Recovery of taste organs and sensory function after PNAS PLUS severe loss from Hedgehog/Smoothened inhibition with cancer drug sonidegib

Archana Kumaria, Alexandre N. Ermilovb, Marina Grachtchoukb, Andrzej A. Dlugoszb,c,1, Benjamin L. Allenc,1, Robert M. Bradleya,1, and Charlotte M. Mistrettaa,1,2

aDepartment of Biologic and Materials Sciences, University of Michigan School of Dentistry, Ann Arbor, MI 48109; bDepartment of Dermatology, University of Michigan Medical School, Ann Arbor, MI 48109; and cDepartment of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, MI 48109

Edited by Linda M. Bartoshuk, University of Florida, Gainesville, FL, and approved October 11, 2017 (received for review July 19, 2017)

Striking taste disturbances are reported in cancer patients treated signaling in FP and circumvallate (CV) papillae and tested the with Hedgehog (HH)-pathway inhibitor drugs, including sonidegib hypothesis that morphologic and functional recovery would occur (LDE225), which block the HH pathway effector Smoothened (SMO). following cessation of HH/SMO inhibition by sonidegib treatment. We tested the potential for molecular, cellular, and functional Taste cells turn over continuously in adults (9, 10), but TB are recovery in mice from the severe disruption of taste-organ biology eliminated when essential signaling pathways, including HH, are and taste sensation that follows HH/SMO signaling inhibition. disrupted (11–13). However, the taste cells are resilient in specific Sonidegib treatment led to rapid loss of taste buds (TB) in both contexts, as illustrated by degeneration after nerve cut in adult fungiform and circumvallate papillae, including disruption of TB mammals and regeneration as nerves grow back into the taste progenitor-cell proliferation and differentiation. Effects were selec- papilla epithelium (14). The taste organ therefore is ideally suited tive, sparing nontaste papillae. To confirm that taste-organ effects of to explore fundamental questions about the biology of tissue ho- sonidegib treatment result from HH/SMO signaling inhibition, we meostasis and renewal, the mechanisms that sustain sensation in studied mice with conditional global or epithelium-specific Smo dele- constantly renewing organs comprised of heterogeneous cell types, CELL BIOLOGY tions and observed similar effects. During sonidegib treatment, and the potential restoration of organs and function after TB chorda tympani nerve responses to lingual chemical stimulation were elimination resulting from deregulation of essential controls. maintained at 10 d but were eliminated after 16 d, associated with Although the HH pathway is a major regulator in several organ nearly complete TB loss. Notably, responses to tactile or cold stimulus systems (15), there is sparse fundamental knowledge about the modalities were retained. Further, innervation, which was maintained function and timing of HH signaling in taste organs, especially the in the papilla core throughout treatment, was not sufficient to sustain mechanisms of cell regulation, the regulation across anterior and TB during HH/SMO inhibition. Importantly, treatment cessation led to posterior tongue tissues, and the effects on taste sensation per se. Whereas transcriptional-level regulation of HH/GLI signaling has rapid and complete restoration of taste responses within 14 d asso- Gli2 ciated with morphologic recovery in about 55% of TB. However, been studied by genetic manipulation of (11), the conse- although taste nerve responses were sustained, TB were not restored quences of HH signal disruption at the cell surface remain largely in all fungiform papillae even with prolonged recovery for several unexplored, although most pharmacologic HH inhibitors act at this level (16). months. This study establishes a physiologic, selective requirement for HH/SMO signaling in taste homeostasis that includes potential for sensory restoration and can explain the temporal recovery Significance after taste dysgeusia in patients treated with HH/SMO inhibitors. Hedgehog pathway-inhibitor drugs effectively treat basal cell taste receptor cell | fungiform papilla | circumvallate papilla | carcinoma, a common skin cancer. However, many patients taste regeneration | chorda tympani nerve taking such drugs report severe taste disturbances that impair their quality of life. To understand the biology behind these ancer patients treated with Hedgehog (HH) pathway in- adverse effects, we studied the consequences of Hedgehog Chibition (HPI) drugs experience severe taste disturbances (1– pathway inhibition on taste organs and neural sensation in mice. 5). The Food and Drug Administration-approved HPI drug Taste bud progenitor-cell proliferation and differentiation were sonidegib (LDE225) blocks HH signaling at the Smoothened altered, resulting in taste bud loss. Nerve responses to lingual taste stimuli were also eliminated, while responses to touch and (SMO) receptor (Fig. 1A) (6). We had reported that oral gavage cold stimuli remained. After stopping Hedgehog pathway in- with sonidegib in mice for 16 or 28 d results in progressive fungi- hibition, taste buds and sensory responses recovered. This study form papilla (FP) taste-organ disruption, taste bud (TB) loss, and advances our understanding of Hedgehog signaling in taste elimination of taste nerve responses to chemical stimuli (7). Now homeostasis and the reported taste recovery after clinical we present data for the initial time course of HPI effects on taste treatments with Hedgehog pathway-inhibiting drugs. organsandcelltypesandonsensation;studyofSmo deletion; and the presence of the HH ligand in the nerve fibers of taste organs. Author contributions: A.K., A.N.E., A.A.D., B.L.A., R.M.B., and C.M.M. designed research; Importantly, the potential for and nature of recovery from HPI A.K., A.N.E., R.M.B., and C.M.M. performed research; M.G. contributed new reagents/ effects in taste organs and taste neurophysiology are demonstrated. analytic tools; A.K., A.N.E., A.A.D., B.L.A., R.M.B., and C.M.M. analyzed data; and A.K., Lingual taste organs are composed of (i) the TB, about 80– A.A.D., B.L.A., R.M.B., and C.M.M. wrote the paper. 100 modified epithelial/neuroepithelial cells of several types; The authors declare no conflict of interest. (ii) the sensory innervation to the TB and surrounding epithe- This article is a PNAS Direct Submission. lium; and (iii) the papilla residence, a stratified epithelium over a Published under the PNAS license. core of stromal cells and connective tissue elements (8). Precise 1A.A.D., B.L.A., R.M.B., and C.M.M. contributed equally to this work. controls within each compartment are essential to integrate the 2To whom correspondence should be addressed. Email: [email protected]. cell processes that support taste organs and sensation. Here we This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. investigated homeostatic lingual taste organ regulation by HH/SMO 1073/pnas.1712881114/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1712881114 PNAS Early Edition | 1of10 Downloaded by guest on September 25, 2021 Fig. 1. Sonidegib alters FP and TB morphology and reduces all TB cell types. (A)TheHHpathwayinOFF and ON signaling and INHIBITED at SMO by sonidegib. (B) Schematic: FP and filiform papillae with HH sig- naling components. (C) H&E staining for FP/TB cate- gories. Dotted lines demarcate TB or regions of TB remnants. Blue dashed surface lines indicate epithelial keratinization in FP; a red dashed line indicates epi- thelial keratinization in filiform papilla in type III atypical FP/no TB. (Scale bar: 50 μm.) (D)Percentageof FP/TB categories: typical FP/TB (category I), atypical FP/ TB (category II), and atypical FP/no TB (category III), after vehicle or sonidegib treatment. Bars are mean ± SEM. Numbers of tongues per group are in paren- theses. Brackets indicate significant differences for treatment durations (two-way ANOVA and Tukey’s HSD post hoc tests); ###P < 0.001 for vehicle vs. soni- degib treatments. Complete F and P values are given in Fig. S1A.(E) Antibody detection of TB cell types: NTPdase2 (type 1), PLCβ2 (type 2), SNAP25 (type 3) after vehicle or sonidegib treatment. Cell nuclei are identified with DAPI (blue). (Insets) TB cell types 2 and 3 were eliminated after 28 d of sonidegib treatment. Type 1 cells (NTPdase2) were eliminated after 36 d. (Scale bar: 50 μm.) (F) Number of TB cell types after 16 or 28 d of sonidegib treatment compared with vehicle treatment. Data are mean ± SEM. Numbers of tongues are in parentheses in bars. Brackets indicate significant differences (one-way ANOVA with Tukey’s HSD post hoc tests). Complete F and P values are given in Fig. S1C.

