Wnt-responsive Lgr5⁺ globose basal cells function as multipotent olfactory epithelium progenitor cells

Abstract

Persistent neurogenesis in the olfactory epithelium provides a unique model to study neural stem cell self-renewal and fate determination. In the olfactory neuroepithelium, globose basal cells (GBCs) are considered to be the direct progenitors of olfactory neurons. However, the study of neurogenesis from GBCs has been impeded by the paucity of GBC-specific markers. Here we report that Lgr5, a recently discovered adult stem cell marker, is exclusively expressed in GBCs in neonatal and adult mice. Lgr5(+) cells display characteristics of cycling stem cells, including Ki67 expression and EdU incorporation. Lineage tracing analysis demonstrates that Lgr5(+) GBCs regenerate multiple cell types under normal turnover condition or after olfactory lesion. Furthermore, upregulation or downregulation of Wnt signaling in vivo indicates a key role of Wnt signaling not only in maintaining Lgr5(+) cell proliferation and promoting neuroregeneration, but also in delaying sensory neuron maturation. Together, our observations provided new insights into the dynamics of neurogenesis in the olfactory epithelium.

Keywords: Lgr5; Wnt; globose basal cells; neural stem cells; olfactory epithelium; regeneration.

Figure 1.

Lgr5 marks GBCs in the olfactory epithelium. A, Quantitative analysis of Lgr5 expression in the olfactory epithelium by real-time PCR. B, C, Confocal images of Lgr5-EGFP⁺ cells in the olfactory epithelium of mice at P2 (B) and 3 months (3M) of age (C). Images presented are from olfactory epithelium coronal sections stained with an anti-EGFP antibody to amplify the endogenous EGFP signal. Lgr5-EGFP⁺ cells in the basal layer are marked by arrowheads. D, E, Coimmunohistological staining of EGFP with HBC markers, K14 (D) and ICAM-1 (E). Lgr5-EGFP⁺ cells always rest directly on the ICAM-1⁺ or K14⁺ HBCs. F–I, Coexpression of GBC markers Ascl1 or NeuroD1 with Lgr5-EGFP in P2 (F, H) and adult olfactory epithelium (G, I). Many Lgr5-EGFP-positive cells lost Ascl1 expression (G), and NeuroD1 could hardly be detected in 3-month-old animals (I). Boxed areas are highlighted on the right. Lgr5-EGFP⁺Ascl1⁺ cells and Lgr5-EGFP⁺NeuroD1⁺ cells are marked by arrowheads. J, The percentage of Ascl1⁺ and NeuroD1⁺ cells in Lgr5-EGFP⁺ population. The mean ± SD of three independent experiments is presented. Scale bars: B–I, 50 μm.

Figure 2.

Lgr5⁺ cells proliferate and maintain olfactory epithelium renewal. A, B, Coexpression of Lgr5-EGFP and the proliferation marker Ki67 in P2 (A) and adult (B) olfactory epithelium. Lgr5-EGFP⁺ Ki67⁺ cells are marked by arrowheads. C, EdU labeling demonstrates that the Lgr5⁺ GBCs are proliferating cells in neonatal pups. D, In adult olfactory epithelium, EdU⁺ cells are restricted to the basal layer and mostly are Lgr5-EGFP⁺ GBCs. Pups (P2) or adults (2 months old) were injected with EdU two times and analyzed directly 6 h after the last injection. Arrowheads mark representative Lgr5-EGFP⁺EdU⁺ cells. E, In normal olfactory epithelium, EdU⁺ cells migrated apically after a 2 week chase. F, The percentage of Ki67⁺ and EdU⁺ cells in Lgr5-EGFP⁺ population. Data are given as mean ± SD. G–I, LacZ⁺ cells exhibit morphology of olfactory sensory neurons (G, H), supportive sustentacular cells (H), and Bowman's gland and duct cells (I). Olfactory epithelium cryosections from Lgr5 ^{EGFP-Ires-CreERT2}; Rosa26-LacZ mice were stained with X-gal 1 month after tamoxifen induction. The arrowhead in H marks a sustentacular cell. Typical morphology of Bowman's gland and duct cells was indicated by arrows and arrowheads (I). J, K, Confocal images show that β-gal-positive cells coexpress OMP and CK18. Scale bars: A–E, G–K, 50 μm.

Figure 3.

