?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 119 Number 7 July 2009
Heparan sulfate deficiency leads
to Peters anomaly in mice by disturbing
neural crest TGF-β2 signaling
Keiichiro Iwao,1,2 Masaru Inatani,1 Yoshihiro Matsumoto,3,4 Minako Ogata-Iwao,1
Yuji Takihara,1 Fumitoshi Irie,4 Yu Yamaguchi,4 Satoshi Okinami,2 and Hidenobu Tanihara1
1Department of Ophthalmology and Visual Science, Kumamoto University Graduate School of Medical Sciences, Kumamoto, Japan.
2Department of Ophthalmology, Faculty of Medicine, Saga University, Saga, Japan. 3Department of Orthopaedic Surgery,
Kyushu University School of Medicine, Fukuoka, Japan. 4Burnham Institute for Medical Research, La Jolla, California, USA.
Developmental glaucoma is a congenital blinding disease associ-
ated with elevated intraocular pressure (IOP) because of anoma-
lies of the drainage structure for the aqueous humor in the eye.
The major drainage structure for aqueous humor consists of the
iridocorneal angle, which is the place in which the iris and cor-
nea meet (1, 2). Developmental studies have revealed that the iri-
docorneal angle originates from the periocular mesenchyme and
mainly consists of neural crest cells (3, 4). During ocular morpho-
genesis, these neural crest cells migrate into the eye to create the
anterior chamber, which is a small space within the anterior ocu-
lar segment (3, 4). The migrating neural crest cells cover the ante-
rior chamber and differentiate into the iridocorneal angle, the
corneal endothelium, and the anterior portion of the iris (5–9).
Several of the transcriptional factor genes, such as forkhead
box C1 (Foxc1), paired-like homeodomain transcription factor
2 (Pitx2), and paired box gene 6 (Pax6), have been identified as
the causative genes for developmental glaucoma, and they have
been demonstrated to be responsible for the differentiation of
the neural crest cells (10–18). In addition to the iridocorneal
angle, developmental glaucoma is also highly associated with
anomalies in the neural crest–derived tissues such as the corneal
endothelium defect (Peters anomaly; OMIM #604229), iris col-
oboma (Axenfeld-Rieger syndrome; OMIM #180500, %601499,
and #602482), cleft palate, jaw defect, and ear deformity (15, 17,
19). Thus, failed differentiation of the neural crest cells causes
anterior chamber dysgenesis, which ultimately results in devel-
Both the orderly migration and differentiation of the neu-
ral crest cells are controlled by cues from various cell guidance
factors, morphogens, and extracellular matrices. The TGF-β2
superfamily has been found to be especially associated with
mediating the differentiation of the ocular neural crest cells
(20–22). The genetic disruption of TGF-β2 causes anterior
chamber dysgenesis (20), with TGF-β2 signaling regulating the
expression of the causative genes for developmental glaucoma,
Foxc1 and Pitx2 (22). On the other hand, extracellular matrix
molecules, such as fibronectin, laminin, peanut agglutinin-
binding molecules, and chondroitin sulfate proteoglycans, are
thought to affect the behavior of the neural crest cells (23–27).
However, despite the influence of these molecules in vitro, it has
yet to be determined in vivo whether these extracellular matrices
are critical factors for neural crest cell development. The drain-
age structure has been found to have abundant extracellular
matrices (28). Both the accumulation and changes of the com-
ponent in the human trabecular meshwork are thought to
affect the aqueous outflow resistance. Abnormal expression of
glycosaminoglycans and their glycoproteins, along with proteo-
glycans in the trabecular meshwork, have been found in human
glaucoma (29–31). Heparan sulfate (HS) is a glycosaminogly-
can component of the proteoglycans that is expressed in the
Conflict?of?interest: The authors have declared that no conflict of interest exists.
Nonstandard?abbreviations?used: Ext1flox mice, transgenic mice carrying the loxP-
modified Ext1 allele; Foxc1, forkhead box C1; HS, heparan sulfate; HSPG, HS pro-
teoglycan; IOP, intraocular pressure; Pitx2, paired-like homeodomain transcription
factor 2; Wnt1-CreExt1flox/flox mice, the mutant carrying homozygous floxed alleles of
Ext1 and an allele of Wnt1-Cre.
Citation?for?this?article: J. Clin. Invest. 119:1997–2008 (2009). doi:10.1172/JCI38519.
1998?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 119 Number 7 July 2009
extracellular matrix of the iridocorneal angle and on cell sur-
faces of the trabecular meshwork cells (32, 33). Interestingly,
genetic studies in Drosophila showed that the defect of a HS pro-
teoglycan (HSPG) homolog, dally, disturbs signaling mediated
by the TGF-β homolog, dpp, suggesting that TGF-β–mediated
morphogenesis may be dependent upon HSPGs (34). Moreover,
a genetic interaction between the HSPG homolog, UNC-52, and
the TGF-β homologs is observed during the development of the
Caenorhabditis elegans gonad arm (35). Therefore, we hypothe-
sized that HS/HSPGs may contribute to the development of the
ocular neural crest cells. HS synthesis is governed by a series of
enzymes. Exostosin 1 (EXT1) is indispensable for HS synthesis,
because it has a critical role in the polymerization process of the
alternating d-glucuronic acid and N-acetyl-d-glucosamine that
ultimately provide the HS sugar chain backbone (36–38). Ext1-
disrupted cells exhibit a complete loss of HS (37). In the cur-
rent study, we found that the disruption of Ext1 in the neural
crest cells leads to the Peters anomaly phenotype in the anterior
chamber and, thus, affects the TGF-β2–mediated morphogen-
esis. We also found that gene reduction of Ext1 and Tgfb2 results
in developmental glaucoma with an elevated IOP. This suggests
that the neural crest cells require HS for TGF-β2–dependent iri-
docorneal angle development.