SMO is the core signal transduction component of HH signaling tion of oral sensory modalities of taste, touch, and temperature, (Fig. 1A) (17, 18). In HH signaling, pathway activity is repressed in and the recovery of taste organs and sensation. Our data provide the absence of ligand via the transmembrane receptor PTCH, insight into the regenerative biology and clinical consequences in which represses the pivotal HH signaling protein SMO (19–21). patients treated with sonidegib who experience dysgeusia. HH binding to PTCH1 blocks its inhibition of SMO; downstream signaling is then initiated, resulting in the modulation of GLI Results transcription factors (GLI1, GLI2, GLI3) and leading to the Treatment with HPI Drug Sonidegib Alters FP Taste-Organ Morphology transcription of target genes, including Gli1 and Ptch1 (Fig. 1A). Within 10 D. Before testing recovery from HPI drug treatment, In the tongue, HH pathway components are in distinct compart- it was important first to determine the temporal aspects of ments of adult taste papillae and TB (Fig. 1B), including actively HH/SMO signaling inhibition in mice gavaged with sonidegib for proliferating TB progenitor cells (8, 13). The secreted SHH ligand 5–36 d. We quantified effects by characterizing FP and TB mor- in epithelium is produced principally within TB cells (13, 22), phology as category I (typical FP/TB), II (atypical FP/TB), or III whereas the HH targets, Ptch1 and Gli1, are expressed within (atypical FP/no TB) (Fig. 1C). Compared with vehicle-treated perigemmal cells around the TB, in basal cells of the stratified mice, there were no differences in FP/TB categories after 5 d of epithelium that lines the papilla core, and in stromal or connective sonidegib gavage (Fig. 1D). However, after just 10 d of sonidegib tissue cells of the papilla core (Fig. 1B)(11,13).Paracrinesig- treatment the proportion of category I FP (typical FP/TB) FP was naling from SHH in TB cells to surrounding epithelial and con- reduced by half, and category II FP (atypical FP/TB) had in- nective tissue cells has been proposed (13, 23). creased several fold to 40% of all FP, compared with ∼2% in Sonidegib and other HPI drugs block the HH pathway at SMO vehicle treatment. After 16 d of sonidegib gavage only ∼1% of all and are effective in treating patients with advanced BCC (3, 16, FP were category I (typical FP/TB) FP; category II (atypical FP/ 24–26). However, these patients experience dysgeusia and ageusia TB) FP comprised almost 70%, and category III (atypical FP/no that compromise treatment adherence and quality of life (4, 5, 27, TB) FP accounted for ∼30% of all FP (Fig. 1D). With 28 d of 28). There are reports that patients have some recovery 2–3mo sonidegib gavage, category I (typical FP/TB) were essentially after stopping treatment (5), but the nature and extent of this eliminated, replicating our prior results (7); notably, after 36 d, recovery are unclear. Regenerative potential after HPI drug virtually all FP were category III, atypical FP/no TB (Fig. 1D). treatment has not been explored in animal models, so the basic Therefore, in a detailed time course, HPI drug duration of 10–16 d biology of patient recovery is not known. eliminated typical FP and TB, whereas 5 d was not effective, and Here we used the cancer drug sonidegib in mice, in parallel with in progressive effects by 36 d only atypical papillae that lack TB conditional Smo deletion targeting the whole body or epithelium, (category III) remained on the tongue. (Statistics for data in Fig. to test the main site of inhibitory effects and discern the mecha- 1D are given in Fig. S1A.) nisms for HH/SMO inhibition in FP and CV taste organs and in Importantly, we quantified all FP numbers in a standard 600-μm sensory responses from the chorda tympani nerve that innervates region of vehicle- and sonidegib-treated tongues and found no TB in the FP. Further, we assessed taste organs and nerve re- reduction in papilla density across treatments (Fig. S1B). That is, sponses for periods of several months after cessation of HPI drug FP were not eradicated, and we were not categorizing fewer FP at treatment to determine whether recovery is possible. We demon- long treatment durations. Rather, these duration-specific results strate coordinated cell proliferation and differentiation regulated suggest that HPI drug effects are related to the disruption of by HH/SMO signaling in taste papillae and TB, selective regula- physiologic cell renewal in taste organs.

