Mash1-expressing cells may be relevant totype III cells and a subset of PLCβ2-positive
cell differentiation in adult mouse taste
buds
Chia-Chien Hsu, Yuji Seta, Kae
Matsuyama, Shinji Kataoka, Mitsushiro
Nakatomi, Takashi Toyono, Kaori
K. Gunjigake, Kayoko N. Kuroishi, et al.
Cell and Tissue Research
ISSN 0302-766X
Volume 383
Number 2
Cell Tissue Res (2021) 383:667-675
DOI 10.1007/s00441-020-03283-w

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Author's personal copy
Cell and Tissue Research (2021) 383:667–675
https://doi.org/10.1007/s00441-020-03283-w

REGULAR ARTICLE

Mash1-expressing cells may be relevant to type III cells and a subsetof PLCβ2-positive cell differentiation in adult mouse taste buds
Chia-Chien Hsu 1,2 & Yuji Seta 2 & Kae Matsuyama 2 & Shinji Kataoka 2 & Mitsushiro Nakatomi 2 & Takashi Toyono 2 &Kaori K. Gunjigake 1 & Kayoko N. Kuroishi 1 & Tatsuo Kawamoto 1
Received: 10 February 2020 / Accepted: 14 August 2020 / Published online: 22 September 2020
# Springer-Verlag GmbH Germany, part of Springer Nature 2020

Abstract
Mammalian taste bud cells have a limited lifespan and differentiate into type I, II, and III cells from basal cells (type IV cells)(postmitotic precursor cells). However, little is known regarding the cell lineage within taste buds. In this study, we investigated thecell fate of Mash1-positive precursor cells utilizing the Cre-loxP system to explore the differentiation of taste bud cells. We found that
Mash1-expressing cells in Ascl1CreERT2::CAG-floxed tdTomato mice differentiated into taste bud cells that expressed aromatic Lamino acid decarboxylase (AADC) and carbonic anhydrase IV (CA4) (type III cell markers), but did not differentiate into most ofgustducin (type II cell marker)-positive cells. Additionally, we found that Mash1-expressing cells could differentiate into phospholipase
C β2 (PLCβ2)-positive cells, which have a shorter lifespan compared with AADC- and CA4-positive cells. These results suggest that
Mash1-positive precursor cells could differentiate into type III cells, but not into most of type II cells, in the taste buds.
Keywords Mash1 . bHLH transcription factor . Taste bud . Fate maping . Cell lineage

Introduction
Mash1 (Ascl1) encodes a basic helix-loop-helix transcription
factor that plays an important role in the differentiation ofneurons in the nervous system, neuroendocrine cells, and sensory organs (Johnson et al. 1990, Lo et al. 1991, Guillemot
et al. 1993). Previously, we reported that Mash1 is expressedin basal cells and a subset of type III cells, but not in type II
cells, within mature taste buds (Seta et al. 2006). Additionally,
we revealed using Mash1 knockout mice that Mash1 plays animportant role in the expression of aromatic L-amino aciddecarboxylase (AADC) and glutamate decarboxylase 67(GAD67) in type III cells in the taste buds (Seta et al. 2011;
Kito-Shingaki et al. 2014).

* Yuji Setayujiseta■kyu-dent.ac.jp
1

Division of Orofacial Functions and Orthodontics, Kyushu Dental
University, 2-6-1 Manazuru, Kokurakita-ku, Kitakyushu 803-8580,
Japan

2

Division of Anatomy, Department of Health Improvement, Kyushu
Dental University, 2-6-1 Manazuru, Kokurakita-ku,
Kitakyushu 803-8580, Japan

