Annals of Anatomy 229 (2020) 151482

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Annals of Anatomy
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RESEARCH ARTICLE

Morphological differences between regenerating salivary glands after

salivary gland duct ligation and embryonic salivary glands
Hitomi Ono Minagi a,b , Yu Usami c , Manabu Sakai a,d , Takayoshi Sakai a,∗
a
Department of Oral-facial Disorders, Osaka University Graduate School of Dentistry, Osaka, Japan
b
Research Fellow of Japan Society for the Promotion of Science, Japan
c
Department of Oral Pathology, Osaka University Graduate School of Dentistry, Osaka, Japan
d
Department of Clinical Laboratory, Osaka University Dental Hospital, Osaka, Japan

a r t i c l e i n f o a b s t r a c t

Article history: Background: Most animals and organs have regenerative capabilities. Whether regeneration is a devel-
Received 21 August 2019 opmental process or a distinct phenomenon that is independent of development is debatable.
Received in revised form 9 December 2019 Method: We examined the differences between developing and regenerating salivary glands using duct-
Accepted 9 January 2020
ligation models. We performed morphological analyses comparing submandibular gland regeneration
and development. To reveal the proliferation processes that occur during salivary gland regeneration
Keywords:
and development, we counted the number of Ki67-positive cells over time. In addition, we examined the
Salivary gland
expression of the following markers: aquaporin 5, smooth muscle actin, cytokeratin 7, and tubulin beta
Development
Regeneration
3.
Result: The proliferation patterns seen during regeneration differed from those observed during devel-
opment. Different salivary gland marker expression patterns were seen during development and
regeneration.
Conclusion: This study showed that regenerating salivary glands do not follow the same growth process
as developing salivary glands.
© 2020 Elsevier GmbH. All rights reserved.

1. Introduction
Regeneration, ẗhe replacement of lost body parts¨, is a
widespread phenomenon in both animals and plants, and it has
been studied by biologists for a long time (Sakai, 2016; Wang et al.,
2013). Regenerative medicine aims to restore dysfunctional organs
to a normal state. Understanding cell-division dynamics, cell-fate
decisions, and the spatial organization of cells is crucial for under-
standing regeneration. In addition, by using various methods, it has
been demonstrated that the molecular mechanisms and molecules
that are important for developmental processes are also involved
in regenerative processes (Baron and van Oudenaarden, 2019).
Advances in developmental biology and regenerative biology are
required to make such goals possible (Ahuja et al., 2007). On the
other hand, salivary gland hypofunction and xerostomia, which
can be caused by local surgery, radiotherapy for head and neck
cancer, Sjögren’s syndrome, and certain medications, can result in
severe dental caries, oral ulcers, swallowing and speech difficul-
∗ Corresponding author.
E-mail address: sakai@dent.osaka-u.ac.jp (T. Sakai).
ties, and taste disorders (Harunaga et al., 2011; Patel and Hoffman,
2014). However, the current therapies for salivary gland hypo-
function and xerostomia are palliative and temporary, and they
cannot completely abrogate salivary gland dysfunction (Minagi
et al., 2018). Therefore, new treatment strategies for these condi-
tions are required.
Salivary gland regenerative therapies involving tissue repair and
whole salivary gland replacement are regenerative therapies that
have the potential to recover the salivary gland function. To facili-
tate the development of such regenerative therapies, there has been
increasing interest in the identification of stem cell populations
(and their regulators) that could be used for salivary gland repair or
regeneration. Preliminary studies in this field have focused on elu-
cidating the steps involved in salivary gland development (Ogawa
and Tsuji, 2017). Many researchers have examined the mecha-
nisms responsible for the regeneration and development of salivary
glands (Sakai et al., 2003). Regressive tissue changes and regenera-
tion have been investigated by subjecting animal models to salivary
gland duct ligation. However, the precise mechanisms responsible
for the healing of salivary glands and acinar cell regeneration are
not fully understood.

