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First published online 15 April 2009
doi: 10.1242/dev.034082
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1 Department of Ophthalmology and Visual Sciences, Washington University, St
Louis, MO 63130, USA.
2 Department of Cell Biology and Physiology, Washington University, St Louis, MO
63130, USA.
3 Developmental Biology Program, Childrens Hospital Los Angeles, Departments of
Pathology and Surgery, Keck School of Medicine, University of Southern
California, Los Angeles, CA 90027, USA.
4 Molecular Developmental Biology Group, Laboratory of Reproductive and
Developmental Toxicology, National Institute of Environmental Health Sciences,
Research Triangle Park, NC 27709, USA.
5 Genetics of Development and Diseases Branch, National Institute of Diabetes
and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD
20892, USA.
6 Laboratory Molecular Biology (Celgen), Department for Molecular and
Developmental Genetics, VIB, B-3000 Leuven, Belgium.
7 Laboratory Molecular Biology (Celgen), Center for Human Genetics, KU Leuven,
B-3000 Leuven, Belgium.
8 Laboratory of Cell Regulation and Carcinogenesis, National Cancer Institute,
National Institutes of Health, Bethesda, MD 20892, USA.
* Author for correspondence (e-mail: Beebe{at}vision.wustl.edu)
Accepted 9 March 2009
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SUMMARY |
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Key words: Eyelid closure, Conjunctival cell fate, c-Jun nuclear transport, BMP signaling, FGF signaling, Mouse
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INTRODUCTION |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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). At
E11.5, invagination of the dorsal and ventral periocular ectoderm signals the
beginning of the period of eyelid growth. The resulting eyelid folds grow
towards each other across the surface of the eye between E11.5 and E15.5. At
E15.5, a projection of the outer, peridermal layer of the ectoderm extends
from the eyelid margins across the cornea until the periderm extensions meet
and fuse. The two lids separate at `eye opening' on about postnatal day 10
(P10) (Findlater et al.,
1993). The closing eyelids are constituted by a loosely organized mesenchyme and the overlying epithelium. The eyelid epithelium differentiates into the palpebral epidermis (outer surface of the eyelid) and the palpebral conjunctiva (inner surface of the eyelid). The palpebral conjunctiva is continuous with the bulbar conjunctiva (the epithelium covering the anterior periphery of the globe), which is continuous with the corneal epithelium on the most anterior surface of the globe (Fig. 1).
After eyelid closure, the palpebral epidermis differentiates as part of the
skin. Stratification and keratinization begin, and the regularly spaced hair
follicles of the eyelashes form at the margins of the lids. However, the
conjunctival epithelium does not stratify until after the eyelids re-open and
it remains non-keratinized throughout life. The mature conjunctival epithelium
contains abundant goblet cells, which produce mucus that is important for the
properties of the tear film. Soon after birth, the Meibomian glands, which
produce a lipid component of the tears, form by in growth of the conjunctival
epithelial cells near the inner surface of the lid margin
(Findlater et al., 1993).
