key: cord-0873167-otweegs9
authors: Wang, Zhonghou; Zhou, Jingqi; Marshall, Brett; Rekaya, Romdhane; Ye, Kaixiong; Liu, Hong-Xiang
title: SARS-CoV-2 Receptor ACE2 Is Enriched in a Subpopulation of Mouse Tongue Epithelial Cells in Nongustatory Papillae but Not in Taste Buds or Embryonic Oral Epithelium
date: 2020-07-23
journal: ACS Pharmacol Transl Sci
DOI: 10.1021/acsptsci.0c00062
sha: aab168c8f72908771e585b222ed0e3e1d0d93627
doc_id: 873167
cord_uid: otweegs9

[Image: see text] As a result of the COVID-19 pandemic, evidence revealed that SARS-CoV-2 infection caused taste loss at a rate higher than that of influenza. ACE2, the entry receptor of SARS-CoV-2, has been identified in the oral epithelium; however, it is unclear at what developmental stage ACE2 expression emerges and whether ACE2 is expressed in taste buds. To identify the specific developmental stage, we analyzed RNA-Seq data from embryonic and newborn mouse oral tissue. We found that robust ACE2 expression was observed in the newborn oral epithelium. In contrast, only extremely low levels, if any, of ACE2 transcripts in the embryonic stage oral tissue were found (E12.5 and E14.5). Analyses of three public scRNA-seq data sets of adult mouse tongue epithelial cells showed that receptors for various viruses were enriched in distinct clusters of tongue epithelial cells. ACE2 was enriched in a subpopulation of epithelial cells in the basal region of nongustatory filiform papillae but not in the taste papillae or taste buds. Expression of ACE2 was detected in a small proportion of type III taste cells. Our results indicate that when applied across species, nongustatory papilla epithelial cells are the prime targets for SARS-CoV-2 infection in the tongue; thus, taste loss in COVID-19 patients is likely not caused by a direct infection of SARS-CoV-2 to taste bud cells. Additionally, fetuses at different stages of development may have distinct susceptibility to SARS-CoV-2 infection.

T aste buds are the sensory organs for taste that transduce gustatory stimuli into neural signals conveyed to the central nervous system. In mammals, taste buds are located in the oral cavity, primarily in the lingual taste papillae and the soft palate. Mammalian taste bud cells are post-mitotic and have a short life span (on average 10−12 days in mice). 1−5 Thus, it is essential that progenitor cells in the surrounding tissue compartments continuously differentiate into the specific types of taste cells (types I, II, and III) for taste bud homeostasis and intact taste sensation.

Deficiencies of taste buds and/or their progenitors cause taste dysfunctions. These deficiencies may be caused by genetic factors, injuries, medical treatments (e.g., radiotherapy and chemotherapy), and illness, including infections in the oral cavity (see review by Feng et al.) . 6 Upper respiratory and oral cavity viral infections are among the most common causes of taste dysfunction and are frequently associated with taste loss. 7−11 For example, hypogeusia and dysgeusia are often a complaint in patients following an influenza-like illness. 11 Evidence collected during the COVID-19 pandemic revealed that taste and smell loss occurs at a higher rate in patients with COVID-19 symptoms compared to those with influenza-like symptoms. 12−17 Angiotensin-converting enzyme 2 (ACE2), the receptor that mediates the entry of SARS-CoV-2 into cells, 18−22 is expressed in the oral mucosa and highly enriched in the tongue epithelial cells in humans, thus providing evidence for the infectious susceptibility of the oral cavity. 23, 24 More recent data showed that the ACE2-expressing (ACE2 + ) cells in the tongue of humans are a subpopulation of keratinocytes. 23 In mice, ACE2 is detected in both gustatory and nongustatory tongue epithelium. 25 Quantitative data from cellular analyses are currently unavailable. In spite of the valuable knowledge gained recently about ACE2 and COVID-19 infections, several relevant questions remain unanswered: (1) Where are ACE2 + cells localized in the tongue epithelium? (2) Is ACE2 expression more enriched in taste bud cells such that viral infection directly causes taste bud cell death and subsequently taste loss? (3) How does ACE2 expression in oral epithelium change during different stages of embryonic development?

