This project focuses on the genetic and epigenetic factors controlling the regenerating olfactory neuroepithelium in vertebrates. The olfactory system is distinctive in that it maintains a steady state turnover of sensory neurons by retaining, into adulthood of the animal, a stem cell population. This system is capable of regenerating the neuroepithelium and its sensory neurons after lesion or other significant insult. A classic experimental strategy is to perturb a system by lesion and then analyze the biological response to the challenge. We have chosen to use this proven approach utilizing transgenic mouse lines to generate genetic mutants for the analysis of """"""""pseudoallele"""""""" perturbations of the olfactory neuroepithelium. The transgenic lines would include both the targeted expression of an exogenous gene to the olfactory neuronal lineage and the attenuated expression of a known gene endogenous to the olfactory neuronal lineage by antisense technology. Through these models, we can begin to assess the consequences of genetic perturbations within the contest of cell turnover within the olfactory neuroepithelium. Hopefully, the phenotypes observed will prove useful in answering several biological questions concerning olfactory neuroepithelial cell dynamics and, perhaps, raise new questions. Specifically, we intend to construct transgenes driven by the Olfactory Marker Protein (OMP) promoter which will selectively direct transgene expression to the olfactory neuronal lineage. The first targeted gene expression will be the viral oncogene SV40 T-ag which should provide a positive influence on cell genesis. Another transgene will generate pseudoalleles for a gene strongly implicated in the developmental process - GAP 43, a growth associated protein thought to be involved in neuronal process formation. This gene is known to be expressed within the olfactory neuroepithelium in the population of neurons that are relatively immature and likely to be actively extending neurites. Through the use of antisense expression we will generate pseudoalleles for GAP 43 where expression levels will be significantly attenuated - effectively creating null alleles of varying degrees specifically within the olfactory neuroepithelium. The phenotypic consequences of transgenic perturbation will be assessed over an extended time course. Specifically, we are interested in changes within the morphology of the olfactory neuroepithelium, mitotic cell populations (the stem cells), and patterns of expression for several specific and relevant genes. These analyses will involve in situ hybridization histochemistry/immunocytochemical analysis of relevant genes and proteins diagnostic for cell populations within the olfactory neuroepithelium.