2of10 | www.pnas.org/cgi/doi/10.1073/pnas.1712881114 Kumari et al. Downloaded by guest on September 25, 2021 To further understand the progression of TB loss in FP, we treated tongues (Fig. S2 A and B). That is, accelerated or altered PNAS PLUS used immunostaining to test whether all TB cell types were cell death did not contribute to TB cell loss in HPI. susceptible to sonidegib treatment or whether specific taste cell Cell differentiation. When TB were eliminated from the FP after + + types (type 1: NTPDase2 ; type 2: PLCβ2; type 3: SNAP25 ) sonidegib gavage, as seen in category III FP (atypical FP/no TB), (29) were lost in a particular sequence or time course or were the papilla apex acquired a particular conical shape (Figs. 1C and retained. Whereas cell types 2 and 3 were essentially eliminated 2A,28d,Inset). To learn whether TB progenitor cells were within this conical FP region, we used immunostaining for the basal cell in TB after 28 d, type 1 cells were not completely lost until 36 d + + E F marker keratin 5 (K5). K5 /K14 cells are among the HH- of sonidegib gavage (Fig. 1 and ). The type 1 cells are most + + responding progenitors for TB cells in FP (13, 32), and K5 /K14 numerous in the TB and have a long turnover time, up to 24 d + (29–31), which could account for their longer retention after HPI cells in CV differentiate into mature K8 TB cells (33). In vehicle- treatment. However, turnover for no TB cell type was resistant to treated mice a few cells within the TB were dual-labeled for the TB cell marker K8 (34) and K5 (Fig. 2C). After sonidegib treatment for the HPI drug, suggesting effects on progenitor(s) for all TB cells. + 5, 10, and 16 d there was a reduction in K8 TB cells and an in- (Complete statistics for data in Fig. 1F are in Fig. S1C.) + Cell proliferation and cell death. crease in K5 cells in the perigemmal region; a few cells within the To test whether HPI altered turn- C Insets over in lingual epithelium, we studied cell proliferation within FP TB were dual-labeled for K8 and K5 (Fig. 2 , ). However, at 28 d in category III FP (atypical FP/no TB), where there were no and the nontaste filiform papillae. In FP we designated three re- + TB or TB remnants, the FP had K5 cells in suprabasal layers gions, basal, apical, and perigemmal zones of FP basal cells (Fig. + + throughout the conical apex (Fig. 2C, 28 d sonidegib). Thus, K5 2A, vehicle), and quantified Ki67 cells for proliferation. Cells in cells occupy tissue territory where TB had been located. all these regions include TB progenitors (13, 32). In basal cells of + + + We also assessed the expression of K14 and p63 cells in the the apical FP wall there were substantial reductions in Ki67 cells + + – A B FP apex and observed both K14 and p63 cells in suprabasal as early as 3 5 d of HPI treatment (Fig. 2 and ), that is even cells of the apex after 28 d of sonidegib treatment (Fig. S2C), as before the FP/TB organs had noticeable morphological disruption + + with K5 cells. Ki67 cells were sometimes found in the FP apex D + + (Fig. 1 ). This early decrease in the number of Ki67 TB pro- but generally were not suprabasal (Fig. S2D). As the K5 /K14 / + genitors in the FP was sustained after 16 and 28 d of drug treat- p63 cells in the tongue differentiate to TB, their expressions are A B ment (Fig. 2 and ). Alterations in proliferating cell numbers lost (32). After sonidegib treatment, at the apex of category III + + were not observed in basal or perigemmal regions of FP epithelial FP (atypical FP/no TB), the suprabasal expression of K5 /K14 / + cells (Fig. 2 A and B). Note that perigemmal counts were made p63 points toward a differentiation defect from a normal pro- + + + CELL BIOLOGY only within category I and category II FP, which have TB or TB gression of K5 /K14 /p63 expression to K8 cells. remnants, because there were no TB in type III FP (Fig. 2A,28d + and Fig. 2B). Notably, there was no reduction in Ki67 cells in the Smo Deletion Mimics HPI Drug Effects on FP Taste Organs. To es- basal cells of filiform papillae (Fig. 2 A and B). tablish that the effects observed in sonidegib-treated mice reflected We did not observe differences in the extent or distribution of the blockade of SMO, the HH signaling effector targeted by the cell death markers [TUNEL assay, Cleaved caspase 3 (CC3) drug, we generated mice to conditionally (doxycycline-regulated) M2rtTA/+ fl/fl antibody] in FP or filiform papillae of vehicle- or sonidegib- delete Smo globally (R26 ;tetO-cre;Smo , referred to as

Fig. 2. Sonidegib treatment reduces apical epithelial cell proliferation in FP and leads to the accumulation + of suprabasal K5 cells. (A) Immunofluorescent anti- body detection of E-cadherin (Ecad, red) and Ki67 (green) in basal epithelial cells of FP and filiform pa- pillae after vehicle or 5 d, 16 d, or 28 d sonidegib treatments. The image for FP, vehicle, shows three + regions for quantifying Ki67 cells (apical, basal, and perigemmal). (Inset) Twenty-eight days sonidegib il- + lustrates the absence of Ki67 perigemmal cells in some category III FP (atypical FP/no TB). The vehicle image for filiform papillae illustrates Ki67+ cells, clus- tered at the base of rete ridges (arrows). White dotted lines indicate the surface of the epithelium. (Scale bar: 50 μm, also applies to C.) (Magnification: Inset,0.9×.) + (B) Number of Ki67 cells in FP regions in vehicle- and sonidegib-treated mice. Counts in the perigemmal region were done only when TB or TB remnants remained in the papilla, i.e., in category I (typical FP/ TB) or category II (atypical FP/TB) FP. In filiform pa- + pillae Ki67 proliferating cells were quantified in a 300-μm length of tongue. Numbers of tongues are in parentheses. Statistical analysis was performed with one-way ANOVA with Tukey’s HSD post hoc tests (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001). F and P values are shown in the table at the right of the graphs. (C) Antibody detection in FP of K5 (green, arrows + point to K5 cells in TB or TB remnants) in TB cells after vehicle or 5, 10, 16, or 28 d sonidegib treatment. (Insets) K5 coexpression with a few K8+ TB cells (red) indicated by arrows. (Magnification: Insets,3.5×.)