Defects in eyelid growth or fusion may cause the eyelids to be open at
birth (EOB). A surprising number of genes and signaling pathways are required
for eyelid closure. An EOB phenotype is seen in mice with germline deletion of
activin β-B (Inhbb), MEK kinase1 (Map3k1), c-Jun
N-terminal kinase (Mapk8), c-Jun (Jun), the epidermal growth
factor (EGF) family members HB-EGF (Hbegf) and transforming growth
factor 
(Tgfa), and their receptor (Egfr), fibroblast
growth factor 10 (Fgf10), its receptor (Fgfr2), the forkhead
transcription factors Foxc1 and Foxc2, and the Wnt
antagonist Dkk2 (Gage et al.,
2008; Kidson et al.,
1999; Kume et al.,
1998; Li et al.,
2001; Li et al.,
2003; Luetteke et al.,
1994; Luetteke et al.,
1993; Miettinen et al.,
1995; Mine et al.,
2005; Smith et al.,
2000; Takatori et al.,
2008; Tao et al.,
2005; Vassalli et al.,
1994; Weston et al.,
2004; Zenz et al.,
2003; Zhang et al.,
2003). Previous studies suggested that activin β-B promotes
eyelid closure by activating a Smad-independent cascade involving MEK kinase1,
Jun N-terminal kinase (JNK) and c-Jun
(Takatori et al., 2008;
Weston et al., 2004;
Zhang et al., 2003). EGF
family members contribute to periderm migration by activating the ERK
signaling pathway (Mine et al.,
2005). Upstream of the EGF cascade, c-Jun increases EGF receptor
expression (Li et al., 2003;
Zenz et al., 2003). FGF10
controls eyelid epithelial proliferation and periderm migration by stimulating
the expression of activin β-B and TGF, and by modulating the
expression of sonic hedgehog (Shh)
(Tao et al., 2005). The
administration of a short-acting Shh antagonist at E9 results in EOB
(Lipinski et al., 2008).
Recently, mice lacking the Wnt antagonist Dkk2, showed EOB,
indicating that Wnt activity must be properly tuned during eyelid
development.
It has not been clear whether bone morphogenetic protein (BMP) signaling
plays a role in eyelid closure. An EOB phenotype was detected in one mouse
strain in which Bmpr1a was conditionally deleted in the ectoderm by
using a keratin 14-driven Cre-recombinase transgene
(Andl et al., 2004). However,
mice overexpressing the BMP antagonist noggin under the control of the human
K14 or K5 promoters had eyelid defects, but no EOB phenotype
(Plikus et al., 2004;
Sharov et al., 2003). Mice
overexpressing the inhibitory Smad Smad7, driven by the bovine K5 promoter,
did have an EOB phenotype (He et al.,
2002). However, whether this phenotype was attributable to
blocking TGFβ, activin, or BMP signaling has not been clarified. In
addition, overexpression of BMP signaling antagonists or deficiencies in the
BMP signaling pathway cause other epithelial defects that may indirectly
result in EOB. For example, in some of these cases, the epidermal,
conjunctival and corneal epithelia were hyperplastic, and sweat glands
transdifferentiated into hair follicles
(He et al., 2002;
Plikus et al., 2004).
To clarify the possible function of BMP signaling in eyelid development, we conditionally deleted two type I BMP receptors, two of the BMP-activated R-Smads or the co-Smad Smad4 in the prospective eyelid epithelium beginning on E9. In each case, the mice showed normal eyelid formation and adequate growth, but the eyelid epithelia did not fuse, resulting in an EOB phenotype. Deletion of the sole type II TGFβ receptor or the two activin- and TGFβ-activated R-Smads did not interfere with eyelid closure. Further analysis suggested that Fgf10 from the mesenchyme activates Fgfr2 in the lid ectoderm. Fgfr2 signaling modulates Shh levels, resulting in Bmp4 expression in the mesenchyme. FGF signaling also inhibits Wnt signaling in the eyelid ectoderm, independently of its effects on BMP expression. BMPs are required for the expression of the transcription factors Foxc1 and Foxc2 in the ectoderm, the nuclear translocation of activated c-Jun in periderm cells, the proper timing of conjunctival epithelial differentiation and the establishment of conjunctival epithelial cell fate. In the absence of BMP signaling, ectopic hair follicles formed on the inner edges of the eyelid at the expense of the Meibomian glands, a feature of human lymphedema-distichiasis syndrome.