Loss of Ext1 and HS in neural crest–derived tissues of the Wnt1-CreExt1flox/flox
mutant. Ext1 mRNA was broadly expressed throughout the
embryo. Similar to a previous report, intense signals for Ext1
were especially observed at E10.5 in the head region and bran-
chial arches of the embryo (Figure 1A) (39). Subsequently, we
demonstrated that Ext1 was particularly localized in the fore-
brain, hindbrain, facial region, and limb buds at E12.5 (Figure
1C). In order to conditionally disrupt Ext1 in the neural crest
cells during embryogenesis, transgenic mice carrying the loxP-
modified Ext1 allele (Ext1flox mice) were bred to transgenic mice
with Cre recombinase driven by the Wnt1 promoter (Wnt1-Cre
mice). The mutant carrying homozygous floxed alleles of Ext1
and an allele of Wnt1-Cre (Wnt1-CreExt1flox/flox mice) exhibited
substantially decreased signals for Ext1 mRNA in the branchial
arches and the facial region, including the optic vesicle at E10.5
(Figure 1B). In spite of the intense signal of the control (Figure
1, C and E), loss of the signal for the periocular mesenchyme of
the mutant occurred at E12.5 (Figure 1, D and F). As compared
with the control tissue, immunohistochemistry with an anti-
HS antibody revealed there was little staining for the mutant
periocular mesenchyme (Figure 1H). The other ocular tissues
(including the lens, presumptive ciliary body, and neural retina)
in the mutant (Figure 1H) showed similar levels of expression to
those in the control (Figure 1G).
HS deficiency causes a Peters-like anomaly. All of the mutants
died within the first day of life. Appearance was grossly normal
except for severe malformation of the craniofacial tissues such
as cleft palate (Figure 2, A–F) and ear deformity (Figure 2, A
and B). Eyes of mutants exhibited multiple anomalies. All the
eyes of mutants displayed eyelid defects (Figure 2, G–I). Of the
67 mutants at E18.5, 66 (98.5% of the total mutants) had ven-
tral iris coloboma (Figure 2H). The coloboma was consecutively
observed in the ciliary body of 39 (58.2%) mutants (Figure 2I).
The anterior chamber structure was also affected in the mutant,
with an abnormally thin cornea and dysgenesis of the irido-
corneal angle (Figure 3, A and B). While a histological study
showed that the values of the mutant corneal thickness and
anterior chamber depth were significantly smaller than those
of the control (Figure 3, A and C), the size of the lens derived
from the surface ectoderm was not affected (Figure 3C). Van
Gieson staining revealed that collagen was not distributed in
the mutant corneal stroma (Figure 3D). Each of the corneal
endothelial cells expressed ZO-1, a tight junction protein, in
Genetic disruption of Ext1 and reduced expression of HS in developing neural crest–derived tissues. In situ hybridization for mRNA of Ext1
at E10.5 (A and B) and at E12.5 (C–F). The Wnt1-CreExt1flox/flox embryos (B and D) exhibited weaker signals for Ext1 mRNA in the head
region (black arrows), the branchial arches (asterisks), and the optic vesicle (arrowhead) as compared with the control embryos (A and C).
Wnt1-CreExt1flox/flox embryos (F) also showed weaker signals in the periocular mesenchyme (yellow dotted lines) as compared with the control
embryos (E). Immunohistochemistry for HS in ocular tissues at E13.5 (G and H). While control embryos (G) showed broad expression of HS,
the Wnt1-CreExt1flox/flox embryos (H) displayed weak staining for HS in the periocular mesenchymal cells (black dotted lines). cb, ciliary body;
le, lens; nr, neural retina; pom, periocular mesenchyme. Scale bar: 50 μm. Original magnification, ×40 (A and B); ×80 (E and F).
?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 119 Number 7 July 2009
the cell margins (Figure 3E). Immunostaining for ZO-1 revealed
loss of ZO-1–positive cells in the mutant corneal endothelium
(Figure 3E). These phenotypes in the anterior chambers are
very similar to the ocular phenotypes of Peters anomaly, which
is associated with disturbed differentiation of the ocular neu-
ral crest cells. Moreover, both a histological study performed
between E11.5 and E18.5 (Figure 3, A and B) and a fate-map-
ping study for the neural crest cells (Figure 3F) demonstrated
that neural crest cells of the mutant migrated to the periocular
mesenchyme as early as E11.5. However, the subsequent devel-
opmental steps did not progress to the point in which they
formed a distinct anterior chamber.
HS deficiency disturbs neural crest cell proliferation in the anterior
chamber. An abnormally thin cornea and hypoplastic iridocor-
neal angle may be associated with diminished proliferation or
enhanced apoptosis of the neural crest cells. In utero BrdU label-
ing revealed that cell proliferation was decreased in the mutant
cornea and iridocorneal angle at E15.5 (Figure 4). However,
there were no differences in the frequency of the apoptotic cells
observed (data not shown).