Kumari et al. PNAS Early Edition | 3of10 Downloaded by guest on September 25, 2021 fl/fl “cSmoKO” mice) or in the epithelium (K5rtTA;tetO-cre;Smo , tively, the efficiency of Smo deletion in stromal cells might be referredtoas“cSmoKO-epi” mice), diagrammed in Fig. 3A.In less than in epithelial cells. cSmoKO mice, the category I FP (typical FP/TB) were reduced to As HH signaling activity was eliminated in the epithelium, + less than 10% of all FP after 16 d of Smo deletion (Fig. 3B). With there was an apparent decrease in Ki67 proliferating cells in the epithelial deletion of Smo,incSmoKO-epi mice, there were no basal cell layer of the apical papilla wall but not in the basal effects at 5 d after gene deletion, but after 16 d only 15% of FP region (Fig. 4B, Ecad/Ki67). This demonstrates a disruption in were category I (typical FP/TB) (Fig. 3B). After 24 d, more than the supply of a subset of TB cell progenitors. 40% of all FP were category III (atypical FP/no TB), which is comparable to the 16-d effects in cSmoKO mice. Therefore the After Smo Deletion, Nerves Remain Within the FP Connective Tissue Core. major target cell population on which sonidegib acts to alter FP Whereas TB cells were absent after Smo deletion in both cSmoKO and TB is likely to be epithelial. Statistical analyses for the data in and cSmoKO-epi mice, innervation remained within the FP core B A from the lingual nerve that typically innervates the FP walls and Fig. 3 are in Fig. S3 . The FP density did not alter with time in + Smo B perigemmal regions (neurofilament, NF , nerve fibers) and from either deletion model (Fig. S3 ). + Overall the data from two cSmoKO models are similar in time the chorda tympani nerve that typically innervates TB cells (P2X3 taste nerve fibers) (Fig. 4C, K8/NF and K8/P2X3). In addition, course, extent, and effects after sonidegib treatment. Further, lacZ when comparing sonidegib and Smo-deletion experiments a sim- X-Gal staining for Gli1 -positive cells plus NF immunofluores- ilar phenotype was seen in category III FP (atypical FP/no TB) cence demonstrates a close physical association of nerves and HH- (Fig. S3C, H&E). That is, a heavily cornified, conical papilla apex responding cells in the FP connective tissue core in control and + was observed with a discernible cell collection where the K8 taste cSmoKO mice (Fig. 4D), suggesting potential interactions between cells had been located. As with sonidegib treatment, in the cate- the papilla innervation and stromal cells. Furthermore, the results + gory III papilla after Smo deletion, K5 cells occupied the conical demonstrate that nerve fibers sustained within the FP are not able + papilla apex (Fig. S3C, K5/K8). With HPI, therefore, K5 cells to maintain TB in the context of HPI effects in lingual epithelium. replace differentiated cells of the apical FP that typically include TB cells and accumulate as suprabasal, stratified epithelial cells. HPI Leads to Decreased TB in CV on Posterior Tongue. We found that sonidegib gavage and Smo gene deletion each substantially re- + + After Smo Deletion, SHH and K8 Cells Are Reduced in FP, and HH- duced TB and altered morphology in ectoderm-derived FP of the Responding Cells Are Eliminated from the Epithelium but Remain in anterior tongue. To test whether HH/SMO inhibition could also the Connective Tissue Core. Associated with the loss of TB cells affect TB and morphology of the CV (an organ with endodermal + (using K8 as a taste-cell marker), the SHH TB cells also were derivation), we extended studies to include a major posterior- reduced in cSmoKO and cSmoKO-epi mice (Fig. 4A, SHH/K8). tongue taste organ. We used H&E staining to quantify CV size Gli1lacZ and TB numbers. TB were identified as cell collections with a taste Furthermore, -positive HH-responding cells were not A Inset retained in the FP epithelium, in either the perigemmal cells or pore (Fig. 5 , , arrows) or as collections of cells with or lacZ “ ” A Inset basal cells of the FP epithelial wall (Fig. 4A, Gli1 ). In the without a taste pore, termed TB profiles (Fig. 5 , , dotted papilla core, however, HH-responding cells remained but were lines encompass TB profiles). CV depth (all sections in which the Smo papilla appeared, multiplied by section thickness) and the length reduced in number after global deletion compared with A controls. The remaining HH-responding cells in the FP core of of papilla walls (Fig. 5 , bars denote wall length measure) were cSmoKO mice might relate to noncanonical signaling; alterna- assessed for size determinations. Across sonidegib-treated and Smo-deletion mice, there was not an apparent gross alteration in CV form (Fig. 5B). Nor were depth or wall length different, except in cSmoKO-epi mice, in which CV depth was reduced over time and there was a trend for wall length to be shorter (Fig. 5C). (Statistical analyses for data in Fig. 5C are presented in Fig. S4A.) Importantly, in pharmacologic and genetic HPI models, numbers of TB profiles were reduced, and numbers of TB with pores decreased by 25–75% after 16 d (Fig. 5D). Notably, the effects were more pronounced in Smo-deletion mice than after sonidegib gavage. In the CV of cSmoKO-epi mice we examined early-time-point effects and found that the number of TB with pores was not reduced after 5 d treatment, but there was a sig- nificant decrease after 16 d (Fig. 5D). This is similar to effects in FP/TB. (Detailed statistics for Fig. 5D are given in Fig. S4B.) Overall, the TB loss is substantial and demonstrates that HPI affects the CV as well as FP taste organs and, therefore, can alter two major sets of TB on the anterior and posterior tongue. Although not all of the more than 100 TB in the CV were totally eliminated, astheywereintheFP,whichhaveasingleTBeach,theeffectsare substantial. In assessing the efficacy of HPI in cSmoKO-epi mice, we lacZ further observed that Gli1 -positive HH-responding cells were eliminated in the CV epithelium but not from the stroma (Fig. 5E and Fig. S4C). Similar to effects in the FP, HH-responding cells were reduced in the stroma in cSmoKO mice (Fig. S4C). Further- more, even with loss of TB cells, innervation to the CV was retained in physical association with HH-responding cells (Fig. S4C). Fig. 3. Smo deletion alters FP morphology and reduces TB. (A)Diagrams showing the conditional deletion of Smo from all tissues (cSmoKO)orfrom Recovery of FP and TB Cells and Molecular Phenotypes When Sonidegib epithelial cells and progeny (cSmoKO-epi). The gray/shaded region on the cSmoKO-epi diagram indicates normal Smo expression. (B)Percentageof Treatment Is Discontinued. To test potential FP taste-organ resto- category I (typical FP/TB), category II (atypical FP/TB), and category III (atypical ration after HPI, we gavaged mice with sonidegib for 16 d and FP/no TB) FP in cSmoKO or cSmoKO-epi mice. Bars are mean ± SEM. Numbers then discontinued treatment and maintained mice for recovery of tongues are in parentheses. Brackets indicate significant differences (two- periods of 7, 14, or 21 d and for 3, 5, or 9 mo (Fig. 6A). A recovery way ANOVA with Tukey’s HSD post hoc tests); ###P ≤ 0.001 for control vs. period of 7 d was not sufficient to restore papillae to category I cSmoKO or cSmoKO-epi. Complete F and P values are given in Fig. S3A. morphology (typical FP/TB); rather most papillae were atypical, in

4of10 | www.pnas.org/cgi/doi/10.1073/pnas.1712881114 Kumari et al. Downloaded by guest on September 25, 2021 Fig. 4. Smo deletion leads to reduced TB, HH ligand, PNAS PLUS HH-responding cells and reduced proliferation in FP, while innervation is retained. (A) Immunofluorescent antibody detection of SHH (red) and K8 (green) in TB cells; X-Gal staining (blue) of Gli1lacZ-positive cells from control, cSmoKO,andcSmoKO-epi mice. (Insets) Merged images for SHH and K8. No TB cells remain in the cSmoKO image (white box, Inset). (B) Anti- body detection of E-cadherin (Ecad, red) and Ki67 + (green) in control and Smo deletions. Ki67 cells were reduced in apical FP walls shown by white bars. (C) Antibody detection of K8+ (red) TB cells and nerves (NF, green or P2X3, green) in cSmoKO or cSmoKO-epi mice. Arrows indicate P2X3 fibers in the FP core. (Scale bar: 50 μm, all images.) (D) Antibody detection of nerves. NF (green) with X-Gal staining (blue) of Gli1lacZ in control and 16-d cSmoKO mice. (Inset) Enlarged image of the boxed region shows the associa- tion of Gli1lacZ-positive, HH-responding cells with nerves + (NF ) in FP stroma (arrows). Dotted lines in in A, C,andD indicate the basal lamina. (Magnification: D, Inset,5×.)