https://doi.org/10.1016/j.aanat.2020.151482
0940-9602/© 2020 Elsevier GmbH. All rights reserved.
2 H. Ono Minagi, Y. Usami, M. Sakai et al. / Annals of Anatomy 229 (2020) 151482
In this study, we used a submandibular gland (SMG) duct lig-
ation model to perform a comparative analysis of salivary gland
regeneration versus salivary gland development, focusing on ductal
and acinar components, in order to reveal the histological differ-
ences between these processes.
2. Materials and methods
2.1. Animals and duct ligation
All animal experiments were performed in strict accordance
with the guidelines developed by the ethics committee of the Fac-
ulty of Dentistry, Osaka University Graduate School of Dentistry
(permit number: 25-004-0). All animal surgery was performed
under sodium pentobarbital-induced anesthesia, and all efforts
were made to minimize pain. Salivary glands were obtained from
female C57/BL6 mice, which were purchased from Japan SLC, Inc.
Unilateral excretory duct ligation was performed as follows: post-
natal day (P) 30 mice were anesthetized via the intraperitoneal
injection of pentobarbital (0.2 mg/g mouse weight), and the main
excretory duct on one side of the neck was dissected and separated
from the surrounding connective tissue under a surgical stereo-
scope (Leica S6D, Nusloch, Germany). The main excretory duct was
ligated using surgical sutures, and then the incision was closed on
P30. After a week, the ligation procedure was reversed in some of
the mice. Specifically, the surgical sutures were removed from the
main excretory duct, and the incision was closed. After three days,
seven days, or two weeks of recovery, the mice were anesthetized
and euthanized, and their SMG were harvested. In the analysis of
the developmental process, three E16.0 to E18.0 mice and three P0
and P30 mice were used.
2.2. Histological analysis, immunohistochemistry, and imaging
The salivary glands were harvested and immediately fixed in
4% (wt/vol) paraformaldehyde/ phosphate-buffered saline at 4
◦ C overnight. The fixed tissues were then embedded in paraf-
fin, and Sections (4 ␮m) were made, mounted onto slide glasses,
and dried overnight at 40 ◦ C. Following the deparaffinization
procedure, the sections were incubated in antigen retrieval H
buffer (LSI Medience, Tokyo, Japan) at 121 ◦ C for five minutes,
before being stained with primary antibodies against E-cadherin
(E-cad, 1:100 dilution; BD Biosciences, New Jersey, USA), Ki67
(MKI67, 1:200 dilution; Abcam, Cambridge, England), cytokeratin
7 (CK7, 1:200 dilution; Abcam), aquaporin 5 (AQP5, 1:200 dilu-
tion; Alomone Labs, Jerusalem, Israel), tubulin beta 3 (TUBB3,
1:200 dilution; R&D Systems, Minnesota, USA), smooth muscle
actin (SMA, 1:200 dilution; Sigma, Missouri, USA), and cytoker-
atin 5 (CK5, 1:200 dilution; Bio Legend, California, USA) and then
incubated with fluorophore-conjugated secondary antibodies; i.e.,
Cy2-labeled donkey anti-mouse, Cy3-labeled donkey anti-rabbit,
and Cy5-labeled donkey anti-rat or anti-goat IgG antibodies (1:100
dilution; Life Technologies, Massachusetts, USA). Examinations
of fluorescence-labeled cells were performed using a confocal
microscope (Leica SP-8, Leica Biosystems, Inc., Nusloch, Germany)
after DAPI (4’,6-diamidino-2-phenylindole; GeneCopoeia, Mary-
land, USA) staining, and images of the cells were captured.
Isotype-matched immunoglobulins for rats and rabbits were used
as controls. Unless otherwise indicated, we analyzed sections from
at least three mice in independent experiments, and representative
images are shown. The histology of the sections was analyzed using
hematoxylin and eosin staining. The histological method was per-
formed as described previously (Ono et al., 2015). The histological
analysis was conducted in a blind manner by expert pathologists.
2.3. Statistical analyses
The differences among means were evaluated using one-way