To exclude the possibility that decreased numbers of BrdU-posi-
tive cells in the mutant corneal stroma were due to limited number
of cells in the thinner mutant corneal stroma at E15.5, we count-
ed both the number of BrdU-positive cells and the total corneal
cells during the earlier embryonic stage (E13.5). No differences
for the corneal thickness or the distribution of neural crest cells
(β-gal–positive cells traced by Rosa26R mouse strain) were noted
between the mutant and control at E13.5. However, there was a
significant (Figure 4J) reduction in the number of BrdU-positive
cells in the mutant corneal tissue. These results indicated that
reduced mutant neural crest cell proliferation is associated with
poor anterior chamber development.
HS deficiency is associated with the inactivation of TGF-β2 signaling
and downregulation of Foxc1 and Pitx2. The characteristics of the
phenotypes in the anterior chamber were similar to those of the
mutant genes for the TGF-β2 signaling molecules (Figure 5, A
and B) (20, 22). Binding assays demonstrated that TGF-β2 had
an affinity to HS, whereas epidermal growth factor did not bind
to HS (Figure 5C). If there is disturbance of the TGF-β2 signal-
ing in the HS-deficient eye, then there should be activation of
downstream mediators of TGF-β2. It has been previously report-
ed that TGF-β2 expression in the ocular anterior segment peaks
between E13.5 and E15.0 (22). In the current study, while no
major changes were noted for TGF-β2 distribution in the mutant
eye at E13.5 (Figure 5, D and I), there was specific inhibition, in
the periocular mesenchyme of the mutant, of the phosphoryla-
tion of Smad2, which is a downstream molecule of TGF-β2 (Fig-
ure 5, E, F, J, and K). Previous studies have demonstrated that
Foxc1 and Pitx2 encoding transcriptional factors in the neural
crest cells are the causative genes for developmental glaucoma
(12, 13, 15, 17). The expression of these factors is dependent
upon the activation of the TGF-β2 signaling (22). At E15.5, Pitx2
was observed in the cornea (Figure 5H), and there was localized
expression of Foxc1 in the corneal endothelium and primitive
trabecular beam (Figure 5G). In the mutant eye, however, a dra-
matic downregulation of Foxc1 and Pitx2 expression was noted
(Figure 5, L and M). This reduced expression of the downstream
molecules suggests that the HS deficiency is responsible for the
disturbances in the TGF-β2 signaling.
In addition, it has also been shown that Tgfb2-disrupted
mutants exhibit cleft palate (40). In the palatal tissue of these HS-
deficient embryos, a dramatic downregulation of the immunore-
activity of phosphorylated Smad2 was observed (Supplemental
Figure 1; supplemental material available online with this article;
FGFs are known to be HS-binding morphogens, which are
essential factors for HS-mediated lens development (41). Thus,
it is possible that FGF signaling might be involved with anterior
chamber morphogenesis. To further study this, we examined the
expression of phosphorylated ERK1/2, which are downstream
effectors of the FGF-MAPK pathway (Supplemental Figure 1).
However, no differences in the phosphorylated ERK1/2 immu-
noreactivity in the anterior chamber were found between the
mutant and control.
TGF-β2 signaling in neural crest cells requires cell-autonomous expres-
sion of HS. The transfection of the adenovirus that encodes the Cre
recombinase gene showed there was expression of Cre recombi-
nase in the cultured periocular neural crest cells with the Ext1flox/flox
alleles (Figure 6A). Immunocytochemistry with the anti-HS
antibody showed that the transfection of the Cre recombinase
gene caused a loss of HS in the neural crest cells (Figure 6A). In
primary cultures of periocular neural crest cells, TGF-β2–depen-
HS deficiency causes craniofacial malformation. Stereomicroscopy for control embryos (A, C, E, and G) and Wnt1-CreExt1flox/flox embryos (B, D,
F, H, and I) at E18.5. The mutant embryos showed ear deformity (black arrow in B), severe cleft palate (white arrows in B and D), and the lack
of a hard palate (open arrow in F). The eyes of mutants also displayed eyelid defects (black arrowheads) and iris coloboma (white arrowhead in
H). Some colobomas affected both the iris and ciliary body (white arrowhead in I). Original magnification, ×3.2 (G–I).
2000?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 119 Number 7 July 2009
? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 119 Number 7 July 2009
dent BrdU incorporation was observed. However, HS-deficient
neural crest cells showed significantly less TGF-β2–dependent
incorporation of BrdU than HS-positive cells did (Figure 6C). In
addition, without TGF-β2–stimulation, these HS-deficient cells
were found to have the same level of BrdU-positive cells as did
HS-positive cells (Figure 6, B and C). Moreover, immunoblotting
that used the anti-Smad2 antibody revealed that the HS-defi-
cient neural crest cells exhibited a loss of activation of Smad2
after TGF-β2 stimulation (Figure 6D).
The reduction of TGF-β2–dependent BrdU incorporation
in HS-deficient neural crest cells suggested that the HS that
is expressed on the cell surface is critical in order for TGF-β2–
induced cell proliferation to occur. To further confirm this
hypothesis, HS-deficient neural crest cells were cocultured with
HS-positive neural crest cells. The TGF-β2–induced BrdU incor-
poration was then analyzed. As shown in Figure 6, E and F, the
HS deficiency in the cocultured neural crest cells resulted in a
reduced BrdU incorporation after TGF-β2 stimulation. Thus,
these data indicated that TGF-β2 signaling in neural crest cells
requires cell-autonomous expression of HS.
