Blocking GLI function in epithelial cells leads to fungiform papilla disruption and taste bud loss

Blockade of Gli activity using a dominant-negative GLI repressor.

C-terminal deletion of the GLI2 transactivation domain yields proteins with potent GLI dominant-negative activity [39,40], and expression of a GLI2ΔC4 mutant in mouse skin epithelia mimics the phenotype seen with epithelial deletion of Smo to block HH signaling [41,42]. To conditionally block HH-driven transcriptional responses in tongue we produced transgenic mice carrying the Gli2ΔC4 mutant downstream of tetO response elements, and generated either K5-rtTA;tetO-Gli2ΔC4 (K5GliR) or K5-Cre;R26-lsl-rtTA;tetO-Gli2ΔC4 (epiGliR) mice. In doxycycline-treated K5GliR mice, GLI activity is blocked by GLI2ΔC4 in K5-expressing basal cells; whereas in epiGliR mice, Gli2ΔC4 is expressed and GLI activity blocked throughout the lingual epithelium (Fig 1A).

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Fig 1. Inhibition of epithelial HH/GLI signaling leads to alterations in fungiform papillae and epithelium and loss of taste buds.

A. Fungiform papilla and taste bud diagrams to illustrate cells types and location of HH pathway signaling elements. Canonical HH signaling is restricted to cells expressing both Gli1 and Ptch1 (Control Papilla and Taste bud, left). Center and right panel diagrams illustrate cell/tissue areas (blue) of dominant-negative transgene expression in doxycycline-inducible K5GliR and epiGliR mouse models. B. Three types of Fungiform Papilla and Taste Bud (FP/TB) used for quantifying effects of HH repression: I, Typical FP/TB: Typical papilla morphology with intact apical taste bud; II, Atypical FP/TB: Atypical, mis-shapen papilla morphology and taste bud remnant or cluster of distinctly staining cells; III, Atypical FP/No TB: Atypical papilla morphology with keratinized apical point and no discernable taste bud cells. C. H&E sections illustrate FP/TB morphology for Control and K5GliR and epiGliR mouse tongues at indicated time points after transgene induction. Scale bar applies to all micrographs except low power and insets. Low power image for epiGliR Control illustrates overall tongue morphology. Inset at Day 11 indicates disruption of fungiform and filiform papillae but at Day 35 K5GliR, inset illustrates intact filiform. Graphs present quantification of TYPE I, II and III taste organs at increasing durations of transgene activation. TYPE I (Typical) FP/TB are substantially reduced (K5GliR) or eliminated (epiGliR) whereas TYPE III (Atypical/No TB) organs accumulate. Numbers of tongues at each time point are included in parentheses in graph legends. Full statistics for ANOVA are presented in S1A and S1B Fig.

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To characterize the phenotypic response of taste organs to HH/GLI blockade, we classified fungiform papillae (FP) and taste buds (TB) as TYPE I (Typical FP and Typical TB), TYPE II (Atypical FP and Atypical TB), or TYPE III (Atypical FP and No TB) (Fig 1B), and we quantified the proportion of fungiform papilla/taste bud phenotypes at multiple time points after GLI blockade. No phenotypic alterations were detected in either K5GliR or epiGliR mice 1 day after starting doxycycline treatment (Fig 1C). In contrast, after 5 days of transgene activation in K5GliR mice only about 50% of taste organs were typical (TYPE I); after 35 days, the proportion of typical taste organs was reduced to about 35%. Notably, by 35 days about 40% of taste organs had no detectable taste bud (Fig 1C K5GliR, TYPE III). In epiGliR mice, with GLI function blocked in all epithelial cell compartments of the tongue, the effect was faster and more robust. After 5 days very few typical taste organs (TYPE I) remained; by 11 days almost all taste organs had no detectable taste bud (TYPE III) (Fig 1C epiGliR). In both models the remaining aberrant fungiform papillae had acquired multiple layers of cornified cells at the papilla apex, and in epiGliR mice, many fungiform papillae did not retain the deep epithelial down-growths into the stroma that characterize their typical rectangular papilla shape (Fig 1C, Day 11). (Complete ANOVA data and posthoc analyses are provided in S1A and S1B Fig)

Gli2 gene deletion.

To confirm the requirement for HH/GLI signaling function in FP/TB maintenance, we conditionally deleted Gli2 in the epidermis of K5-rtTA;tetO-Cre;Gli2^fl/fl mice (referred to as Gli2cKO) up to 35 days. In these animals, taste bud cells were gradually lost and FP apices became progressively more heavily cornified (Fig 2A Gli2cKO). Comparable to timing in K5GliR and epiGliR mice, after 5 days of doxycycline there was a significant reduction in Typical FP/TB (TYPE I); after 16 days, TYPE I were reduced to about 15% of all FP and were virtually eliminated after 28 days (Fig 2A). After 35 days, more than 65% of FP had no TB (TYPE III). (ANOVA data and posthoc analyses are included in S1C Fig).

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Fig 2. Conditional deletion of Gli2 in tongue epithelia leads to loss of Typical fungiform papillae and taste buds and accumulation of Atypical taste organs.

H&E sections illustrate loss of Typical FP/TB morphology after 16 and 28 days of epithelium-targeted deletion of Gli2 in Gli2cKO (A) and Gli2cKO;Gli1KO (B) mouse tongues, compared to Control. Scale bar in A refers to all images. Graphs present quantification of TYPE I, II and III taste organs at increasing durations of gene deletion, demonstrating essential elimination of TYPE 1, Typical FP/TB after about one month with progressive accumulation of TYPE III FP/No TB. Numbers of tongues at each time point are included in parentheses in graph legends. Full statistics for ANOVA are presented in S1C and S1D Fig.

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In mice with an epithelial Gli2- and global Gli1-deletion (K5-rtTA;tetO-Cre;Gli2^fl/fl;Gli1^lacZ/lacZ, referred to as Gli2cKO;Gli1KO) the extent and timing of FP/TB alteration were similar to those in mice with Gli2 deletion alone (Fig 2B Gli2cKO;Gli1KO). These data support the concept that GLI2 is the primary transcriptional activator mediating responses to HH signaling in peripheral taste organs, with little if any contribution from GLI1. Typical FP/TB (TYPE I) were reduced to less than 10% after 16 days of doxycycline administration, and after 35 days more than 60% of FP had no TB (TYPE III) (Fig 2B). Continuing gene induction through 45 days led to virtual elimination of TYPE I FP/TB (Fig 2B). Similar to FP effects in Gli2cKO tongues, the apical fungiform papilla epithelium where a taste bud would normally reside acquired a spinous cap with expanded epithelial cell layers (Fig 2B). (ANOVA data and posthoc analyses are provided in S1D Fig).

