Imaging cell-type-specific transcription in living mammalian brain

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Gene expression is a fundamental problem at the core of life science. All cells in an adult organism contain an identical set of genes, but their expression varies widely depending on the context, such as cell type and environmental queues. Transcription is the critical first step in gene expression, which is intricately controlled by complex regulations and the disruption is pathogenic for many diseases. Much knowledge about the mechanism has been gained using fixed cells and sequencing assays. Nonetheless, we still do not understand how the variability of transcription is regulated in living mammals, dynamically in space and time, as the animal performs development, physiology, and cognitive tasks such as learning. In particular, the role of the cis- regulatory elements (i.e., enhancers) plays in the context-dependent transcriptional dynamics is obscure. Here we will develop technology for addressing the current bottleneck. Over the past years, our laboratory has advanced methods for visualizing transcriptional dynamics in the brain of live mouse (‘intravital MS2 technology’). Single molecules of mRNA of a gene of interest have been imaged in a specific cell type by intravital multiphoton microscopy (MPM), revealing previously unknown properties of nascent transcripts in vivo. For the next five years, we will demonstrate more versatile intravital MS2 technology geared toward the functional annotation of the genome. Taking inspirations from the prior success of illuminating the structure and function of the neuronal network in vivo, we will fuse intravital MPM, smart molecular sensors, and genomic engineering to dissect the function of the transcriptional regulatory network in live murine brains. Our research will lay a foundation for unraveling the origin of diverse cell types, plasticity, and neurodegenerative disorders of the mammalian brain.