Although mouse ACE2 is not susceptible to SARS-CoV-2, 26 its expression may still provide insights into the development of pathological changes in humans given that mice and humans share similar gene expression patterns. In this study, analyses of available RNA-Seq data from our lab and recent publications 27, 28 indicate that ACE2 expression was detected in the oral epithelium at birth and not in embryos at E12.5 and E14.5. In adult mice, ACE2 is enriched in a subpopulation of epithelial cells in the nongustatory filiform papillae but not in taste papillae or taste buds. Our mouse data, together with other reports, if true in humans, suggest that (1) taste loss in COVID-19 patients is likely not directly caused by the transfection of SARS-CoV-2 to taste bud cells, (2) embryos at different stages of development have distinct susceptibility to the disease, and (3) the risk of a maternal−fetal transmission exists given the expression of ACE2 in the organs of late embryos and newborns, placenta, and the female reproductive system.

Expression Dynamics of SARS-CoV-2, Influenza Virus, and Inflammation-Associated Genes in Mouse Oral Cavity at Early Stages (E12.5, E14.5, and P1). To predict how early oral tissues can be susceptible to SARS-CoV-2 and influenza virus, we retrieved our bulk RNA-Seq data regarding the expression of genes associated with SARS-CoV-2 (ACE2, TMPRSS2), 29 influenza virus (St3gal4 and St6gal1), 30 and inflammation (If ngr1 and Tnf rsf1a). Four tissue compartments (epithelium and underlying mesenchyme in the tongue and soft palate) were collected at three developmental stages (E12.5, E14.5, and P1). The selected genes showed distinct expression patterns across the four tissue compartments and during the different stages of development. In general, the expression level increased for all genes during tissue development except for St6gal1, which was decreased at later development stages. Among the examined genes, ACE2 was Figure 1 . Histograms of fragments per kilobase million reads (FPKM) values (mean ± SE, n = 3) to illustrate the expression of genes related to SARS-CoV-2 (A and B), influenza (C and D), and inflammation (E and F). The epithelium (Epi) and mesenchyme (Mes) were separately collected from E12.5, E14.5, and P1 mouse tongue (T) and soft palate (SP), the two main regions that host taste buds. Dots in each histogram represent the individual data points of the biological replicates. Statistical analysis of differential expression in the same tissue compartment across stages was performed based on read counts using DESeq2. *, P < 0.05; **, P < 0.01 (adjusted P value). exclusively found in the epithelium, while all other genes were detected in both the epithelium and the mesenchyme.

The expression of ACE2 was rarely detected in any of the four tissue compartments in embryos (E12.5 and E14.5); however, robust expression of ACE2 transcripts was found in the epithelium at P1 but not the mesenchyme of the soft palate and tongue ( Figure 1A) . Expression of the protease TMPRSS2, which mediates the initial viral entry of SARS-CoV-2, showed a progressive increase over developmental stages in both the epithelium and mesenchyme of the soft palate and in tongue epithelium. Furthermore, the transcripts of TMPRSS2 were the most abundant in the soft palate epithelium ( Figure 1B ). St3gal4 and St6gal1, which are essential for synthesis of sialic acid recognized by influenza virus, 30 were detected across all tissue compartments at all three developmental stages. The pattern of expression of these two genes varied across the different developmental stages characterized by a general increased expression for St3gal4 and decreased abundance of St6gal1 transcripts ( Figure 1C ,D). If ngr1 and Tnfrsf1a, genes that encode two essential receptors for inflammatory cytokines, were detected in all examined tissue compartments as early as E12.5 and their expression increased during development ( Figure 1E ,F).