Overall these data indicate that disruption of GLI transcriptional activity in lingual epithelium using several genetic approaches leads to rapid and significant loss of typical fungiform papillae and taste buds.

K5-targeted GLI blockade leads to loss of HH-responding cells in fungiform papilla epithelium

To assess effectiveness of signaling blockade in our models we stained for lacZ positive cells in mice carrying a Gli1^lacZ allele, which serves as a sensitive and specific reporter for HH pathway activity [18]. In Control mice, HH-responding cells were detected in the basal cell layer of the FP epithelial walls, in perigemmal epithelial cells around the TB, and in stromal cells of the connective tissue core (Fig 3 Control Gli1^lacZ). As early as 5 days after transgene activation in epiGliR mice, HH-responding cells were no longer detected in the fungiform papilla walls and perigemmal epithelial cells but were retained in the FP connective tissue core (Fig 3, epiGliR Day 5, Day 11), confirming repression of HH signaling selectively in the targeted cell populations. Similar results were obtained in K5GliR, Gli2cKO and Gli2cKO;Gli1KO mice (S2A Fig).

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Fig 3. HH-responding cells are lost from the papilla epithelium but retained in connective tissue, and taste bud size, and SHH expression, decrease with duration of HH/GLI repression.

TOP ROW: X-Gal staining to detect β-gal-positive, HH-responding cells in Control Gli1^lacZ/+ mice is typically in perigemmal cells (P), basal epithelial cells of the FP wall (W), and cells of the stromal core (ST). After epiGliR transgene activation for 5 days, and continuing at 11 days, there is loss of detectable HH signaling in epithelium with lacZ-positive cells in the FP stroma only. BOTTOM ROW: SHH is expressed principally within taste buds cells (K8) in Control FP. During HH/GLI repression in epiGliR tongues, after 5 days remaining taste buds are progressively reduced in size and SHH ligand is reduced in association with taste cell loss. After 11 days very few TB remnants are observed (see Fig 1 data) and these are very small cell collections. Inset at Day 11 illustrates the nature of the cell remnants that remain in a very small percentage of FP.

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Taste bud cell loss and reduced SHH expression in fungiform papillae

Having established effective blockade of epithelial HH/GLI signaling activity we examined expression of SHH, which in the FP is typically restricted to a subset of taste bud cells [27] that express the taste bud cell marker Keratin 8 (K8) [43]. After only 5 days of transgene expression/GLI blockade in epiGliR mice, taste bud cells were fewer in number, did not span the full thickness of the epithelium, and apical taste pores were missing (Fig 3, SHH/K8 Control and Day 5). By 11 days (epiGliR) or 35 days (K5GliR) of transgene expression, discernible taste bud cell collections were detected only occasionally and they were localized to the lower third of lingual epithelium (Fig 3, Day 11 and inset; S2B Fig, Day 35 K5GliR). Overall, within the TB remnants at various stages of “deterioration” associated with HH signaling repression, there were some SHH expressing cells in the TB base as is typical in adult FP/TB (Fig 3, Day 5, Day 11 and inset; S2B Fig, Day 35 K5GliR).

The residual SHH in some FPs may be capable of signaling to HH-responsive stromal cells retained in the FP after HH repression and could potentially participate in maintaining epithelial integrity and/or FP structure. There are, however, TYPE III FP/No TB in all models (Figs 1 and 2) and because these have no TBs they have no SHH in the FP.

Proliferation effects in a sub-set of fungiform papilla epithelial cells

The effects of HH signaling on proliferation in different cell types within tissues are highly context-dependent. Whereas HH signaling drives proliferation in several settings [1], in others the HH pathway maintains quiescence [2]. Altered proliferative activity in papilla organs could contribute to the profound morphological alterations we detected following GLI blockade. Using antibodies to the basal layer marker, p63, the proliferation marker, Ki67, and EdU labeling to detect cells in S phase, we confirmed previously identified domains of high proliferation at the base of the FP and in perigemmal cells (27) in Control tongues (Fig 4A–4E). The marker p63 was expressed uniformly within basal cells of the FP walls and perigemmal cells (Fig 4A; S3A and S3B Fig) whereas the intensity of Ki67 immunostaining was typically reduced in basal cells as they ‘ascended’ the papilla wall toward the apex (Fig 4D).

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Fig 4. Inhibition of epithelial HH/GLI signaling leads to decreased cell proliferation in specific domains of the fungiform papilla.

A,B. p63-positive proliferating cells are continuous in basal epithelium of the FP (A, arrows) and in perigemmal cells around the taste bud (B,arrows). C. Proliferating cells labeled with EdU are prominent at the base of the FP (arrow) and in the perigemmal region (arrow). D,E. Ki67 positive, proliferating cells are numerous in basal epithelial cells at the FP base, are decreasing in number toward FP apical half (D, arrows), and are in the perigemmal region (E, arrows). F. Section through a TYPE I, Typical FP/TB with labels to indicate regions for Ki67-positive cell counts in APICAL and BASAL halves of the FP walls and in the PERIGEMMAL region. G. Region-specific, proliferation (Ki67+ cells) in Control epiGliR mice; reduced proliferation after 5 days transgene activation (Day 5); and graph for quantified data. Bars between pairs in graph indicate statistical significance at p < 0.001 (***). After only 5 days, cell proliferation was reduced in the basal FP wall (BASAL: t = 6.21, p< 0.001) and in the perigemmal region (PERI: t = 4.57, p<0.001). H. Cell proliferation in Gli2cKO mice in Control; after 16 days of gene deletion (Day 16); and graph for quantified data. Bars between pairs in graph indicate statistical significance at p < 0.001 (***). Cell proliferation was reduced in the perigemmal region (PERI: t = 4.34, p < 0.001). Numbers in parentheses for graphs refer to number of FP analyzed. E- cadherin (Ecad, red) immunoreactions were used to label the epithelium throughout images. Scale bar in D applies to A,C,D,F,G,H. Scale bar in E applies to B,E.

