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.
The adult olfactory epithelium (OE) is a regenerative tissue consisting of many specialized cells, including horizontal basal cells (HBCs), a reserve stem cell population, which replenish the OE following severe injury. However, the signaling pathways that control HBC-mediated OE regeneration remain poorly understood. This proposal will investigate the contribution of GLI proteins, the transcriptional effectors of the Hedgehog signaling pathway to the maintenance and regeneration of the adult OE.