Enrichment of SARS-CoV-2 Receptor Gene ACE2 Occurs in a Subpopulation of Tongue Epithelial Cells of Nongustatory Papillae but Not in Taste Papillae or Buds. Recent studies indicated that ACE2 is enriched in the tongue epithelial cells in humans. 23 To understand which subpopulation(s) of tongue epithelial cells are the likely target of SARS-CoV-2, we scoured the data in the recently published Figure 2 . Distribution of SARS-CoV-2-associated genes, ACE2 and TMPRSS2, in the cell clusters of anterior tongue epithelium. (A) t-SNE map to illustrate the 13 cell clusters that were identified in a scRNA-seq data set (7538 anterior tongue epithelial cells in total) from Schaum et al. 27 Each dot represents a single cell in this data set. (B) Heatmap to show the 13 cell clusters identified with the most significantly different gene from other clusters. The colors represent expression (log(UMIs count/10 000 + 1)) of marker genes as the legend indicates. (C, D) t-SNE maps and violin plots to illustrate the expression (log(UMIs count/10 000 + 1)) of ACE2 (C) and TMPRSS2 (D). In the t-SNE maps, each dot represents an individual cell in the data set, and the color gradients of dots represent the expression levels of ACE2 (C) or TMPRSS2 (D) in cells. In the violin plots, each dot represents an individual cell that expressed ACE2 (C) or TMPRSS2 (D). mouse scRNA-seq atlas. 27 Using the best high-throughput data set (7538 cells in total), we identified 13 anterior tongue epithelial cell clusters ( Figure 2A ) and their associated signature genes ( Figure 2B , Table S1 ) using a machine learning approach. Consistent with human data, 23 SARS-CoV-2 entry receptor coding gene, ACE2, is detected in mouse tongue epithelium and ACE2 + cells are specifically enriched in 4 of the 13 clusters (Nos. 1, 2, 4, and 11) of anterior tongue epithelial cells ( Figure 2C ). In contrast, the expression of TMPRSS2 is broadly scattered at a low frequency across all cell clusters ( Figure 2D ).

To identify the type(s) of ACE2 + cells and answer the question of whether taste bud cells express ACE2, we empirically analyzed the ACE2 + cell-enriched clusters using markers for taste bud cells (Krt8 + ), taste bud cell progenitors (Lgr6 + ), and basal epithelial cells in fungiform papillae (Gli1 + ) in three independent scRNA-seq data sets. In the first data set (7538 cells in total from the anterior tongue epithelium), Krt8 + , Lgr6 + , and Gli1 + cells were not enriched in ACE2 + cell- To further confirm that ACE2 is not expressed in taste bud cells, a third independent scRNA-seq data set (52 taste bud cells in total) 28 was analyzed. As shown in Figure 3H , most taste bud cells from distinct types (Gad1 + type III, Gustducin + type II, T1r3 + sweet and umami type II, and manually selected type III) and taste bud progenitors (Lgr5 + ) did not express ACE2 ( Figure 3H ). Out of the 52 analyzed cells, only 8% showed robust ACE2 transcripts (higher than 160 FPKM), and all of them were type III cells. Collectively, ACE2 is not enriched in most taste bud cells or the surrounding taste bud progenitors and taste papilla epithelial cells.

To further characterize the identity of ACE2-expressing cells, we examined the genes that have been reported to be exclusively expressed in the nongustatory filiform papillae of the dorsal surface of the tongue. Hoxc13 + cells, distributed in the basal region of filiform papillae, 31 were enriched in 3 out of 13 cell clusters (Nos. 2, 10, and 11). It is noteworthy that Hoxc13 + and ACE2 + cell enrichment overlapped in two (Nos.

2 and 11) of the cell clusters ( Figure 4A ). An additional two genes (Krt36 and Krt84) that are expressed in filiform papillae were enriched in the same cell clusters as Hoxc13 and ACE2. Jointly, our data suggest that a proportion of ACE2 + cells are distributed in the epithelium at the basal region of filiform papillae.

Potential for Distinct Tropism of Multiple Viruses in the Tongue Epithelium. To understand the relevance of the distribution of viral receptor expression in oral epithelium and the vulnerability of taste sensation to infectious diseases, we performed a head-to-head comparison of the frequency and the abundance of key viral entry factors for SARS-CoV-2, HCoV-229E, influenza virus, and MERS-CoV45 across all 13 cell clusters identified in the first scRNA-seq data set. 27 The two most TMPRSS2-enriched cell clusters (Nos. 2 and 11) were also ACE2-enriched, although the proportion of TMPRSS2 + cells was generally low. The HCoV-229E's entry factor Anpep 32 shared one cell-enriched cluster (No. 4) with ACE2. However, the St3gal4-enriched clusters (essential for influenza virus entry) were totally separate from those for ACE2. The expression of Dpp4, which is essential for MERS-CoV infection, 32 is very low in cell clusters throughout the tongue epithelium ( Figure 5 ).