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MATERIALS AND METHODS
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DISCUSSION
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),
Acvr1 flox (Dudas et al.,
2004), Bmpr1a flox
(Gaussin et al., 2002),
Smad4 flox (Yang et al.,
2002), Smad1 flox
(Huang et al., 2002),
Smad5 flox (Umans et al.,
2003), Smad2 flox
(Piek et al., 2001),
Smad3 germline knockout (Roberts
et al., 2006), Fgfr2 floxed
(Yu et al., 2003),
Tgfbr2 floxed (Chytil et al.,
2002), presenilin 1 floxed (Yu
et al., 2001), presenilin 2 germline knockout
(Steiner et al., 1999) and
TOPGAL, a Wnt reporter strain (DasGupta and
Fuchs, 1999). Matings between mice that were homozygous for the
floxed allele, only one of which was Cre-positive, resulted in litters in
which about half of the offspring were Cre-positive (conditional knockout,
CKO). The others were Cre-negative (wild type). Noon of the day when the
vaginal plug was detected was considered as embryonic day (E) 0.5 of
development. Embryos were collected at the desired stages (n=3 to 5
for each genotype and stage).
Histology
Embryo heads were fixed in 4% paraformaldehyde/PBS overnight at 4°C,
dehydrated through a series of ethanol concentrations, embedded in paraffin
and sectioned at a thickness of 4 µm. For morphological studies, sections
were stained with Hematoxylin and Eosin (Surgipath, Richmond, IL, USA).
In situ hybridization
Frozen sections were fixed in 4% paraformaldehyde/PBS, treated with
proteinase K (10 ug/ml), post-fixed in 4% paraformaldehyde/PBS and acetylated
in triethanolamine-acetic anhydride solution. Samples were pre-hybridized in
50% formamide, 5xSSC, 5 mM EDTA, 1xDenhardt's, 100 ug/ml heparin,
0.3 mg/ml yeast tRNA and 0.1% Tween-20, incubated in the same solution with
riboprobes overnight, washed with 0.2xSSC, blocked in 10% lamb serum and
incubated with anti-digoxigenin antibody overnight. The color reaction was
developed using NBT and BCIP in the dark. After the reaction was completed,
the slides were washed in PBS, fixed in 4% paraformaldehyde/PBS and mounted in
100% glycerol.
Digoxigenin-labeled riboprobes were synthesized from cDNA generated from RNA isolated from wild-type E15.5 eyelids using the following PCR primer pairs:
Probe for patched 1 was a kind gift from Dr David Ornitz (Washington University, St Louis, MO, USA). Gene expression patterns were compared between CKO and wild-type littermates and each in situ hybridization was performed at least twice.
Immunofluorescence staining
Frozen sections were warmed to room temperature and then fixed in 4%
paraformaldehyde/PBS. After three washes in PBS, the samples were treated with
3% H2O2 in methanol to quench endogenous peroxidase
activity, blocked in 5% goat serum/0.1% Triton X-100, incubated in primary
antibody overnight, washed and processed with tyramide amplification. The
antibodies for pSmad1/5/8 and p-c-Jun were from Cell Signaling Technology
(Danvers, MA, USA). The keratin 14 and keratin 10 antibody was from Covance
Research Products (Denver, PA, USA). The keratin 4 antibody was from
Sigma-Aldrich (St Louis, MO, USA). The Dkk2 antibody was from Santa Cruz
Biotechnology (Santa Cruz, CA, USA).
X-gal staining
Staged embryos expressing a lacZ reporter gene were fixed 4% in
paraformaldehyde/PBS at 4°C for 30 minutes, washed twice in PBS with 2 mM
MgCl2, 0.02% NP-40/0.01% deoxycholate (DOC), and stained with X-gal
solution [5 mM K3Fe(CN)6, 5 mM K4Fe(CN)6, 1 M
MgCl2, 0.02% NP-40/0.01% DOC NP-40, 1 mg/ml X-gal in PBS] for 5
hours at 37°C, post-fixed with 4% paraformaldehyde for 1 hour,
cryoprotected and, when required, 10-µm sections were prepared.
