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Anterior segment dysgenesis correlation
with epithelial-mesenchymal transition in Smad4 knockout mice
Jing
Li1, Yu Qin1, Fang-Kun Zhao1, Di Wu2,
Xue-Fei He3, Jia Liu1, Jiang-Yue Zhao1, Jin-Song
Zhang1
1Department of Ophthalmology, the Fourth Affiliated
Hospital of China Medical University, Eye Hospital of China Medical University,
Key Lens Research Laboratory of Liaoning Province, Shenyang 110005, Liaoning Province,
China
2The Shenyang Fourth People’s Hospital, Shenyang Eye
Research Institute, Shenyang 110005, Liaoning Province, China
3Department of Ophthalmology, Ningbo No.2 Hospital, Ningbo
315010, Zhejiang Province, China
Correspondence to: Jin-Song Zhang and
Jiang-Yue Zhao. Department of
Ophthalmology, the Fourth Affiliated Hospital of China Medical University,
No.11 Xinhua Road, Heping District, Shenyang 110005, Liaoning Province, China.
zhangjs35015@163.com; zhaojiangyue@hotmail.com.
Received: 2015-11-30
Accepted: 2016-02-19
Abstract
AIM: To explore the molecular mechanisms in lens
development and the pathogenesis of Peters anomaly in Smad4 defective mice.
METHODS: Le-Cre transgenic mouse line was employed to
inactivate Smad4 in the surface ectoderm selectively. Pathological techniques
were used to reveal the morphological changes of the anterior segment in Smad4
defective eye. Immunohistochemical staining was employed to observe the
expression of E-cadherin, N-cadherin and α-SMA in anterior segment of Smad4
defective mice and control mice at embryonic (E) day 16.5. Real-time
quantitative polymerase chain reaction (qPCR) was performed to detect the
expression of Snail, Zeb1, Zeb2 and Twist2 in lens of Smad4 defective mice and
control mice at E16.5. Statistical evaluations were performed using the
unpaired Student’s t-test
(two-tailed) by SPSS 11.0 software.
RESULTS: Conditional deletion of Smad4 on eye surface ectoderm
resulted in corneal dysplasia, iridocorneal angle closure, corneolenticular
adhesions and cataract resembling Peters anomaly. Loss of Smad4 function
inhibited E-cadherin expression in the lens epithelium cells and corneal
epithelium cells in Smad4 defective eye. Expression of N-cadherin was
up-regulated in corneal epithelium and corneal stroma. Both E-cadherin and
N-cadherin were down-regulated at the future trabecular meshwork region in
mutant eye. The qPCR results showed that the expression of Twist2 was increased
significantly in the mutant lens (P<0.01).
CONCLUSION: Smad4 is essential to eye development and likely a
candidate pathogenic gene to Peters anomaly by regulating
epithelial-mesenchymal transition. Twist2 can be regulated by Smad4 and plays
an essential role in lens development.
KEYWORDS: Peters anomaly; anterior segment dysgenesis; Smad4;
N-cadherin; Twist2
DOI:10.18240/ijo.2016.07.02
Citation: Li J, Qin Y, Zhao FK, Wu D, He XF,
Liu J, Zhao JY, Zhang JS. Anterior
segment dysgenesis correlation with epithelial-mesenchymal transition in Smad4
knockout mice. Int J Ophthalmol
2016;9(7):943-947
INTRODUCTION
Peters anomaly is referred to a range of a
congenital abnormality of the anterior segment of the eye, such as corneal
opacity, shallow anterior chamber, corneolenticular adhesions, cataract and so
on[1-2]. Over 50% of Peters anomaly cases
have glaucoma and more than 15% of cases accompanied lenticular malformations[1-2]. Right now there is no effective
treatment for the disease, and visual loss is inevitable. Although surgical
techniques have been constantly improved to cure the disease, the rate of
surgical success still remains low.
Organogenesis of the eye is a complicated
process. The surface ectoderm becomes thickened and invaginates to form the
lens vesicle. The lens vesicle gradually develops into the mature lens, while
the remained surface ectoderm develops into corneal epithelium. The cranial
paraxial mesoderm and mesenchymal cells of neural crest origin migrate into the
space between the lens vesicle and the remained surface ectoderm, and give rise
to corneal stroma, corneal endothelium, ciliary muscle as well as the
trabecular meshwork. It has been proposed that abnormal development of surface
ectoderm and disturbed neural crest cells migration during eye development are
responsible for Peters anomaly, but the precise pathogenesis still remains
unknown[1-2].
