Epidermal Differentiation and DNA Demethylation of the Epidermis in Late Gestation of the Mouse Fetus

23
OBJECTIVE: To investigate the dynamic changes of
epidermal differentiation and demethylation in Kunming
mice during late gestation fetal development.
STUDY DESIGN: The fetuses were obtained from preg-
nant Kunming mice at embryonic (E) days 12.5, 14.5,
16.5, and 18.5. Hematoxylin and eosin staining and
immunohistochemistry were used to detect the epidermal
differentiation and demethylation on the back, abdomen,
hind leg, and face of the late-stage murine fetuses.
RESULTS: Cutaneous keratinization did not occur in
Kunming mice until E18.5. The facial epidermis was
thicker than the other parts of the epidermis. Cytokeratin
(K) 15 was strongly expressed in 4 parts of the epider-
mis at E16.8. K19 was stronger in the abdominal and
hind limb epidermis at E18.5. Involucrin appeared in
the epidermis of the abdomen, hind limb, and face from
E16.5 to E18.5 but in the back epidermis only at E18.5.
5-methylcytosine (5mC) and 5-hydroxymethylcytosine
(5hmC) began to be detectable at E12.5, but they were
undetectable in all parts of the epidermis except the
facial skin at E18.5. The expression of Tet methylcytosine
dioxygenase 2 (TET2) and TET3 appeared mainly at
E14.5 and E16.5.
CONCLUSION: The differentiation of epidermis in dif
ferent parts of Kunming mice before birth is related to
DNA demethylation, which is related to the function of
epidermis in different parts. (Anal Quant Cytopathol
Histpathol 2020;42:23–33)
Keywords: 5-hydroxymethylcytosine; differentia-
tion; DNA demethylation; ten-eleven translocation
enzymes; skin.
Skin is the first defense against harmful external
factors, such as UV light, microbial pathogens, haz-
ardous substances, mechanical stress, and the loss
of internal bodily fluids.1 These essential functions
are mediated by the epidermis, the outermost layer
of the skin. During embryonic development the
epidermis originates from the ectoderm, and a
layer of epithelial cells differ
entiates into the strat-
ified squamous epithelium.2 The cuticle layer is
Analytical and Quantitative Cytopathology and Histopathology®
0884-6812/20/4201-0023/$18.00/0 © Science Printers and Publishers, Inc.
Analytical and Quantitative Cytopathology and Histopathology®
Epidermal Differentiation and DNA
Demethylation of the Epidermis in Late
Gestation of the Mouse Fetus
Fangfang Xie, M.B., Ziwei Luo, M.B., Anli Weng, M.B., Xiangjun Li, M.M.,
Dongfeng Chen, M.D., and Aijun Liu, M.D.
From the Department of Anatomy, Guangzhou University of Chinese Medicine, Guangzhou, P.R. China.
Fangfang Xie and Ziwei Luo are co-first authors and contributed equally to this work.
Fangfang Xie, Ziwei Luo, and Anli Weng are Postgraduate Students.
Xiangjun Li is Medical Master.
Dongfeng Chen is Professor.
Aijun Liu is Professor.
Fangfang Xie and Ziwei Luo are co-first authors and contributed equally to this work.
This study was supported by the Natural Science Foundation of Guangdong Province (grant no. 2017A030312009).
Address correspondence to: Dongfeng Chen, M.D., No. 232 Waihuandong Road, Panyu, Guangzhou 510006, P.R. China (cdf27212@21cn.
com), or to: Aijun Liu, M.D., No. 232 Waihuandong Road, Panyu, Guangzhou 510006, P.R. China (aijunliu@gzucm.edu.cn).
Financial Disclosure: The authors have no connection to any companies or products mentioned in this article.