BrdU and TUNEL staining and quantification
Pregnant female mice were injected with 50 mg/kg of a mixture of 10 mM BrdU
(Roche, Indianapolis, IN, USA) and 1 mM 5-fluoro-5'-deoxyuridine (Sigma,
St Louis, MO, USA) and sacrificed after 1 hour. A monoclonal anti-BrdU
antibody (diluted 1:250; Dako, Carpinteria, CA, USA) was used with a
Vectastain Elite Mouse IgG ABC kit. Sections were counterstained with
Hematoxylin. Terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine
triphosphate nick end-labeling (TUNEL) was done with an Apoptag kit (Chemicon,
Temecula, CA, USA). The deparaffinized slides were treated with 3%
H2O2 in methanol for 30 minutes, followed by proteinase
K treatment (20 µg/ml) for 15 minutes. Slides were incubated with TdT
enzyme in equilibration buffer for 1 hour at 37°C. The reaction was
terminated with wash buffer provided by the manufacturer for 10 minutes at
room temperature. Anti-digoxigenin-peroxidase conjugate was added for 30
minutes at room temperature, followed by DAB and H2O2
treatment. Slides were counterstained with Hematoxylin.
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MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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).
The ocular surface epithelia targeted by the transgene include the palpebral
epidermis, palpebral conjunctiva, bulbar conjunctiva and corneal epithelium
(Ashery-Padan et al., 2000)
(Fig. 1). Le-Cre mice were
mated to mice with floxed alleles of two of the three type I BMP receptors
(Acvr1 and Bmpr1a), two of the BMP-activated R-Smads
(Smad1 and Smad5), the two activin/TGFβ-activated
R-Smads (Smad2 and Smad3), the sole type II TGFβ
receptor (Tgfbr2) or the co-Smad Smad4. Matings were between
Cre-positive homozygous flox and Cre-negative homozygous flox animals,
assuring that about half of the offspring were Cre-positive and no animals
received two copies of the transgene. In each knockout targeting the BMP pathway (Acvr1/Bmpr1a, Smad1/5 and Smad4), Cre-positive animals had an eyelid-open-at-birth (EOB) phenotype (Fig. 2B-D). Offspring with conditional deletion of one allele of the BMP pathway genes (Acvr1/Bmpr1a, Smad1/5 and Smad4; not shown), both Tgfbr2 alleles (Fig. 2G), or both Smad2 and Smad3 alleles (data not shown) had normal-appearing, closed eyelids at birth and normal-appearing conjunctival epithelium between E15.5 and birth. By examining embryos between E16.5 and birth, we found that eyelids from the Cre-positive embryos with EOB never closed, indicating that the phenotype resulted from the failure of eyelid closure, not from premature eyelid opening (not shown). Because only knockouts in the canonical BMP-Smad pathway had an EOB phenotype, in the remainder of the studies described we show only the phenotype of Smad4CKO mice to represent the function of BMP signaling in eyelid closure. The `control' eyelids shown are all from homozygous flox, Cre-negative littermates.
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BMP signaling activates the expression of transcription factors that are required for eyelid closure
Expression of the forkhead transcription factors Foxc1 and Foxc2 is
required for eyelid closure (Kidson et
al., 1999; Kume et al.,
1998; Smith et al.,
2000). Foxc1 mRNA was present in wild-type upper and
lower eyelid epithelia (Fig.
4A), but undetectable in Smad4CKO eyelids
(Fig. 4C). Similarly, in
wild-type embryos, Foxc2 mRNA was expressed in palpebral conjunctival
epithelial cells (Fig. 4D), but
could not be detected in Smad4CKO palpebral conjunctiva
(Fig. 4F).
BMP signaling is required to promote the translocation of c-Jun into the nuclei of migrating periderm cells
c-Jun and the signaling cascade that leads to its phosphorylation are
required for eyelid closure (Li et al.,
2003; Zenz et al.,
2003). In wild-type mice, a shelf of periderm cells begins to
extend from the margin of the eyelids at E15.0
(Fig. 5A), covering much of the
cornea by E15.5 (Fig. 5B). The
nuclei of these periderm cells were strongly stained by an antibody to
phosphorylated c-Jun (Fig.
