Horizontal basal cells self-govern their neurogenic potential during injury-induced regeneration of the olfactory epithelium

Abstract

Horizontal basal cells (HBCs) residing within severely damaged olfactory epithelium (OE) mediate OE regeneration by differentiating into odorant detecting olfactory sensory neurons (OSNs) and other tissue supporting non-neuronal cell types. Within various regenerative tissues, the Notch signaling pathway can either positively or negatively regulate resident stem cell activity and potentially vary with tissue integrity. Although Notch1 specifies HBC dormancy in the uninjured OE, little is known about how HBCs are influenced by the Notch pathway following OE injury. Here, we show that HBCs depend on a functional inversion of the Notch pathway to appropriately mediate OE regeneration. At 24 hours post-injury, HBCs enhance Notch1-mediated signaling. Moreover, at 3 days post-injury when the regenerating OE is composed of multiple cell layers, HBCs enrich both Notch1 and the Notch ligand, Dll1. Notably, HBC-specific Notch1 knockout increases HBC quiescence and impairs HBC differentiation into neuronal progenitors and OSNs. Interestingly, complete HBC knockout of Dll1 only decreases differentiation of HBC-derived OSNs. These data underscore the context-dependent nature of Notch signaling. Furthermore, they reveal that HBCs regulate their own neurogenic potential after OE injury.

Spatiotemporal dynamics exhibited by horizontal basal cells reveal a pro-neurogenic pathway during injury-induced olfactory epithelium regeneration

“…At 2 and 3 days post-injury (dpi), distinct layers of cells begin to comprise the regenerating OE. Within these layers reside p63 + HBCs and p63 - non-HBCs 30 ( Figure S2 ). Using the CK5 promoter to drive expression of tamoxifen-inducible Cre recombinase and lineage trace tdTomato + (tdTom + ) HBCs and their progeny ( K5CreER T2 ; Rosa26R(tdTomato) ), p63 - /tdTom + non-HBCs within these bi-genic mice at 2 dpi express 25.5% more Rac1 relative to p63 + /tdTom + HBCs ( Figures S2 A–S2E).…”

“…Hopx -expressing HBCs primarily give rise to Sus cells and olfactory sensory neurons (OSNs), 10 with the former identifiable by CK8 and interleukin-33 (IL33) located apically within the regenerated tissue 10 , 38 while immature and mature OSNs are marked by PGP9.5. 30 , 39 , 40 , 41 , 42 Following OE injury, HBC differentiation into CK8 + /apical IL33 + /tdTom + Sus cells was unaffected (17.1 HBC-derived Sus cells per 100 μm Rac1 fl/fl OE vs. 18.3 HBC-derived Sus cells per 100 μm Rac1 WT OE) ( Figures 6 A–6K). Interestingly, however, HBC-specific Rac1 cKO attenuated HBC differentiation into PGP9.5 + /tdTom + OSNs at 28 dpi by nearly half (14.6 HBC-derived OSNs per 100 μm Rac1 fl/fl OE vs. 25.7 HBC-derived OSNs per 100 μm Rac1 WT OE) ( Figures 6 L–6V).…”

“…While it has been known for more than a decade that HBCs can help repair the OE following tissue injury, 1 the transcriptional and environmental mechanisms that enable HBC-mediated OE regeneration have only relatively recently been explored. 5 , 6 , 8 , 30 However, it still remains unknown whether HBCs in situ exhibit processes that are spatiotemporally dynamic across subcellular domains soon after tissue injury and, if so, whether any of these early molecular players facilitates appropriate HBC contribution to OE reconstitution. We show here that HBCs reorganize Rac1 apically following OE injury.…”

“…From an evolutionary perspective, the need to restore barrier function may explain why Sus cells can differentiate directly from HBCs without the need for intermediary progenitors, 10 which could represent possible failure points. Moreover, in light of unperturbed Sus cell differentiation following various HBC-specific manipulations 30 ( Figures 6 A–6K), the question of whether a subset of HBCs is already determined during OE development to become Sus cells upon their activation deserves examination.…”