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[page 8] [Dermatology Reports 2019; 11:7853]
Trps1-deficient transplanted
skin gave rise to a substantial
amount of hair:
Trps1 is unnecessary for hair
development
Yingzhe Zhang,1
Tomoyuki Nakamura,2
Fukumi Furukawa,2
Yasuteru Muragaki1
1Department of Pathology; 2Department
of Dermatology, Wakayama Medical
University School of Medicine, Japan
Abstract
Trps1 is considered as an important
gene involved in the interactions between
the epithelial and mesenchymal cells during
hair follicle morphogenesis. The number of
hair follicles in Trps1 Knockout (KO) new-
born mouse skin was significantly lower
than that in wild-type (WT) newborn skin.
To gain insight into the functional role of
Trps1 in hair development, we transplanted
Trps1 KO newborn mouse skin on the backs
of nude mice and examined hair growth at
day 42 after transplantation. Surprisingly,
transplanted skin from Trps1 KO newborn
mice gave rise to a substantial amount of
hair, although the hair was softer than that
of WT mice. Histological examination
revealed that the diameter of both hair folli-
cles and hair shafts were significantly
lower, whereas the density of hair follicles
showed no significant difference between
the Trps1 KO and WT mice. We introduce
mouse hair follicles as a fascinating model
to study the functions of Trps1 in mouse
hair growth and pathology. This model sug-
gests that the function of Trps1 is unneces-
sary for the development of normal hair fol-
licles and hair shafts, although the loss of
Trps1 affects the diameters of hair follicles
and hair shaft.
Introduction
It has been reported that heterozygous
germ line mutations in tricho-rhino-pha-
langeal syndrome (Trps1) on chromosome
8q23 in humans result in autosomal domi-
nant inheritance of tricho-rhino-phalangeal
syndrome type I (TrpsI) and III (TrpsIII),
characterized by sparse and slow growing
scalp hair, as well as craniofacial and skele-
tal abnormalities.1,2 Trps1 Knockout (KO; it
is a genetically modified mouse,
Musmusculus, in which researchers have
inactivated, or knocked out, an existing
gene by disrupting it with an artificial piece
of DNA) mice were reported to have fewer
hair follicles, with craniofacial and skeletal
defects that mirror the phenotypic charac-
teristics of human patients. Patients with
TRPS I have an orbicular nose, a long and
even philtrum, a thin upper lip, sparse scalp
hair that grows slowly, and protruding
ears.3,4
These findings proved that Trps1 was
required for specific aspects of hair growth
regulation. In addition to the apparent
defects in facial soft tissues, male patients
were particularly affected by hair loss, with
many being nearly or completely bald soon
after puberty. Some children with this dis-
ease have loose skin, although the skin
becomes tighter over time. Individuals with
TRPS I may experience excessive sweating,
although the clinical description is often
incomplete.5-8
Skin development is a complex dynam-
ic process that includes formation of epider-
mis, a layered self-renewing epithelium,
and several skin auxiliaries such as hair fol-
licles (HF), hair nails and sweat glands. HF
morphogenesis is driven by bidirectional
ectodermal-mesenchymal interactions
between epidermal keratinocytes and a spe-
cialized population of dermal fibroblasts,
resulting in formation of the hair bulb, in
which epithelial progenitor cells proliferate
and differentiate into cell lineages to form
hair shafts and their supporting layers in the
inner root sheath. HF morphogenesis is
governed by a well-balanced mutual effect
among cell proliferation, differentiation,
and apoptosis, all of which are controlled at
several levels including signalling/tran-
scription factor-mediated and epigenetic
regulatory mechanisms.9,10
We previously demonstrated that tran-
scription factor Trps1 played an important
role in the morphogenesis of secondary HFs
via an interaction with the BMP inhibitor
Noggin.11 We found that development of
secondary hair follicles in mutant Trps1
embryos was inhibited compared to their
wide-type counterparts. Additional analysis
revealed that Trps1 activated Wnt inhibitors
and other transcription factors essential for
follicle morphogenesis in mice.12 While this
study demonstrated a requirement for Trps1
during early HF formation, it did not
address the mechanisms underlying hair
follicle degeneration in subsequent stages
after birth. In addition, since Trps1 KO
mice die within a few hours due to respira-
tory failure, we do not know whether or not
hair grows with total loss of Trps1. In this
study, to observe hair follicle growth after
birth, we transplanted Trps1 KO newborn
mouse skin to the back of nude mice.