5A,B, insets). In the Smad4CKO eyelid
epithelium, the appearance of the lid margin was comparable to that of wild
type at E15.0 (Fig. 5C),
although, by E15.5, fewer migrating periderm cells were present than in
control eyes (Fig. 5D). In the
Smad4CKO embryos, the levels of phosphorylated c-Jun
appeared to be lower than in wild-type periderm cells and p-c-Jun staining was
present in the perinuclear cytoplasm, but not in the nuclei
(Fig. 5C,D, insets). Thus, BMP
signaling is required for the full activation of c-Jun and for its
translocation into the nucleus to exert its function as a transcription
factor.
BMP expression and function is regulated by FGF signaling during eyelid closure
Fgf10 signaling via Fgfr2 is essential for eyelid growth and closure
(Li et al., 2001;
Tao et al., 2005). To
determine whether there is a relationship between FGF and BMP signaling, we
deleted Fgfr2 in the prospective eyelid epithelium using Le-Cre. Mice
deficient in Fgfr2 in the ectoderm showed an EOB phenotype, as
described previously (Garcia et al.,
2005), and deficiencies in BMP expression and function. In
wild-type lower eyelids at E15.5, Bmp4 mRNA was expressed in a
cluster of mesenchymal cells underlying the palpebral conjunctival epithelium
(Fig. 6A, arrows), but
Bmp4 transcripts were undetectable in the lower eyelids of
Fgfr2CKO mice (Fig.
6C). Bmp4 mRNA was expressed in two groups of mesenchymal
cells in the wild-type upper eyelid: in a cluster corresponding to those found
in the lower eyelid (Fig. 6A,
arrows) and in a cluster underlying the palpebral epidermis
(Fig. 6A, arrowheads). In the
Fgfr2CKO upper eyelid, Bmp4 mRNA accumulation in
the mesenchyme underlying the palpebral conjunctiva was not affected, but
Bmp4 transcripts were not detectable in the mesenchyme underlying the
palpebral epidermis (Fig.
6B).
A previous study found that Fgf10 maintains Shh expression in the
eyelid mesenchyme (Tao et al.,
2005). We found that hedgehog function in the mesenchyme, as
measured by the expression of the hedgehog receptor patched 1
(Ptch1), a direct target of Shh signaling, was remarkably similar to
the distribution of Bmp4 transcripts
(Fig. 6D). Moreover, the
pattern of residual Ptch1 expression in the
Fgfr2CKO eyelid was similar to the pattern of residual
Bmp4 expression. Although Ptch1 expression was preserved in
the upper lid ectoderm and mesenchyme, it diminished greatly in the lower lid
ectoderm and mesenchyme of Fgfr2CKO mice
(Fig. 6E). Ptch1
expression was not affected in Smad4CKO mice
(Fig. 6F).
In agreement with the dependence of Bmp4 expression on Fgfr2, nuclear staining for phosphorylated Smad1/5/8, the receptor-activated Smads that transduce BMP signals, was strong in wild-type epithelial cells (Fig. 6G,H), but greatly diminished in the upper and lower eyelids of Fgfr2CKO mice (Fig. 6I,J). As in the Smad4CKO eyelids, Foxc1 and Foxc2 mRNA was not detectable in Fgfr2CKO conjunctival epithelia (Fig. 4B,E). Thus, FGF signaling controls eyelid closure, at least in part, through the activation of BMP signaling.
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). To
further assess the regulation of Wnt pathway signaling in the ocular surface
epithelia, we produced Fgfr2 and Smad4 conditional knockouts
in the ectoderm in a TOPGAL background, in which canonical Wnt signaling
activates a β-galactosidase reporter transgene
(DasGupta and Fuchs, 1999). In
wild-type (Cre-negative) eyelids at E15.5, β-galactosidase staining was
abundant in hair follicles of the epidermis and in the conjunctival epithelium
in a band near the edge of the upper eyelid. Weaker staining was present in a
band along the edge of the lower eyelid
(Fig. 7A,D). In
Fgfr2CKO eyes, TOPGAL reporter activity increased in
intensity and spread over the conjunctival epithelium of the upper and lower
eyelids (Fig. 7B,E). In
Smad4CKO eyelids, β-galactosidase expression was not
increased, but had a different distribution from wild type. Instead of
localizing in a continuous band in the peripheral conjunctival epithelium,
staining was present in an extra row of ectopic hair follicles in the upper
and lower eyelids (Fig. 7C,F).