exfoliated from the skin surface and is continual-
ly replaced by differentiating basal cells moving
outward. The epidermal cells are constantly self-
renewing throughout the animal’s life.
Stepwise differentiation of epidermal cells is
essential for the development of a stratified epi-
thelium. Cytokeratin (K) belongs to the family of
intermediate filament proteins and indicates the
differentiation of the epidermis.3 Epidermal stem
cells in the basal layer play an important role in
the division, proliferation, differentiation, and kera-
tinization of keratinocytes. K15 and K19 are con
sidered markers of epidermal stem cells.4 The main
function of K14 is to provide structural support to
the basal layer of stratified epithelia and initiate a
program of stratification, and to eventually under-
go terminal differentiation to form the mature adult
epidermis.5 K10 is involved in the formation of a
mechanically elastic cytoskeleton, which is essen
tial for the epidermal integrity of skin.6 Involucrin
is a known marker of keratinocyte terminal differ-
entiation.7 However, the differentiation mechanism
of epidermal cells during embryonic development
remains unclear.
DNA methylation is one of the essential epi-
genetic mechanisms after DNA replication during
mammalian embryonic development. DNA meth-
ylation, an important marker of gene silencing, is
an enzymatic reaction in which the 5th carbon atom
of DNA cytosine is catalyzed by DNA methyltrans-
ferase to form 5-methylcytosine (5mC). During em-
bryonic development, DNA methylation poses a
fundamental epigenetic barrier that guides and re-
stricts cell differentiation and prevents cell regres-
sion into an undifferentiated state.8 The elimination
of DNA methylation is called demethylation. There
are 2 widely accepted demethylation modes in
mammals. One is passive demethylation, which is
lost due to the absence of methylation modification
mechanisms during DNA replication. The other is
active demethylation, which removes DNA meth-
ylation modifications under the action of specif-
ic demethylases to form 5-hydroxymethylcytosine
(5hmC). Postnatal DNA demethylation plays an
important role in tissue maturation.9 Enzymes of
the ten-eleven translocation (TET) proteins are re-
lated to active demethylation.10 There are 3 mem-
bers of the TET family, including TET1, TET2, and
TET3. We have previously reported that TET2 reg-
ulates the demethylation of major visceral organs
in mice during late-gestation fetal development.11
One of the important purposes of this study is to
explore the demethylation of the epidermis in the
late-gestation Kunming mouse fetus.
The structure and function of the epidermis in
different parts of the body are not identical. It is
necessary to explore the relationship between epi-
dermal differentiation and demethylation in dif-
ferent parts of the fetal skin in mice. Despite the
increasing use of novel molecular techniques in
pathology, histology remains the standard method
for monitoring tissue alterations and for assessing
pathology.12 In the present study the fetuses were
obtained from pregnant Kunming mice at embry-
onic (E) days 12.5, 14.5, 16.5, and 18.5. Hematoxylin
and eosin (HE) staining showed dynamic devel
opment of the epidermis on the back, abdomen,
hind leg, and face of the late murine fetuses. Im
munohistochemistry is an important method for
determining overall changes in embryos,13 and it
has obvious advantages in tracking methylation
changes in the fetus.14 Therefore, immunocyto-
chemistry was used to test the expression of K15,
K19, K14, K10, and involucrin. Additionally, the
spatiotemporal expression of 5mC, 5hmC, TET1,
TET2, and TET3 was detected by the same meth-
od. The results of this study will be helpful in
further study of the epigenetic regulation mechan-
isms of epidermal differentiation in different parts
of the epidermis during late-gestation Kunming
mouse fetal development.
Materials and Methods
Mouse Fetus Collection
The Kunming mice used in this study were spe-
cific pathogen-free and were purchased from the
Animal Centre of Guangzhou University of Chi-
nese Medicine (Guangzhou, China). The Kunming
mice were maintained in an environment of con-
trolled humidity, temperature (22°C), and lighting
(artificial light between 7:00 a.m. and 7:00 p.m.) and
with constant access to breeding diet and water.
Animal procedures were approved by the Care of
Experimental Animals Committee of Guangzhou
University of Chinese Medicine. All animal studies
were conducted in accordance with the ARRIVE
(Animal Research: Reporting in Vivo) guidelines
for reporting experiments involving animals. The
permit number for the mice was SCXK 2013-0020.
All experimental mice were healthy and not given
any drugs in the present study. There were 12 preg-
nant mice obtained after mating. The pregnant mice
were sacrificed by cervical dislocation at set exper-
imental time points (E12.5, E14.5, E16.5, or E18.5).
24 Analytical and Quantitative Cytopathology and Histopathology®
Xie et al