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To quantify proliferation we counted Ki67-positive cells in three epithelial compartments: the apical and basal halves of the fungiform papillae walls, and the perigemmal compartment surrounding the taste bud (Fig 4F). In the basal papilla domain of epiGliR mice, after 5 days there was a 66% reduction in Ki67-positive cells and a trend toward reduction in the apical domain that did not reach significance (Fig 4G APICAL, BASAL). In Gli2cKO tongues, after 16 days, there was again a non-significant trend toward fewer Ki67+ cells in the apical FP but there was no difference in the basal papilla (Fig 4H APICAL, BASAL). The difference in basal papilla proliferation in epiGliR compared to Gli2cKO FP could relate to the more profound effects on papilla structure in the epiGliR model (see Fig 1).

Strikingly, GLI blockade led to a reduction in Ki67-positive perigemmal FP cells both in epiGliR and Gli2cKO mice (Fig 4G epiGliR PERI; H Gli2cKO PERI). This reflects the fact that the number of perigemmal cells in the region that neighbors taste bud ‘remnants’ is radically reduced in GLI-inhibited tongues, arguing in favor of a pivotal role for the HH pathway in maintaining this epithelial cell population. This concept was further examined by immunostaining for the HH target Cyclin D1. We quantified Cyclin D1-positive cells in apical and basal papilla walls and in perigemmal cells, as we did for Ki67 expression (Fig 4H). In the Gli2cKO tongue, there was a trend to a reduced number of cells in the apical FP wall and in perigemmal cells. Cyclin D1-positive cells in apical and perigemmal regions were reduced by about 20% from Control (S3C, S3D and S3E Fig). Overall the data are similar to results with Ki67+ cell counts, but do not reach statistical significance.

Thus, GLI blockade leads to fewer proliferating cells in epithelial compartments that contribute to taste cells during homeostasis [27,32], suggesting that the loss of taste buds following HH pathway blockade is due at least in part to a deficiency in taste cell renewal.

Apoptosis does not appear to contribute to loss of taste cells in GLI-inhibited mice

In an effort to gain insight into the mechanism of cell loss after HH blockade, we examined apoptosis in tongues from Control and GLI-inhibited mice. Immunostaining for cleaved caspase 3 did not reveal differences from Controls in any of our models (S3F and S3G Fig) and cell labeling was infrequent as predicted from other studies in taste bud [44,45]. TUNEL-positive cells are occasionally seen in epidermis and they are up-regulated after UV-irradiation [46] and also in tongue after X-ray irradiation [47]. We used H&E sections in Control and Gli2cKO tongues (S3H, S3I and S3J Fig) to directly compare with TUNEL reactions. There were multiple TUNEL-positive cells in Control and Gli2cKO FP epithelium (S3K, S3L and S3M Fig), with an accumulation of positive cells in more apical layers of Gli2cKO TYPE II and in TYPE III FP that had taste bud cell loss and acquisition of a conical, filiform-like cap (S3L and S3M Fig). Accumulated TUNEL-positive cells in the FP apex in GLI-inhibited models resembled those in filiform papillae in Control mice (S3H Fig FILI). These are unlikely to represent classical apoptotic cells because we did not detect cells expressing cleaved caspase 3; moreover, it is known that cell death in keratinocyte differentiation does not follow classic apoptotic pathways [48,49,50]. Overall, increased apoptotic cell death is not likely to be a principal factor in taste organ alterations during HH/GLI blockade.

Innervation to fungiform papillae and taste buds is retained following epithelial GLI blockade

We demonstrated that FP and TB are disrupted or lost with HH/GLI blockade, and with fewer taste cells, SHH expression is much reduced in taste bud remnants. Further, proliferation was reduced in the TB perigemmal cells. Given the requirement for innervation in taste organ homeostasis [51], we examined innervation to the tongue and to taste buds specifically in Gli2 repressor (K5GliR, epiGliR) and Gli2cKO mice. Innervation to the tongue (combined chorda tympani/lingual nerve) and to taste buds specifically (chorda tympani only) was studied (Fig 5).

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Fig 5. Innervation to fungiform papillae and taste buds is retained in epithelial HH/GLI suppression models.

A,C,E,G. Chorda/Lingual: K8 immunostaining to label taste bud cells and neurofilament (NF) to label fibers of the chorda tympani/lingual nerve innervation to FP and TB. A. In Control the Chorda/lingual innervation is throughout the FP core and forms a dense ‘basket’ under the TB at the apex of the papilla core. With HH/GLI suppression at specified time points in K5GliR (C), epiGliR (E), and Gli2cKO (G) mice, NF-positive fibers are retained in the FP even though papillae are of atypical morphology and TBs are lost (E) or much reduced (G). At the illustrated time points in all models there are substantial morphological effects, as seen in Figs 1 and 2. B,D,F,H. Chorda tympani: K8 immunostaining to label taste bud cells and P2X3 to label fibers of the chorda tympani nerve innervation to FP and TB. B. In Control for Chorda tympani innervation the P2X3-positive label is within fibers in the papilla core (arrow) and within the TB. With HH/GLI suppression at specified time points in K5GliR (D), epiGliR (F), and Gli2cKO (H) mice, P2X3-positive fibers (arrows) are retained in the FP and TB cells even though papillae are of atypical morphology and TBs are lost (F) or much reduced (H). Scale bar in H applies to all panels. * in H indicate areas of nonspecific staining in the surface epithelial cells.

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The connective tissue core of the FP typically contains neurofilament (NF)-positive fibers of the combined chorda tympani/lingual nerve that distribute to the taste bud and lateral wall regions of the papilla core (Fig 5A, Chorda/Lingual Control). A distinctive ‘basket’ of fibers encircles the basal lamina region just under taste bud cells. After 5 days of transgene activation in lingual epithelium of K5GliR or epiGliR mice, despite taste bud disruption (Fig 1C), there is a robust innervation within papillae as seen with NF label (Fig 5C, K5GliR Day 5). NF-positive nerve fibers are also detected in epiGliR mice despite severe disruption of taste buds and papillae (Fig 5E), and in Gli2cKO mice (Fig 5G).