A double row of eyelashes is called distichiasis. Because distichiasis occurs
in humans and mice haploinsufficient for FOXC2
(Fang et al., 2000;
Kriederman et al., 2003), this
finding is consistent with our observation that BMP signaling was required for
Foxc2 expression.
Although loss of Dkk2 expression causes EOB
(Gage et al., 2008),
Dkk2 mRNA or protein expression was not affected in
Fgfr2CKO or in Smad4CKO eyelid
epithelial cells (Fig. 8A-F).
FGF signaling modulates Shh expression in the eyelid and, in other
tissues, hedgehog signaling induces the expression of the Wnt antagonist,
secreted frizzle-related protein 1 (Sfrp1)
(He et al., 2006;
Katoh and Katoh, 2006). We
examined the levels of Sfrp1 mRNA in wild-type,
Fgfr2CKO and Smad4CKO eyelids. In
wild-type eyelids, Sfrp1 mRNA was expressed in the entire eyelid
epithelium, with strongest expression in the conjunctival epithelia
(Fig. 8G). Consistent with the
effects of FGF and BMP signaling on TOPGAL activity, Sfrp1
transcripts were undetectable in Fgfr2CKO eyelids
(Fig. 8H), but were present at
normal levels in Smad4CKO eyelids
(Fig. 8I). Thus, FGF signaling
suppresses Wnt signaling, at least in part, through the activation of
Sfrp1. However, the control of Sfrp1 expression by Fgfr2 is
independent of BMP signaling.
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BMP signaling suppresses the differentiation of conjunctival epithelial cells prior to eyelid closure and specifies conjunctival epithelial cell fate
The conjunctival epithelium in mice deficient in BMP signaling developed
features that were reminiscent of epidermis, including keratinization and
ectopic hair follicles. We, therefore, examined the levels of the epithelial
cell differentiation marker keratin 14 (K14) at E15.0, before eyelid closure,
and at E15.5, during closure. We also stained for a specific epidermal
differentiation marker, keratin 10 (K10), and a conjunctival epithelial
differentiation marker, keratin 4 (K4), at E17.5, after eyelid closure. In
wild-type mice, the conjunctival epithelium first expressed K14 at about the
time of eyelid closure (Fig.
9A). However, in conjunctival epithelial cells deficient in BMP
signaling, expression of K14 was precocious
(Fig. 9C), with the mutant
conjunctiva differentiating at the same time as the epidermis. In wild-type
E17.5 eyes, K10 is expressed by epidermis, and K4 is expressed by conjunctiva
(Fig. 9D,G). However, in
Smad4CKO conjunctival epithelium, we found ectopic K10
expression, with K4 staining detected only in a few residual cells
(Fig. 9F,I), suggesting that
most mutant conjunctival cells transdifferentiated into epidermal cells.
Although conjunctival differentiation in Fgfr2CKO eyes was
premature, as determined by K14 expression
(Fig. 9B), the
transdifferentiation of conjunctiva to epidermis was not evident, because
Fgfr2CKO conjunctiva did not express K10
(Fig. 9E,H). It seems possible
that residual BMP signaling in the Fgfr2CKO conjunctiva
maintained conjunctival cell fate, but was unable to suppress the premature
differentiation of this tissue.
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SUMMARY
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MATERIALS AND METHODS
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DISCUSSION
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). Our
results are consistent with this interpretation, as deletion of the
activin/TGFβ-stimulated R-Smads Smad2 and Smad3 did not cause an EOB
phenotype. Deletion of the TGFβ type II receptor also did not show an
eyelid phenotype, indicating that the Smad4 phenotype was due to defects in
signaling through the canonical BMP pathway, and not in another of the
TGFβ superfamily pathways that share Smad4 function.