Given that a strong innate immune response was activated by SARS-CoV-2 infection 33 and that the resulting inflammation that may affect taste bud homeostasis, 2,34 the response of the innate immune system of the host may be involved in taste loss in COVID-19 patients. Strikingly, tumor necrosis factor receptor 1 gene, Tnf rsf1a, displays abundant expression across all cell clusters. Moreover, the expression of IFN receptor genes If ngr1 and Ifngr2 (but not the Il1 receptor gene Il1r1) was detected broadly in the tongue epithelium. Expression of Tlr3, which is essential for the recognition of viral pathogen and activation of the immune system, was very low ( Figure 5 ).

COVID-19, caused by the infection of SARS-CoV-2, 35 is highly contagious and often life-threatening due to severe acute respiratory syndrome and the failure of other organs. 36, 37 The entry of the virus into cells is through the obligatory receptor ACE2 18−22 and the protease TMPRSS2. 29 Several studies showed that COVID-19 viral infections occur through multiple potential routes including respiratory, oral, and contact. 38−40 The symptoms vary among patients including sore throat, cough, fever, difficulty breathing, and loss of taste and smell. 36 In this report, we provide a new understanding of loss of taste in COVID-19 patients and a potential correlation of the susceptibility with the gestational stages. We acknowledge that mouse ACE2 is, unlike humans, not susceptible to SARS-CoV-2; 26 however, given that mice and humans share similar gene expression patterns, ACE2 expression in mice may still provide insights into how pathological changes develop in humans.

Taste Loss in COVID-19 Patients Is Likely Not Due to an Initial Direct Viral Infection in Taste Bud Cells. A matter of particular interest is that taste loss, often together with smell loss, is one of the common symptoms of COVID-19, even when there are no other apparent symptoms. 16, 41 Recently reported data showed that ACE2 is enriched in tongue epithelial cells in humans, 23 and ACE2 mRNA is detected in both the gustatory and nongustatory tongue epithelium in mice. 25 However, it remains unclear what specific types of cells highly express ACE2 and where they are localized in the tongue epithelium.

Taste buds are primarily located in the tongue, and lingual taste buds reside in the epithelium of the taste papillae. The three types of taste papillae (fungiform, foliate, and circumvallate) are stereotypically distributed on the tongue, i.e., fungiform located among the nongustatory filiform papillae on the anterior 2/3 oral tongue, foliate on the two lateral edges of the posterior oral tongue, and circumvallate papillae (8−12 in humans and 1 in rodents) in the border between the oral and pharyngeal tongue. 42 Mammalian taste bud maintenance and homeostasis require the surrounding progenitor cells to continuously renew the differentiated taste bud cells (types I, II, and III) for intact taste function. 6 Multiple lineages of taste bud cell progenitors have been reported including Krt14 + , 43 Lgr5 + , 44 Lgr6 + , 45 SOX2 + , 46 and Gli1 +47 epithelial cells that surround taste buds.

To dissect the heterogeneity of gustatory epithelial cells, rapidly developing scRNA-seq techniques provide a powerful tool. In this report, the mining of two independent sets of data from a single cell RNA-Seq of anterior tongue epithelium provides insights into the molecular heterogeneity of these cells (Tables S1 and S2 ). Our results indicate that ACE2 is expressed in a subpopulation of tongue epithelial cells. The ACE2 + cells are enriched in 4 out of 13 clusters of these tongue epithelial cells. Two of the four clusters highly express markers (Hoxc13, Krt36, and Krt84) that are restricted to a subpopulation of epithelial cells in the basal region of filiform (nongustatory) papillae. 31, 48 However, ACE2 + cell-enriched clusters do not frequently overlap with those clusters in which markers are expressed in taste bud cells and the surrounding taste papilla epithelial cells, i.e., Krt8, 49 Lgr6, 45 and Gli1. 47 Furthermore, scRNA-seq data of taste bud cells 28 confirm that most taste bud cells have no or extremely low expression level of ACE2. Expression of ACE2 was detected in a small proportion of type III taste cells, which is consistent with mRNA detection in taste buds using RT-PCR. 25 Collectively, these scRNA-seq data provide a more precise information than the previously reported 24 regarding ACE2 + cell types and enriched tissue compartments. The enrichment of ACE2 in nongustatory papillae but not taste buds indicates that taste loss in COVID-19 patients is not, at least initially, primarily due to a direct viral infection of taste bud cells.