FP innervation from the chorda tympani nerve to taste buds specifically, and not to the papilla walls, can be identified with immunostaining for P2X3 (Fig 5B, arrow, Chorda tympani, Control). P2X3 is strongly expressed in peripheral sensory, including gustatory neurons [52,53]. In contrast to NF, P2X3 immunostaining therefore detects chorda tympani nerve and also some fibers within the taste bud, with cells that co-express K8 (Fig 5B). Taste specific P2X3-positive innervation is retained in the papilla core of K5GliR mice and into the apical papilla epithelium and taste bud cell remnants (Fig 5D, K5GliR, Day 5, arrow). In day 11 epiGliR mice, P2X3-positive fibers are detected even though most K8-positive taste bud cells are lost (Fig 5F arrows). In FP of Gli2cKO tongues over a prolonged period of 28 days of doxycycline administration, taste nerves remain although FPs are disrupted and few retain intact TB remnants (Fig 5H arrow).

Importantly, we did not observe any re-patterning or displacement of lingual innervation from the FP to aberrant sites; that is, fibers remained directed to the appropriate ‘target’ region in K5GliR, epiGliR and Gli2cKO mouse tongues. In tongue, NF-positive fibers typically course within the reticular lamina propria under the epithelium (S4A Fig, Control arrows) and turn to innervate FPs (S4B Fig, Control). A similar pattern of nerves under the epithelium is retained in K5GliR and epiGliR tongues (S4C and S4E Fig arrows) and is in a directed course to densely innervate the FP (S4D and S4F Fig). Significantly, innervation is retained in FPs without K8-positive taste bud cells at a comparable extent to FPs with taste buds (S4B, S4D and S4F Fig).

The NF-positive bundles of the chorda/lingual nerve in the tongue body and coursing into the FP are surrounded by lacZ-positive, HH-responding cells (S4G, S4H and S4I Fig). Furthermore, S100-positive immunohistochemistry, to mark nerve Schwann cells, demonstrates a close association with HH-responding cells in the FP core (S4J Fig). Thus, any sources of SHH ligand from taste bud cell remnants or from other cell elements in the FP tissue core have direct access to nerve-associated HH-responding cells.

Our data demonstrate that overall there is no gross elimination or misdirection of innervation in tongue, FP and/or TB cells with HH pathway suppression over long periods and profound target organ disruption.

Cells of the fungiform papilla core are retained and not grossly redistributed after HH/GLI repression

Although taste buds and papillae are severely disrupted or lost in the K5GliR, epiGliR and Gli2cKO mouse tongues, FP-like structures can be identified that retain a somewhat rectangular shape, albeit more pointed apically, with epithelial down-growths (Figs 1 and 2). Also, HH-responding, Gli1^lacZ-positive cells remain within the FP connective tissue (Fig 3). Because HH interacts with connective tissue cells and can be chemotactic [2,54], we used antibodies against vimentin, the principal intermediate filament of stromal fibroblast cells and a pan-fibroblast marker, to test for potential effects of HH suppression on FP stromal cells. In Control FP, vimentin-positive cells are: 1) in the central stromal core; 2) distributed along the papilla walls; 3) in the neurofilament-positive ‘basket’ niche just under the taste bud cells; and, 4) in cells that send filopodia extensions to contact the basal lamina (Fig 6A, 6B and 6C). In Gli2cKO tongues vimentin-positive stroma cells are not disrupted or redistributed but are retained in the basket region at the FP apex and have filopodia extending into the basal lamina (Fig 6D, 6E and 6F). Vimentin-positive cells were not observed within the taste bud in Control or Gli2cKO tongues; a few cells were observed in the epithelium. Overall there was no evidence for deregulated stromal cell activity in HH repression.

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Fig 6. Stromal core of the fungiform papilla retains vimentin-positive fibroblasts with filopodia extensions after epithelial HH/GLI suppression.

A,B,C. In Control FP, VM-positive cells (Vimentin) are throughout the FP core and in close association with basal lamina at FP walls and under the taste bud (A). B,C are enlarged images from A (regions with arrow and arrowhead). The epithelium is demarcated with DAPI. HSPG immunostaining delineates the basal lamina. Filopodia extend from vimentin-positive cells into basal lamina of FP walls (C). D,E,F. With HH/GLI suppression in Gli2cKO tongues, vimentin-positive cells remain in the FP core and filopodia extend into basal lamina of FP walls (D). E,F are enlarged images from D (regions with arrow and arrowhead).

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Other stromal cell types were labeled with antibodies for the macrophage marker, F4/80, and for smooth muscle actin (α-SMA). Labeled macrophages were in relatively small numbers in the FP core (S5A, S5B and S5C Fig) and there was no evidence of extensive macrophage infiltration associated with HH suppression. SMA-positive stromal cells were distributed in reticular lamina propria under the epithelium, within the filiform papilla core and the FP central core (S5D and S5E Fig) and were not numerous compared to vimentin-positive cells.

The data demonstrate that following epithelial repression of HH signaling and loss of taste organ integrity, major cell types in the papilla core are retained.

The circumvallate papilla loses taste buds but retains size and shape and innervation after HH/GLI repression

Whereas anterior tongue FPs have an ectodermal embryonic origin, the posterior tongue tissues and large circumvallate papilla (CV) have endodermal derivation [55,56] and could be affected differently by HH/GLI suppression. In contrast to the single TB in rodent FP, the CV contains a few hundred taste buds in dense physical juxtaposition in walls of the papilla trenches [57]. In Control and Gli2cKO mice, the general structure of the CV was similar (Fig 7A and 7C). To assess size of the CV we quantified length of CV walls (Fig 7A, bars on left wall) and computed depth of the CV from the number of dorsal to ventral serial sections. In Gli2cKO tongues these papilla measurements were not different from Control tongues across 5 to 35 days of gene deletion (Fig 7E). However, in contrast to the maintained overall CV structure, the numbers of TB profiles/remnants, or complete TBs with a taste pore, were markedly reduced compared to Control (Fig 7B, 7D and 7F). After 16 days of epithelial Gli2 deletion, TB profiles were reduced by about 60% and full TBs (with pores) were almost eliminated (Fig 7F). (ANOVA data and posthoc analyses are presented in S1E and S1F Fig)

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Fig 7. Circumvallate papilla structure is retained but taste buds are lost with conditional Gli2 deletion.