category II or III (Fig. 6B). Remarkably, however, 14 d were TB with pores were not different from the numbers in vehicle- sufficient to restore about 50% of all papillae to category I mor- treated mice. Therefore, there was recovery from HPI effects in phology, and only 5% or fewer of all FP were category II FP the CV as well as in the FP. + (atypical FP/TB). About 40% of all papillae were category Innervation in the FP. Innervation, shown with NF and TB-specific + III (atypical FP/no TB). P2X3 fibers, was retained within the FP core during sonidegib We used prolonged recovery times to determine whether an in- treatment and also was obvious and robust during recovery from A B

creased proportion of FP was restored to category I after several HPI (Fig. 7 and ). The nerve fibers were observed even in CELL BIOLOGY months. Notably, the proportions of typical FP/TB ( category I) and category III papillae (atypical FP/no TB) that have no taste buds atypical FP/no TB (category III) did not change from 14 d through (Fig. S7A) and in FP after prolonged HPI for 36 d (Fig. S7B). 9 mo of recovery (Fig. 6B). This suggests that a proportion of FP is There were no detectable differences in the extent of innervation not resilient and cannot recover after HPI drug treatment, whereas in papillae treated with vehicle or the HPI drug (Fig. S7C). another FP subset recovers and maintains TB after sonidegib However, nerves alone could not maintain or restore TB in FP treatment is discontinued. Details of FP/TB morphology during with epithelial effects of HH/SMO inhibition. treatment and the recovery transition period from 7 to 14 d after We found that HH-responding cells and nerve fibers remain stopping sonidegib treatment, and after 9 mo of recovery are seen within the FP connective core after HPI and continue in recovery with H&E staining in Fig. S5A. The category III (atypical FP/no periods (Figs. 6C and 7 A and B). Nerve fibers and neurons in the TB) phenotype persists throughout treatment and recovery. This dorsal root ganglion are a source of SHH in signaling to Gli1- category, which loses all TB cells, apparently cannot recover from expressing cells in the hair follicle (35). To determine whether HPI effects, whereas category II FP (atypical FP/TB), which retain fibers in the FP and their soma express SHH, we studied genic- TB cell remnants, presumably can recover. ulate ganglion (GG) and trigeminal ganglion (TG) neurons in Shh Statistical analysis for FP/TB categories across recovery in Fig. reporter mice. SHH expression has been reported in TG neurons 6B is presented in Fig. S6A, and additional data are included to (36), and we confirmed that finding (Fig. 7C). Importantly, we + demonstrate that FP numbers per tissue region do not differ further observed Shh expression within GG neurons and Shh across sonidegib treatment and recovery periods (Fig. S6B). fibers within the tongue and FP and projecting into TB (Fig. 7C). + To test whether all taste cell types in the FP/TB recover after To test whether SHH neurons were altered after HH pathway discontinuing sonidegib treatment or whether some types were inhibition, we used immunoreactions for SHH in ganglia after + resistant to recovery, we used immunostaining and quantified cell vehicle or sonidegib treatment and quantified SHH neurons with types. As noted, all cell types were eliminated after the HPI drug NeuN coexpression in the GG. No differences were detected (Fig. (Fig. 1D), but when treatment was discontinued the three cell types 7D). Although not quantified, similar results were seen in the TG were restored, with similar time courses, after 14 d in category I (Fig. 7E). This indicates that a GG ganglion source of SHH ligand (typical FP/TB) and category II (atypical FP/TB) FP to the values remains during pathway inhibition, although the lingual epithelial seen in vehicle-treated FP (Fig. S6C). Category III FP (atypical FP/ SHH associated with TB cells is eliminated. The GG- and TG- no TB), were not quantified because no TB were present. derived SHH sources could potentially maintain HH signaling in During recovery from HPI treatment, which occurs between papilla stroma via secretion from nerves. 7 and 14 d after stopping sonidegib gavage, there was an increase + + in SHH and K8 cells in the reconstituted taste buds (Fig. 6C, Responses to Chemical Stimuli from the Chorda Tympani Nerve lacZ SHH/K8), associated with recovery of Gli1 -positive cells in Recover After Sonidegib Treatment Is Stopped, and Tactile and lacZ the perigemmal region and FP epithelial walls (Fig. 6C, Gli1 ). Cold Responses Are Maintained Throughout. lacZ To determine early A gradual restoration of Gli1 -positive cells was apparent in sensory effects after HPI and to test whether taste sensation is the varied expression patterns seen at 7 d recovery, when HH- restored in concert with taste-organ recovery, we recorded from responding cells were observed in perigemmal regions only or the chorda tympani nerve that innervates TB in FP and stimu- partially patterned in perigemmal cells and papilla walls (Fig. 6C, lated the tongue with chemicals including salts, acids, sucrose, 7 d recovery and Inset). Overall, the HH ligand and responding and quinine to elicit taste responses. Compared with vehicle cells returned to FP/TB in concert at 14 d after drug cessation. treatment, after 10 d of sonidegib treatment the taste responses Recovery of TB in the CV. TB in the posterior tongue CV papillae were fully maintained (Fig. 8). Therefore, although 10 d of HPI were decreased after 16 d of sonidegib treatment (Fig. 5). When drug treatment effectively reduced the proportion of category I sonidegib gavage was discontinued, there was no restoration of (typical FP/TB) FP to about 60% of the proportion in vehicle TB with pores after 7 d (Fig. S5B), but there was a trend toward treatment (Fig. 1D), neurophysiological responses were not al- increased numbers at 14 d. By 3–9 mo of recovery the numbers of tered. However, after 16 d of sonidegib, responses to chemicals

Kumari et al. PNAS Early Edition | 5of10 Downloaded by guest on September 25, 2021 Fig. 5. Pharmacologic and genetic HH pathway blockade decreases TB in CV papillae. (A) H&E staining for CV/TB morphology in vehicle-treated mice. Bars delimit the CV walls, for measuring wall length. (Inset) An enlarged image of the boxed region illustrating TB profiles (dotted lines) and TB pores (arrows). (Scale bars: 50 μm.) (B) H&E staining for CV/TB morphology after 16 d of HPI by sonidegib or whole-body (cSmoKO) or epithelial Smo (cSmoKO-epi) deletion. (Scale bar: 50 μm for all images in A and B.) (C and D) Graphs for depth and wall length (C) and TB profiles and TB pores (D). Brackets in D denote significant differences (one-way ANOVA with Tukey’s HSD post hoc tests). Bars are mean ± SEM. F and P values are given in Fig. S4 A and B.(E) X-Gal staining (blue) for Gli1lacZ illustrates HH- responding cells in epithelium and stroma in control and 24-d cSmoKO-epi mice. Dotted lines outline the epithelium. (Scale bar: 50 μm for both images.)