Smad4 is a key intracellular effector of
the transforming growth factor β (TGF-β) superfamily of secreted ligands, which
plays an essential role in organogenesis and tissue homeostasis during
developmental process. Previous studies have shown that Smad4 is expressed in
both the lens vesicle and presumptive corneal ectoderm, and conditional
deletion of Smad4 in the eye surface ectoderm leads to severe abnormality in
the anterior segment [3-5]. However,
the precise role of Smad4 in anterior segment development and the underling
mechanism are still unclear. Here we present data that Smad4 in the ocular
surface ectoderm is required for cornea, lens and anterior chamber angle
development. Conditional deletion of Smad4 on eye surface ectoderm resulted in
corneal dysplasia, iridocorneal angle closure, corneolenticular adhesions and
cataract resembling Peters anomaly. Mechanistically, Smad4 in the eye surface
ectoderm affected the epithelial-mesenchymal transition, and regulated the
expression of Twist2.
MATERIALS AND METHODS
Animals All animal experiments followed the guidelines of the
Association for Research in Vision and Ophthalmology Statement for the Use of
Animals in Ophthalmic and Vision Research. Le-Cre transgenic mice [6] and mice carrying floxed Smad4
alleles (Smad4fl/fl)[7] were kindly
gifted from Dr. Yi-Hsin Liu (University of Southern California, Los Angeles,
USA). The Le-Cre; Smad4fl/fl mice were acquired as the mating
picture shown (Figures 1A), and littermate mice carrying Smad4fl/fl
or Smad4fl/+ were used as controls. Polymerase chain reaction (PCR)
was performed to establish the genotype with the primers as previous described
(Figures 1B, 1C) [6-7].
Figure 1 Generation of conditional deleted
of the Smad4 gene in surface ectoderm
A: Mice mating
procedure to acquire Le-Cre; Smad4fl/fl mice; B, C: The detection of Le-Cre (B) and Smad4 (C) allele by PCR. A fragment of 350 bp indicated the existence of
Cre gene. The fragment of 438 bp indicated the Smad4 floxed allele and 385 bp
of wild-type Smad4 gene.
Hematoxylin and Eosin Staining and
Immunohistochemical Staining Pregnant mice were sacrificed to get the
embryos. The embryos were fixed in 4% paraformaldehyde overnight at 4℃, then
dehydrated through graded alcohols and embedded in paraffin. The 4 μm sections
were cut for hematoxylin and eosin (HE) staining and immunohistochemical
staining. Immunohistochemical staining was performed as previously described[8]. Primary antibodies were used as
follows: rabbit polyclonal anti-E-cadherin (Santa Cruz, CA, USA), rabbit
polyclonal anti-N-cadherin (Santa Cruz, CA), rabbit polyclonal anti-α-SMA
(Santa Cruz, CA, USA). Nine sections, obtained from three independent animals
of different litters, were employed for each antibody staining.
Real-time Quantitative Polymerase Chain
Reaction of Lens Tissue Three RNA specimens were extracted from
control and Le-Cre; Smad4fl/fl lens of three independent litters
respectively at embryonic (E) day 16.5 using RNeasy micro kit (Cat#74004,
QIAGEN, GmBH, Germany). Quantitative PCR (qPCR) was carried out using SYBR
Premix Ex TaqTM II (Takara, Dalian, China) and repeated thrice with each
independent specimen. The qPCR procedure was 95℃ for 30s, followed by 40 cycles
at 95℃ for 5s and at 60℃ for 34s. The results were analyzed based on the
equation RQ= 2−ΔΔCT.
The sequences of qPCR primers were listed
as follows: Snail: forward 5’-ATTTGTCCTGGTGACACCTGTTT-3’, reverse 5’-ACTTGGCCCCTAACAAGTGATG-3’;
Zeb1: forward 5’-TCCCTTTCCCCAGTTTTTAATAGGA-3’, reverse
5’-GTTATGGCTGGGCCAACTCT-3’; Zeb2: forward 5’-CATGCCCAACCATGAGTCCT-3’, reverse
5’-TTGCAGAATCTCGCCACTGT-3’; Twist2: forward 5’-CTGCTCAGCTAGCCGTGTTT-3’, reverse
5’-TCCTGGGTGTGGAGCGTTAT-3’.