The EOB phenotype in eyelids lacking epithelial BMP receptors and
BMP-activated R-Smads is consistent with the results of Bmpr1a
deletion using a K14-Cre transgene (Andl et
al., 2004) and the overexpression of the inhibitory Smad Smad7
using the bovine K5 promoter (He et al.,
2002). The inability of K14-driven noggin overexpression to cause
EOB may be due to the late expression of K14 in the conjunctiva, just as
eyelid closure is occurring, giving insufficient time for noggin to prevent
the activation of BMP receptors. EOB in mice deficient in BMP signaling was
not due to decreased cell proliferation or increased cell death in the
palpebral epithelia. On the contrary, increased cell proliferation was found
in the conjunctival epithelia, consistent with the hyperplasia observed in the
BMP receptor knockouts or when Smad7 is driven by the K14 promoter
(Andl et al., 2004;
He et al., 2002). Thus, BMPs
normally inhibit the proliferation and differentiation of the conjunctival
epithelium, and specify conjunctival epithelial cell fate.
FGF-regulated BMP signaling is required for the nuclear localization of phosphorylated c-Jun and the transcription of Foxc1 and Foxc2
The phosphorylation and function of c-Jun in the eyelid epithelium depends,
at least in part, on Smad-independent signaling by activin β-B
(Zhang et al., 2003). However,
in Acvr1;Bmpr1aDCKO, Smad1/5DCKO and
Smad4CKO eyelids, c-Jun appeared to be more weakly
phosphorylated than in wild type and failed to translocate into the nuclei of
periderm cells. Previous studies showed that Smads can bind to c-Jun when it
is not associated with DNA, that c-Jun is phosphorylated after treatment of
cells with TGFβ and that Smad-c-Jun complexes promote AP-1-dependent
transcription (Liberati et al.,
1999; Qing et al.,
2000; Verrecchia et al.,
2001; Zhang et al.,
1998). Our data suggest that BMPs, acting through the canonical
R-Smad/Smad4 pathway, cooperate with activin β-B to promote maximal
phosphorylation of c-Jun, and that association with the R-Smad/Smad4 complex
mediates the translocation of phosphorylated c-Jun to the nucleus. To our
knowledge, this is the first report suggesting that activated Smads are
required for the nuclear translocation of c-Jun. Further studies are required
to determine whether this function of BMP signaling is important in other
examples of epithelial fusion, such as closure of the neural tube and ventral
closure of the optic cup, for which proper function of the JNK pathway is
essential (Xia and Karin,
2004).
|
;
Kume et al., 1998;
Smith et al., 2000). Foxc1 and
Foxc2 are also expressed in periocular mesenchyme
(Gage et al., 2005) and limb
bud mesenchyme (Nifuji et al.,
2001), and treatment with Bmp4 and Bmp7 increases the
transcription of Foxc2 in limb bud mesenchyme in organ culture
(Nifuji et al., 2001). The
function of Foxc1 and Foxc2 in eyelid closure is not clear. However, it has
been shown that Foxc1 and Foxc2 control somitogenesis by regulating Notch
signaling (Kume et al., 2001).
Foxc transcription factors also directly stimulate the production of the Notch
ligand Dll4, to activate Hey2 accumulation in vascular endothelial cells
(Hayashi and Kume, 2008). In
experiments undertaken for another study, we found that deletion in the
surface ectoderm of the presenilins Psen1 and Psen2 resulted
in an EOB phenotype (see Fig. S1 in the supplementary material). Because, in
most tissues, presenilin activity is required for Notch activation, Foxc1 and
Foxc2 might promote Notch signaling during eyelid closure. However, the
presenilins are also required for the activity of other transmembrane
proteins. For this reason, the function of Notch signaling in eyelid closure
requires further exploration.