analysis of variance followed by Bonferroni’s correction. P-values
of <0.05 and <0.01 were considered significant.
3. Results
In the analysis of development, the embryonic salivary glands
exhibited dynamic changes. In this study, we examined each rep-
resentative stages, the canalicular stage (E16), the terminal bud
stage (E18), birth (P0), and early adulthood (P30), according to a
previous study (Larsen et al., 2011). The following representative
stages were observed after staining. In the analysis of regenera-
tion, we analyzed the following four stages (App-Fig. 1A): one week
post-ligation, which was considered to be when the severe dam-
age would be seen (1w); three days after the release of the ligature
(1w3d), a week after the release of the ligature (1w1 w), and two
weeks after the release of the ligature (1w2w). Note, the state of
the SMG at developmental stage P30 represents its state before it
was ligated. Duct ligation reduced the weights of the excised SMG.
The weights of these glands subsequently recovered to the level of
the unligated salivary glands (App-Fig. 1B). In 1w and 1w3d groups,
duct-like structures were observed in the atrophied salivary gland
tissue. In the 1w3d group, dilation of the ductal lumen, interlobular
connective tissue, and other signs of atrophied salivary gland tissue
were evident, but they were less prominent than in the 1w group.
In addition, small aggregates of acinar cells were observed in some
areas (Fig. 1A, B). In the 1w2w group, atrophy of salivary gland tis-
sue was not clear, and the aggregates of acinar cells were larger than
those seen in the 1w3d group (Fig. 1D) (Watanabe et al., 2017). To
examine the differences in cell proliferation between regenerating
and developing salivary glands, regenerating salivary glands were
subjected to Ki67 staining. Ki67 is a cell division-related protein
and is used as a marker of proliferating cells (Scholzen and Gerdes,
2000). While the nuclei of the mesenchymal cells were flat, those
of the salivary duct and acinar glands were round (App-Fig. 1C, E).
Salivary ducts were identified using CK7 (App-Fig. 1E). The total
number of cells increased immediately after the salivary duct lig-
ation, and it subsequently returned to its original state (Fig. 1I–L,
2E). The proliferation of salivary duct, acinar, and mesenchymal
cells was induced by ligation. The number of Ki67-positive cells
was increased among the mesenchymal cells, and it is considered
that such cells contribute to tissue repair through the inflammatory
reactions (Fig. 2A, D). The salivary duct and acinar cells exhibited
different growth patterns. At two weeks after the duct ligation,
the salivary glands were in more stable state. During regeneration,
Ki67-positive cells were mainly found in the acini in the 1w2w
group (Fig. 1L, 2A, C). On the other hand, during development Ki67-
positive cells were present throughout the whole salivary gland and
decreased in number when the salivary gland developed (Fig. 1M–P,
2I–L). Throughout development, a large number of Ki67-positive
cells were seen in the acini (Fig. 2I). Furthermore, there were more
Ki67-positive cells in the acini than in the ducts or mesenchymal
tissue (Fig. 2J–L).
Immunohistochemistry was used to clarify the differences
between salivary gland regeneration and development. E-cad was
used as a framework marker (Ray et al., 2013). During prenatal
development, AQP5 expression increased in the apical membranes
of the salivary glands (Fig. 3F). Similar results of E-cad staining were
obtained at E18 and 1w3d. AQP are membrane water-channel pro-
teins (Agre, 2006). However, when damage was induced via salivary
duct ligation, AQP5 expression increased in the basal, lateral, and
apical membranes (compared with that observed in the non-ligated
P30 tissue) (Fig. 3A, H). Then, the expression of AQP5 decreased, and
 H. Ono Minagi, Y. Usami, M. Sakai et al. / Annals of Anatomy 229 (2020) 151482 3