Hematoxylin and eosin sections of the circumvallate papilla from Control (A,B) and conditional epithelial gene deletion, Gli2cKO (C,D) tongues. Papilla structure was measured from the number of serial sections that encompassed the papilla (depth) and length of each ‘wall’ marked by two bars in A. Whereas papilla shape is retained (A,C) numbers of taste buds are much reduced after gene deletion (B,D). Taste bud remnants (dotted ovals) or with complete pores (arrows) are numerous in Control papilla (B, enlarged from Box in A) but few are found with HH/GLI suppression (D, enlarged from Box in C). One scale bar applies for A and C; or B and D. E,F. Graphs for circumvallate structure (E) and numbers of taste bud profiles/remnants and pores (F) at increasing durations after Gli2 gene deletion. CV depth and wall length did not change across time points in Gli2cKO tongues (E). Profile measures, used to indicate cell collections, were decreased by 28 days after Gli2 gene deletion (F) and TB pore measures, to indicate complete TBs, also decreased (F). Taste buds with pores are essentially eliminated after 16 days gene deletion (F). Numbers of tongues studied at each time point after gene deletion are in parentheses in the legends. Full statistics for ANOVAs are presented in S1E and S1F Fig.

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As in FP, NF-positive innervation to the CV, via the glossopharyngeal nerve, was not eliminated after epithelial gene deletion for 28 days which led to profound taste bud reduction (S6A and S6B Fig). Furthermore, comparable to results for FP, proliferating Ki67+ cells in the CV basal epithelium were substantially reduced after HH/GLI suppression (S6C and S6D Fig). To determine whether cell death was a major contributor to TB cell loss in the CV after HH inhibition, we performed TUNEL staining and did not discern a difference from Control tongue (S6E and S6F Fig). Again this is similar to the FP.

Similar to Gli2cKO mice (Fig 7F), in K5GliR tongues after 35 days of HH/GLI blockade, the number of CV taste pores was reduced to a mean of 10 compared to the control mean of 50 (S6G Fig). An even more profound effect on CV taste buds was observed in epiGliR mice: taste bud remnants and/or complete taste buds were essentially eliminated after 5 days (S7A, S7B, S7C and S7D Fig), and the CV shape was altered, with increased keratinization after 11 days (S7E and S7F Fig).

HH responding cells and SHH expression are reduced in circumvallate papilla epithelium after K5-targeted GLI blockade

In Control mice carrying a Gli1^lacZ allele, X-Gal-positive HH-responding cells were detected in basal epithelial and perigemmal cells in the CV and in the stroma around taste bud-bearing CV walls (Fig 8A and 8B Control). However, in Gli2cKO mice, there was a sustained loss of HH-responding X-Gal-positive epithelial cells in the CV, as early as 5 days after gene deletion, with variable numbers of X-Gal-positive cells remaining in the surrounding stroma particularly near taste bud remnants (Fig 8C and 8D Day 5; Fig 8E and 8F Day 35).

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Fig 8. HH-responding cells are lost from circumvallate papilla epithelium but remain in stroma after epithelial Gli2 deletion, and taste bud size, and SHH expression, decrease with duration of HH/GLI repression.

A,B. Gli1lacZ-positive cells in Control and C,D;E,F after gene deletion, in Gli2cKO CVs, at 5 and 35 days. B,D,F are enlargements for boxed regions in paired panels. A,B. In Control CV, HH- responding cells are in epithelial cells (perigemmal) surrounding the taste buds and in stroma of the papilla surround, including just under the basal lamina. C,D; E,F. From 5 days of gene deletion and continuing through 35 days, Gli1lacZ-positive cells are lost from the papilla epithelium but remain in stroma just under the taste bud- bearing epithelium. Dotted ovals are used to denote taste buds (B) or taste bud profiles/remnants (D,F). G,I,K. SHH and K8 Immunoreactions in Control, and at Day 5 and Day 35 after gene deletion illustrate reduction in K8-positive taste bud cells and associated loss of SHH expression. H,J,L. Same sections as those in merged panels (G,I,K) to illustrate SHH ligand alone in taste bud cells and remnants. After 35 days SHH expression is retained in reduced cell collections.

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In Control CV, SHH expression is clustered in the basal cell region of taste buds (Fig 8G and 8H). Associated with the loss of taste bud cells seen as early as Day 5 after gene deletion, there was a reduction in SHH expression within the taste buds of the CV (Fig 8I and 8J Day 5). With taste bud profiles or remnants some SHH-expressing cells remained, shown at Day 35 (Fig 8K and 8L Day 35).

In summary, the CV papilla epithelium was severely disrupted after HH/GLI blockade and was unable to sustain a full complement of TBs with pores, although papilla innervation remained. In association with taste bud cell loss, Ki67-positive basal epithelial cells were decreased, and epithelial HH-responding cells and SHH expression were reduced. These data indicate that both ectoderm-derived FP and endoderm-derived CV papillae require epithelial HH signaling for proper tissue maintenance.

Loss of taste papillae and taste buds after HH/GLI blockade is reversible

To determine whether effects of epithelial HH/GLI blockade on papillae and taste buds were reversible, we induced transgene expression in K5GliR mice by treating with doxycycline for 16 days, and then withdrew doxycycline for a subsequent 7, 14 or 30 days. At 16 days of transgene expression, K5GliR FP were disrupted and taste bud cells were reduced or lost (Fig 9A and 9B). TYPE I FP/TB were reduced to about 25% of Control values and TYPE III Atypical FP/No TB papillae were about double those in Control, or 40% of all FP (Fig 9D). When doxycycline treatment was stopped to shutdown transgene expression, 7 days were not sufficient to induce recovery of FP/TB organs (Fig 9D). However, after treatment withdrawal for 14 days, epithelial integrity was restored (Fig 9C) and TYPE I Typical FP/TBs were recovered to about 80% of Control (Fig 9D). TYPE III Atypical FP/No TB papillae, on the other hand, also remained at a substantial proportion or about 40% of FP over 7–30 day recovery periods (Fig 9D), without apparent recovery. (All ANOVA data are provided in S8A Fig).

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Fig 9. Recovery from effects of epithelial HH/GLI repression in fungiform and circumvallate papillae and taste buds.

