Elucidating the molecular mechanisms of sensory regeneration

National Institute Of Neurological Disorders And Stroke

Investigators

Abstract

The Molecular Neurobiology Section uses a variety of approaches to elucidate the molecular and cellular mechanisms regulating olfactory stem cells and olfactory neurogenesis in the mouse. In the lab's fourth year at NIH, several projects have neared completion, and new projects that build on these findings have been initiated. 1) Genetic investigation of EGFR signaling: 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 signaling pathways -- EGFR and canonical Wnt signaling. Ongoing projects in the lab rely on a combination of approaches in genetic mouse models to identify the roles these pathways play in regulating stem cell proliferation and differentiation: clonal lineage tracing is used to determine the effect of the pathway on cell fate; proliferation and birth-dating analyses are used to determine the pathway's role in cell cycle control, and single-nucleus multiomics (sequencing of the transcriptome/RNA-seq and accessible chromatin/ATAC-seq in a single nucleus) is used to elucidate gene regulatory networks and identify how these networks are altered by genetic perturbation. To this end, the lab has optimized a pipeline for generating high quality multiome sequencing libraries completely in-house, from tissue dissection and FACS purification of stem cells through single nucleus capture, library preparation, and quality control. Over the past two years, we have generated a complete multiome dataset consisting of multiple biological replicates of nine different conditions. Using this complete dataset, consisting of over 100,000 nuclei, we have established a data processing pipeline and are currently developing approaches for analyzing the data to understand EGFR signaling in regeneration of the olfactory epithelium (OE). Similarly, we have generated single nucleus libraries from beta-catenin conditional knockout and conditional over-activation mice before and after injury. Preliminary findings from the complete EGFR dataset indicate that EGFR plays a role in the proliferation of multipotent OE stem cells, termed horizontal basal cells (HBCs), after injury and may help determine the timing of cell cycle exit. Related studies seek to understand the role of EGFR signaling in olfactory metaplasia after repeat injury to the OE and a potential genetic interaction with genes involved in mTOR signaling. In addition, the lab has established a cell culture system for analyzing HBCs isolated from genetically modified mice. This approach provides an alternative to the in vivo model, allowing for complementary studies evaluating potential regulators of OE proliferation and differentiation. For example, HBCs isolated from EGFR-conditional knockout mice appear to form smaller clusters in vitro than HBCs isolated from wild-type mice. 2) Evaluation of the stem cell niche over perinatal development: An additional project in the lab nearing completion is an investigation of the cell fate competence of stem cells in the OE from birth to weaning (postnatal day 0-22 in mice). Using single cell sequencing and genetic lineage tracing of the OE between embryonic day 14 and postnatal day 22 in the mouse, we have traced the developmental lineages of multiple cell types in the OE and observed that plasticity of the stem cell niche gradually decreases over developmental time. A manuscript summarizing the results of this study is currently in preparation for submission for publication. 3) Role of immune signaling in HBC activation: A specific area of interest for the lab is the role of immune signaling in stem cell activation. A new study in the lab addresses the hypothesis that cytokines released after injury act as activation signals to HBCs. Using multiplex fluorescent in situ hybridization (FISH), we have identified the specific expression of cytokine receptors in the OE before and after injury. We are currently using a genetic approach to test whether specific cytokines are required for HBC activation and/or cell fate determination. 4) Epigenetic priming of quiescent HBCs: Finally, the lab is investigating the hypothesis that there is a sub-population of quiescent HBCs that is primed for activation. 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 FISH has confirmed increased expression of these genes in HBCs 1-2 days after injury. The lab is actively interested in further untangling the gene regulatory networks that prime these cells for rapid expression of specific genes. A manuscript describing these findings, currently available as a preprint, is in revision for publication.