Fig. 1. Histological comparison of regenerating and developing salivary glands.

The morphological changes seen in regenerating (A–D) or developing submandibular glands (SMG) (E–H) are shown. Images of ligated SMG obtained at one week after
ligation (A) or at three days (B), one week (C), or two weeks (D) after the removal of the ligature are shown. All SMG were located the border of the tissue. SMG at the
canalicular stage [E (embryonic day) 16] (E), the terminal bud stage (E18) (F), at birth [P (postnatal day)0] (G), or early adulthood (P30) (H), are shown. Scale bars: 400 ␮m.
The black arrowheads indicate immature acinus cells. The white arrowhead indicates acinus cells that exhibited polarity.
Confocal images of protein expression obtained during salivary gland regeneration (I–L) or development (M–P) are shown. Ki67 (green), E-cadherin (E-cad) (red), Cytokeratin
7 (CK7) (blue); scale bars: 75 ␮m

similar AQP5 expression patterns were noted in the E18 and 1w3d
groups (Fig. 3B, F). Finally, AQP5 expression returned to the state
seen before the ligation procedure. Thus, different AQP5 expres-
sion patterns were observed during development and regeneration
(Table 1).
The expression of SMA increased in the initial stages of devel-
opment (Fig. 4E–H). On the other hand, during regeneration SMA
expression was seen in the basal, lateral, and apical membranes
immediately after the ligation (Fig. 4A–D). Based on the above
findings, salivary glands display different SMA expression patterns
during development and regeneration. The CK5 protein was mainly
expressed in acini and demonstrated the same expression patterns
as SMA (Weng et al., 2018). However, CK5 began to be expressed
earlier than SMA (data not shown). The SMA expression pattern
seen during development was different from that observed during
regeneration (Table 2).
CK7 was used as a ductal marker in the present study. In the
E16 salivary glands, CK7 was restricted to the salivary duct cells
and was not observed in the terminal epithelial bud cells (App-Fig.
2E). At P0, CK7 was detected throughout the examined ducts. In the
postnatal period, strong CK7 signals were seen in the salivary duct
cells (App-Fig. 2G). Similar CK7 localization patterns were observed
4 H. Ono Minagi, Y. Usami, M. Sakai et al. / Annals of Anatomy 229 (2020) 151482

Fig. 2. Cell proliferation in ligated salivary and salivary glands that had been released from ligation.
The changes in cell proliferation seen in regenerating A–H or developing SMG I–P are shown. The total number of Ki67-positive cells (Ki+ cells) A–D, I–L and the total number
of cells (Total cells) E–H, M–P are shown. The number of all cells A, E, I, and M and the numbers of cells in each structure, acinus, duct, or mesenchymal tissue B–D, F–H, J–L,
N–P are shown. Each slide shows an area of 15,000 ␮m2 . *P < 0.05, **P < 0.01, according to the Bonferroni method

Fig. 3. Comparison of the expression of the acinus marker aquaporin 5 (AQP5) between salivary gland regeneration and development.
AQP5 expression during regeneration (A–D) or development (E–H) of the salivary glands is shown.
AQP5 (green), E-cad (red); acinus: a, intercalated duct: id, striated duct: sd, excretory duct: ed; scale bars: 25 ␮m
 H. Ono Minagi, Y. Usami, M. Sakai et al. / Annals of Anatomy 229 (2020) 151482 5

Fig. 4. Comparison of the expression of the myoepithelial marker smooth muscle actin (SMA) between salivary gland regeneration and development.
SMA expression during salivary gland regeneration (A–D) or development (E–H) is shown. SMA (green), E-cad (red); acinus: a, striated duct: sd; scale bars: low-power images
obtained at E18 (F): 250 ␮m, low-power images obtained at P30 or 1w3d (B, H): 50 ␮m, others: 25 ␮m

Fig. 5. Histological differences between regeneration and development.

The schema shows the observed changes in the levels of each marker. The control shows the state of the salivary gland before it was ligated. AQP5 (red), SMA (brown), CK7
(beige), Tubb3 (green)

Table 1 Table 2
The localization of AQP5 during regeneration or development. The localization of SMA during regeneration or development.

Regeneration 1w 1w3d 1w1w 1w2w Regeneration 1w 1w3d 1w1w 1w2w

Acini +A, L, B +B +B (L, A) +B (L) Acini +A (L) +B, A, L +A +A

Intercalated duct – Intercalated duct +A
Striated duct – – Striated duct
Excretory duct – – – – Excretory duct +(A) +A

Development E16 E18 P0 P30 Development E16 E18 P0 P30

Terminal bud – Terminal bud –
Pro-acini – +B +B (L) Pro-acini +(A) +(A)
Acini +B (L) Acini +A
Intercalated duct – Intercalated duct – +A
Striated duct – – – Striated duct – – –
Excretory duct – Excretory duct –

-; negative staining, +; positive staining, (); weak staining, A; apical staining, B; -; negative staining, +; positive staining, (); weak staining, A; apical staining, B;
basal staining, C; cytoplasmic staining, L; lateral staining, empty slots: structure not basal staining, C; cytoplasmic staining, L; lateral staining, empty slots: structure not
present. present.