Statistical Analysis Statistical evaluations between control and mutant
samples were performed using the unpaired Student’s t-test (two-tailed).
RESULTS
Targeted Disruption of Smad4 on Eye Surface
Ectoderm Results in Anterior Segment Dysgenesis Resembling Peters Anomaly We used the Le-Cre transgenic mouse line to inactivate
Smad4 in the surface ectoderm selectively. The mutant mice showed abnormal
development of anterior segment. At E12.5 mass of neural crest cells
migrated into the space between the lens vesicle and the remained surface
ectoderm, leading to thickened corneal stroma and excessive neural crest cells
around lens in Smad4 defective mice (Figure 2A, 2E). At E16.5, the corneal
stromal cells appeared loosely arranged and displayed highly variable shapes
and sizes in Smad4 deficient mice (Figure 2B, 2F). The mutant eye showed congenital
cataract with lens epithelium cells lost its polarity and distributed diffusely
in the lens capsule, and numerous vacuole appeared (Figure 2C, 2G). By E16.5,
the anterior chamber angle had not formed with failed separation between cornea
and iris stroma, and the cornea attached severely to the lens (Figure 2C, 2D,
2G, 2H).
Figure 2 Loss of Smad4 on eye surface
ectoderm leading to anterior segment dysgenesis resembling Peters anomaly A, E: Photographs of the mutant and control eye section at
E12.5 by HE staining. B-D, F-H:
Photographs of the mutant and control eye section at E16.5 by HE staining to
show the developmental changes in cornea (B, F), lens (C, G)
and the anterior chamber angle (D, H)
between the Smad4 defective mice and control mice. Corneolenticular adhesions
appeared (arrow).
Loss of Smad4 Function Down-regulates E-cadherin
Expression and Up-regulates N-cadherin Expression Immunostaining was performed to detect the proteins
related to cell-cell adhesion, namely E-cadherin and N-cadherin. The expression
of E-cadherin was down-regulated in the lens epithelium cells and corneal
epithelium cells in mutant eye, and the expression of N-cadherin was up-regulated
in corneal epithelium cells (Figure 3A, 3B, 3D, 3E, 3G, 3J). The lens
epithelium cells showed lower expression of N-cadherin probably due to massive
loss of lens epithelium cells in the mutant eye (Figure 3H, 3K). In addition,
the expression of N-cadherin was up-regulated in corneal stroma in Smad4
defective eye, while the expression of E-cadherin was absent in corneal stroma
which showed no difference between the two groups (Figure 3A, 3B, 3D, 3E). Both
E-cadherin and N-cadherin expression were down-regulated at the future
trabecular meshwork region in the mutant eye (Figure 3M, 3N, 3P, 3Q).The
expression of α-SMA was also detected which showed no difference between the
two groups (Figure 3C, 3F, 3I, 3L, 3O, 3R).
Figure 3 Detect of E-cadherin, N-cadherin
and α-SMA on Smad4 defective eye and control eye at E16.5 A-F: Detect of E-cadherin, N-cadherin and α-SMA
on Smad4 defective cornea and control cornea. G-L: Detect of E-cadherin, N-cadherin and α-SMA on Smad4 defective
lens and control lens. M-R: Detect
of E-cadherin, N-cadherin and α-SMA on Smad4 defective anterior chamber angle
and control anterior chamber angle. Both E-cadherin and N-cadherin were
down-regulated at the future trabecular meshwork region in mutant eye (arrows).
Loss of Smad4 Function Up-regulates Twist2
Expression We performed real-time qPCR to detect the
expression of E-cadherin repressors, namely Snail, Zeb1, Zeb2 and Twist2, with
lens isolated from control and Smad4 defective mice, respectively. The results
showed that the expression of Twist2 was increased significantly in the mutant
lens, but the expression of Snail, Zeb1 and Zeb2 showed no difference between
the two groups (Figure 4).
Figure 4 Real-time qPCR to detect the
expression of Snail, Zeb1, Zeb2 and Twist2 with lens isolated from control and
Smad4 defective mice at E16.5. n=9, bP<0.01.