Mammalian taste buds are multicellular sensory organs comprising several distinct taste cells. Morphological and immunohistochemical studies revealed that taste buds possess four distinct cell
types (Murray 1986; Finger and Simon 2000). In this classificationof taste cells, types II and III cells serve as taste receptors, whereas
type I cells are glia-like cells (Lindeman 1996; Yang et al. 2004).
Type II cells express different immunohistochemical markers, including phospholipase C β2 (PLCβ2), gustducin, and protein
gene product 9.5 (PGP9.5) (Yang et al. 2004; Yee et al. 2001;
Kim et al. 2003; Clapp et al. 2004). Type III cells have also been
distinguished using immunohistochemical markers. AADC, carbonic anhydrase IV (CA4), Synaptosome-Associated Protein 25(SNAP25), and GAD67 are utilized as type III cell markers (Yeeet al. 2001; Clapp et al. 2004; Takagi et al. 2018). These taste cells
differentiate from basal cells (type IV cells) that arise from the local
epithelium, and they are maintained through continuous cell renewal (Beidler and Smallman 1965; Farbman 1980; Delay et al.
1986; Stone et al. 1995). There are two hypotheses for the differentiation of taste cells: (1) basal cells (type IV cells) (postmitotic
precursor cells) are restricted to individual cell types (Farbman
1965a, b; Fujimoto and Murray 1970; Pumplin et al. 1997), and(2) basal cells (type IV cells) that express Sonic hedgehog (Shh)give rise to all taste cell types (types I, II, and III) (Miura et al.
2006, 2014). However, little is known about the mechanism ofbasal cell development within taste buds.

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In this study, we utilized transgenic mouse strains(Ascl1CreERT2 and CAG-floxed tdTomato) for tracing the fateof Mash1-expressing cells in taste buds. The characteristics ofAscl1CreERT2::CAG-floxed tdTomato mice (MT mice) aresuch that we could label Mash1-expressing cells temporarily
by using the Cre-loxP system after tamoxifen induction. Prior
to tamoxifen induction, CreERT2 was localized in the cytoplasm of Mash1-expressing cells. When tamoxifen is administered, CreERT2 is activated by transfer to the nucleus toinduce the expression of tdTomato as a reporter red fluorescent protein. Perea-Martinez et al. (2013) reported that type II
mature taste bud cells are differentiated from type IV basal
cells after 2 days, and type III mature taste bud cells are differentiated after 3 days. To evaluate the relationship between
Mash1-expressing cell fate and mature taste bud cells differentiation, we harvested MT mice at 2, 5, and 10 days later after
3 days of oral tamoxifen induction. The time point of
2 days was set because type II mature taste bud cells
differentiated. We attempted to study the average
lifespan of the total number of taste bud cells, which
is 10 days, and 5 days is the intermediate time.
As mentioned above, this study investigated the cell fate ofMash1-positive precursor cells utilizing transgenic mousestrains (Ascl1CreERT2 and CAG-floxed tdTomato) to explore
the differentiation of taste bud cells.

Fig. 1 tdTomato expression was
identified after TAM induction incircumvallate papillae. tdTomato
expression was exclusively
identified in taste buds after TAM
induction in Ascl1CreERT2::CAGfloxed tdTomato mice (a–c).
tdTomato expression was not
identified in taste buds after TAM
induction in CAG-floxed
tdTomato mice (d). Scale bars:
10 μm. e The number of
tdTomato-expressing cells gradually declined over time postadministration of tamoxifen. It
reveals significantly decreased
tendency during three continuous
harvest time. Error bars indicate
as mean ± SE (n = 3 for each harvest time). The individual resultsfor each animal indicate as dots.
Results were analyzed by using
one-way ANOVA and TukeyKramer post hoc test. * P < 0.05,** P < 0.001

Cell Tissue Res (2021) 383:667–675

Materials and methods
Animals
All of the animals used in this study were maintained
and handled according to the protocols approved by
Kyushu Dental University Animal Care (Certification
No: 17–010). Adult Ascl1 C r e E R T 2 knock-in (TheJackson Laboratory, Stock No: 012882) and CAGfloxed red fluorescent protein variant (tdTomato) mice
(The Jackson Laboratory, Stock No: 007905) were usedin the study. Ascl1CreERT2::CAG-floxed tdTomato mice
were generated by breeding Ascl1CreERT2 knock-in and
CAG-floxed tdTomato mice and were referred to as MT
mice. The genotyping of mice was performed using thefollowing primers:&&&&

Ascl1CreERT2 mutant sense, 5′-AACTTTCCTCCGGG
GCTCGTTTC-3′
Ascl1CreERT2 mutant antisense 5′-CGCCTGGCGATCCCTGAACATG-3′
CAG-floxed tdTomato sense, 5′-AAGGGAGCTGCAGTGGAGTA-3′
CAG-floxed tdTomato antisense, 5′-CCGA
AAATCTGTGGGAAGTC-3′