Haploinsufficiency of Ext1 and Tgfb2 causes developmental glaucoma.
If TGF-β2 signaling in the anterior chamber development is
dependent upon HS, then the ocular phenotypes of the Tgfb2
mutants should be enhanced when there is a reduction of 1
allele of Ext1. Compound mutants that had a haploinsufficiency
of Ext1 and Tgfb2 were able to survive and reached adulthood.
The eyes of these mutants exhibited a significantly reduced cell
number in the trabecular beam and a defect of Schlemm canal,
both of which are major components of the aqueous humor
drainage structure in the iridocorneal angle (Figure 7, A, D, and
E). In contrast, there were no major changes in the iridocorneal
angles of the mutants when there was heterozygotic deletion of
Ext1 (Figure 7B) or Tgfb2 (Figure 7C). Moreover, the compound
mutants showed significant IOP elevations (Figure 7F). This was
in contrast to each of the heterozygotes, which had IOP levels
that were the same as the wild type (Figure 7F). These results
indicated that iridocorneal angle development depends on the
interaction between HS and TGF-β2 signaling.
In the present study, genetic disruption of Ext1 in neural crest
cells caused anterior chamber dysgenesis, similar to that which is
observed in Peters anomaly in humans. In addition, the anterior
eye segment exhibited reduced phosphorylation of Smad2 and
downregulated expression of Pitx2 and Foxc1, which are the down-
stream molecules of TGF-β2. Moreover, in vitro BrdU assays indi-
cated that the TGF-β2 signaling in neural crest cells was dependent
on the HS that is expressed by the cell itself (cell autonomous).
When there is a reduced interaction between TGF-β2 and HS, this
can lead to an elevated IOP that is associated with decreased cell
numbers in the trabecular beam and the loss of Schlemm canal.
Overall, the present study demonstrated that in order for morpho-
genesis to occur in the anterior chamber of the eye, neural crest
cells require an interaction between the TGF-β2 and HSPGs that
are expressed cell autonomously. When there is impaired interac-
tion, this leads to developmental glaucoma.
In humans, the major phenotypes found for Peters anomaly
include defects of the corneal endothelium, Descemet mem-
brane, and posterior corneal stroma (42, 43). Patients with
Peters anomaly also have a high association with anomalies in
other craniofacial neural crest–derived tissues such as ear defor-
mities and cleft palates (19, 44). This association strongly sug-
gests that the pathogenesis of Peters anomaly depends upon the
maldevelopment of the craniofacial neural crest cells. In some
pedigrees, the inheritance has been described as being autoso-
mal dominant (11) or autosomal recessive (45, 46), while in oth-
ers it has been stated as being sporadic (47). This suggests that
there are multiple causative genes for Peters anomaly. Although
large subsets of Peters anomaly cases occur without any molecu-
lar characterization, a mutation of PITX2 is highly associated
with Peters anomaly. When there is a loss of PITX2, this leads to
a defect of the corneal endothelium and corneal stroma, which
indicates an essential role for this gene in corneal development
(48). PITX2 promotes collagen synthesis via the activation of
the protocollagen lysyl hydroxylase (14). In the current study,
the HS-deficient mutant manifested ocular phenotypes asso-
ciated with Peters anomaly, presenting both downregulated
expression of Pitx2 and reduced collagen in the corneal stroma.
These data provided support for a role for HS in the regulation
of the expression of Pitx2 in ocular development that leads to
corneal phenotypes. However, in order to regulate the expres-
sion of intracellular Pitx2 in the neural crest cells, extracellular
HS is required for an interaction with a morphogen, which in
this case is the upstream molecule of Pitx2. For anterior cham-
ber development, TGF-β2 is the most critical morphogen (20).
In mammals, there are 3 members of the TGF-β family that are
known to exist: TGF-β1, TGF-β2, and TGF-β3 (49, 50). Since
TGF-β2 predominates in the eye during embryonic develop-
ment, no anomalies are seen in the eyes of either TGF-β1– or
TGF-β3–null mice (20). However, TGF-β2–null embryos exhibit
Peters ocular phenotypes (20) and cleft palate (40), similar to the
phenotype seen for the HS-deficient embryo. Lyon et al. (51) per-
formed affinity chromatography and demonstrated that there
are 2 TGF-βs, TGF-β1 and TGF-β2, that can bind to heparin
and highly sulfated HS. The binding assay in the current study
confirmed this affinity between HS and recombinant TGF-β2.
Moreover, HS-deficient neural crest cells in vitro failed to show
TGF-β2–dependent proliferation without the phosphorylation
of Smad2, which is a downstream mediator specific to TGF-β.
HS deficiency causes Peters-like anomaly. (A and B) Thionin staining
of the eyes of Wnt1-CreExt1flox/flox and control embryos during embry-
onic development. At E15.5 and E18.5, Wnt1-CreExt1flox/flox embryos
exhibited abnormal thinning of the central cornea (arrows) and dys-
genesis of the iridocorneal angle (open arrowheads). While the con-
trol eye shows lid closure (black arrowheads in B), the mutant embryo
lacked eyelids. Boxed regions in B indicate the areas shown at higher
magnification in A. (C) Impaired ocular growth in Wnt1-CreExt1flox/flox
embryos. The central cornea thickness and the anterior chamber
depth in the mutant embryos were significantly smaller, as compared
with the control eyes. However, the lens thickness was not affected in
the eyes of mutants. (D) Van Gieson staining revealed collagen accu-
mulation in the control corneal stroma. In contrast, Wnt1-CreExt1flox/flox
embryos lacked collagen matrix. (E) Immunohistochemistry using
anti–ZO-1 antibody exhibited a defect of the endothelial layer in the
mutant cornea. (F) Fate mapping for neural crest cells. Cre-positive
neural crest cells had already migrated to the periocular region as early
as E11.5, while the neural crest cells remained distributed at E18.5.