Lgr5⁺ cells regenerate multiple olfactory epithelium lineages after methimazole injury. A–C, Robust proliferation of Lgr5-EGFP⁺ cells was activated in the early stage of regeneration. Two-month-old Lgr5^{EGFP-Ires-CreERT2} mice were injected with methimazole to induce olfactory epithelium damage. At day 2, Lgr5-EGFP⁺ cells were activated to proliferate and usually coexpressed Ki67. At postlesion days 5–7, the number of Lgr5-EGFP⁺ cells reached a peak, and abundant proliferation continued to day 14 (A). B, Lgr5-EGFP⁺ cells colabeled with GBC marker Ascl1 in the very early stage of regeneration, but a significant reduction of Ascl1⁺ cells was observed at postlesion day 14 (B). C, Lgr5-EGFP is expressed exclusively by the GBCs that are located above the K14⁺ HBCs. Arrowheads mark Lgr5-EGFP⁺Ki67⁺ cells in A and Lgr5-EGFP⁺Ascl1⁺ cells in B. D, Quantitative analysis of Lgr5-EGFP⁺ cells during regeneration. E, The percentage of Ki67⁺ and Ascl1⁺ cells in the Lgr5-EGFP⁺ population. The data are shown as mean ± SD from three experiments. F, Genetic lineage tracing using 1-month-old Lgr5^{EGFP-Ires-CreERT2}; Rosa26-LacZ mice. G–I, Lgr5⁺ cells regenerate multiple cell types in the olfactory epithelium. LacZ⁺ cells are present in basal layer, olfactory sensory neurons, sustentacular cells (G), Bowman's gland and ducts (H), and microvillous cells (I). J–L, Confocal images of olfactory epithelium sections double stained with β-gal and OMP (J) or CK18 (K, L). β-gal signal colocalizes with both OMP and CK18. M, No colocalization was observed between β-gal and CK14 in HBCs. N, EdU labeling (arrow) remained within Lgr5-EGFP⁺ GBCs after methimazole-induced lesion followed by a 2 week EdU chase. The arrowhead marks an Lgr5-EGFP⁺EdU⁺ cell. O, LacZ⁺ cells are retained in basal layer 6 months after tamoxifen induction. Scale bars: A–C, G, H, J–O, 50 μm; I, 20 μm.

Figure 4.

Proliferation and differentiation of Lgr5⁺ olfactory epithelium cell in vitro. A–C, In NSC medium, olfactory epithelium cells formed compact adhesive colonies (A) or neurosphere (B). The majority of cells in these colonies was Lgr5-EGFP⁺ and coexpressed the proliferation marker Ki67(C). Some Lgr5-EGFP⁺Ki67⁺ cells are highlighted by arrowheads (C). D–G, Sorted Lgr5-EGFP⁺ cells generated different cell types of the olfactory neuroepithelium. After differentiation in neural medium for 5 d, progeny of sorted Lgr5-EGFP⁺ cells expressed Tuj1 (E), DCX (F), and CK18 (G), markers of differentiated olfactory epithelium cells. Scale bars: A, B, F, G, 50 μm; C, E, 100 μm.

Figure 5.

Wnt signaling maintains Lgr5⁺ cell proliferation. A, Confocal images of β-catenin staining in the olfactory epithelium. Boxed region was magnified at the bottom. Compared with upper adjacent cells (marked by arrows), Lgr5-EGFP⁺ GBCs (marked with arrowheads) have increased nuclear accumulation of β-catenin. B, Confocal images of Lgr5-EGFP⁺ GBC colony stained with Ki67. Arrowheads mark Lgr5-EGFP⁺Ki67⁺ cells. C, Quantification of Ki67 and Lgr5-EGFP double positive cells in the presence and/or absence of Wnt3a in culture. D–F, Confocal images of the olfactory epithelium stained with antibodies for Ascl1 and β-gal in early stage of regeneration. G, Quantification of Ascl1 and β-gal double positive cells in the olfactory epithelium of mice as shown in D–F. Wnt activation significantly increased the percentage of Lgr5⁺ GBC-derived Ascl1⁺ transit-amplifying neural progenitors (*p = 0.000). H, Confocal images of olfactory epithelium stained with antibodies for OMP, Ki67, and β-gal. Deletion of APC maintained Lgr5⁺ GBC progeny in a proliferative state in the basal layer 1 month after lesion. I, Quantification of β-gal⁺ clusters in the olfactory epithelium of the three mice strains 1 month after injury. Data presented are the numbers of clusters that contains 6 or >6 β-gal⁺ cells (*p = 0.003). Scale bars: A, 25 μm; B, D–F, H, 50 μm. WT, wild-type.

Figure 6.

Sustained Wnt activation delays olfactory sensory neuron maturation. A–C, Confocal images of olfactory epithelium stained with antibodies specific for OMP and β-gal. Compared with wild-type control (A), downregulation of Wnt activity by β-catenin deletion obviously reduced the number and size of β-gal⁺ clusters (B). In APC-deleted olfactory epithelium, β-gal⁺ clusters were located in the immature or basal layer, and the majority of these cells was OMP negative or had weak OMP expression as highlighted by arrowheads in C. D, Quantitative analysis of OMP⁺ olfactory sensory neurons in β-gal⁺ clusters (*p = 0.001). Due to the low frequency of labeling, we did not calculate the β-gal⁺ cells after β-catenin deletion. E–G, Confocal images of the olfactory epithelium stained with GAP43 and β-gal. β-gal⁺/GAP43⁺ cells were highlighted by arrowheads in G. H, Quantification of GAP43⁺ immature neuron in β-gal⁺ population (*p = 0.003). I–K, Confocal images of the olfactory epithelium stained with antibodies specific for CK18 and β-gal. Compared with wild-type control mice (I), Wnt activation significantly reduced the number of β-gal⁺ sustentacular cells located on the surface layer (K). L, Quantification of CK18⁺ sustentacular cells that express β-gal (*p = 0.008). M, Confocal images of sorted Lgr5-EGFP⁺ GBCs in the presence or absence of Wnt3a. N, Quantification of the percentage of Tuj1-positive and EGFP-positive cells in culture (*p = 0.000; **p = 0.002). Scale bars: A–C, E–G, I–K, 25 μm; M, 50 μm.