A,B,C. H&E sections of FP in Control (A), K5GliR after 16 days HH repression (B), and with 16 days HH repression followed by 14 days doxycycline withdrawal (C). D. Graph for FP recovery data. Whereas TYPE I Typical FP/TB are substantially reduced after 16 days of transgene activation, there is recovery after 14 days to more than 60% of TYPE I FPs. E,F,G. H&E sections of CV to illustrate papilla size and shape in Control (E) and in K5GliR tongue after 16 days HH repression (F) and with 16 days HH/GLI repression followed by 14 days doxycycline withdrawal (G). H. Graph for CV structure. There were no significant differences in CV size across Control, after HH/GLI repression, or recovery from HH/GLI repression. I,J,K. Enlargements, to illustrate taste bud pores and taste bud remnants, of boxed regions from Control (E), K5GliR after 16 days HH repression (F) and with 16 days HH repression followed by 14 days doxycycline withdrawal (K). L. Graph to demonstrate CV taste bud recovery. Taste bud profiles/remnants and taste buds with pores were much reduced after 16 days of HH repression but recovered to more than 60% Control levels with doxycycline withdrawal for 14 days, or up to 95% after 30 days. Dotted ovals are used to denote taste buds or taste bud remnants/profiles (I,J,K); arrows point to taste pores (I,K). Numbers of tongues at each time point are indicated in parentheses in graph legends.

https://doi.org/10.1371/journal.pgen.1006442.g009

After 7–14 days recovery from 16 days of HH repression, X-Gal-positive, HH responding cells were again detected in perigemmal and basal epithelial cells in regenerated TYPE I Typical FP/TB (S9A, S9B and S9C Fig) but were only in the connective tissue stroma of TYPE III Atypical FP/No TB taste organs (S9G, S9H and S9I Fig). In 32% of TYPE II Atypical FP/Atypical TB after 7 days recovery there were some X-Gal-positive cells already appearing in the papilla epithelium (S9F Fig). Note that after 7 days recovery from HH repression, 25% of TYPE I Typical FP/TB have a distribution of lacZ-positive cells in FP epithelial walls and the stromal core (S9C Fig), whereas Type III Atypical FP/No TB have no lacZ-positive cells in the epithelium (S9I Fig).

To probe for recovery of proliferating cells we examined Ki67 expression in apical, basal and perigemmal cell regions of the FP. In tongues from Controls and mice with K5GliR transgene expression for 16 days, the results replicated those shown in Fig 4H for cell proliferation in Gli2cKO mice. In tongues from 16 day-induced K5GliR mice followed by 30 days treatment withdrawal, there was recovery of Ki67+ cell numbers in apical and perigemmal regions (S8G, S8H, S8I and S8J Fig). The extent of this recovery, significant in perigemmal cells, directly matched that for recovery of Type I FP/TB after treatment withdrawal (S8K Fig).

Similarly, CV papillae and taste buds also recovered after release of HH/GLI blockade. Sixteen days after GLI repression in K5GliR mice, the CV retained structural integrity compared to Control mice (Fig 9E, 9F and 9H). However, there was a clear and substantial loss of CV taste buds (Fig 9I, 9J and 9L), comparable to that seen in Gli2cKO mice (Fig 7). The CV taste bud profiles in K5GliR tongues were reduced to about 50% and pores were reduced to about 20% of Control values (Fig 9L). After 14 days off doxycycline treatment, the CV structure was still intact (Fig 9G and 9H) and taste bud profiles recovered to about 66% of Control values and to about 95% after 30 days (Fig 9K and 9L). Taste pores recovered to more than 80% of Control values after 14 or 30 days (Fig 9L). Compared to loss of HH-responding cells in the epithelium around the taste buds after HH repression (Fig 8), after 14 days recovery from treatment, X-Gal-positive cells were in the CV stroma and surrounding the taste buds (S10 Fig). (All ANOVA data are in S8B and S8C Fig)

With a shorter HH/GLI suppression period of 5 days, there was full recovery of TYPE I Typical FP/TBs, and of TB profiles and pores in the CV, within 14 days. Therefore with a shorter period of suppression and less extensive taste organ effects, recovery was complete (S8D, S8E and S8F Fig).

Thus, even after a prolonged period of HH/GLI repression, there was recovery of taste organ integrity in over half of FP papillae, and FP and CV taste buds, after only two weeks of doxycycline withdrawal. Therefore, during the period of epithelial HH/GLI blockade, progenitor cells that could reconstitute the papilla epithelium and taste buds upon reversal from HH/GLI repression survived or were rapidly re-formed. In TYPE III Atypical FP/No TB recovery was not detected, suggesting that with elimination of TB cells and therefore epithelium-derived HH ligand, progenitors for intact papillae and taste buds were irreversibly eliminated. The data suggest that HH-expressing taste bud cells are necessary for epithelial recovery from HH/GLI blockade.

Recovery from HH signaling suppression

Our data demonstrate effects of HH signaling block in K5-expressing cells in two very different gustatory papillae: the FP with a single taste bud in the apex and the CV with a few hundred taste buds that are dense and in physical juxtaposition in walls of the papilla trenches [57]. On withdrawal of doxycycline to stop transgene expression in K5GliR mice, a large proportion of FP and CV epithelia and taste buds recover. In FP perigemmal cells, the Ki67 cell expression that is reduced after HH/GLI suppression also recovers. Therefore TB progeny are not eliminated in HH signaling repression in the epithelium of these papillae but are poised to regenerate TBs, with innervation and papilla organ connective tissue core elements already in place, once HH/GLI signaling is restored. SHH-positive cells within TBs are proposed as a possible obligate stage in differentiation of all TB cell types, as post-mitotic precursors not stem cells [35].

However, we find that a subset or about 40% of TYPE III Atypical FP/No TB taste organs do not recover after HH/GLI blockade. Because these FPs lack TBs, they lack SHH ligand in the epithelium and do not attain reconstitution of HH-responding cells in the papilla epithelium. Therefore we suggest that epithelium-derived HH is essential to maintain and re-activate TB cell progenitors which may reside within TBs.