Data represent mean ± SEM. *P < 0.05, **P < 0.01, Student’s t test
(n = 6). c, cornea; ICA, iridocorneal angle; KO, Wnt1-CreExt1flox/flox.
Scale bar: 50 μm.
2002? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 119 Number 7 July 2009
When taken together, our results indicated that the loss of HS
disturbs the TGF-β2 signaling pathway in the ocular neural crest
cells and, thus, leads to the Peters phenotypes.
There are 2 modes of action that HS employs to control the
TGF-β2 signaling. First, HS modulates the diffusion and the gra-
dient of TGF-β2 within the local environment. For example, the
Drosophila TGF-β homolog, Dpp, moves along the cell surface via
restricted extracellular diffusion that involves HSPGs (52). When
there is a loss of HSPGs, this disturbs Dpp-dependent wing disc
development. However, in the present study, we found that the
distribution of TGF-β2 was not affected in the mutant periocular
mesenchyme, suggesting that there is a smaller contribution of
the morphogen gradient for TGF-β2–induced morphogenesis of
the anterior chamber. The second mode of action involves pro-
motion of the interaction between TGF-β2 and the receptors by
the HSPGs on the cell surface. For example, another morphogen,
FGF, requires cell-surface HS as a coreceptor for FGF signaling,
which combines to form a 2:2:1 ratio that consists of a FGF/FGF
receptor/HS ternary complex (53). Our present data strongly sug-
gested that HS on the cell surface of neural crest cells modulates
the ligand-receptor interaction for TGF-β2 (Figure 8).
Because several HS-binding morphogens and growth factors
can contribute to the craniofacial development, interactions
between morphogens/growth factors other than TGF-β2 and HS
may also be impaired in HS-deficient ocular neural crest cells.
Previous studies have reported that genetic disruption of Fgf8
and Shh in the neural crest caused facial dysmorphism (54–56).
In addition, the loss of Fgf8 has been shown to induce defects
of the eyelids and outer ears (55). However, these ocular phe-
notypes are not similar to Peters anomaly. A variety of ocular
abnormalities, including the iridocorneal angle hypoplasia and
elevated IOP, have been reported in heterozygous Bmp4 mutants
(21). However, reduced corneal stroma or corneal endothelial
defects were not found in the Bmp4 mutant (21). We also did
not find any significant disturbance of the phosphorylation of
Smad1/5/8 (data not shown) or the phosphorylation of ERK1/2
BrdU-labeled neural crest cells in the embryonic anterior ocular segments. At E13.5, there were no differences found for the distribution and the
number of β-gal–positive neural crest cells between the Wnt1-CreExt1flox/flox embryos (B and I) and control embryos (A and I). There was a sig-
nificantly smaller number of BrdU-labeled cells in the central cornea (arrows) in the stroma of the Wnt1-CreExt1flox/flox embryos (D) as compared
with the control embryos (C). Moreover, only a small number of BrdU-labeled cells were present in the corneal endothelium (arrowheads in E and
F) at E15.5. (G and H). In mutant embryos at E15.5, BrdU-positive cells in the iridocorneal angle (open arrows) were also decreased. Original
magnification, ×20 (A–H). (J) The total number of BrdU-positive cells in the central cornea and iridocorneal angle in the mutant embryos was
significantly smaller, as compared with the control eyes. Data represent mean ± SEM. *P < 0.01, Student’s t test (n = 6).
?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 119 Number 7 July 2009
(Supplemental Figure 1), which are downstream mediators of
BMP4 (57) and FGFs, respectively, in the HS-deficient anterior
ocular segment. Taken together, even though we were not able
to completely exclude the possibility that other HS-binding
morphogens were affected in the HS-deficient anterior cham-
ber, the phenotypical similarities strongly suggested there is a
predominant role for HS in the TGF-β2–dependent development
of ocular neural crest cells.
In the compound mutant, haploinsufficiency of Ext1 and
Tgfb2 causes hypoplasia of the iridocorneal angle, resulting in
IOP elevation. Phenotypes of the compound mutant mice sug-
gest that an impaired TGF-β2–HS interaction leads to develop-
mental glaucoma. Recent reports have indicated that TGF-β2 is
involved in the pathogenesis of primary open-angle glaucoma
(POAG) (58). It has also been reported that there are elevated
concentrations of TGF-β2 in the aqueous humor of eyes with
POAG (59–61) and in developmental glaucoma (61). The tra-
becular tissue in developmental glaucoma is associated with an
abnormal distribution of HS that exhibits an abnormal accu-
mulation (29) or a significant loss (31). These findings may indi-
cate that improper expression of TGF-β2 and HS can promote
the immaturity of the anterior chamber angle in developmental
glaucoma or cause impaired cellular function in the trabecu-
lar meshwork and Schlemm canal of POAG. To determine the
role of HS in human developmental glaucoma, further genomic
analyses designed to examine the HS-synthesizing enzymes in
patients with developmental glaucoma are required.
