，我们将依靠类似的实验方法结合RNA-seq和Gli2 芯片顺序所获得的数据将有助于理解和说明味觉紊乱背后的机制 在接受HH途径抑制剂药物的患者中，并可能导致发现新的开发靶点 治疗方法。主要是定义不同口味中的基因表达和调控 (Gli1+；Gli2+)和无味觉(Gli1-；Gli2+)上皮细胞将提供关于 舌头动态平衡。总体而言，这一建议将为深入了解基因、 FP和FILIF基底层上皮的分子和功能构筑及其HH信号调控 特殊的味觉和非味觉的语言环境。

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.