Horizontal basal cells are multipotent progenitors in normal and injured adult olfactory epithelium

Abstract

The mammalian olfactory neuroepithelium provides a unique system for understanding the regulation of neurogenesis by adult neural stem cells. Recently, mouse horizontal basal cells (HBCs) were identified as stem cells that regenerate olfactory receptor neurons (ORNs) and non-neuronal cell types only after extensive injury of the olfactory epithelium (OE). Here we report a broader spectrum of action for these cells. We show that even during normal neuronal turnover, HBCs actively generate neuronal and non-neuronal cells throughout adulthood. This occurs in a temporally controlled manner: an initial wave of HBC-derived neurogenesis was observed soon after birth, and a second wave of neurogenesis was observed at 4 months of age. Moreover, upon selective depletion of mature ORNs by olfactory bulbectomy, HBCs give rise to more neurons. Our findings demonstrate a crucial role for HBCs as multipotent progenitors in the adult OE, acting during normal neuronal turnover as well as in acute regeneration upon injury.

Figure 1

Horizontal basal cell (HBC)-specific Cre expression in K5.CrePR1. K5.CrePR1 tg/+ mice at P26 (A–D) and 6m (E–H) were examined for Cre and K14 expression by double immunofluorescence. The majority of HBCs were Cre⁺whereas a subset was Cre⁺ (arrows). No Cre⁺/K14⁺ cells were seen. The intracellular localization of Cre was primarily cytoplasmic, with a subset of cells showing Cre in the nucleus as well (arrowheads). Scale bars = 55 µm (A, E) and 55 µm (B–D, F–H). Abbreviations: m, months; P, postnatal day.

Figure 2

Horizontal basal cells (HBCs) give rise to neuronal cells in the olfactory epithelium (OE). (A–H): K5.CrePR1 tg/+: R26R^+/− mice at P3 (A, B), P10 (C, D)1m (E, F), and 4m (G, H) were examined for β-gal activity by X-gal staining. Insets in (A, C, E, G) are enlarged in (B, D, F, H), respectively. Arrowheads in (B, D, F, H) indicate the position of the basal lamina. (A, B): At P3, infrequent β-gal⁺ HBCs were observed (arrows). (C, D): By P10, the number of β-gal⁺ HBCs, as well as the intensity of β-gal activity in HBCs, had increased (straight arrow). Notably, β-gal⁺ cell clusters containing tentative globose basal cells (GBCs) (bracket) and olfactory receptor neurons (ORNs) (curved arrow) were observed. (E, F): By 1m, the majority of HBCs were β-gal⁺ (straight arrow). β-Gal⁺ tentative ORNs were observed (curved arrows). (G, H) At 4m, β-gal⁺ cell clusters containing many tentative ORNs were observed. (I, J, K): K5.CrePR1 tg/+: R26R+/− mice at 2–3m were examined for β-gal/TuJ1 (I)β-gal/OMP (J), and β-gal/Mash1 (K) expression by double immunofluorescence. Dotted lines indicate the position of the basal lamina. The presence of β-gal⁺/TuJ1⁺ (I) and β-gal⁺/OMP⁺ cells (J) demonstrated that HBCs had generated ORNs. The presence of β-gal⁺/Mash1⁺ cells (K) demonstrated that HBCs had generated Mash1⁺ GBCs. Arrowheads point to Mash1⁺ GBCs that are negative for β-gal (K). The red signal in the apical region of the OE corresponds to nonspecific binding of the secondary antibody (K). Scale bars = 60 µm (A, C, E, G)60 µm (B, D, F, H), and 55 µm (I, J, K). Abbreviations: β-gal, β-galactosidase; m, months; OMP, olfactory marker protein; P, postnatal day; TuJ1, neuronal class III β-tubulin.

Figure 3

Horizontal basal cells (HBCs) give rise to non-neuronal cell types in the olfactory epithelium (OE). Four-month-old K5.CrePR1 tg/+: R26R^+/− mice were examined for β-galactosidase (β-gal) activity by X-gal staining. Straight arrows and brackets indicate the position of HBCs and globose basal cells, respectively. (B) and (C) are adjacent sections. In addition to multiple olfactory receptor neurons, sustentacular cells positive for β-gal were observed ([A], curved arrows). These cells have large, columnar cell bodies located in the most apical layer of the OE. β-Gal⁺ Bowman’s gland cells ([B], asterisk) and duct cells ([C], asterisk) were also observed. Gland cells are located in the lamina propria beneath the OE and extend a duct-like structure that traverses the OE. Scale bar = 50 µm (A–C).

Figure 4

Two waves of horizontal basal cell (HBC)-derived neurogenesis. K5.CrePR1 tg/+: R26R^+/− mice at various ages ranging from P10 to 12m were examined for the number of β-galactosidase (β-gal⁺) neuronal clusters in the olfactory epithelium per animal (clusters/animal), the cellular composition of individual clusters (cluster composition), and the total number of β-gal⁺ cells per animal (total cells/animal) (A). All numbers represent the average ± SEM of data obtained from five animals per age. In parentheses is shown the range of each data set. (B, C): Graphical presentations of (A), regarding cluster composition and total cells per animal, respectively. For simplicity, the data on SUSs are excluded. The first wave of HBC-derived neurogenesis started at P10, and the second wave peaked at 4m of age. Abbreviations: GBC, globose basal cell; m, months; ORN, olfactory receptor neuron; SUS, sustentacular cell.

Figure 5

Olfactory bulbectomy enhances horizontal basal cell-derived neurogenesis. Unilateral olfactory bulbectomy was performed on 2-month-old K5.CrePR1 tg/+: R26R^+/− mice. Thirty days later, olfactory epithelium (OE) on the contralateral side of the lesioned bulb (control) and on the ipsilateral side of the lesioned bulb (lesioned) was examined for β-gal activity by X-gal staining (A–F). Both control (A, C, E) and lesioned (B, D, F) tissues shown are from the same animal. (A, C, E) represent every third section through the largest cluster found in control OE, whereas (B, D, F) represent every third section through the largest cluster found in lesioned OE. Arrows point to sustentacular cells, and asterisks indicate cells of Bowman’s glands and ducts (B, D, F). Lesioned OE was examined for β-gal/OMP (G) and β-gal/Notch 2 (H) expression by double immunofluorescence. Dotted lines indicate the position of the basal lamina. The arrow points to a β-gal⁺/OMP⁺ olfactory receptor neuron (G). Arrowheads point to β-gal⁺/Notch 2⁺ sustentacular cells (H). Scale bars = 60 µm (A–F) and 60 µm (G, H). Abbreviations: β-gal, β-galactosidase; OMP, olfactory marker protein.

Figure 6

Model of HBC differentiation pathways. HBCs undergo self-renewal or differentiation mainly into GBCs, which eventually produce ORNs. HBCs can also proceed toward differentiation into SUSs or BG cells, albeit at a lower frequency. Abbreviations: BG, Bowman’s gland and duct; GBC, globose basal cell; HBC, horizontal basal cell; ORN, olfactory receptor neuron; SUS, sustentacular cell.