We hypothesize that taste loss in COVID-19 patients may result indirectly from multiple potential causes. First, local and/or systematic immune responses to SARS-CoV-2-induced inflammation may disrupt taste bud homeostasis. 6 It has been recently reported that deletion of TNFα prevents taste bud loss caused by the chronic inflammation. 50 Thus, abundant expression of Tnf rsf1a, the gene encoding tumor necrosis factor receptor 1 (TNFR1), in single-cell transcriptomic 34 Detection of both If ngr1 and If ngr2 expression in our single cell-level analysis of anterior tongue epithelium indicate the involvement of IFN-mediated signaling in regulating fungiform taste bud homeostasis in the anterior tongue. Second, infections of SARS-CoV-2 in the nervous system may damage the innervation of taste buds which leads to taste bud degeneration. 51 Finally, if ACE2 expression in taste buds can be activated by IFN 40 after the virus first enters into a host as recently reported in the olfactory epithelium in humans, 40 then there is a possibility that SARS-CoV-2 in the blood and saliva 52 may infect taste bud cells via the subsequently expressed ACE2 in these cells. This possibility requires further experimental testing.

Fetuses May Have Developmental-Stage-Dependent Susceptibilities to the Infection of SARS-CoV-2. The available pandemic data shows that elderly people are more susceptible to SARS-CoV-2 infection and are more likely to have a poor prognosis, including death. 53 A recent report showed that the ACE2 protein level in some organs, e.g., olfactory epithelium, increases with age, 54 thus providing a possible explanation as to why the severity and mortality rate are higher in older people. Though young people are also susceptible to infection when exposed to the virus, the symptoms are mostly mild. 55 The youngest COVID-19 patient reported was only 4 weeks old. 56 To understand whether fetuses are also susceptible to COVID-19 through maternal transmission, thorough examinations on ACE2 expression in the body of fetus and placenta are needed. It has been reported that ACE2 is highly expressed in early ovum-phase human embryos (2-and 4-cell stages), and the expression level drops afterward. 57 Furthermore, the presence of ACE2 in the urine of 23 week old human fetuses suggests the expression in the body. 58 In mouse pancreas, ACE2 is detected in embryos and peaked at E16.5. 59 In the present study, we analyzed the expression of ACE2 receptor and coreceptor TMPRSS2 in different tissue compartments of the oral cavity in mouse embryos and newborns. Although the transcripts of TMPRSS2 for SARS-CoV-2 entry were detected in the lingual and palatal epithelium of both embryos and newborns, those of the receptor ACE2 were only detected in newborns, not in embryos at examined developmental stages (E12.5 and E14.5). Together, these findings indicate that the susceptibility of fetuses to SARS-CoV-2 infections is likely to be stage-dependent, i.e., a high level immediately after fertilization and during late fetal stages. Given that ACE2 is highly expressed in the fetal part of placenta (labyrinth and basal zones) 60 and the female reproductive tract including uterus and vagina, 61 the potential risk of maternal−fetal transmission exists before and during birth, although SARS-CoV-2 infections were not found in neonates of infected pregnant mothers in a recent report. 62 Different Viral Infections Transmit through Distinct Cell Targets in the Tongue Epithelium. It is known that various viral infections affect taste function to different extents, e.g., at distinct incidence and severity levels of taste dysfunction. 6 Taste loss occurs more frequently in COVID-19 patients than in other patients with influenza-like symptoms. 12 The underlying mechanisms in these infectious disease conditions remain elusive at the cellular level. In this study, scRNA-seq profiling of anterior tongue epithelial cells that are heterogeneous structurally, molecularly, and functionally revealed that molecules for various viruses' entries into cells are enriched in different cell clusters. We hypothesize that different viral infections may cause impairments of different subpopulations of tongue epithelial cells and may trigger various immune responses leading to distinct taste deficiencies. To better understand how taste bud cells and/or progenitors are affected in various disorders, further experimental studies are needed. The available data from our analysis and others 27 will be useful in these future studies.

