The long-term objective of this application is to understand how single cell diversity is generated in the nervous system, and to determine the function of this diversity in neural circuit assembly. This objective will be pursued through the analysis of the clustered protocadherins (Pcdhs), which are cadherin-family cell surface proteins that engage in strictly homophilic interactions. A unique feature of the Pcdh system is the potential for generating enormous cell surface diversity through stochastic promoter choice, alternative pre-mRNA splicing, and a combination of mono- and bi-allelic expression at a single-cell level. To address the mechanisms involved in promoter choice, the functional organization of the Pcdh gene cluster will be studied through the identification and characterization of transcriptional regulatory sequences (promoters and enhancers), and the identification of the proteins that bind specifically to these sequences through whole genome Chromatin Immuno-Precipitation and deep sequencing (ChIP-Seq) experiments. In addition, Chromosome Conformation Capture (3C) methods will be used to identify specific interactions between regulatory sequences. The central focus will be on correlating binding and DNA looping interactions with the transcriptional activation or repression of specific Pcdh alternative isoform. In addition, the role of chromatin structure and DNA methylation will be studied using whole genome ChIP-Seq and whole methylome technologies. These studies will be carried out using well-characterized human neuroblastoma cell lines, as well as in the olfactory sensory system of the mouse, which affords the opportunity to study Pchd gene expression and chromatin structure during development in vivo. The function of Pcdhs in neural circuit assembly will be addressed using a comprehensive, well-characterized set of Pcdh deletion mutants in the mouse. Much of the effort will focus on the Pcdha gene cluster and its possible role in serotonergic neuron projection and function. Selected mutants will be subjected to extensive behavior analyses, and the effects of mutations on neural circuit assembly will be studied in vivo using in situ hybridization, confocal imaging, electron microscopy and genetic methods for studying cell lineage and cell-specific knockouts. The question of the function of Pcdh diversity will be approached using a 'uni-identiy' a,b,g specifically in the olfactory sensory system. The effects of expressing this uni-identity construct on olfactory neural circuit assembly will be studied using optical imaging methods and antibody staining. Taken together, these studies should provide significant new insights into the generation and function of single cell diversity i the brain.
Understanding the basis of neural connectivity in the brain is critical to the understanding and ultimate treatment of a variety of neurological disorders, ranging from autism, to bipolar disease and schizophrenia, as well as mood disorders such as depression and anxiety. In addition, inappropriate expression of the clustered protocadherins has been implicated in a variety of human cancers, including kidney, colorectal and breast cancer. The studies proposed here should provide important new insights into the function of the clustered Pcdhs in neural circuit assembly, and in their role in mental health disorders and cancer.
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