in the striated ducts and intercalated ducts of the adult salivary
glands (App-Fig. 2H) (Adhikari et al., 2018). Many duct-like struc-
tures were seen after the ligation procedure. In the analysis of CK7
staining during development, basal membrane staining of the ducts
for CK7 produced a positive result (App-Fig. 2E–H). On the other
hand, although it was weak, positive CK7 expression was detected
in the apical and lateral membranes of the regenerating salivary
glands. Therefore, it is considered that the CK7 expression patterns
of regenerating and developing salivary glands are also different
(App-Fig. 2A–H). TUBB3 was used as a nerve marker in the current
6 H. Ono Minagi, Y. Usami, M. Sakai et al. / Annals of Anatomy 229 (2020) 151482
Table 3
Expression strength of each salivary gland markers.
Regeneration Development
AQP5 AQP5 expression decreased AQP5 expression gradually
once and then recovered increased from the terminal
bud stage
SMA SMA expression increased after SMA expression was first seen
the de-ligation procedure and after the postnatal stage
subsequently decreased
CK7 CK7 expression remained CK7 expression gradually
constant became strong
Tubb3 Low-level Tubb3 expression Tubb3 expression expanded
was restored from the main nerve to the thin
peripheral nerve
Ki67 Ki67 was mainly expressed in The Ki67 expression ratio was
mesenchymal tissue the same in ducts, acini, and
mesenchymal tissue
study (Knox et al., 2013a). In each salivary gland, the main nerve and
a thin peripheral nerve were seen (App-Fig. 2I–P). Histological anal-
ysis revealed that TUBB3 expression increased during development
and regeneration. However, the other examined markers exhibited
different expression patterns between salivary gland development
and regeneration (Fig. 5, Table 3).
4. Discussion
This study examined whether the proliferation and protein
expression patterns seen during the regeneration of salivary glands
matched those observed during the development of salivary glands.
In the morphological analysis, salivary glands that had started to
recover from damage (1w3d) displayed similar findings to devel-
oping salivary glands (E18). However, pattern of the proliferating
cells seen during development and regeneration were different.
Salivary glands are formed via branching morphogenesis, which
involves coordinated cell proliferation, clefting, differentiation,
migration, and apoptosis, and reciprocal interactions between
epithelial and mesenchymal cells (Knosp et al., 2012). This pro-
cess was first described in salivary glands by Elio Borghese in 1950
(Borghese, 1950). To analyze the process underlying salivary gland
regeneration, we used a duct ligation model. Our histopathologi-
cal analysis of salivary gland weight loss showed that salivary duct
ligation caused the disappearance of acinar cells and an increase in
fibrous tissue in the intralobular regions. The loss of secretory gran-
ules from the cytoplasm of tubules and duct cells might be related to
gland dysfunction. These findings are similar to those obtained in a
previous study, in which salivary gland weight and functions recov-
ered after removal of salivary duct ligatures (Cotroneo et al., 2008;
Nagai et al., 2014). To reveal the histological differences between
the development and regeneration of salivary glands, we used vari-
ous markers of salivary gland structures, such as the acinus marker
AQP5, the muscle marker SMA, the duct marker CK7, and the nerve
marker TUBB3.
CK7 is a member of the keratin gene family and is classified
into the type II cytokeratins, which consist of basic or neutral pro-
teins. CK7 is specifically expressed in the simple epithelia lining
the cavities of the internal organs, gland ducts has been used to
identify luminal areas within ducts, as was previously reported for
developing salivary glands (Walker et al., 2008). A previous study
showed that CK7 was expressed in less differentiated ductal cells in
the pancreas (Bouwens, 1998). However, in our histological anal-
ysis CK7 expression was seen in the salivary ducts in each stage
of development and regeneration. Therefore, CK7 can be used as
a salivary duct marker during salivary gland development. More-
over, it was revealed that CK7 was still expressed in salivary duct
cells after one week of salivary duct ligation. When the examined
structures were classified according to their CK7 expression, it was
found that the cell proliferation patterns of the salivary duct, acinar,
and mesenchymal cells are different. In the proliferation analysis,
many differences were detected between regeneration and devel-
opment. Regeneration is a healing process. Therefore, it starts just
after duct ligation, and Ki67-positive cells in mesenchymal tis-
sue contribute to healing. On the other hand, Ki67 expression was
observed throughout the whole salivary gland during development
and decreased afterwards. The ratios of the Ki67 expression levels
of each structure did not change during development. As for prolif-
eration patterns, salivary gland regeneration was shown to involve
different proliferation patterns from salivary gland development.
We performed immunostaining analysis involving salivary
gland markers to reveal the differences between salivary gland