were essentially eliminated, replicating our prior data for 16-d tympani nerve taste responses, responses to tactile and cold mo- treatment (7), and category I (typical FP/TB) papillae were re- dalities were retained (Fig. 8), replicating our prior data (7). This duced to less than 3% of all FP (Fig. 1D). particularly noteworthy result demonstrates that HH differentially We further tested whether neural responses could be restored regulates oral sensory modalities and suggests that the receptor after HPI that had effectively eliminated intact FP taste organs. organs for touch and cold are not within the TB cells per se. When sonidegib treatment was stopped after 16 d, a recovery pe- The tactile and cold responses were sustained throughout all riod of 7 d was not sufficient to restore chorda tympani nerve re- the recovery periods, from 7 d through 9 mo (Fig. 8). We have + sponses to lingual taste stimuli (Fig. 8). Nor is this recovery period observed Shh nerve fibers in ShhCre reporter mice that extend sufficient for restoring category I(typicalFP/TB)papillaetothe in apical FP outside the bounds of TB cells (Fig. 7C) and pro- tongue in large proportion (Fig. 6B). In contrast, after 14 d recovery pose that these might represent fibers that respond to touch and/ and continuing through 9 mo, chorda tympani nerve responses to or cold FP stimulation or fibers that connect to epithelial re- taste stimuli were fully restored. However, during this period the ceptors for tactile and temperature. category I (typical FP/TB) papillae were ∼55% and category III Response/concentration series and chloride salt stimuli. We challenged (atypical FP/no TB) taste organs were ∼40% of all papillae (Fig. the taste organs and neurophysiological properties of the chorda 6B). Therefore, normal chorda tympani nerves responses can be tympani nerve with ability to generate graded responses to a series obtained with only about 55% of intact FP taste organs recovered. of NaCl (from 0.05–1.0 M) during HPI and with recovery. After (Quantified analysis for data in Fig. 8 is presented in Fig. S8A.) sonidegib treatment for 16 d, nerve responses were essentially not Modality-specific effects. Importantly, we also stimulated the ante- discernible, except as very small responses to 0.5 and 1.0 M NaCl rior tongue with tactile (stroking with a wooden rod) and cold (Fig. S9A). A 7-d recovery period after sonidegib treatment was (water at 4 °C) stimuli. Although HPI for 16 d can eliminate chorda not sufficient for restoration of responses. However, after 14 d

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Fig. 6. Cessation of sonidegib treatment results in the recovery of FP/TB morphology, SHH ligand, and HH-responding cells within 14 d and continuing up to 9 mo. (A) Time line for studying recovery from the effects of 16 d sonidegib treatment. (B) Percentage of category I (typical FP/TB), category II (atypical FP/ TB), and category III (atypical FP/no TB) FP after 16 d of sonidegib treatment followed by recovery periods of 7, 14, or 21 d and 3, 5, or 9 mo. Examples of FP/TB

morphology are shown in Fig. S5A. Bars are mean ± CELL BIOLOGY SEM. Numbers in parentheses are number of mice analyzed. Brackets denote significant differences for treatment durations (two-way ANOVA with Tukey’s HSD post hoc tests); ###P ≤ 0.001 for vehicle vs. sonidegib treatments. Complete F and P values are given in Fig. S6A.(C) Antibody detection of SHH (red) and K8 (green, Inset) for TB cells and X-Gal staining (blue) for Gli1lacZ after 16 d of sonidegib treatment and recovery for 7, 14, or 21 d. The Gli1lacZ Inset at 7 d recovery is included to illustrate the variability in the recovery phenotype at 7 d. Dotted lines demarcate the basal lamina. (Scale bar: 50 μm.) (Magnification: C, Lower, Inset,0.6×.)

graded responses to lingual stimulation with increasing concen- recovery of taste nerve responses. TB elimination based on the trations of NaCl were apparent (Fig. S9A). The timing of recovery, deregulation of essential HH signaling controls for cell pro- between 7 and 14 d, matches that for responses to a broad range liferation and differentiation and loss of all TB cell types is rapid, of taste stimuli (Fig. 8). Further, response/concentration functions within 2 wk. Whereas nerves remain during HH/SMO inhibition demonstrated typical curves after 14 d of recovery and through and recovery, they are not able to sustain or restore TB in the 9mo(Fig. S9B). context of selective taste papilla epithelial effects. However, when We also used a series of 0.5 M chloride salts, which have sonidegib treatment is stopped, the taste organs are restored, and distinctive “tastes” and receptor mechanisms, after HPI and with neural taste sensation returns to typical response patterns within recovery. After sonidegib treatment the chorda tympani nerve 2 wk. Although restoration of a full complement of TB numbers is + + + + did not respond to stimulation with Mg2 ,Ca2 ,K ,NH ,or not achieved, the recovery of chorda tympani nerve responses can + 4 Na chloride salts (Fig. S8B). However, recovery in responses to be supported by about 55% of all FP taste buds. Redundancy in values comparable to vehicle treatment was apparent after the taste periphery has long been noted (37, 38) as being crucial sonidegib had been discontinued for 14 d. for life-essential sensations but has not previously recognized as Therefore, chorda tympani nerve responses to a range of chemical important in recovery from HPI drug effects. We suggest that the taste qualities, a concentration series of NaCl, and high-concentration reversible taste disruption in HPI-treated patients is, therefore, a disparate chloride salts were eliminated by HPI but recovered by specific, directed effect reflecting the physiologic requirement for 2 wk after treatment was discontinued. On the other hand, re- HH/SMO signaling in taste-organ homeostasis. sponses to lingual stimulation with touch or temperature modal- Whereas HH signaling is required for TB maintenance and ities were not affected by treatment with the HPI drug sonidegib. restoration, it is not clear why some papillae can recover but others cannot after HPI is stopped. We suggest that as long as Discussion some TB cells remain, and some HH-responding cells are in the We tested the potential for restoration of taste homeostasis after epithelium (in category II, atypical FP/TB papillae), there is HH signaling deregulation and show that taste organs and sen- potential for TB reconstitution. However, if TB are eradicated sory responses can recover after severe loss from HH/SMO in- along with putative TB stem cells, then there is no TB restoration hibition with the cancer drug sonidegib. Indeed, after elimination even up to 9 mo after stopping treatment. This places TB stem of TB and of chorda tympani nerve responses to taste stimuli, cells within the TB. In fact, basal cells of the TB have been in- there is a remarkable restoration of TB in taste organs and full dicated as stem/progenitor cells (13, 22, 30). In addition, after

