Fig 1.
Lgr5 expression by developing circumvallate papillae of Lgr5-GFP knock-in mice.
(A) E14.5. A layer of GFP-labeled epithelial cells is observed covering the papilla dome (bracket) and lining the invaginations of the nascent trenches (bars). (B) E18.5. Labeling within the papilla epithelium is interrupted by non-expressing cells in both the dome and trench areas. Lgr5+ cells are also found deeper in the mesenchyme, at potential sites of salivary duct formation. At birth (C), GFP fluorescence can be seen in the medial and lateral walls of the papilla trenches (e.g., bar) and in the epithelium of the forming ducts (between arrows). The boxed area in (C) is shown at higher magnification in (D). (E and F) P7. Lgr5 labeling in the papilla itself appears mainly in the layer around the base of taste buds arranged in the trench walls. Fluorescence is most intense at the terminations of salivary ducts, three of which are indicated by arrowheads in (F), which is a higher magnification image of the boxed area in (E). Images represent maximal projections of 3-5 single 1 μm confocal scans. Scale bar in (A) also applies to (B) and (C). These images were selected from tongue sections (20 μm) obtained from 3-5 animals at each time point.
Fig 2.
Expression of Lgr5 in posterior tongue of adult Lgr5-LacZ reporter mice.
(A and B) Circumvallate papilla. In the low power photomicrograph (A), lacZ reaction product is evident in the taste bud-containing epithelium lining the trenches and extending into extrapapillary tissue near the base of the papilla. In the boxed area, shown at 2X magnification in (B), three salivary ducts (brackets) can be seen near the base of the trench. LacZ staining is intense where von Ebner’s gland ducts intersect the papilla (arrowheads) and around the base of taste buds (asterisks) in the adjacent extragemmal taste epithelium. (C and D) Foliate papillae. (C) Lgr5 expression is shown at low power for three foliate papillae. At higher power (D), two salivary ducts (brackets) can be seen to merge as they contact the base of one papilla. Lgr5 labeling is evident in the ducts (arrowheads) and around taste buds (asterisks), as in the circumvallate papilla in (A) and (B). Scale bars in (A) and (B) pertain to (C) and (D), respectively. Images are representative of processed tissues from 6 mice.
Fig 3.
Characterization of Lgr5+ cells in circumvallate papillae of Lgr5-GFP reporter mice co-labeled for epithelial and ductal markers.
(A-D) Immunolabeling for Krt14. In the low power confocal overlay image (A), expression of this epithelial cell marker (red) is demonstrated in von Ebner’s salivary glands (vEG), their ducts (arrows), the epithelium of the papilla and the basal layer of the surface epithelium of the tongue. A zone of high GFP fluorescence corresponding to Lgr5 expression (green) is evident in the terminal portion of the salivary duct on the left, where it contacts the base of the papilla and its lumen opens into the papilla trench. In higher magnification images of the boxed region in A, Lgr5 expression (B) and Krt14 immunoreactivity (C) are seen to partially overlap in the distal, excretory portion of the duct (D). (E) Immunolabeling for Krt8. Anti-Krt8 reactivity (red) is located throughout the single-layered epithelium of the salivary duct (arrow) and in the internal (luminal) layer of the bi-layered excretory segment of the duct proximal to the base of the papilla. Staining for Krt8 is also seen within taste buds (asterisks). GFP fluorescence is limited to the external (basal) layer of the excretory segment of the duct (arrowheads) surrounding the Krt8-immunoreactive cells of the internal layer. Lgr5 expression appears to extend into basal portions of the adjacent papilla epithelium, but fluorescence falls off in intensity with distance from the duct. (Ei-Eviii) Images of subsequent serial sections through this circumvallate papilla further illustrate the consistent association of high Lgr5-GFP expression with segments of at least 5 excretory ducts (numbered arrowheads) and not with the distal, single-layered portions of the ducts (red labeling only). (F) Immunolabeling for the nuclear marker Sox9. Co-localization of Lgr5 labeling (green) and Sox9 immunoreactivity (red) is seen in the excretory segment of the salivary duct where the two overlap (yellow nuclei, arrowheads). The insets show the respective single channel images for the overlay. (A-D): Single optical sections. (E and Ei-Eviii) Maximum projection of 3 optical sections. (F): Single optical sections. Images were selected from immunoreacted tissues of 5 (anti-Krt14), 6 (anti-Krt8) and 3 (anti-Sox9) animals.
Fig 4.
Lineage tracing of Lgr5-expressing cells in circumvallate papillae.
Lgr5-GFP-IRES-CreERT2;mTmG mice were injected with a single dose of tamoxifen (day 0) and examined at the indicated time intervals for induction of Cre-mediated expression of membrane GFP (mG). (A-D) 1 day post-injection. (A) is a low power image of one side of a papilla (dashed boundary line) with associated ducts of von Ebner’s glands (arrows). Boxed areas are seen at 2X magnification in (B), (C) and (D). Cells with induced membrane labeling are prominent at the terminations of salivary ducts, where they can be identified by the distinct circular mG profile (open arrowheads in B) surrounding their less intense cytoplasmic Lgr5-GFP expression. These cells, as well as uninduced Lgr5+ cells displaying cytoplasmic labeling only (closed arrowheads), are restricted to the basal layer of the bi-layered ductal epithelium (dots indicate luminal layer cells). Induced cells are also present in the perigemmal region in (C) and (D) (open arrowheads). In (C), asterisks indicate two longitudinally sectioned taste buds while in (D), taste buds are cut in cross section. (E) 2 days post-injection. The progeny of GFP-expressing cells are first seen in taste buds at this time point, illustrated by the surface-marked cell at upper left (open arrowhead). Cells with induced labeling appear at all levels within the taste epithelium, including at the tissue surface. At 3 days post-injection (F), membrane-labeled cells continue to accumulate within the epithelium. A continuous line of surface-marked cells (open arrowheads) can be seen extending from the basal layer of the duct (arrow) to the basal region of the papilla below an adjacent taste bud. (G) 7 days post-injection. Duct cells with induced labeling remain (open arrowheads), with their progeny occupying taste buds as well as the surrounding papilla epithelium. Representative images were selected from processed tissues of 5 (1 Day), 3 (2 Days), 5 (3 Days) and 6 (7 Days) tamoxifen-injected mice.
Fig 5.
Diagram illustrating the niche and cell relationships of putative stem/progenitor cells mediating renewal of taste bud cells in the circumvallate papilla.
The upper drawing represents the junction between terminal portions of salivary gland ducts and taste epithelium at the base of the papillary trench (see schematic below). Stem cells (Lgr5+high) occupy primarily the basal layer of the stratified columnar epithelium of excretory ducts. Arrows indicate the proposed pathway of cell transfer of descendent cells from the ductal niche, via Lgr5+low cells, to the basal layer of taste epithelium.