In conclusion, we have demonstrated that HS is an essential
factor required for proper differentiation and proliferation of
the ocular neural crest cells in the anterior chamber. When there
HS deficiency leads to the inactivation of TGF-β2 signaling and downregulation of the transcription factors Foxc1 and Pitx2. A thin cornea
(black arrows in A) and iridocorneal dysgenesis (black arrowheads in B) were observed in the mutant embryos that lacked the gene for
TGF-β2 (E18.5). (C) Binding assay indicated that HS had an affinity for TGF-β2 but not for EGF. Immunohistochemical staining of TGF-β2
(D and I), Smad2 (E and J), and phosphorylated Smad2 (F and K) in the iridocorneal angle of the E13.5 embryo, and Foxc1 (G and L)
and Pitx2 (H and M) in the anterior eye segment of the E15.5 embryo. A disturbance was noted for the phosphorylation of Smad2 in the
periocular mesenchyme (dotted lines in F and K) in the Wnt1-CreExt1flox/flox embryos. In the eyes of mutants, the expression of Foxc1 was
substantially reduced in the corneal endothelial layer (white arrows) and in the iridocorneal angle (open arrows), while Pitx2 expression was
hardly detected in the corneal stroma or endothelium. en, corneal endothelium; epi, corneal epithelium; str, corneal stroma; *, presumptive
ciliary body. Scale bar: 20 μm.
2004? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 119 Number 7 July 2009
is HS deficiency, this leads to severe anterior chamber dysgen-
esis, which is reminiscent of Peters anomaly in humans. In addi-
tion, the impaired interaction between TGF-β2 and HS causes
developmental glaucoma. These findings suggest that distur-
bances in the synthesis of HS might contribute to the pathology
of developmental glaucoma.
Mice. The mutant mouse strains used in this study have all been reported
previously (6, 22, 39, 62). To produce mutant mice with the Ext1-defi-
cient periocular mesenchymal cells during embryogenesis, Wnt1 pro-
moter-driven Cre-transgenic mice (The Jackson Laboratory) were mated
with mice carrying the Ext1flox allele. Subsequently, in order to obtain
mutants with a Wnt1-CreExt1flox/flox genotype, the Wnt1-CreExt1flox/wild
male mice were crossed with female mice that were homozygous for the
Ext1flox allele. Littermates carrying Ext1flox/flox or Ext1flox/wild were used as
controls. To detect the neural crest cells in the anterior eye segment,
Wnt1-Cre transgenic mice were crossed with Rosa26R mice (a gift from
H. Okita, National Research Institute for Child Health and Develop-
ment, Tokyo, Japan, and P. Soriano, Mount Sinai School of Medicine,
New York, New York, USA) (B6.129-Gt(ROSA)26Sor, The Jackson Labora-
tory), as they express β-gal following Cre-mediated recombination (63).
Tgfb2 transgenic mice (The Jackson Laboratory) were generated via gene
targeting, as has been previously reported (40). Tgfb2 heterozygous mice
were mated with Ext1 heterozygous mice. Genotyping of the mice was
performed by PCR-based methods that used DNA prepared from tail
biopsies. All of the mice strains used in this study were backcrossed with
C57BL/6 more than 10 times. All of the procedures involving the mice
were performed in accordance with the Association for Research in Vision
and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision
Research and the guidelines were approved by the Kumamoto University
Committee on the Use and Care of Animals.
Whole-mount in situ hybridization. To study the expression pattern of Ext1
mRNA in developing embryos, in situ hybridization with a digoxigenin-
conjugated riboprobe for Ext1 was performed, as has been previously
reported (39). Briefly, the riboprobe was hybridized at 55°C overnight, fol-
Disturbed TGF-β2–stimulated proliferation of HS-deficient neural crest cells. (A) Immunostaining with anti-HS and anti-Cre recombinase antibod-
ies indicated that there were Cre-transfected periocular neural crest cells with Ext1flox/flox alleles that expressed Cre recombinase while having
lost HS. (B and C) BrdU proliferation assay. While the periocular neural crest cells showed TGF-β2–dependent BrdU incorporation (orange-
colored cells), the HS-deficient neural crest cells had a low rate of BrdU-positive cells. There was a statistical significance found for the TGF-β2–
dependent proliferation between the HS-positive and HS-deficient cells. (D) Western blot for Smad2 and phosphorylated Smad2 (p-Smad2)
was analyzed in the cultured cells. While TGF-β2 enhanced the phosphorylation of Smad2, there was no phosphorylation of Smad2 in the HS-
deficient neural crest cells, even after TGF-β2 stimulation. (E and F) BrdU proliferation assay in cocultures of HS-positive and HS-deficient cells.
Since the HS, BrdU, and nucleus were labeled by FITC (green), Alexa Fluor 568 (red), and Hoechst 33258 (blue), respectively, the BrdU-posi-
tive cells exhibit a white-colored nucleus (white arrows) in HS-positive cells, while they have a magenta-colored nucleus in HS-deficient cells.
A significant reduction of the BrdU-positive cells was seen in the HS-deficient cells (yellow arrows). (F) The comparison of the percentage of
BrdU-positive cells between HS-positive and HS-negative cells in coculture. Data represent mean ± SEM. *P < 0.01, Student’s t test (n = 9).
Original magnification, ×10 (A, B, and E).
?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 119 Number 7 July 2009
lowed by stringent washes. The embryos were then treated with an alkaline
phosphatase-conjugated antidigoxigenin antibody (Roche). Hybridization
signals were visualized with BM purple AP substrate (Roche).