DISCUSSION
Peters anomaly is a congenital and
developmental eye disease, which can lead to severe visual impairment. Until
now there is no efficient treatment and the pathogenesis of Peters anomaly
still remains ambiguously. In the present work, we found that conditional
deletion of Smad4 on surface ectoderm led to corneal dysplasia, failed
development of anterior chamber, corneolenticular adhesions and cataract
resembling Peters anomaly, which suggested that Smad4 was essential to eye
development and likely a candidate pathogenic gene to Peters anomaly.
The TGF-β signaling can induce
epithelial-mesenchymal transitions (EMT) in lens epithelial cells. The EMT
plays an important role in the pathogenesis of anterior subcapsular cataract
and posterior capsular opacification, characterized by inducing expression of
numerous extracellular matrix proteins, such as α-SMA[9-13].
On the other hand, EMT also plays a vital role in embryogenesis[14-15]. The transitions process
includes changes in cellular morphology and a loss of cell polarity. Loss of
E-cadherin expression is a hallmark of EMT, and several E-cadherin repressors
have been reported such as Snail, Zeb1, Zeb2, Twist2 and so on[14-18]. In our present work, loss of
Smad4 resulted in congenital cataract, and lens epithelium cells lost its
polarity and distributed diffusely in the lens capsule. The mutant lens showed loss
of E-cadherin expression in lens epithelial cells and up-regulation of Twist2
expression, which indicated that the EMT process might be activated. But the
expression of α-SMA showed no difference in Smad4 defective lens compared to the
control. These results are opposite to the TGF-β-induced EMT process in the anterior
subcapsular cataract and posterior capsular opacification. Therefore, it can be
demonstrated that different mechanisms of EMT may be existed in TGF-β inducing
cataract and lens development, which requires further exploration. Moreover,
immensely up-regulation of Twist2 expression in Smad4 defective mice manifests that
Twist2 plays an essential role in the lens development and pathogenesis of
congenital cataract. It has been reported that Twist2 can directly act on the
promoter of E-cadherin and silence E-cadherin genes[18],
thus the inhibition of E-cadherin expression in Smad4 defective eye is probably
due to increase of Twist2 expression.
Proper development of the eye relies on
coordinated interactions of the neuroepithelium, overlying surface ectoderm,
and neural crest mesenchyme. Lens has been reported crucial in the development
of the cornea and anterior chamber[8]. In our
present work, conditional deletion of Smad4 on surface ectoderm led to corneal
dysplasia and failed development of anterior chamber, further demonstrating
that the lens can influence the development of surrounding tissues. In the
Smad4 defective mice, the expression of N-cadherin was up-regulated in corneal
stroma and expression of both E-cadherin and N-cadherin were down-regulated at
the future trabecular meshwork region. Moreover, TGF-β signaling and ablation
of p120 catenin can induce iridocorneal angle closure and corneal dysplasia in
mice and rat, and affect the expression of cadherins[19-21].
Therefore, it can be deduced that conditional deletion of Smad4 on surface ectoderm
led to corneal dysplasia and failed development of anterior chamber by
interrupting TGF-β signaling pathway and cadherins expression in surrounding
tissues.
In conclusion, our works elaborate the
possible pathogenesis of Peters anomaly in Smad4 defective mice and demonstrate
the role of E-cadherin and N-cadherin in this process. We also propose for the
first time that the Twist2 is a downstream target of Smad4 and plays an
essential role in the lens development. These results may well pave the way to
improve the clinical diagnosis and therapeutic schedule of Peters anomaly.
ACKNOWLEDGEMENTS
The whole
research was performed in the key lens research laboratory of Liaoning
province, China. Jing Li
conceived and carried out experiments, interpreted data and wrote the
manuscript. Yu Qin and Fang-Kun Zhao carried out real-time qPCR. Di Wu and Jia
Liu carried out immunostaining experiment. Xue-Fei He carried out the animal
raising and mating. Jiang-Yue Zhao and Jin-Song Zhang conceived experiments,
obtained research funding and modified the manuscript.
Foundations: Supported by the National Natural Science Foundation
of China (No.81470617; No.81371003); Colleges and Universities Scientific
Research Project of Liaoning Province, China (No.L2014305).
Conflicts of Interest: Li J, None;
Qin Y, None; Zhao FK, None; Wu D, None; He XF, None; Liu
J, None; Zhao JY, None; Zhang JS, None.
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