Materials and Methods
Trps1 Knockout mice and tissue
preparation
Trps1 KO mice were generated as pre-
viously described.13 The principles of ani-
mal care and use were followed as directed
by the Committee of Wakayama University.
We crossed male and female heterozygous
mice to obtain wild-type and homozygous
newborns.11 When newborn mice were har-
vested, genotyping was performed. Dorsal
skin was carefully peeled off newborn mice
and skin (6-mm in diameter) was taken with
a tissue puncher and transplanted to the
back of nude mice. 40 skin grafts from
wild-type, heterozygous, and homozygous
newborn, respectively, were transplanted to
20 nude mice (6 skin grafts per nude
mouse). The skin graft was affixed to the
recipient skin by adding Aron Alfa (KON-
ISHI, Japan) at the margin.
Preparation of histological sections
and immunohistochemistry
At day 42 after transplantation, trans-
planted skin tissue was obtained and fixed
with 4% paraformaldehyde overnight. The
Dermatology Reports 2019; volume 11:7853
Correspondence: Yasuteru Muragaki,
Department of Pathology, Wakayama Medical
University School of Medicine, 811-1
Kimiidera, Wakayama 641-0012, Japan.
Tel.: 073.447.2300 - Fax: 073.447.2300.
E-mail: ymuragak@wakayama-med.ac.jp
Key words: Hair follicle; Trps1; Hair develop-
ment; Hair shaft.
Contributions: YZ, TN, FF data collecting and
analyzing; YZ, manuscript writing and refer-
ences search.
Conflict of interest: the authors declare no
potential conflict of interest.
Funding: this work was supported in part by a
Grant-in-Aid for Scientific Research
(15K08430) from the Ministry of Education,
Science, Sports, and Culture of Japan (to
Y.M.). It was also supported by CSC scholar-
ship (NO. 201406220175).
Received for publication: 28 August 2018.
Revision received: 18 December 2018.
Accepted for publication: 18 December 2018.
This work is licensed under a Creative
Commons Attribution-NonCommercial 4.0
International License (CC BY-NC 4.0).
©Copyright Y. Zhang et al., 2019
Licensee PAGEPress, Italy
Dermatology Reports 2019; 11:7853
doi:10.4081/dr.2019.7853
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next day, it was embedded in paraffin
blocks. The skin was sectioned longitudi-
nally into 5-μm sections for HE staining or
immunostaining.
Statistical analysis
The data were analyzed by Student’s t-
test with a Student-Newman-Keuls test
(SPSS, 13.0). P<0.05 was considered to be
statistically significant.
Results
Abnormalities in appearance and
morphogenesis of hair follicles in
newborn Trps1 knockout mice
As we reported in a previous paper,
newborn Trps1 KO mice showed a
decreased size of the maxillary region com-
pared with that of wild-type (WT; it refers
to the phenotype of the typical form of a
species as it occurs in nature) mice.11 Hair
follicles in Trps1 KO mice were present at
postnatal day1, but were reduced in number,
irregularly spaced and smaller than those of
WT mice (Figure 1).
Trps1 knockout mice showed dis-
tinct appearance differences at day
42 after transplantation
To investigate whether or not hair folli-
cles develop and give rise to hair, and if
Trps1 KO mice could survive, we trans-
planted newborn mouse skin to nude mice.
Surprisingly, at day 42 after transplantation,
transplanted skin from Trps1 KO newborn
mice produced hairs with the same length
and density as those of WT mice although
they were softer than those of WT mice
(Figure 2).