Histology. Embryos were fixed with Carnoy’s fixative solution (6 parts
EtOH [absolute or 99.5%], 3 parts chloroform, and 1 part glacial acetic
acid) at 4°C for 3 hours, embedded in paraffin, and processed for histo-
logical examination. After preparing 4-μm-thick sections, samples were
stained with thionin and then histologically examined by light microscopy.
Photographs were taken with a DP50 digital camera (Olympus). Samples
were stained with van Gieson solution to determine the accumulation of
collagen in the cornea. To analyze the ocular size, the thickness of the cen-
tral cornea and the axial length of the anterior chamber and lens were mea-
sured in 5 Wnt1-CreExt1flox/flox and 5 control embryos. For analysis of the
iridocorneal angle of the Tgfb2 and Ext1 double-mutant mice, 6-week-old
mice were sacrificed and used to prepare 4-μm-thick paraffin sections. Cell
nuclei in the trabecular beam area were counted within a 200-μm-width
sampling window, located within the iridocorneal angle. A total of 9 mice
were analyzed for each genotype strain.
Immunohistochemistry. Four-micrometer-thick sections fixed with Carnoy’s
solution or frozen 15-μm-thick sections fixed with 4% paraformaldehyde
solution were incubated with primary antibodies. Anti–TGF-β2 (Lab
Vision), anti-Smad2 (Santa Cruz Biotechnology Inc.), anti-phosphorylated
Smad2 (Chemicon), anti-phosphorylated ERK1/2 (Cell Signaling Technol-
ogy), anti-Pitx2 (Santa Cruz Biotechnology Inc.), anti-Foxc1 (Santa Cruz
Biotechnology Inc.), and anti-HS, HepSS1 (Seikagaku Corp.) were used
as the primary antibodies. For the anti-HS analysis, we used biotin-con-
jugated anti-mouse IgM as the secondary antibody, the Vectastain Elite
ABC kit (Vector Laboratories), diaminobenzidine substrate (Vector Labo-
ratories), and the Entellan New mounting solution (Merck). Fluorescence
immunohistochemistry was performed using fluorescein anti-mouse IgM
(Vector Laboratories), Alexa Fluor anti-rabbit IgG, and Alexa Fluor anti-
goat IgG polyclonal (Molecular Probes Inc.) antibodies as the secondary
antibodies. After mounting using ProLong Gold Antifade Reagent (Molec-
ular Probes), the sections were examined using confocal laser microscopy
(FV300 and FV500-IX; Olympus).
Fate mapping of the neural crest cells. To analyze the distribution of the neu-
ral crest cells in the anterior eye segment, we used the Rosa26R mouse strain
B6.129-Gt(ROSA)26Sor (The Jackson Laboratory), as it expresses β-gal fol-
lowing Cre-mediated recombination (63). Female Ext1flox/floxRosa26R mice
were crossed with male Wnt1-CreExt1flox/wild mice to produce mutants with
Wnt1-CreExt1flox/floxRosa26R. The mutant and control mice with Rosa26R
were stained with X-gal (Sigma-Aldrich). Samples were also counterstained
by Contrast RED solution (KPL) for cell counting. We subsequently pre-
pared frozen sections for the analyses.
BrdU labeling of the embryos and TUNEL staining. Timed pregnant female
mice (E13.5 and E15.5) were injected intraperitoneally with 200 μg/g body
weight of BrdU and then sacrificed 1 hour later. Embryos were removed,
fixed in Carnoy’s solution at 4°C for 3 hours, and then embedded in par-
affin. Samples were cut into 4-μm horizontal sections and stained with
anti-BrdU antibody (Chemicon). Samples then underwent sequential
incubation with biotin-conjugated anti-mouse IgG antibody, followed by
examination using a Vectastain Elite ABC kit (Vector Laboratories) and
diaminobenzidine substrate (Vector Laboratories). The numbers of BrdU-
positive cells were counted within two 100-μm-width sampling windows
(per section), located in the center of the corneal stroma and on both side of
the iridocorneal angle. The analyses were conducted in 5 Wnt1-CreExt1flox/flox
and 5 control embryos. TUNEL assays were performed using the Dead-
End Fluorometric TUNEL system (Promega). The 4-μm sections from the
embryos at E15.5 were treated with 0.85% NaCl solution and 20 μg/ml pro-
teinase K solution after deparaffinization. After equilibrating, fluorescein-
Haploinsufficiency of Ext1 and Tgfb2 leads to aberrant iridocorneal development. (A) There were no major anomalies noted in Schlemm canal
(black arrows) or the trabecular beam (white arrowheads) for either the controls (A) or the Ext1 (B) or Tgfb2 (C) haploinsufficient mutants. As
compared with the wild-type mice, the double heterozygote mice (D) had a hypoplastic trabecular beam, and they lacked a Schlemm canal. The
double heterozygote mice also exhibited a significant reduction of the total cell number for the iridocorneal angle (E), in addition to an elevated
IOP (F). Data represent mean ± SEM. *P < 0.01, Student’s t test (n = 9). Scale bar: 50 μm.
2006?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 119 Number 7 July 2009
12–dUTP was then incorporated at the 3′-OH DNA ends through the use
of terminal deoxynucleotidyl transferase recombinant enzyme. Sections
were then analyzed by confocal laser microscopy (Olympus).