Pharmacologic blockade of HH signaling and epithelial HH suppression

We note that after 14–30 days to restore HH/GLI signaling after HH repression, the recovery of Typical FP/TB and CV taste buds is substantial but incomplete in the FP/TB. Notably, in a pharmacological block of systemic HH signaling with the HH pathway inhibitor drug LDE225, there also is substantial loss of Typical FP and taste buds after 16 days [31]. These experiments uncovered the likely mechanisms underlying taste loss in cancer patients treated with systemic HH pathway inhibition [29,30]. Although with LDE225 pathway inhibition the taste buds and associated SHH were lost, a robust innervation remained within aberrant fungiform papillae. As in our genetic models to block epithelial HH signaling, there also is recovery after 14 days without the inhibitor drug, of about 50% of Typical FP/TB [60]; and these typical organs demonstrate a full complement of X-Gal-positive, HH responding cells in papilla epithelial walls and perigemmal cells. However, in LDE225 HH inhibition, and all genetic models, about 40% of taste organs are Atypical FP/No TB, and in these papillae HH-responding cells are only in the papilla core. We are studying the nature of the FP/TB that apparently are incapable of regeneration.

Papilla and taste bud disruption in epithelial HH repression does not eliminate nerves or alter innervation pattern

Whereas we do not rule out a possible decrease in nerve fibers, at the light microscopic level there is no general elimination or misdirection of innervation in anterior tongue, FP and/or TB cells even after epithelial HH pathway suppression over long periods. Also, innervation remains robust in the CV in Gli2 repressor and cKO mice. Because SHH is much reduced in the taste bud, in parallel with the loss of SHH-expressing taste bud cells, the retained innervation indicates that SHH is not a major, taste bud, target-derived maintenance factor for innervation to the papilla and TB.

Further, because P2X3 fibers remain in FP in Gli2 mutant and deletion tongues there is apparently not a major destruction of geniculate ganglion neurons that project via the chorda tympani to taste buds. In a pharmacologic block of HH signaling with the HH pathway inhibitor drug LDE225 we also demonstrated that although taste buds were lost, thus reducing available taste bud-derived SHH, a robust innervation remained to taste bud cell remnants and aberrant fungiform papillae [31].

However, we have localized HH-responding, Gli1^lacZ-positive cells adjacent to nerve bundles and S100-labeled Schwann cells within the FP. Furthermore, within the body of the tongue Gli1^lacZ-positive cells are seen along the perimeter of large bundles of the chorda tympani/lingual nerve. Overall our data put Gli1^lacZ-positive cells in contiguity with nerve fibers and nerve-associated Schwann cells, possibly even in cells of the perineurium [61]. We propose that in addition to SHH in taste bud cells, there are possible sources of SHH from the geniculate ganglion and trigeminal ganglion, and via innervating nerve fibers could potentially provide local SHH ligand to Gli1^lacZ-positive HH-responding cells in the taste papilla organ. SHH has been reported in trigeminal ganglion neurons in studies of pulp innervation [62]. If sensory nerve endings are a source of HH ligand from trigeminal or geniculate ganglia these could activate HH signaling in stromal cells of the FP core, as seen in hair follicle innervation [3]. There is, then, ligand from SHH released from taste buds in epithelium, much reduced with HH suppression; and, potentially from trigeminal and geniculate ganglia at nerve endings within the FP. Thus, HH-responding cells in multiple domains of the papilla organ could have access to ligand from at least two sources.

Not only do we find that SHH in taste bud cells is apparently not a major target-derived support factor for FP/TB lingual innervation but also, even the sustained lingual and FP innervation is not sufficient to maintain or regenerate intact taste buds in the face of epithelial HH-repression. Innervation is necessary to maintain taste buds but on anterior tongue of mouse with combined chorda tympani and lingual nerve cut a number of taste buds remain [51]. Therefore whereas nerves are essential to transmit taste responses to the CNS and to maintain taste buds, there apparently is not a complete or sole sensory, chorda tympani nerve dependence of all adult FP taste bud cells. In mice with SHH over-expression, K8-positive, taste bud-like cells can form in lingual epithelium outside of FPs but these are not innervated and therefore are not able to transmit taste sensation [28]; this indicates a taste bud cell-like phenotype in the absence of taste innervation and chemosensory function. Therefore, the sensory nerve dependence of taste bud cells is complex [63].

Stromal cells are retained in HH suppression

The importance of stromal cells in the papilla core, adjacent to taste bud cells and their supporting epithelium, has been essentially ignored in the taste field. Stromal fibroblasts with the intermediate filament vimentin are important in cell adhesion, migration and signaling [64] and SHH can be chemotactic and interact with fibroblasts in SHH trafficking [54]. There also is ample evidence that HH signaling regulates fibroblast activity, e.g., in kidney interstitium [65], in lung [2,5] and during pancreas tumorigenesis [6]. However, despite the profound alterations in taste organs that arise after blockade of HH/GLI signaling in epithelial cells, immunostaining for fibroblasts did not reveal appreciable differences compared to Control mice. This is in contrast to mesenchymal cell proliferation when HH signaling is downregulated after injury in adult lung epithelial cells [2]. Nor did we observe a massive influx of macrophages in the papilla core. The retention of multiple cell populations, and nerve endings, in the papilla core, despite disruption of overall papilla structure, may provide a crucial microenvironment needed for recovery of taste organs after HH/GLI blockade.

Taste homeostasis and HH signaling

Sensory homeostasis demands balanced cell physiology, and physiological adjustments in major signaling pathways can alter taste function and create risk for diet selection, toxin avoidance and proper nutrition. We showed that HH/GLI signaling is essential to preserve homeostasis in taste papillae and resident TBs; this pathway operating in peripheral taste organs is, therefore, crucial for taste sensation and nutrient regulation. Our findings contribute to understanding the biological basis of taste alterations in patients who take HH Pathway Inhibitors (HPIs) for treatment of basal cell carcinoma [29,30]. The responses to genetic HH/GLI repression that we observed in FP have a similar time course to FP/TB alterations in mouse tongue after HPI administration [31]. HH repression in the lingual epithelium leads to profound loss of taste buds but nerves and connective tissue cells are maintained within taste papilla organs. Therefore, taste bud progenitor cells are strictly dependent on epithelial HH signaling, and can function to regenerate taste buds when HH signaling is restored as long as some residual TB cells remain. However, if HH and HH-responding cells are eliminated from the fungiform papilla epithelium through taste bud loss, papillae and taste buds do not recover from HH suppression. This suggests that HH in taste bud cells, acting through paracrine signaling to responding cells, is in a principal role for taste bud and papilla maintenance and restoration. To keep taste bud cell homeostasis at a necessary steady state for sensory function, epithelial HH/GLI signaling is required.