Comparison between Trps1
knockout and wild-type mice at day
42 after transplantation
Histological examination revealed that
hair follicles and hairs were thinner in the
transplanted skin from Trps1 KO newborn
mice than those from WT mice (Figure 3A).
Diameters of both hair follicles and hair
shafts were significantly smaller in trans-
planted skin from Trps1 KO mice than
those from WT mice (Figure 3B-D).
Discussion and Conclusions
In this study, we investigated whether
hair could develop in a condition of Trps1-
deficient hair follicles. We expected that
few hairs would emerge from transplanted
Article
Figure 2. Appearance comparison of WT and Trps1 KO mice after 42 days of transplan-
tation. (A) -/- shows the KO mouse transplanted skin, +/- shows the WT mouse trans-
planted skin; (B) Appearance of WT (left) and KO (right) mouse skin sample at 42 days
after transplantation by naked-eye observing. Trps1 KO mouse sample showed a thinner
and softer hair.
Figure 1. H&E staining showing wild-type (WT) and Trps1 Knockout (KO) mice display
differences during hair growth. 20× magnification. Histology of newborn dorsal skin got
from WT and KO newborn mice. Note that Trps1 KO mice lack a number of hair follicles
in newborn mice.
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[page 10] [Dermatology Reports 2019; 11:7853]
skin from Trps1 KO newborn mice, because
hair follicles in Trps1 KO newborn mice
showed decreased number and diameter
compared to those of WT mice. At day 42
after transplant, however, transplanted skin
from Trps1 KO newborn mice produced
soft, otherwise comparable amounts of hair
as that of WT mice. Histological examina-
tion revealed smaller diameter of a hair fol-
licle in Trps1 KO mice than that of WT
mice.
In our previous papers, we provided
evidence that Trps1 expression was essen-
tial for normal hair follicle growth using
Trps1 KO mice.11 Trps1 KO mice die short-
ly after birth due to respiratory insufficien-
cy. These developmental abnormalities may
be related to disrupted Wnt signalling.14
How disrupted Wnt signalling caused this
abnormality led us to investigate the func-
tion of Trps1in hair follicle growth. The
extensive phenotypic outcomes in Trps1
KO mice suggested that the structure of
Trps1 protein might be required in the
embryonic development of complex organ
systems. In addition, a recent study of com-
prehensive transcriptome profiling of bald-
ing and non-balding scalps in TRPS I
patients demonstrated that Trps1 indeed
plays a vital role in human hair loss.6
Specifically, we found that Trps1 controls
gene expression and plays a fundamental
role in the interaction between epithelial
and dermal papilla cells during hair follicle
morphogenesis. We sought to identify alter-
ation of the target genes of TRPS1, focusing
on Wnt and androgen/androgen receptor
signalling in HF development.
Wnt signalling is required for crosstalk
between follicular keratinocytes and dermal
papilla cells during hair follicle develop-
ment. Activation of the Wnt signalling path-
way requires accumulation of β-catenin that
translocates into the nucleus, where it acts
as a coactivator of T cell-factor proteins to
regulate gene expression. A number of
recent studies demonstrated that androgen
is essential for hair follicle morphogenesis;
paracrine secretion is involved in this
crosstalk at various hair cycle stages and
some signaling pathways are affected.15,16
These findings demonstrate that the
processes required for hair follicle growth
are complicated. Wnt/β-catenin signalling is
essential for the initiation and maintenance
of hair morphogenesis.17 In promoting der-
mal papilla cells properties to maintain HF
regeneration, Wnt signalling through the β-
catenin pathway played an important role.18
Trps1 may maintain mouse HFs through
inhibition of canonical Wnt signalling and
may operate as a major molecular regulator
of HF regression. Remarkably, Wnt
inhibitors, Wif1, Apcdd1 and Dkk4, were
down-regulated in Trps1 KO mouse skin
according to microarray hybridization
analysis, suggesting that Trps1 may repress
the Wnt signalling pathway to develop nor-
mal hair growth.12 Therefore, we hypothe-
sized that the miniaturization of hair folli-
cles and hair shafts seen in Trps1 KO mice
may be directly caused by inhibition of Wnt
signalling. Nevertheless, to date, we have
failed to detect any significant alteration in
gene expression of Wnt inhibitors such as
WifI, apcdd1 and Dkk4.