Binding assay. To study the affinity of morphogens for HS, we per-
formed a binding assay that used the ELISA method, as has been previ-
ously reported (64). Briefly, TGF-β2 and epidermal growth factor (R&D
Systems) were applied to the polystyrene ELISA tray and incubated for
1 hour at 37°C. After blocking with BSA/PBS, biotinylated HS (Celsus)
was then applied to the wells and incubated for 1 hour at 37°C. The
bound HS was detected by HRP-conjugated streptavidin and the ELISA
kit (BioSource, Invitrogen). The absorbance after the reaction was mea-
sured using a Model 550 plate reader (Bio-Rad).
Cell culture and transfection. Periocular mesenchymal tissues were removed
from Ext1flox/flox embryos at E11.5 by microdissection. The mesenchymal
cells were cultured in DMEM/F12 medium (Gibco, Invitrogen) containing
10% fetal bovine serum (Sigma-Aldrich) at 37°C in 5% CO2. To disrupt
the HS synthesis, an adenovirus, which included the sequence (65) of the
Cre recombinase that was generated from the cosmid, pAxCANCreit2/Pac I
(Nippon Gene) was added to cultured periocular mesenchymal cells. The
virus solution (2.3 × 109 PFU/ml) was diluted accordingly and then added
to the cultured cells for 1 hour at 37°C. For the coculture study, transfected
cells were additionally cultured with HS-positive periocular mesenchymal
cells for an additional day. After virus transfection, the cells were cultured in
DMEM/F12 with 10% fetal bovine serum for 16 hours. To detect the expres-
sions of HS and Cre recombinase, we performed immunohistochemistry
using anti-HS and anti-Cre recombinase antibody.
Protein extraction and Western blot analysis. Following a 16-hour incubation
in DMEM/F12 containing 0.1% BSA at 37°C, cells were treated with 5 ng/ml
of TGF-β2 (R&D Systems) for 90 minutes at 37°C, as has been previously
described (22). Proteins were extracted from cells with RIPA buffer (Thermo
Fisher Scientific) that contained protease and phosphatase inhibitor cock-
tails (Thermo Fisher Scientific). Proteins in the cell extracts were separated by
SDS-PAGE and electrotransferred to nitrocellulose membranes (Whatman)
at 30 V for 60 minutes using the XCell SureLock Mini-Cell (Invitrogen).
The membranes were blocked with 5% normal rabbit serum/Tris-buffered
saline containing 0.1% Tween20 (TBST) and 5% skim milk/TBST for the
anti-Smad2 (Santa Cruz Biotechnology Inc.) and the anti-phosphorylated
Smad2 (Chemicon) antibody reactions, respectively. After the primary anti-
body reaction, target proteins were detected with HRP-conjugated anti-
goat IgG (Jackson ImmunoResearch Laboratories Inc.) and anti-rabbit IgG
(Amersham Biosciences) antibodies. Immunopositive bands were visualized
by chemiluminescence with the ECL Western Blotting Detection Reagents
(Amersham Biosciences) and LAS 4000 Mini UV (Fujifilm).
BrdU proliferation assay in vitro. Following 16 hours of incubation in DMEM/
F12 containing 0.1% BSA at 37°C, cells were incubated in DMEM/F12
containing 0.1% BSA and BrdU (final concentration of 10 μM) with or
without TGF-β2 (5 ng/ml) for 12 hours at 37°C. Cells incorporating BrdU
were identified by staining with anti-BrdU monoclonal antibody (Chemi-
con) and 3,3′-diaminobenzidine. After analysis of each of the 9 samples, a
Student’s t test was used to determine the statistical significance.
IOP measurement. IOP was measured using the TonoLab rebound tonom-
eter for rodents (M.E. Technica), according to the manufacturer’s recom-
mended procedures. After a few minutes of acclimation, IOPs of conscious
mice at P42 were measured between 11:30 AM and 12:30 PM. Statistical
significance was determined by Student’s t test.
Statistics. For statistical comparison of 2 samples, we used a 2-tailed
Student’s t test. P values of less than 0.05 were regarded as statistically
Hypothetical schema of the HS-dependent TGF-β2 signaling on the cell surface of neural crest cells. (A) HS has an affinity for TGF-β2, which
enhances the ligand presentation to the TGF-β receptors. Subsequently, transduction of the signal to the nucleus occurs via phosphorylation of
the Smads. (B) For the HS defect, there is deterioration of the efficiency of the interaction between TGF-β2 and the receptors, which leads to a
disturbance of the Smads phosphorylation. The loss of phosphorylated Smads then inhibits the expression of Foxc1 and Pitx2.
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We thank H. Okita and P. Soriano for their gift of the Rosa26R
mice and Y. Fukuchi, E. Otsubo, and C. Naito for their experimen-
tal assistance. This work was supported in part by Grants-in-Aid
for Scientific Research (KAKENHI) (S) 19679008 from the Minis-
try of Education, Culture, Sports, Science and Technology, Japan
(to M. Inatani) and NIH grants R01 NS49641 and P01 HD25938
(to Y. Yamaguchi).
Received for publication January 9, 2009, and accepted in revised
form April 22, 2009.
Address correspondence to: Masaru Inatani, Department of Oph-
thalmology and Visual Science, Kumamoto University Graduate
School of Medical Sciences, 1-1-1, Honjo, 860-8556 Kumamoto,
Japan. Phone: 81-96-373-5247; Fax: 81-96-373-5249; E-mail:
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