Six normal fetuses from each time point were
sacrificed by cervical dislocation. The fetuses were
washed several times with ice-cold phosphate-
buffered saline (PBS) (pH 7.4, Boster, Wuhan,
Hubei, China) and then fixed with 4% paraformal-
dehyde for 24 hours and embedded in paraffin wax.
Histological Specimens
Fetal specimens were cut into 2-μm slices, and then
HE staining was carried out in accordance with
routine protocols. First, the tissue sections were
de-waxed according to standard procedures and
stained with a hematoxylin solution (Boster) for
8 minutes, followed by 1% acid ethanol (1% HCl in
70% ethanol) and then rinsed in ultrapure water.
Next, the samples were stained with an eosin
solution (Boster) for 5 minutes, dehydrated with
graded alcohol, and cleared in xylene. Finally, all
of the samples were sealed with neutral gum.
Immunohistochemistry for K15, K19, K10, K14, and
Involucrin
For immunohistochemistry, the tissue sections
were de-waxed according to standard procedures
and immunostaining following a standard pro-
tocol. The samples were immersed in a 0.01 M
citrate buffer solution (pH 6, Boster) and micro-
waved at full power for 10 minutes in order to
repair anti
gen sites. The samples were covered
by 0.5% Triton X-100 for 15 minutes, and then
2N HCl for 1 hour. The samples were blocked in
5% bovine serum albumin (BSA) for 30 minutes.
The primary antibodies, including anti-keratin 15
(K15), anti-keratin 19 (K19), anti-keratin 10 (K10),
anti-keratin 14 (K14), and anti-involucrin (1:100
dilution; all purchased from Boster), were used 8
hours at 4°C. After washing with PBS, the samples
were incubated with a horseradish peroxidase–
conjugated goat antibody against mouse IgG (Im
munohistochemistry Reagent Kit; Boster) for 30
minutes and then incubated with a streptavidin-
biotin complex (SABC) (Boster) for 30 minutes.
Finally, the samples were incubated with diami-
nobenzidine (DAB) peroxidase substrate (Boster)
for 2 minutes. The samples were counterstained
in hematoxylin solution (Boster). Control staining
Volume 42, Number 1/February 2020 25
DNA Demethylation During Epidermal Development
Figure 1
Histological structure and
differentiation of the dorsal
epidermis during late gestation
of the Kunming mouse fetus.
Tissue structure of the dorsal
epidermis at different stages,
as shown by HE staining
(A–D). Immunohistochemical
staining showed the expression
of K15 (E–H), K19 (I–L), and
involucrin (M–P) in the dorsal
epidermis at different stages.
Scale bars=50 µm.

without the primary antibody produced no detect-
able signal.
Immunohistochemistry for 5mC, 5hmC, TET1, TET2,
and TET3
Tissue sections were treated in the same way as
described above. All the samples were de-waxed
according to standard procedures, followed by a
standard immunostaining protocol. The primary
antibodies included 5mC (1:500 dilution; Abcam,
Cambridge, UK) and 5hmC (1:500 dilution; Ac-
tive Motif, Carlsbad, California, USA). Corre
sponding to these 2 primary antibodies, a sec-
ondary horseradish peroxidase–conjugated goat
antibody against rabbit IgG (Immunohistochemis-
try Reagent Kit, Boster) was used. The other pri-
mary antibodies included TET1, TET2, and TET3
(1:500 dilution; Santa Cruz Biotechnology, Santa
Cruz, California, USA). Corresponding to these
primary antibodies, a secondary horseradish per-
oxidase–conjugated goat antibody against rabbit
IgG (Immunohistochemistry Reagent Kit, Boster)
was used. All other reagents used were the same
as those mentioned above. Images were acquired
using an Olympus IX71 microscope (Olympus,
Tokyo, Japan).
Image Capture and Processing
HE and DAB immunohistochemistry staining were
Xie et al
Figure 2
The expression of
demethylation and TETs
in the dorsal epidermis
during late gestation of the
Kunming mouse fetus.
Immunohistochemical staining
showed the expression of 5mC
(A–D), 5hmC (E–H), TET2 (I–L),
and TET3 (M–P) in the dorsal
Scale bars=50 µm.
Table I Semiquantitative Analysis of Immunohistochemistry
in the Dorsal Epidermis During Late Gestational Age
Kunming Mouse Fetus
E12.5 E14.5 E16.5 E18.5
K15 – – ++ +
K19 – – + +
Involucrin – – – +
5mC + + + –
5hmC + + – –
TET2 – + + –
TET3 – + + –
5mC = 5-methylcytosine, 5hmC = 5-hydroxymethylcytosine, E = embryon-
ic days, K = cytokeratin, TET2 = Tet methylcytosine dioxygenase 2.
– Absence of staining.
+ Low density.
++ High density.