On the other hand, androgen (dihy-
drotestosterone: DHT) is believed to have
an important role in transformation of scalp
hair to vellus hair through dermal papilla
morphogenesis. It has been reported that the
dermal papilla from HF of a balding scalp
contains higher levels of DHT than those
from non-balding scalp.19 In addition, it has
been shown that DHT disturbs the balance
of Wnt agonist/antagonist in dermal papilla
cells, down regulating Wnt10b mRNA.20
It is possible that Trps1 may act as a
regulator of hair shaft formation. Trps1
functions in the normal hair cycle as a key
molecule of HF regression by inhibiting
Wnt signalling via androgen expression.
This is in line with a previous report in
which DHT abrogated the ability of DP
cells to induce HF stem cells differentiation
into a hair follicle lineage via inhibition of
Wnt signaling.21
Although we could not show any signif-
icant change in androgen receptor expres-
sion in the transplanted skin from Trps1 KO
mice compared with WT mice skin by
immunofluorescence, it is possible that AR
expression may be up regulated in Trps1
KO skin.
We have reported that Trps1 plays an
important role in the morphogenesis of sec-
ondary but not primary HFs via an interac-
tion with the BMP inhibitor Noggin.
However, there was no significant differ-
ence in the density of hair follicles in the
transplanted skin. To date, although we can-
not explain the discrepancy clearly, it is
possible that the interaction of the trans-
planted skin with normal dermal tissue in
Article
Figure 3. Hair follicles are attenuated in Trps1 KO mice skin compared with those of WT
mice at day 42 after transplantation. (A) H&E staining of P42 WT and KO dorsal skin
after transplant. Compared with WT (left), KO (right) displayed thinner hair shaft and
shrunken hair follicles. 20× magnification. (B) Comparative analysis of diameter of hair
follicles (n=3 per group, error bars represent SD; *P<0.001. **P<0.005) Trps1 KO (right)
mice showed significantly reduced diameter of hair follicles compared with those of WT
mice (P=0.001). (C) Comparative analysis of diameter of hair shafts (n=3 per group, error
bars represent SD; *P<0.001. **P<0.005) Trps1 KO (right) mice showed significantly
reduced diameters of hair shaft compared with those of WT (left) mice (P=0.005). (D)
Comparative analysis of density of hair follicles (n=3 per group, error bars represent SD;
*P<0.001. **P<0.005) Density of hair follicles in WT (left) and Trps1 KO mice were not
significantly different.
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nude mice might have increased the hair
follicle density. Injury to the recipient
mouse skin could cause an upregulation in
Wnt, resulting in signaling that promotes
hair growth in the KO skin. Another possi-
bility would be a mechanism by which
Ambras syndrome is caused. Koa mice, a
mouse model of AS, display hypertrichosis,
which is an opposite phenotype of Trps1
KO mice although Trps1 expression levels
are reduced.22 This molecular mechanism
might explain the discrepancy between
Trps1 KO and the transplanted skin. To fur-
ther study the mechanism of how Trps1 reg-
ulates hair follicle growth after birth, we
ought to investigate follicle growth using
Trps1 conditional KO mice where Trps1 is
specifically deficient in the skin.
In conclusion, soft hairs grew out of the
transplanted skin from Trps1-deficient new-
born mice. These hairs were thin, but were
otherwise comparable with normal hairs,
suggesting that Trps1 is unnecessary for
hair growth itself, perhaps regulating only
the diameter of hair shaft. Further study
should be undertaken to elucidate the pre-
cise function of Trps1 on hair development.
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