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First published online April 10, 2009
doi: 10.1242/10.1242/dev.029090

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Fate mapping of mammalian embryonic taste bud progenitors

¹ Department of Cell and Developmental Biology and the Rocky Mountain Taste and Smell Center, University of Colorado School of Medicine, Aurora, CO 80045, USA.
² Department of Anatomical Sciences and Neurobiology, University of Louisville School of Medicine, Louisville, KY 40292, USA.

View larger version (121K): [in this window] [in a new window]   Fig. 1. Shh-descendent cells restrict to taste placodes. (A) In intact and sectioned tongues from an E14.5 embryo tamoxifen dosed at E12.5, X-gal stained Shh-descendent cells are present in two bilateral rows of placodes (arrow) and numerous epithelial cells (arrowhead). (B) In an E15.5 tongue from an embryo tamoxifen treated at E13.5, labeled cells are predominantly within taste papillae (arrow) and there is less extra-placodal labeling (arrowhead). (C) +/+;R26RLacZ embryos lack β-gal activity. (D,E) E14.5 tongue cryosections from embryos tamoxifen treated at E12.5 reveal placodal (red arrows) and extra-placodal (arrowheads) X-gal-positive cells. Extra-placodal cells were distant from [in the apical (D; red arrowhead) or basal (D,E; black arrowheads) epithelium] or adjacent to (E; black arrows) taste placodes. (F,G) Cryosections of E15.5 embryos given tamoxifen at E13.5 had consolidated placodal labeling (F; arrow); labeled extra-placodal cells were fewer in number and were present in the apical layer (G; arrowheads). (H) Placode-descendent cells at E15.5, from embryos tamoxifen treated at E13.5, stained with anti-β-gal (red) and the pan-neuronal marker PGP9.5 (green) show innervation of Shh-descendent cells. Scale bars: 10 µm in D-H.

View larger version (72K): [in this window] [in a new window]   Fig. 2. Shh-expressing taste placode cells continue to express Shh, whereas extra-placodal descendents cease Shh expression. (A-C) Tongue cryosections from E15.5 embryos tamoxifen treated at E12.5 and immunostained for β-gal (B; red) and Shh (C; green) show co-labeling in taste placodes (yellow in A; merge). Occasional peripheral cells in placodes were β-gal-IR only (yellow arrowheads). Broken lines indicate epithelial basement membrane. (D,E) Double immunostained tongue cryosections from the same embryo as in A-C show that extra-placodal cells labeled at E12.5 but distant from placodes, and in basal (D) and apical (E) epithelial layers, turned off Shh by E14.5. Scale bars: 20 µm in A-C; 10 µm in D,E.

View larger version (72K): [in this window] [in a new window]   Fig. 3. At P0, Shh-expressing placodes give rise primarily to immature taste buds, and not to papillae or to general epithelium. (A-C) X-gal-stained P0 tongue sections, tamoxifen treated at E12.5 (A,C-G) or at E14.5 (B), show placode-descendent cells in taste buds (white asterisks), but not papillae. Occasional labeling was detected in the keratinized acellular layer above immature taste buds (red arrows in B,C). (C) In P0 tongues from mice treated at E12.5, X-gal is evident in filiform papillae in basal (white arrows) and keratinized (black arrows) epithelial layers. Black asterisks indicate non-gustatory filiform papillae. (D-G) Cryosectioned P0 tongues from mice tamoxifen treated at E12.5 and immunostained at P0 for β-gal (green) and CK8 (red) reveal that some placodally descendent cells are CK8-IR (D; black and white asterisks indicate double-labeled taste cell; white arrowhead, CK8-IR only cell; white arrow, β-gal-IR only cell). (G) Double immunolabeling for β-gal (green) and CK8 (red) shows that at P0 not all taste buds have CK8-IR taste cells descendent from placodes. One taste bud (white asterisk) has a double-labeled cell (white arrowhead). Arrow indicates an epithelial clone labeled at E12.5. Blue indicates nuclear counterstain. Scale bars: 20 µm in A-F; 50 µm in G. (H) Number of labeled taste buds and epithelial clones at P0 with respect to time of tamoxifen treatment. Labeled taste bud number does not differ with respect to time of treatment (blue bars; n=3), whereas epithelial labeling is diminished with later tamoxifen (brown bars; n=3; *P<0.05).

View larger version (111K): [in this window] [in a new window]   Fig. 4. Taste placodes give rise to type I and II, but not to type III cells in adult taste buds. Mice treated with tamoxifen at E12.5 were examined for β-gal (green) and differentiated taste cell marker (red) immunoreactivity 6 weeks postnatally. (A-C) Single confocal planes of adult tongue cryosections stained with anti-β-gal (green; cytoplasmic) and anti-NTPDase2 (red; membrane associated) showed many double-labeled cells (white asterisks). Intragemmal basal cells were also labeled by anti-β-gal (white arrowheads). (D-F) Confocal single plane image of a cryosection immunostained for β-gal and PLCβ2 (red; cytoplasm) shows numerous co-expressing cells (white asterisks). An intragemmal basal cell (white arrowheads) and perigemmal edge cell (white double arrows) were also β-gal-IR. (G-I) A taste receptor cells (trc) immunopositive for PGP9.5 (red) was not β-gal-IR. Anti-PGP9.5 also labels nerve fibers (n) in and around buds. (J-L) Staining for β-gal (green) and anti-NCAM (red) did not reveal double-labeled taste receptor cells (trc). Anti-NCAM also labels nerve fibers (n) innervating taste buds. The single white arrow in each panel indicates the taste pore. Scale bars: 20 µm.

View larger version (70K): [in this window] [in a new window]   Fig. 5. Neural crest does not contribute to taste placodes or adult taste buds. (A,B) Sections of E13.5 Wnt1cre;R26LacZ tongues stained with X-gal and counterstained with Neutral Red confirm the absence of neural crest cells in lingual epithelium. White arrows in A indicate X-gal-negative tongue muscle. In B, an X-gal-negative taste placode is bracketed. (C,D) Sections of a 6-week-old Wnt-1cre;R26LacZ tongue stained for anti-β-gal (green) and anti-PLC β2 (red) reveal neural crest cells in taste papilla mesenchyme. (E,F) In 4-month-old Wnt-1cre;R26LacZ tongue sections immunostained for a cocktail of type I, II and III taste cell markers (red) and β-gal (green), neural crest cells are found only in lingual mesenchyme. (D,F) Blue is Hoechst nuclear counterstain. Arrows indicate the taste pore. Scale bars: 50 µm in A,C,E; 10 µm in B,D,F.

View larger version (63K): [in this window] [in a new window]   Fig. 6. Embryonic development of taste bud progenitors is normal without Bdnf-dependent gustatory innervation. (A,B) At E14.5, Shh-expressing taste bud progenitors do not differ between Bdnf+/+ (A) and Bdnf-/- (B) littermates. (C,D) In transverse sections of E14.5 tongues immunostained with anti-Shh (green) and anti-Gap43 (red), Shh-IR taste progenitors are comparable regardless of genotype, although Gap-43-IR fiber contact occurs in wild types (C), but is absent in Bdnf-/- mice (D). (E-G) The number (E) and size (F) of taste progenitors in Bdnf+/+ versus Bdnf-/- embryos are not affected by loss of Bdnf-supported innervation (G). (H,I) CK8-IR immature taste buds in sections of E18.5 tongues do not differ between (H) wild-type and (I) Bdnf-/- embryos. Scale bars: 10 µm in C,D,H,I.

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