imaged using an Olympus IX71 microscope. All
figures were produced using Photoshop CS5.1
(Adobe, San Jose, California, USA). Semiquantita-
tive immunohistochemistry assays were processed
by ImageJ v.1.8.0 software (National Institutes of
Health, Bethesda, Maryland, USA).
Results
Spatiotemporal Changes of Dorsal Epidermal
Differentiation and DNA Demethylation in
Late-Gestation Kunming Mouse Fetus
At E12.5, the dorsal epidermis was comprised of
1–2 layers of epithelium and the cytoplasmic stain-
ing was bright (Figure 1A). After that, the dorsal
epidermis became thicker (Figure 1B–C). At E18.5,
there were 4–5 layers of epithelial cells in the dor
sal epidermis containing a few keratohyalin gran-
ules, and the stratum corneum was distinct on the
surface of the dorsal epidermis (Figure 1D). The
positive expression of K15 and K19 were strong
at E16.5 (Figure 1G, K) and weak at E18.5 (Figure
1H, L). Involucrin exhibited weak positive expres-
sion only at E18.5 (Figure 1P). 5mC was in the basal
layer nucleus from E12.5 to E16.5 (Figure 2A–C),
and 5hmC was detectable at E12.5 and E14.5 (Fig-
ure 2E, F). The expression of TET2 and TET3 was
present in the cytoplasm at E14.5 and E16.5 (Figure
2J, K, N, O). Table I showed the semiquantitative
analysis of immunohistochemistry in the dorsal
epidermis from E12.5 to E18.5.
Spatiotemporal Changes of Abdominal Epidermal
Differentiation and DNA Demethylation in
The abdominal epidermis was comprised of 1–2
layers of epidermal cells at E12.5, which increased
to 4–5 layers at E18.5. Cytoplasmic staining was
basophilic from E12.5 to E16.5 (Figure 3A–C). At
E18.5, only the basal layer cell cytoplasm was
basophilic, and the keratohyalin granules and the
stratum corneum were distinct (Figure 3D). The
expression of K15 and K19 was stronger at E18.5
than at E16.5 (Figure 3G, H, K, L). Conversely,
involucrin was stronger at E16.5 than at E18.5
(Figure 3O, P). The expression of 5mC increased
from E12.5 to E16.5 (Figure 4A–C) and then dis
Figure 3
differentiation of the
abdominal epidermis in late
gestation of the Kunming
mouse fetus. Tissue structure
of the abdominal epidermis
at different stages, as shown
by HE staining (A–D).
showed the expression of K15
(E–H), K19 (I–L), and
involucrin (M–P) in the
abdominal epidermis at
different stages. Scale bars=
50 µm.

appeared at E18.5 (Figure 4D). 5hmC was detect-
able from E12.5 to E16.5 (Figure 4E–G). The ex-
pression of TET2 and TET3 was stronger at E16.5
than at E14.5 (Figure 4J, K, N, O). Table II showed
the semiquantitative analysis of immunohisto
chemistry in the abdominal epidermis from E12.5
to E18.5.
Spatiotemporal Changes of Epidermis Differentia-
tion and DNA Demethylation of the Hind Leg in
The epidermis with basophilic cytoplasm of the
hind leg became gradually thicker from E12.5
and E16.5 (Figure 5A–C). At E18.5, there were
4–5 layers of epidermis with more keratohyalin
granules, and the stratum corneum had appeared
(Figure 5D). Positive expression of K15 was
strong at E16.5 and weak at E18.5 (Figure 5G–
H). Furthermore, the expression of K19 was weak
at E16.5 and strong at E18.5 (Figure 5K–L). In-
volucrin exhibited weak expression at E16.5
and E18.5 (Figure 5O–P). 5mC was detectable at
E12.5 and E14.5 (Figure 6A–B). The expression of
5hmC was detectable from E12.5 to E16.5 (Fig-
ure 6E–G). The positive expression of TET2 and
TET3 gradually increased from E14.5 to E16.5
(Figure 6J, K, N, O). Table III showed the semi
quantitative analysis of immunohistochemistry in
the hind leg epidermis from E12.5 to E18.5.
Xie et al
Figure 4
The expression of
demethylation and TETs in
the abdominal epidermis
and TET3 (M–P) in the
abdominal epidermis at
different stages. Scale bars=
50 µm.
Table II Semiquantitative Analysis of Immunohistochemistry
in the Abdominal Epidermis During Late Gestational
Age Kunming Mouse Fetus
E12.5 E14.5 E16.5 E18.5
K15 – – ++ ++
K19 – – + ++
Involucrin – – ++ +
5mC + + ++ –
5hmC + + + –
TET2 – + ++ –
TET3 – + ++ –
+ Low density.
++ High density.

