Elucidating the molecular mechanisms of sensory regeneration

National Institute Of Neurological Disorders And Stroke

Investigators

Abstract

Renovation of lab facilities in the Porter Research Building were completed at the end of September 2021, at which point experiments were able to resume, having been delayed by pandemic- and other construction-related setbacks. Experiments were again delayed for two months at the beginning of 2023 by catastrophic flooding and flood-related remediation efforts in Porter/Building 35. Two years on at NIH, the Molecular Neurobiology Section continues to grow and build on past accomplishments. The lab's expertise involves using a variety of approaches to elucidate the molecular and cellular mechanisms regulating olfactory stem cells and olfactory neurogenesis in the mouse. Previous studies in the lab, prior to its move to NIH, used single cell sequencing to identify potential genes involved in olfactory stem cell activation and landed on two promising candidate intracellular signaling pathways -- EGFR and Wnt signaling. In one current area of inquiry, the lab is using genetic gain- and loss-of-function (GOF and LOF) mouse models to investigate the roles of Wnt and EGFR signaling in regulating stem cell proliferation and differentiation. These projects have relied on clonal lineage tracing of GOF and LOF cells to determine the respective roles of these pathways in olfactory stem cell proliferation and fate after acute injury. A related study seeks to understand the role of EGFR signaling in olfactory metaplasia after repeat injury to the OE. A major goal of the lab is to use single-cell multiomics (sequencing of the transcriptome/RNA-seq and accessible chromatin/ATAC-seq) to elucidate the gene regulatory networks involved in olfactory stem cell dynamics and to identify how these networks are altered by perturbation of EGFR signaling or Wnt signaling. To this end, the lab has acquired its own fluorescence activated cell sorter (FACS) and single cell capture device (10x Chromium X) and has established its own in-house pipeline for purifying, capturing, and generating sequencing libraries of single cells from genetically modified mice. Pilot studies in the lab have confirmed the ability to generate high quality sequencing data from these cells, including both uninjured cells and cells from injured genetically altered olfactory epithelium. These studies provide the foundation for upcoming sequencing of lineage traced genetically altered olfactory epithelial cells at various time points after injury. In addition, the lab has established an in vitro model of olfactory epithelium regeneration. This approach provides an alternative to the in vivo gain- and loss-of-function models, allowing for higher throughput analysis of potential regulators of olfactory stem cell proliferation and differentiation. The lab has confirmed that primary olfactory epithelial cells survive in culture for up to a month and are amenable to transient transfection of reporter plasmids. Finally, the lab is investigating the hypothesis that there is a sub-population of quiescent olfactory stem cells that are primed for activation and may be fate restricted. Using statistical analysis of previously generated RNA-seq and ATAC-seq data, the lab has identified candidate genes that are not expressed but appear to occupy accessible chromatin in uninjured stem cells, which then become expressed in stem cells upon injury. Validation of these candidates using multiplex fluorescent in situ hybridization and has identified several genes that are expressed heterogeneously throughout the olfactory stem cell population shortly after injury. The lab is also actively interested in the gene regulatory networks that prime these cells for rapid expression of specific genes. A manuscript describing these findings is in final stages of preparation; we anticipate posting to BioRxiv and submission for publication this Fall. Throughout the coming fiscal year, the lab's focus will be on studies that employ clonal analysis in vivo and in vitro to determine whether and how Wnt and EGF signaling regulate stem cell proliferation and fate. In addition, the lab will continue to use single cell sequencing of lineage-traced FACS-purified olfactory cells to elucidate the gene regulatory networks important for distinct developmental lineages of cells as they transition from early stem cell states and eventually reach terminal differentiation.