We are aware of the seemingly different expression levels of some genes between our study and literature reports. Il1r1, the gene encoding interleukin 1 receptor type 1, and Tlr3, the gene encoding Toll-like receptor 3, were detected at a low level in the scRNA-seq data set of anterior mouse tongue epithelium. However, detection of proteins IL1-β and IL1-RI receptor has been reported in taste buds in rats. 63 Moreover, multiple TLRs were detected in taste buds from posterior tongues using RT-PCR. 64 These discrepancies may be due, in part, to the difference of innate immune system components between the anterior vs posterior tongue. Furthermore, loss-of-function analysis of one or more innate immune components under different conditions will be beneficial for understanding the effects of inflammation on taste bud maintenance and function.

Overall, our results show that different viral infections may affect distinct cell targets in the tongue epithelium. We identified the basal region of epithelial cells in nongustatory filiform papillae as the prime targets for SARS-CoV-2 infection in tongue epithelium. We revealed that ACE2 was detected in newborns, but little to no expression was found in mouse fetal oral tissues. Thus, the results in mouse tissues suggest when applied across species and organs that taste loss in COVID-19 patients is likely not caused by an initial direct infection of taste bud cells by SARS-CoV-2, that fetal susceptibility to SARS-CoV-2 is stage-dependent during prenatal development, and that risks of maternal−fetal transmission exist.

Animals. Animal use was approved by The University of Georgia Institutional Animal Care and Use Committee and was in compliance with the National Institutes of Health Guidelines for care and use of animals in research.

Timed pregnant and newborn mice (C57BL/6J, The Jackson Laboratory, stock no. 000664,) were used to collect tissues for the in-house bulk RNA-Seq. Noon (12 pm) on the day in which a vaginal plug was detected was designated embryonic day (E)0.5. The day when the pups were born was noted as postnatal day (P)1.

Transcriptomic Profiling of Oral Tissues (Bulk RNA-Seq) in Mouse Embryos and Newborns. Pregnant mice at E12.5 and E14.5 were euthanized with CO 2 , followed by cervical dislocation, and newborn (P1) mouse decapitation. Tongues and soft palates were dissected. To separate the epithelium and underlying mesenchyme, E12.5 tissues were incubated with 2.5 mg/mL dispase II (no. 04942078001, Roche Diagnostics); a 1 mg/mL collagenase A (no. 10103578001, Roche Diagnostics) was added for the E14.5 tissues. In the P1 tissues, the enzyme mixture was injected into the subepithelial space. After enzyme incubation at 37°C for 30 min, the epithelium and mesenchyme of tongue and soft palate were separated for RNA extraction using Trizol and RNeasy Plus kits (Qiagen). RNA Quality was assessed on an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). Each group of tissues included 3 biological replicates and each sample of the replicate contained up to 10 (E12.5), 3 (E14.5), and 1 (P1) tongues or soft palates for sufficient RNA.

cDNA libraries were prepared with Kapa Stranded mRNAseq kit (KAPA Biosystems, Wilmington, MA). Library quality and quantity were measured by Fragment Analyzer Automated CE (Advanced Analytical, Evry Cedex, France) and Qubit (Thermo Fisher) systems, respectively. Libraries were subsequently subjected to 2 × 75 base pair (bp) paired-end sequencing on a NextSeq 500 system (Illumina). All samples were pooled and sequenced on one lane. The RNA quality check, library preparation, and sequencing were conducted at the University of Georgia Genomics and Bioinformatics Core (Athens, GA). The raw RNA-Seq data of each sample was aligned to mouse reference genome (GRCm37.1) via STAR, 65 and the transcripts were quantified via StringTie. 66 Gene expression was calculated in FPKM and presented as FPKM (X ± SE, n = 3). Raw read counts were quantified by HTSeq and subsequently analyzed for differentially expressed genes (DEGs) between stages via DESeq2. 67 Significance was declared when the adjusted P value was less than 0.05.