Spatiotemporal Changes of Epidermis Differentiation
and DNA Demethylation on the Face in
At E12.5, the facial epidermis was comprised of
1–2 layers (Figure 7A). Epithelial cell numbers in-
creased gradually (Figure 7B–C). At E18.5 there
were 5–6 layers of epithelial cells with strong
basophilic cytoplasm in the facial region, and the
keratohyalin granules and the stratum corneum
were distinct (Figure 7D). The expression of K15
was strong at E16.5 and weak at E18.5 (Figure
7G–H). K19 was detectable at E16.5 and E18.5
(Figure 7K–L). Involucrin was also detectable at
E16.5 and E18.5 (Figure 7O–P). The positive
products of 5mC were located in the nucleus of
the facial epidermis from E12.5 to E16.5 (Figure
8A–C). However, 5hmC was present at all stages
(Figure 8E–H). The expression of TET2 was de-
tected from E12.5 to E16.5 (Figure 8I–K), and TET3
was found at E14.5 and E16.5 (Figure 8N–O). Table
IV showed the semiquantitative analysis of im-
munohistochemistry in the facial epidermis from
E12.5 to E18.5.
Discussion
The skin protects the body from the outside world.
The epidermis originates from the embryonic ec-
toderm. However, the mechanisms of epidermal
development during the embryonic period still
need to be explored. In this paper, the epidermal
differentiation and demethylation during late-stage
Kunming mouse fetal development were studied.
The main findings were as follows: epidermal
keratinization was rapidly completed before the
birth of the Kunming mouse; the facial epidermis
was thicker than other parts of the epidermis; and
keratohyalin granules were gradually increased in
the epidermis of the back, abdomen, hind leg, and
face. The expression of epidermal keratins, DNA
demethylation, and TETs were not identical in the
4 parts of skin during the late gestational period of
the Kunming mouse fetus.
Keratinization of the epidermis was rapidly
completed before the birth of the Kunming mouse.
At E12.5, the epidermis of the Kunming mouse
fetus consisted of only 1–2 layers of epithelial cells.
After that, the epithelial cells proliferated to form
Figure 5
differentiation of the hind leg
epidermis during late gestation
Tissue structure of the hind leg
epidermis at different stages, as
shown by HE staining (A–D).
showed the expression of
K15 (E–H), K19 (I–L), and
involucrin (M–P) in the hind
leg epidermis at different
stages. Scale bars=50 µm.

multiple layers. At E18.5, the facial epidermis was
thicker than the other parts of the epidermis, which
may be related to the complex structure of the
face. Meanwhile, the keratohyalin granules had
gradually increased in the epidermis of the back,
abdomen, hind leg, and face. The keratinization
of the epidermis is affected by keratohyalin gran-
ules.15 Keratohyalin granules primarily exist within
the stratum granulosum, with some present in the
stratum spinosum. These granules are insoluble in
water and located within the cytoplasm where they
can prevent water loss in the body.16 The cuticle is
formed by crosslinking hard keratin, which plays
an important role in resisting friction, mechanical
resistance, and foreign body invasion.17,18 In this
study, cutaneous keratinization did not occur in
the Kunming mouse fetus until E18.5 and was in-
tended to prepare for adaptation to the external
environment after birth.
Epidermal keratins were expressed differently in
the 4 parts of the late-gestation Kunming mouse
fetus. During epithelial cell differentiation, the
cytoskeleton was remodeled and enhanced signifi-
cantly until keratin was finally formed. Keratins
are the main components of the intermediate fila-
ment cytoskeleton of epithelial cells.19 K15, a type
I keratin, has been used extensively as a biomark-
er for epidermal stem cells and hair follicle stem
cells.20,21 K15 was strongly expressed in 4 parts of
Xie et al
Figure 6
The expression of
demethylation and TETs of
the hind leg epidermis
and TET3 (M–P) of the hind leg
Scale bars=50 µm.
Table III Semiquantitative Analysis of Immunohistochemistry
in the Hind Leg During Late Gestational Age
Kunming Mouse Fetus
E12.5 E14.5 E16.5 E18.5
K15 – – ++ +
K19 – – + ++
Involucrin – – + +
5mC + + – –
5hmC + + + –
TET2 – + ++ –
TET3 – + ++ –
+ Low density.
++ High density.