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Tissue preparation
MT mice were administered tamoxifen (100 mg/kg; Sigma,
T5648) in sunflower seed oil (Sigma, S5007), which induces the translocation of Cre-ERT2 to the nucleus, inwhich it excises the reporter gene, via oral gavage. A 22gauge feeding needle was used for oral gavage.
At the age of 4–11 months, MT and CAG-floxed
tdTomato (as the control) mice of both sexes were given
tamoxifen once every day for 3 days. CAG-floxed
tdTomato (as the control) mice were also given

Fig. 2 tdTomato-positive cellswith taste cell markers in taste
buds at 2 days post-induction.
The upper six panels were type II
cell markers (gustducin, a–c;
PLCβ2, d–f) results. The lower
six panels were type III cellmarkers (AADC, g–i; CA4, j–l)
results. Coexpression cells wereindicated by arrow. Noncoexpression cells were indicatedby arrowhead. Scale bars: 10 μm

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tamoxifen. After 2, 5, and 10 days of tamoxifen administration, mice were anesthetized via intraperitoneal pentobarbital (50 mg/kg) and perfused with a fixative containing 4% paraformaldehyde in 0.1 M phosphate buffer,
pH 7.4. The posterior portion of the tongue containing
the circumvallate papillae were removed and fixed overnight in the same fixative. The tissues were embedded in
Tissue-Tek OCT compound (Sakura Finetechnical,
Tokyo, Japan). Sections (6–8 μm) were cut using a cryostat, thaw-mounted onto MAS-coated Superfrost slides(Matsunami, Tokyo, Japan), and stored at − 80 °C.

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Immunohistochemistry
The sections were rehydrated using phosphate-bufferedsaline (PBS), and followed by incubation with rabbit
anti-AADC (1:200; Gene Tex), goat anti-CA4 (1:200;
R&D Systems), rabbit anti-gustducin (1:1000; Santa
Cruz), and rabbit anti-PLCβ2 (1:1000; Santa Cruz) as
primary antibodies overnight at 4 °C in a humidified
chamber. After washing with PBS, the sections were incubated with CF488A conjugated donkey anti-rabbit IgG(1:1000; Biotium) or CF488A conjugated donkey antigoat IgG (1:1000; Biotium) as secondary antibodies forFig. 3 tdTomato-positive cellswith taste cell markers in taste
buds at 5 days post-induction.
The upper six panels were type II
cell markers (gustducin, a–c;
PLCβ2, d–f) results. The lower
six panels were type III cellmarkers (AADC, g–i; CA4, j–l)
results. Coexpression cells wereindicated by arrow. Noncoexpression cells were indicatedby arrowhead. Scale bars: 10 μm

Cell Tissue Res (2021) 383:667–675

1 h at room temperature. Slides were washed with PBS
and coverslipped with Vectashield (Vector Laboratories,
USA). Negative controls were created by substitution of
PBS for the primary antibodies. Antibodies in taste buds
have been previously validated (anti-AADC, DeFazio
et al. 2006; anti-CA4, Chandrashekar et al. 2009; antigustducin, Boughter et al. 1997; anti-PLCβ2, Rössler
et al. 1998). All immunostained sections were viewed
using a fluorescence microscope (BZ-9000; KEYENCE,
Osaka, Japan). Immunostained images were contrasted
and color-adjusted and plates were created using Adobe
Photoshop CS5 for Macintosh.

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Statistical analyses

ResultAll statistical analyses were performed using Bell Curvefor Excel (version 2.13). One-way ANOVA was used
for evaluating population normality. After analysis ofvariance, the Tukey-Kramer method was used to evaluate the difference of tdTomato-expressing cell percentages per taste bud between the groups at each harvest
time. The same procedure was also performed to evaluate the difference of coexpression cell percentages per
taste bud between the groups at each harvest time. The
significance level was set at 0.05 for each test.