regeneration and development. TUBB3 is widely known as a neu-
ronal marker (Dráberová et al., 1998), and it is associated with the
parasympathetic submandibular ganglion (PSG), which has previ-
ously been shown to affect epithelial progenitor cell maintenance
(Knox et al., 2010, 2013b). The expansion of the CK5-positive cell
population during duct growth via tubulogenesis is regulated by
PSG innervation (Garcia-Gallastegui et al., 2014; Knox et al., 2010).
TUBB3 is one of the earliest-appearing markers of neuronal differ-
entiation. Reciprocal interactions among the epithelium, the neural
crest-derived mesenchyme, nerves, and blood vessels regulate the
early events of salivary gland development (Patel and Hoffman,
2014). It was reported that radiation-induced damage reduced the
expression of TUBB3, and ligation-induced damage also reduced
TUBB3 expression in the current study. As TUBB3 exhibited sim-
ilar expression patterns in both salivary gland regeneration and
development, it is considered that nerves are less likely to recover
from damage than other tissues. In a previous study, salivary glands
recovered to the control state within two weeks of being de-ligated
(Nagai et al., 2014). In the present study, although hematoxylin and
eosin staining produced the same findings at 1w2w as were seen
at P30, immunostaining indicated that the expression of SMA was
slightly weaker at 1w2w than at P30.
There are other characteristic markers whose expression pat-
terns differ between development and regeneration. For example,
AQP5 is expressed in the basolateral and apical membranes of aci-
nar cells in the mouse salivary gland (Matsuzaki et al., 2012). AQP5
expression was previously reported to be increased in regenerating
salivary glands (Larsen et al., 2011; Zinzen et al., 2004). As water
transfer is a ubiquitous phenomenon, AQP are widely distributed
among bacterial, plant, and animal species (Matsuzaki et al., 2012).
In the current study, increased AQP5 expression was detected in
atrophied acinar cell-like cells in the 1w group. In addition, AQP5
expression was decreased in the 1w3d group and returned to a
normal state afterwards. In the 1w2w group, AQP5 was expressed
in the cellular membranes of the acinar cells. The expression lev-
els of SMA and CK5 seen during regeneration differed from those
observed during development, as was the case for AQP5. SMA is
a marker of various hollow structures and a constituent of blood
and lymph vessels (Petschnik et al., 2010). Previous studies have
shown that the external layers of salivary and sweat glands are
composed of myoepithelial cells (Foschini et al., 2000; Petschnik
et al., 2010). It is considered that the expression of SMA starts after
the establishment of an extensively branched system of cellular
cords, each of which terminates as a spherical cell mass, a terminal
bud. At the next developmental stage, myoepithelial cells acquire
cytoplasmic microfilaments and plasma membranes. After devel-
opment, acinar progenitor cells and salivary duct progenitor cells
appear to reside in acini and salivary ducts, respectively, where they
contribute to tissue homeostasis (May et al., 2018; Ogawa, 2003).
A relationship has been suggested to exist between SMA expres-
sion and stem cells. CK5 is used as a stem cell marker in several
tissues (Leung et al., 2007; Rock et al., 2009), and it is expressed in
embryonic salivary gland progenitors. In adult salivary glands, CK5
 H. Ono Minagi, Y. Usami, M. Sakai et al. / Annals of Anatomy 229 (2020) 151482
expression is restricted to intercalated and basal excretory ducts
and myoepithelial cells (Knox et al., 2010). Our study demonstrated
that similar CK5 and SMA expression patterns were seen during
salivary gland development and regeneration. Development is the
set of processes by which tissues form and reach a fixed state after
fertilization (Patel et al., 2006). On the other hand, regeneration is a
process by which a tissue returns to its original state (Galliot et al.,
2017). The fact that these two phenomena have similarities and
differences is self-evident. This study revealed that there are many
differences between regenerative and developmental processes in
the SMG. The confirmation of these findings in a model of regener-
ation and more definitive information about the embryonic stages
are required to study the processes involved in organ regeneration.
In summary, we performed a morphological analysis comparing
salivary gland development with salivary gland regeneration. The
expression patterns of salivary gland markers differed markedly
between salivary gland development and regeneration. By sum-
marizing the changes in cell proliferation and marker expression
that occurred over time, it was shown that the regeneration pro-
cess was different from the developmental process. Our results
demonstrated that developing salivary glands do not follow the
same processes as regenerating salivary glands.
Acknowledgements
This study was supported by JSPS KAKENHI grant numbers
15H02575 and 16K20573.
Appendix A. Supplementary data
Supplementary material related to this article can be found,
in the online version, at doi:https://doi.org/10.1016/j.aanat.2020.
151482.
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