the epidermis at E16.8 and then weakened at E18.5
except for in the abdomen, suggesting that abdom-
inal epidermal stem cells exist longer than those
in other parts of the body. K15 was not expressed
in the hair follicle epithelium until E18.5. These
results indicate that K15 participates in the devel-
opment of the epidermis first and then in follicle
formation during embryonic development. K19 is
also considered to be a marker of epidermal stem
cells and hair follicle stem cells.22,23 At E16.5, K19
began to appear in all parts of the epidermis. At
18.5, the expression of K19 was stronger in the
epidermis of the abdomen and hind limb. K19
also appeared in hair follicles at E18.5. A previous
study reported that K15-positive cells represented
a more undifferentiated state, while K19-positive
cells preferentially proliferated.24 The results of
the present study indicated that K19 was more
closely related to epidermal keratinization and
follicle formation. Neither K10 nor K14 was ex
pressed in this study, which may be due to the
non-expression of these 2 keratins in the late-
gestation fetus of Kunming mice, or related to
species differences. Involucrin is considered as a
marker of spinous cells which participates in the
outermost layer of the mantle and provides at-
tachment sites for other cornified envelope struc-
tural proteins.25 In the present study, involucrin
appeared in the epidermis of the abdomen, hind
limb, and face from E16.5 to E18.5 but only in the
dorsal epidermis at E18.5, which demonstrated that
the formation of the cornified envelope in the back
occurred slightly later than in other parts of the
epidermis in prenatal Kunming mice.
The expression of epidermal DNA demethyla
tion was not identical in different parts of the
late-gestation Kunming mouse fetus. DNA meth-
ylation is essential for proper mammalian devel-
opment, crucial for imprinting, and plays a role in
maintaining genomic stability.26 However, active
DNA demethylation is related to DNA methyla-
tion clearance, pluripotency, and cell differentia-
tion control, the maintenance of cell characteristics
and nuclear reprogramming in early embryos.27
5mC and 5hmC began to be expressed in the epi-
dermis at E12.5; nearly all of them disappeared at
Figure 7
differentiation of the facial
epidermis in late gestation
Tissue structure of the facial
epidermis at different stages, as
shown by HE staining (A–D).
showed the expression of
K15 (E–H), K19 (I–L), and
involucrin (M–P) in the facial
Scale bars=50 µm.

Xie et al
TETs in the epidermis during the late gestational
period of the Kunming mouse fetus. The detailed
epigenetic regulation of the epidermis in Kunming
mice during embryonic development remains to be
further studied.
E18.5, except 5hmc, which was still expressed in
the facial epidermis. Combined with the above
experimental results, the epidermis and skin ap-
pendages matured rapidly before birth, and the
normal epidermal keratinization process of Kun-
ming mice would begin after birth. The expression
of TET2 and TET3 mainly appeared at E14.5 and
E16.5, which promotes the rapid development and
maturation of the prenatal epidermis in Kunming
mice through active demethylation. However, TET1
was not detected in this study. Notably, expression
of 5hmC in the facial epidermis was detectable
from E12.5 to E18.5. The expression of TET2 and
TET3 in the facial epidermis was positive from
E12.5 to E16.5, which differed from their expres-
sion in the other 3 parts of the epidermis. The
differences are related to the complex structure of
the facial epidermis.
Taken together, the rapid maturation of the pre-
natal epidermis was related to DNA demethylation
in Kunming mice. However, our study showed
only the general changes of demethylation and
Figure 8
The expression of
demethylation and TETs of
the facial epidermis during
late gestation of the
and TET3 (M–P) in the facial
Scale bars=50 µm.
Table IV Semiquantitative Analysis of Immunohistochemistry
in the Facial Epidermis During Late Gestational Age
Kunming Mouse Fetus
E12.5 E14.5 E16.5 E18.5
K15 – – ++ +
K19 – – + +
Involucrin – – + +
5mC + + + –
5hmC + + + +
TET2 + + + –
TET3 – + + –
+ Low density.
++ High density.

during late gestation male germ line development in the rat.
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Stremnitzer C, Buchberger M, Mlitz V, Ballaun C, Sterniczky
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