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Identification of Olfactory Stem Cell Lineage Trajectories and Cell State Transitions


Adult stem cells maintain structure and function of tissues under widely varying conditions, but the molecular and cellular mechanisms that underlie the development of diverse cell types during either tissue maintenance or injury-induced regeneration remain incompletely understood. This dissertation combines lineage tracing with single-cell RNA sequencing and assay for transposase-accessible chromatin (ATAC) sequencing to address the regulation of these phenomena in the olfactory epithelium, one of only a few sites of ongoing adult neurogenesis.

Olfactory stem cells, known as horizontal basal cells (HBCs), contribute to both maintenance and regeneration of the tissue. First, we consider the role of HBCs in homeostatic conditions. Classifying cells on the basis of gene expression, we identify three lineage trajectories arising from HBCs via two developmental bifurcations. We find that sustentacular cells arise by direct fate conversion of HBCs without cell division, as predicted by single-cell RNA-sequencing and validated by clonal lineage tracing. We also establish that multipotency of the HBC population arises from unipotent fate decisions of individual HBCs. Finally, single-cell RNA-sequencing indicates that canonical Wnt signaling promotes neuronal fate choices in HBCs, which we validate in vivo with gain-of-function and loss-of-function experiments.

Second, we consider the contribution of HBCs to injury-induced regeneration of the olfactory epithelium, and whether the HBC injury response differs from homeostatic maintenance. We discovered activated olfactory stem cell states that are both transient and unique to regeneration. The activated stem cells express genes associated with epithelial wound repair in other stem cell niches. These cells are also heterogeneous, giving rise to multiple lineages, including renewed HBCs. Moreover, renewed HBCs are themselves competent to produce differentiated cell types, and do so in a second wave of differentiation several days after injury, bypassing the transient activated states associated with the earliest stages of regeneration.

Finally, we begin investigation of the priming of quiescent HBCs for rapid transcriptional response to injury, using ATAC sequencing to query chromatin accessibility. We find the HBCs to have broadly accessible chromatin at gene promoters corresponding to more differentiated cells in the olfactory lineage, but they maintain a closed conformation over the coding regions of genes that are not actively expressed.

Taken together, these findings contribute to our understanding of how neurogenesis and regeneration are accomplished in a fully-developed, adult sensory tissue. Our integration of single-cell genomics with in vivo lineage tracing lays the groundwork for investigating outstanding questions relating to the development, maintenance, and repair of other tissues. This work may ultimately aid in the development of stem-cell based therapies to replace specialized cell types and tissues lost to damage and disease.

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