Kumari et al. PNAS Early Edition | 7of10 Downloaded by guest on September 25, 2021 Fig. 7. Retained innervation during and after soni- degib treatment and identification of Shh expression in GG and TG and in anterior tongue and FP nerve fibers. (A and B). Immunofluorescent antibody de- tection of TB cells (K8, red) and nerves (NF, green, in A; or P2X3, green, in B). Even in category III (atypical FP/no TB) FP, with no K8+ cells retained during soni- degib exposure or recovery periods, the innervation was intact, as illustrated in Fig. S7A. Dotted lines in- dicate the basal lamina. Arrows in B point to P2X3 fibers. (Scale bars: 50 μm.) (C) RFP (red) expres- sion in constitutive ShhCre;R26RFP mice and after tamoxifen (TAM) administration in ShhCreER;R26RFP mice. In both GG and TG, all cell bodies expressed + Shh. Anterior tongue TB include Shh cells and their + progeny. Arrows denote Shh nerve fibers in the anterior tongue, FP core, and surrounding the TB reaching into the apical epithelium. Dotted lines de- marcate the basal lamina. (Scale bars: 50 μm.) (D) Immunofluorescent antibody detection of SHH (red) demonstrates SHH+ cells in GG in vehicle- or sonidegib-treated mice. Insets show merged and magnified images for SHH and NeuN (green). (Scale bar: 50 μm.) The graph indicates that number of + SHH cells in four noncontiguous 10-μm sections of GG did not change after 16 d sonidegib treatment compared with vehicle (t test; t = 1.32, P = 0.26). Bars represent mean ± SEM. Numbers in parentheses are the number of mice per group. (Magnification: Insets,2×.) (E) Immunofluorescent antibody de- + tection of SHH (red) demonstrates SHH cells in TG. Insets show merged and magnified images for SHH and NeuN (green). (Scale bar: 50 μm.) (Magnification: Insets,3×.)

pathway inhibition we observed the loss of HH-responding cells reduced or eliminated. We propose that in the absence of HH + + + in FP walls, known regions of TB progenitors (13, 32); thus, signaling, the K5 and K14 cells do not differentiate to K8 cells another proposed progenitor/stem cell population is eliminated. but instead occupy the apical FP epithelial layers where the TB had + + We believe that there is more than one source of TB stem/pro- been located. Alternatively, K8 cells could revert to K5 expres- genitor cells and that these are regulated by HH signaling (8, 11). sion in a transdifferentiation processseeninothertissues(40). + Overall, K5 cell differentiation is disrupted when HPI deregulates HH Pathway Regulation of Proliferation and Differentiation. With taste organs. Notably this notion is consistent with a genetic over- complementary pharmacologic and genetic approaches to inhibit expression of SHH, where a role for HH signaling in regulating TB SMO-dependent HH signaling, we found consistent phenotypes differentiation has also been proposed (41). across models. There were rapid effects on TB loss in both The acquisition of a conical apex in the FP that accompanies ectoderm-derived anterior tongue FP taste organs and endoderm- the loss of TB cells is characteristic of reduced HH pathway derived posterior tongue CV taste organs, but there were no dis- activity, as previously reported (7, 8) and as observed here after cernible effects on the nontaste filiform papillae. Thus, HH/SMO sonidegib administration or Smo deletion. This reproducible signaling is an essential and selective regulator of gustatory epi- phenotype with HH signaling suppression or inhibition is remi- thelia and TB that turn over constantly in dynamic papilla organs. niscent of an acquired filiform papilla-like form observed in FP Cell cycles of the three TB cell types range from 3 to 30+ days (9, after prolonged periods of denervation (42). A key feature in all 10, 31), with an average TB cell life span of 10 d (9). TB elimi- these experimental models is the loss of TB accompanied by the nation began after 10 d and derived from the loss of all three taste- loss of HH-responding epithelial cells. This indicates that HH cell types in FP and a reduction in proliferation among TB cell signaling is a principal regulator not only of TB maintenance but progenitors in the apical wall of FP. HH/SMO signaling regulation also of FP as distinct from filiform papilla morphology. of all TB cell progenitors is indicated. Progenitor differentiation to taste cells also is affected in HPI. As Functional Effects of HPI on Lingual Sensation. The rapid effects of TB cells are eliminated, the apical integrity of FP is compromised HPI on taste organs were associated with loss of neurophysio- by the acquisition of a heavily cornified, conical cap over epithelial logical taste responses. Here we found that the altered neural cell clusters that are devoid of orientation and stain intensely with taste responses to stimuli representing all taste qualities relate + + hematoxylin. We identified these cells as K5 and K14 ,charac- directly to the loss of all taste-cell types. Further, effects were teristic of lingual and FP basal epithelial cells that include pro- observed even though the TB were challenged with stimulation + genitors for TB (13, 32). Typically, in stratified epithelia the K5 across high concentrations of varied chloride salts that might cells include transit-amplifying cells that leave the basal cell layer have elicited nonspecific effects. and differentiate to suprabasal cells that are not proliferative and Interestingly, however, typical taste nerve responses were still express different keratins (39). In lingual papillae these differenti- obtained after 10 d of drug HPI, although only about 50% of FP + ated cells include those of the TB, which are K8 cells. With werecategoryI(typicalFP/TB)atthistimepoint.Whereas prolonged HPI and attendant TB loss, the epithelial SHH ligand chorda tympani nerve taste responses were absent after 16 d of production is correspondingly eliminated. HH-responding cells are sonidegib gavage, it is notable that nerve responses to lingual

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Fig. 8. Chorda tympani nerve responses to lingual stimulation with chemical, tactile, and cold stimuli after HPI with sonidegib and recovery. Chemical re- sponse: Integrated electrophysiological whole-nerve recordings in response to chemical stimuli applied to the tongue for 20 s followed by a distilled water rinse for 30 s. The height at the steady-state integrated recording for an individual stimulus above baseline was measured as response. Tactile response: Whole- nerve, not integrated, responses to tactile stimuli, which consisted of five strokes with a wooden rod, on the anterior tongue. Cold response: Integrated re- cordings to water at 4 °C applied for 20 s.