Mash1-expressing cells were only observed within the taste
buds of adult mice (Seta et al. 2006). To trace the fate ofMash1-expressing cells into the mature taste cells, we used
the Cre-flox genetic fate mapping strategy. In
Ascl1CreERT2::CAG-floxed tdTomato (MT) mice, Mash1 lineage cells were labeled with tdTomato following tamoxifen
treatment. A small subset of tdTomato-expressing cells withintaste buds was observed in circumvallate papillae (Fig. 1a–c).
As expected, tdTomato-expressing cells were not observed in
the other papillary elements, such as the papilla groove

Fig. 4 tdTomato-positive cellswith taste cell markers in taste
buds at 10 days post-induction.
The upper six panels were type II
cell markers (gustducin, a–c;
PLCβ2, d–f) results. The lower
six panels were type III cellmarkers (AADC, g–i; CA4, j–l)
results. Coexpression cells wereindicated by arrow. Noncoexpression cells were indicatedby arrowhead. Scale bars: 10 μm

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Cell Tissue Res (2021) 383:667–675

epithelium. The number of tdTomato-expressing cells gradually declined over time after the administration of tamoxifen(Fig. 1e). There were no tdTomato-expressing cells withintaste buds in CAG-floxed tdTomato (T) mice after tamoxifen
treatment as the negative control (Fig. 1d).

To investigate the fate of Mash1-expressing precursor
cells, we performed immunohistochemistry using taste
cell markers on sections of circumvallate papillae fromMT mice administered tamoxifen and then harvested 2,
5, or 10 days later. Gustducin is a marker of a subset of

Fig. 5 The left six panels were type II cell markers (gustducin, a–c;
PLCβ2, a’–c’) results. The right six panels were type III cell markers(AADC, a”–c”; CA4, a”‘–c”’) results. The number of each cell typemarker-–positive cells, tdTomato-positive cells, and coexpression cells

were shown in each Venn diagram. The coexpression rate of each typecell markers was also shown in the overlapped region of Venn diagram at
2, 5, and 10 days post-induction

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Cell Tissue Res (2021) 383:667–675

type II taste cells (Clapp et al. 2004; Yee et al. 2001).
Immunoreactivity for gustducin was observed in a large
population of taste cells. Within circumvallate taste buds,a small number of gustducin-positive taste cells were labeled with tdTomato (Fig. 5; 2 days, 1.8%; 5 days, 6.1%;
10 days, 2.7%). PLCβ2 is a marker of type II taste cells(Clapp et al. 2004). Immunoreactivity for PLCβ2 wasalso present in a large subset of mouse circumvallate taste
buds. Some PLCβ2-positive taste cells were labeledwith tdTomato (Fig. 5; 2 days, 34.6%; 5 days, 36.0%;
10 days, 10.8%). The number of tdTomato-labeled
PLCβ2-positive taste cells was significantly reduced bytamoxifen administration depending on the duration oftreatment (Fig. 6).
AADC and CA4 are type III cell markers (Yee et al.
2003; Seta et al. 2011). Taste cells with AADC and CA4
immunopositivity were found within taste buds (Figs. 2,
Fig. 6 For gustducin, there is no
significant difference among theharvest times (a). For PLCβ2 (b),AADC (c), and CA4 (d), thepercentage of coexpressing cells
per taste bud decreased
significantly between 2 and10 days, and 5 and 10 days. The
percentage of coexpressing cells
in AADC and CA4 wereapproximately 6% at 10 days. In
PLCβ2, the percentage of
coexpression cells was only 1.8%
at 10 days. Error bars indicate
mean ± SE (n = 3 for each harvesttime). The individual results foreach animal are indicated as dots.
Results were analyzed by using
one-way ANOVA and TukeyKramer test. ns no significance *P < 0.05, ** P < 0.001

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3, and 4g–l). Some type III cell marker–positive taste cells
were labeled with tdTomato (Fig. 5; AADC: 2 days,
60.9%; 5 days, 60.2%; 10 days, 46.8%; CA4: 2 days,
72.4%; 5 days, 64.4%; 10 days, 59.3%). The number of
tdTomato-labeled AADC- and CA4-positive taste cells
was significantly reduced by tamoxifen administration depending on the duration of treatment (Fig. 6).