All raw sequencing data and processed data were deposited in Gene Expression Omnibus (GSE151205).

Analysis of Public Data from Single-Cell Transcriptomic Profiling (scRNA-seq) of Adult Mouse Tongue Epithelium. Three independent data sets were used. Two sets of tongue scRNA-seq data were downloaded from Tabula Muris, 27 including one that was based on microfluid platform and contained 7538 adult anterior tongue epithelial cells (for unique molecular identifiers (UMIs)) and another based on the FACS method and contained 1432 tongue epithelial cells (for reads). Genes detected in less than three cells were filtered out. Cells in which less than two hundred genes were detected were removed.

The third set of data (SRP094673) contained 52 available cells: 47 circumvallate taste bud cells from different types (11 Gad1 + cells, 10 Gustducin + cells, 17 type III cells, and 9 Tas1r3 + cells) and 5 taste bud cell progenitors (Lgr5 + ). 28 The raw sequencing data for each cell from Sukumaran et al. 28 was aligned to a mouse reference genome using STAR, 65 and the transcripts of genes were quantified using RSEM. 68 Gene expression was calculated in FPKM.

Analysis of scRNA-seq data sets were performed with R package Seurat (V3.1.5). 69 The expression matrix from Schaum et al. 27 underwent logistic transformation and scaling. Clusters of cells were identified by unbiased machine learning algorithms. Principal component analysis (PCA) was applied for dimension reduction, and t-Distributed Stochastic Neighbor Embedding (t-SNE) was used to visualize data at low dimensions.

Data Visualization. Visualization of bulk RNA-Seq data was conducted using R package ggpubr. For scRNA-seq analyses, t-SNE maps, heatmaps, violin plots, and dot plots were implemented using the built-in functions of R package Seurat (V3.1.5). 69 ■ ASSOCIATED CONTENT * sı Supporting Information Top 10 significant genes for each cluster (13 clusters in total) identified in data set from Schaum et al. (7538 cells in total); top 10 significant marker genes for each cluster (6 clusters in total) identified in data set from Schaum et al. (1432 cells in total) 27 (PDF) 

Functional Cell Types in Taste Buds Have Distinct Longevities

Lipopolysaccharide-induced inflammation attenuates taste progenitor cell proliferation and shortens the life span of taste bud cells

Taste bud contains both short-lived and long-lived cell populations

Renewal of taste bud cells in rat circumvallate papillae

RENEWAL OF CELLS WITHIN TASTE BUDS

Taste bud homeostasis in health, disease, and aging

Smell and taste disorders: a primary care approach

Disorders of smell and taste

Clinical evaluation results from a taste and smell clinic

Hypogeusia, dysgeusia, hyposmia, and dysosmia following influenzalike infection

Association of chemosensory dysfunction and Covid-19 in patients presenting with influenza-like symptoms

Anosmia and Ageusia: Common Findings in COVID-19 Patients

Prevalence and Duration of Acute Loss of Smell or Taste in COVID-19 Patients

Olfactory and gustatory dysfunctions as a clinical presentation of mild-to-moderate forms of the coronavirus disease (COVID-19): a multicenter European study

Loss of smell or taste as the only symptom of COVID-19

A New Symptom of COVID-19: Loss of Taste and Smell

Structural basis for the recognition of SARS-CoV-2 by full-length human ACE2

2020) Structural basis of receptor recognition by SARS-CoV-2

Spike protein recognition of mammalian ACE2 predicts the host range and an optimized ACE2 for SARS-CoV-2 infection

Master Regulator Analysis of the SARS-CoV-2/Human Interactome

On the interactions of the receptor-binding domain of SARS-CoV-1 and SARS-CoV-2 spike proteins with monoclonal antibodies and the receptor ACE2

Knowledge synthesis of 100 million biomedical documents augments the deep expression profiling of coronavirus receptors