tactile or cold stimuli were not altered. Previously we suggested CV Papillae and HPI. In our current experiments, we did not record that the receptors for responses to tactile and cold stimuli must from the glossopharyngeal nerve that innervates taste buds in the not be TB cells per se, because these have been eliminated, and we CV. Because we noted TB loss in the CV after 16 d of HPI drug

indicated that mechanoreceptor or thermal receptor endings treatment and the recovery of about 80% of TB after 14 d, we CELL BIOLOGY might be adjacent to TB (7). Now we have shown nerve endings predict that any loss of posterior tongue sensation would recover + next to TB that are Shh . Studies of receptive fields of GG neu- also. In mice treated with the HPI drug vismodegib for 15 wk rons (soma of chorda tympani nerve fibers) have demonstrated FP (49), statistically significant but biologically small effects were that respond only to tactile or temperature stimuli and not taste seen on TB cell numbers in the CV, whereas we found a sub- (43). This suggests that there are GG/nerve fiber subsets, possibly stantial loss of TB (up to about half of control values) after 4 wk with end organs that are specifically somatosensory. HH signaling of sonidegib gavage. The only other reports of altered HH sig- presumably has different roles in maintaining epithelial sense or- naling on TB in the CV are from HH/GLI suppression in mice, gan specializations vs. receptor nerve endings. where TB were substantially reduced/lost (11). Here we observed effects in TB of both the FP and CV, that is, in two major lingual FP Innervation and HH Signaling. Whereas TB are dependent on an taste regions. Although there were no reported behavioral ef- intact innervation (14), the retention of nerves within FP in the fects in two bottle preference/avoidance tests for sucrose or face of TB elimination indicates that nerves alone are not suf- denatonium benzoate in vismodegib-treated mice (49), we pre- ficient to maintain TB in an epithelium that is devoid of SHH dict behavioral effects after HH/SMO signaling inhibition. ligand and HH-responding cells. Similarly, nerves in the CV la- beled with NF immunostaining are retained in the papilla core Implications for Adverse Taste-Disturbance Effects in Patients Treated but apparently are not able to sustain the full complement of TB. with HPI Drugs. This work is at the confluence of HH/SMO sig- Notably these results substantiate our data after the suppression naling as a principal regulator of taste organs and taste sensation, of HH/GLI signaling (11). deregulated HH signaling in basal cell carcinoma (BCC), and BCC Here we have shown physical association between papilla in- treatment with drugs that inhibit HH signaling with associated lacZ nervation and Gli1 -positive HH-responding cells during ge- severe taste disruption (4, 5, 26, 28, 50) that can alter patient netic HPI in both FP and CV stroma. This affords an opportunity quality of life (27). The demonstrated duration-dependent effects for nerve interactions with HH-responding stromal cells in the on TB in the FP and CV and the profound loss of chorda tympani papilla, not previously proposed. SHH secreted from dorsal root nerve responses to all taste stimuli are the biological bases for + ganglion nerve fibers activates signaling via Gli1 HH-responding severe taste disturbances in patients. We found substantial effects cells in touch domes (44) and maintains homeostasis in the hair in anterior and posterior tongue TB in both the FP and CV, which follicle (35). Because we have observed SHH protein expression in have different innervation and chemical response profiles (51). neurons of the GG and TG that include the soma for nerves in- Effects in patients taking HPI drugs therefore are likely to be wide + nervating FP and have observed Shh expression in fibers within ranging across taste chemical stimuli and oral regions, and the + the FP, we propose interactions between SHH nerve fibers and reported ageusia in >20% of patients (5) is not unexpected. Fur- lacZ Gli1 -positive stromal cells in maintaining the FP structure. ther, based on our neurophysiological data, it is not likely that even + Furthermore, Shh fibers contact basal lamina components of the very strong chemicals would elicit responses in patients during FP and remaining epithelial cells; these could participate in sig- treatment with HPI drugs. However, our modality-specific results naling interactions within a taste-organ niche (8). support consideration of overall lingual sensation, and patient di- SHH likely has varied roles in taste-organ development and ets focused on the texture and temperature characteristics of nu- maintenance. SHH is a morphogen in developing FP placodes (45) trients could temper the loss of taste sensation in overall flavor but might also serve as an axon-guidance molecule or chemo- of meals. attractant via noncanonical signaling (46, 47) to direct growing Notably, a majority of taste organs and TB regain cell in- nerves specifically into the developing papillae (48). In the adult tegrity, and taste nerve responses rapidly return to normal pat- taste organ, HH signaling is required for epithelial cell proliferation terns in the post-HPI drug recovery period in mice. Therefore, and differentiation in FP and TB homeostasis. We suggest that the reported recovery from taste disturbances after treatment is SHH, retrogradely secreted from nerve fibers in the FP core, signals discontinued in patients (5) is related directly to the recovery of to HH-responding cells and tissue elements in the connective tissue. taste organs and sensory responses. Importantly, management of

Kumari et al. PNAS Early Edition | 9of10 Downloaded by guest on September 25, 2021 reported decreased food and fluid intake in patients should ad- Tissue Analyses. Tongues were collected and prepared for tissue analysis, or re- dress flavor perception. We are investigating responses to gus- cordings were made from the chorda tympani nerve and then tongues were dis- tatory and olfactory stimuli in patients treated with HPI drugs to sected. Tissue processing, immunohistochemistry and neurophysiological protocols, learn about possible contrasting effects in taste and smell. and data and quantification analyses are described in SI Materials and Methods.

Materials and Methods Statistics. Animal numbers and statistical tests are included in the graphs and Animals. Animal use and care procedures were performed according to the figure legends. Details are supplied in SI Materials and Methods. guidelines of the National Institutes of Health and approved protocols of the University of Michigan Institutional Animal Care and Use Committee. C57BL/ ACKNOWLEDGMENTS. We thank Ariell M. Joiner in the B.L.A. laboratory 6 mice were treated for 3–36 d by daily oral gavage with sonidegib [NVP- and Libo Li in the C.M.M. laboratory for technical assistance with tissue collec- tion, analyses, and immunostaining, and the staff in the University of Michigan LDE225 diphosphate salt; Chemietek catalog no. CT-LDE225] dissolved in Dentistry Histology Core for technical support. This work was supported by NIH vehicle, PEG 400/5% dextrose in water (75:25 vol/vol), at a dose of 20 mg/kg, funding from the National Institute on Deafness and Other Communication or with vehicle alone. Dose determination was after Kumari et al. (7). For Disorders Multi Principal Investigator Grant R01 DC014428 (to B.L.A., R.M.B., recovery experiments, mice were treated with sonidegib or vehicle for 16 d; A.A.D., and C.M.M.); National Institute of Arthritis and Musculoskeletal and then the drug was discontinued, and animals were maintained for 7, 14, or Skin Diseases Grant R01 AR045973 (to A.A.D.); University of Michigan Compre- 21 d or for 3, 5, or 9 mo in standard housing. Mouse genotypes and sources hensive Cancer Center Core Grant P30 CA046592 (to A.A.D.); and a University are given in SI Materials and Methods. of Michigan Center for Organogenesis Postdoctoral Fellowship (to A.K.).

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