Role of GLI Proteins in Horizontal Basal Cells During Olfactory Epithelium Regeneration

摘要 嗅觉上皮(OE)是一种特殊的神经上皮，由多种细胞类型组成，包括嗅觉 感觉神经元(OSNs)、支持细胞(SUS)和微绒毛细胞(MVC)由两个 假定干细胞群体：快速分裂球状基底细胞(GBCs)，水平相对静止 基底细胞(HbCs)。虽然HBCs和GBCs都有助于OE的再生，但信号通路 对这一过程的控制还不是很清楚。最近的研究表明，HBCs含有初级纤毛，一种细胞 协调来自多条路径的信号的细胞器。值得注意的是，初级纤毛对于 Hhedgehog(HH)在脊椎动物中的信号转导，使HH途径成为控制的一个有吸引力的候选者 对HBC功能的影响。此外，HH信号在另一个化学感觉器官--舌头中也很重要，在那里 药物和遗传的HH通路阻断会导致味蕾的异常维持。Gli蛋白是 HH途径的转录效应物-GLI1仅作为转录激活因子发挥作用，并且是 也是HH信号的靶标；GLI2是HH途径的主要转录激活因子；反之，GLI3 在很大程度上充当转录抑制因子。我使用Lacz报告鼠的初步数据表明，Gli2是 Gli3在所有HbC中表达，而Gli3在HbC的一部分中表达。此外，Gli2表达式在 严重受伤后的OE。为了评估HBCs中可能的GLI功能，我建议使用多西环素诱导的 在HBC中特异性地表达GLI2的构成活性形式以刺激HH途径(K5-RTTA； Teto-Gli2∆N)。我的初步数据表明，GLI2的HBC特异性激活导致了 然后无法逃脱其HBC命运的HBC在他巴唑之后分化和重建OE- 致伤。基于这些初步数据，我假设GLI2和GLI3是OE所必需的 损伤后再生。我将首先通过使用细胞类型来表征GLI1-3的表达来检验这一假设 未受伤的成人OE和受伤的成人OE的特异性标志物(目标1)。要访问GLI函数，我将使用 前述K5-rTTA；TETO-GLI2∆N小鼠检测结构性GLI激活功能的后果 在未受伤和受伤的小鼠中，并反过来使用K5-rtta；Teto-Gli2∆C4小鼠来检测 未损伤和损伤小鼠的结构性GLI抑制功能的后果(目标2)。评估 内源性GliI在HBCs中的作用，我将分别删除Gli2和Gli3，并使用K5-RTTA；Teto- Cre、Gli2fl/fl、Gli3fl/fl小鼠，并评估对损伤后OE维持和再生的影响。这些 实验将阐明控制OE再生的信号通路，并加深我们对 成人神经发生的特征。

ABSTRACT The olfactory epithelium (OE) is a specialized neuroepithelium comprised of several cell types, including olfactory sensory neurons (OSNs), sustentacular cells (SUS), and microvillar cells (MVC), which are replenished by two presumed stem cell populations: rapidly dividing globose basal cells (GBCs), and relatively quiescent horizontal basal cells (HBCs). While HBCs and GBCs both contribute to OE regeneration, the signaling pathways that control this process are not well understood. Recent work indicates that HBCs contain primary cilia, a cellular organelle that coordinates signals from multiple pathways. Notably, primary cilia are essential for proper Hedgehog (HH) signal transduction in vertebrates, making the HH pathway an attractive candidate in the control of HBC function. Further, HH signaling is important in another chemosensory organ, the tongue, where pharmacologic and genetic HH pathway blockade results in abnormal taste bud maintenance. GLI proteins are the transcriptional effectors of the HH pathway – GLI1 functions exclusively as a transcriptional activator and is also a target of HH signaling; GLI2 is the major transcriptional activator of the HH pathway; conversely, GLI3 acts largely as a transcriptional repressor. My preliminary data using lacZ reporter mice suggest that Gli2 is expressed in all HBCs, while Gli3 is expressed in a subset of HBCs. Additionally, Gli2 expression expands in the OE following severe injury. To assess possible GLI function in HBCs, I propose to utilize doxycycline-inducible expression of a constitutively active form of GLI2 to stimulate the HH pathway specifically in HBCs (K5-rtTA; tetO-GLI2∆N). My preliminary data indicate that HBC-specific activation of GLI2 causes hyperproliferation of HBCs that are then unable to escape their HBC fate to differentiate and reconstitute the OE after methimazole- induced injury. Based on these preliminary data, I hypothesize that GLI2 and GLI3 are necessary for OE regeneration post-injury. I will test this hypothesis by first characterizing the expression of GLI1-3 using cell type specific markers in both the uninjured and injured adult OE (Aim 1). To access GLI function, I will use the aforementioned K5-rtTA; tetO-GLI2∆N mice to examine the consequences of constitutive GLI activator function in both uninjured and injured mice, and conversely use K5-rtTA; tetO-GLI2∆C4 mice to examine the consequences of constitutive GLI repressor function in uninjured and injured mice (Aim 2). To assess endogenous GLI function in HBCs, I will delete Gli2 and Gli3 individually and in combination using K5-rtTA; tetO- Cre, Gli2fl/fl, Gli3fl/fl mice and assess effects on OE maintenance and regeneration following injury. These experiments will shed light on the signaling pathways that control OE regeneration and further our understanding of adult neurogenesis.