Discussion
This present study clarified the fate of Mash1-expressing taste
cells within mature taste buds. Using Ascl1CreERT2::CAGfloxed tdTomato mice, we found that Mash1-expressing cells
also expressed AADC and CA4 (type III cell markers). We
additionally observed that Mash1-expressing cells could

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differentiate into PLCβ2-positive cells, and these cells had
shorter lifespans than AADC- and CA4-positive cells.
Mash1 has been demonstrated to play an important role indifferentiation in neural and other tissues (reviewed by
Bertrand et al. 2002). Mash1 is expressed in basal cells andsome elongated taste cells within mature taste buds (Seta et al.
2006), and AADC- and GAD67-expressing type III cells were
not detected in Mash1 mutant mice (Seta et al. 2011; KitoShingaki et al. 2014). Furthermore, we illustrated that the
number of cells expressing type III cell markers was significantly reduced in the circumvallate taste buds of Mash1 miceusing the Cre-loxP system (Takagi et al. 2018). In the present
study, Mash1-expressing cells marked by CreERT2 in
Ascl1CreERT2::CAG-floxed tdTomato mice expressed type
III cell markers within taste buds. These results coincide withthe previous findings and indicate that Mash1-expressing precursor cells differentiate into type III cells in taste buds.
Mammalian taste buds comprise four distinct cell types
according to ultrastructural and immunohistochemical studies(for a review, see Finger and Simon 2000). Within these cell
types, basal cells are assumed to be precursor cells and otherelongated cells are assumed to be mature taste cells (types I–
III cells). Previously, we found that Mash1 was expressed inbasal cells and the majority of differentiated type III taste cells(Seta et al. 2006). These Mash1-expressing elongated cells
coexpressed type III cell markers (NCAM and serotonin),
but not type II cell markers (gustducin and PLCβ2). In thisstudy, we found that Mash1-expressing precursor cells could
differentiate into type III cell marker–expressing cells and
PLCβ2-expressing cells. We have revealed that some
AADC-expressing taste cells were positive for PLCβ2 (Setaet al. 2007). Taken together, Mash1-expressing precursor cells
differentiate into PLCβ2-expressing cells that may express
AADC. Miura et al. (2006) mentioned that Mash1-expressing
cells represent an immature cell state of both types II and type
III cells. Our present results indicate that Mash1-expressing
precursor cells could not differentiate into most gustducinpositive type II cells, suggesting that most gustducinpositive type II cells differentiate from basal cells that do not
express Mash1. However, only a few gustducin-positive cells
were expressed as tdTomato. These results suggest that asmall population of type II cells (gustducin and PLCβ2positive cells) could have possibly differentiated from
Mash1-positve precursor cells. On an average, taste bud cells
have a lifespan of 10–14 days (Beidler and Smallman 1965;
Farbman 1980; Delay et al. 1986; Stone et al. 2002). PereaMartinez et al. (2013) have shown that types II and III cells
have different lifespans. Specifically, type II cells were eliminated with a half-life of 8 days, whereas type III cells survived
much longer, with a half-life of 22 days. Our results indicatedthat type III cell marker–expressing taste cells that differentiated from Mash1-expressing precursor cells have a longer
lifespan than PLCβ2-positive cells that differentiated from

Cell Tissue Res (2021) 383:667–675

Mash1-expressing precursor cells. Another possibility could
be that PLCβ2-expressing type III cells may be immature.
The expression of PLCβ2 in type III cells may represent anerror in the differentiation process, and these cells have ashorter lifespan than normal type III cells caused by somemechanism identifying differentiation errors and removing
such cells from the taste buds.
In conclusion, the present study demonstrated that Mash1expressing precursor cells differentiated into type III cells
within mature taste buds, but not into most of type II cells.
Among taste cells that differentiated from Mash1-expressing
precursor cells, type III cell marker–expressing cells survive
much longer than PLCβ2-positive cells. It remains to be revealed whether PLCβ2-positive cells arising from Mash1-expressing precursor cells are type II or type III cells remains to
be revealed. Further studies using Mash1-expressing precursor cells will provide new insights into taste celldifferentiation.
Acknowledgments The authors would like to thank Enago (www.enago.
jp) for the English language review.
Funding The work was supported by the Japan Society for the Promotion
Grant-in-Aid for Scientific Research number 18 K09525 to Y.S.

Compliance with ethical standards
Conflict of interest The authors state that there is no conflict of interest.
Ethical approval All of the animals used in this study were maintained
and handled according to the protocols approved by Kyushu Dental
University Animal Care (Certification No: 17–010).

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