Expression of Renin-Angiotensin System Components in the Taste Organ of Mice

A pneumonia outbreak associated with a new coronavirus of probable bat origin

Single-cell transcriptomics of 20 mouse organs creates a Tabula Muris

Whole transcriptome profiling of taste bud cells

SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor

N-Glycolylneuraminic Acid as a Receptor for Influenza A Viruses

Hoxc13 mutant mice lack external hair

Human aminopeptidase N is a receptor for human coronavirus 229E

COVID-19: consider cytokine storm syndromes and immunosuppression

Inflammation Activates the Interferon Signaling Pathways in Taste Bud Cells

Naming the coronavirus disease (COVID-19) and the virus that causes it

Analysis of clinical features of 29 patients with 2019 novel coronavirus pneumonia

Clinical Characteristics of Coronavirus Disease 2019 in China

Single-cell RNA-seq data analysis on the receptor ACE2 expression reveals the potential risk of different human organs vulnerable to 2019-nCoV infection

SARS-CoV-2 entry factors are highly expressed in nasal epithelial cells together with innate immune genes

SARS-CoV-2 Receptor ACE2 Is an Interferon-Stimulated Gene in Human Airway Epithelial Cells and Is Detected in Specific Cell Subsets across Tissues

COVID-19 and the chemical senses: supporting players take center stage

Anatomy of the Tongue and Taste Buds. Handbook of Olfaction and Gustation

Cell Lineage Mapping of Taste Bud Cells and Keratinocytes in the Mouse Tongue and Soft Palate

Lgr5-EGFP Marks Taste Bud Stem/ Progenitor Cells in Posterior Tongue

Single Lgr5-or Lgr6-expressing taste stem/progenitor cells generate taste bud cells ex vivo

Genetic Lineage Tracing in Taste Tissues Using Sox2-CreERT2 Strain

Multiple Shh signaling centers participate in fungiform papilla and taste bud formation and maintenance

WNT10A mutation causes ectodermal dysplasia by impairing progenitor cell proliferation and KLF4-mediated differentiation

Keratins as markers of differentiated taste cells of the rat

Inflammation arising from obesity reduces taste bud abundance and inhibits renewal

Fungiform taste bud degeneration in C57BL/6J. mice following chorda-lingual nerve transection

Saliva: potential diagnostic value and transmission of 2019-nCoV

Inflamm-aging: Why older men are the most susceptible to SARS-CoV-2 complicated outcomes

Expression of the SARS-CoV-2 Entry Proteins, ACE2 and TMPRSS2, in Cells of the Olfactory Epithelium: Identification of Cell Types and Trends with Age

Minnesota's youngest COVID-19 patient is 4 weeks old, in hospital

EZH2-mediated H3K27me3 inhibits ACE2 expression

The protective arm of the renin−angiotensin system may counteract the intense inflammatory process in fetuses with posterior urethral valves

The ACE2/Ang-(1−7)/Mas Axis Regulates the Development of Pancreatic Endocrine Cells in Mouse Embryos

ACE2 expression and activity are enhanced during pregnancy

Potential influence of COVID-19/ACE2 on the female reproductive system

from Infected Pregnant Mothers to Neonates: A Review

Functional role for interleukin-1 in the injured peripheral taste system

Inflammation and taste disorders: mechanisms in taste buds

STAR: ultrafast universal RNA-seq aligner

StringTie enables improved reconstruction of a transcriptome from RNA-seq reads

Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2

RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome

Integrating single-cell transcriptomic data across different conditions, technologies, and species

H.X.L. and R.R. designed the experiments for bulk RNA-Seq. H.X.L., Z.W., K.Y., and J.Q. designed the strategy for data analysis. H.X.L. and BM performed tissue harvest. Z.W. analyzed bulk-RNA seq data. Z.W. and J.Q. analyzed the scRNA-seq data. Z.W. performed data visualization and final figure assembling. Z.W. and H.X.L. wrote the manuscript. All authors read and approved the final manuscript.

This study was supported by the National Institutes of Health, grant number R01DC012308 to H.X.L. and the University of Georgia Research Foundation to K.Y.

The authors declare no competing financial interest.

We thank the Georgia Advanced Computing Resource Center (GACRC) for providing high-performance computing (HPC) resources and the Georgia Genomics and Bioinformatics (GGBC) for providing technical assistance for cDNA library preparation. We give thanks to Francisca Burnley for English editing.