Canonical Notch Signaling Directs the Fate of Differentiating Neurocompetent Progenitors in the Mammalian Olfactory Epithelium

The adult olfactory epithelium (OE) has the remarkable capacity to regenerate fully both neurosensory and non-neuronal cell types after severe epithelial injury. Lifelong persistence of two stem cell populations supports OE regeneration when damaged: the horizontal basal cells (HBCs), dormant and held in reserve; and globose basal cells, a heterogeneous population most of which are actively dividing. Both populations regenerate all cell types of the OE after injury, but the mechanisms underlying neuronal versus non-neuronal lineage commitment after recruitment of the stem cell pools remains unknown. We used both retroviral transduction and mouse lines that permit conditional cell-specific genetic manipulation as well as the tracing of progeny to study the role of canonical Notch signaling in the determination of neuronal versus non-neuronal lineages in the regenerating adult OE. Excision of either Notch1 or Notch2 genes alone in HBCs did not alter progenitor fate during recovery from epithelial injury, whereas conditional knock-out of both Notch1 and Notch2 together, retroviral transduction of progenitors with a dominant-negative form of MAML (mastermind-like), or excision of the downstream cofactor RBPJ caused progeny to adopt a neuronal fate exclusively. Conversely, we show that overexpressing the Notch1-intracellular domain (N1ICD) either genetically or by transduction blocks neuronal differentiation completely. However, N1ICD overexpression requires both alleles of the canonical cofactor RBPJ to specify downstream lineage. Together, our results suggest that canonical RBPJ-dependent Notch signaling through redundant Notch1 and Notch2 receptors is both necessary and sufficient for determining neuronal versus non-neuronal differentiation in the regenerating adult OE.

SIGNIFICANCE STATEMENT Despite the substantial reconstitution of the olfactory epithelium and its population of sensory neurons after injury, disruption and exhaustion of neurogenesis is a consequence of aging and a cause of olfactory dysfunction. Understanding the mechanisms underlying the generation of replacement neurons and non-neuronal cells is critical to any therapeutic strategy aimed at rebuilding a functional neuroepithelium. The results shown here demonstrate that canonical Notch signaling determines the balance between neurons and non-neuronal cells during restoration of the epithelium after injury. Moreover, the complexities of the multiple Notch pathways impinging on that decision are dissected in detail. Finally, RBPJ, the canonical Notch transcriptional cofactor, exhibits a heretofore unreported haploinsufficiency in setting the balance among the regenerating populations.