Animals

Maintenance of mice and all experimental procedures were conducted in accordance with NIH guidelines and were approved by the University of Michigan Institutional Animal Care and Use Committee (protocols: PRO00006464, BLA; PRO00006657, AAD; PRO00005851, CMM).

Mouse strains and transgene induction

Gli1^lacZ/+: mice were used to indicate cells responding to HH/expressing the target gene Gli1.

Mice were maintained on mixed backgrounds.

K5-rtTA;tetO-Gli2ΔC4;Gli1^lacZ/+(K5GliR): doxycycline-inducible, reversible, dominant-negative inhibition of Hh/Gli target genes in K5 expressing, basal epithelial cells (littermates negative for K5rtTA and/or tetO- Gli2ΔC4 were used as controls).

K5-Cre;R26-LSL-rtTA;tetO-Gli2ΔC4;Gli1^lacZ/+(epiGliR): doxycycline-inducible, dominant-negative inhibition of Hh/Gli target genes in K5 expressing cells and their progeny (littermates negative for K5rtTA and/or tetO- Gli2ΔC4 and/or R26-LSL-rtTA were used as controls).

K5-rtTA;tetO-Cre;Gli2^fl/fl;Gli1^lacZ/lacZ and K5-rtTA;tetO-Cre;Gli2^fl/fl;Gli1^lacZ/+(Gli2cKO): doxycycline-dependent, Cre-driven deletion of Gli2 in K5-expressing cells, heterozygous or null for Gli1 (littermates negative for K5rtTA and/or tetO-Cre were used as controls).

K5-rtTA;tetO-Cre;Gli2^fl/fl;Gli1^lacZ/lacZ (Gli2cKO;Gli1KO): a Gli2/Gli1 double deletion in K5-expressing cells (littermates negative for K5rtTA and/or tetO-Cre with Gli1^+/+ were used as controls).

K5-rtTA mice [66] were obtained from Adam Glick (Pennsylvania State University). Each of the following strains was obtained from Jackson Labs: Gli1^lacZ/lacZ (Stock No: 008211) [18]; Gli2^fl/fl (Stock No: 007926) [67]; tetO-Cre (Stock No: 006224) [68]. tetO-Gli2ΔC4 mice were generated using a mouse Gli2ΔC4 cDNA [4,40] inserted into pTet-Splice using standard molecular cloning techniques (Ermilov et al in preparation).

Doxycycline was administered in chow at 1 g or 6g doxycycline/kg chow (Bio-Serv), for specified periods. Once started, doxycycline treatment for conditional, epithelium-specific gene deletion studies was continuous throughout the treatment period. For recovery studies using K5GliR mice, doxycycline administration was stopped to extinguish Gli2 expression. Animals were maintained on normal chow to monitor recovery from HH/GLI blockade.

For each experiment and time point, observations were made in at least 3 experimental and 3 littermate control mice, with noted exceptions for very long experimental periods. Actual animal numbers are included in all graphs.

Tissue dissection and processing

Quantification

Cell death

The terminal deoxynucleotidyl transferase dUTP nick ending labeling assay (TUNEL; Millipore Kit S7165) was used with manufacturer protocol to detect apoptotic cell nuclei. We studied TUNEL-positive cells in FP in one experimental and one control tongue from Gli2cKO mice at 28 days after deletion; and for CV in two experimental and two control tongues from Gli2cKO mice at 16 days after deletion and assessed distribution patterns in papilla and surrounding epithelium. We studied Cleaved Caspase 3 in FP in one experimental and one control tongue each for the K5GliR model after 5 days and for the epiGliR model after 11 days induction. Cleaved Caspase 3 in the CV was studied in one Control tongue and one Gli2cKO at 16 days after deletion.

Innervation

Papilla innervation was analyzed in immunoreactions with antibodies to Neurofilament Heavy (NF200) and for specific chemosensory innervation with an antibody to P2X3. We studied 1–2 experimental and one control tongue each for K5GliR and epiGliR models across 3 time points, one control and one experimental tongue from Gli2cKO mice at 28 days post induction. Innervation patterns were analyzed qualitatively to identify nerve fibers in the “basket” region just under the taste bud region (using NF) or within the taste bud (using P2X3) and fibers directed through the middle of the papilla core.

Stromal cell activity in FP

To identify fibroblasts within the FP core and discern how these cells are associated with the epithelium or with basal lamina we used double immunoreactions for vimentin and heparan sulfate proteoglycan (HSPG) to delineate the basal lamina. We studied 8 FP each in Gli2cKO tongues, one experimental and one control, at 28 days post induction. For each of the FP we captured images of four serial sections (or 8 X 4 sections per tongue). We counted FP stromal cells that were in basal lamina contact, that is, contiguous to/touching or crossing/within the basal lamina in each tongue. In these same sections we counted vimentin-positive cells that were in the core of the papilla, that is, in the stroma not in basal lamina contact. We also counted all Vimentin-positive cells within the epithelium for each FP. To ensure that vimentin-positive cells were not macrophages or smooth muscle actin-positive cells we used immunoreactions for F4/80 or αSMA respectively.

Statistics

All data in figures are presented as means and standard errors. For analysis of FP types and CV measures, across time periods, we used One Way Analysis of Variance (ANOVA) for each papilla type, with the Least Significant Difference posthoc test, and a significance level of p<0.05. Numbers of tongues/FPs are in graphs. In Supplemental Figs 1 and 9, complete F and p values are presented for FP and CV quantification data/graphs. For all ANOVA, we pooled data from Control mice for the comparison group against Experimental time points. For Gli2cKO;Gli1KO FP analysis, to overcome small sample sizes at some time points, we compared pooled Control (n = 6) with pooled data from doxy-treated Day 16,28 (n = 4) and from Day 35,45 tongues (n = 4) but all time points are shown in figures.

In analysis of cell proliferation and stromal cells numbers, the independent samples t test, with Levene’s test for equality of variance, was used to compare differences between treatments (significance level = p ≤ 0.05).