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), and the expression of Ptch1, an Shh receptor
that is a direct target of Shh signaling, was greatly decreased in the lid
ectoderm and the underlying mesenchyme in eyelids lacking Fgfr2 in
the ectoderm.
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) (this study). However, a band of Wnt signaling activity is
normally present in the palpebral conjunctiva near the margins of the upper
and lower eyelids. These cells appear to include the precursors of the
Meibomian glands. BMP signaling is required to maintain this domain of Wnt
signaling and Meibomian gland cell fate
(Plikus et al., 2004) (this
study). Thus, FGF signaling is required to suppress Wnt signaling and promote
BMP signaling, yet BMPs maintain local Wnt signaling in the Meibomian gland
precursor cells. The factors that specify the location and extent of BMP and
Wnt signaling in Meibomian gland formation remain to be studied.
Mice deficient in BMP signaling provide a model for the human disease distichiasis
In mice deficient in BMP signaling, conjunctival epithelial cells in both
eyelids formed an extra row of eyelashes, a characteristic called
distichiasis. This phenotype is similar to that of mice that overexpress
noggin in the ectoderm, in which ectopic eyelashes are formed at the expense
of the Meibomian glands (Plikus et al.,
2004). Human distichiasis syndrome is characterized by the
presence of an aberrant second row of eyelashes in place of the Meibomian
glands (Fox, 1962). As a
consequence, patients have Meibomian gland dysfunction, corneal irritation,
conjunctivitis and photophobia. Most families presenting with distichiasis
have lymphedema in common, or lymphedema-distichiasis (LD) syndrome (OMIM
153400). LD syndrome is an autosomal dominant disease caused by mutations in
FOXC2. Foxc2 heterozygous mice mimic LD syndrome, demonstrating
distichiasis and hyperplasia of lymphatic vessels and lymph nodes
(Kriederman et al., 2003). The
distichiasis seen in our studies is consistent with a dependence of Foxc2
expression on BMP signaling.
Dkk2-null mice also develop distichiasis, with decreased
Foxc2 expression in the eyelids
(Gage et al., 2008).
Dkk2 expression is promoted by the transcription factor Pitx2, which
functions in the neural crest-derived eyelid mesenchyme. Surprisingly, we
detected Dkk2 mRNA and protein in the eyelid epithelia and in the
mesenchyme. This observation suggests that another pathway regulates
Dkk2 expression in the ectoderm. Excessive Wnt signaling, whether
resulting from defects in the Pitx2
Dkk2 pathway
(Gage et al., 2008), or the
Fgf10Fgfr2Sfrp1 pathway, appears to be sufficient to suppress
Foxc2 expression, prevent eyelid closure and cause distichiasis.
Because loss of BMP signaling also leads to distichiasis, it seems possible
that excessive Wnt pathway activity inhibits Bmp4 expression in the
eyelid mesenchyme or the function of the BMP pathway in the conjunctival
epithelium. These possibilities remain to be tested.
BMP signaling is required for normal conjunctival epithelial cell fate
Different temporal and spatial expression of keratin intermediate filaments
is an important aspect of the differentiation and function of many epithelia
(Kurpakus et al., 1994). In
wild-type eyelids, the palpebral epidermis expresses K14 before eyelid
closure, whereas the conjunctival epithelia begin expressing K14 as the
eyelids close. However, in mice deficient in BMP signaling, the conjunctival
epithelium expressed K14 at the same time as the epidermis. In addition,
conjunctival cells expressed K10, which is normally restricted to the
epidermis, and K4 expression was reduced. Together with the
transdifferentiation of the Meibomian gland precursor cells to hair follicles,
these observations suggest that BMP signaling normally prevents conjunctival
cells from adopting the epidermal cell fate
(Fig. 10A).
The cross-talk between FGF and BMP signaling may be mediated by Shh
Previous studies and the results described here reveal complex
epithelial-mesenchymal interactions in eyelid development. Activation of Fgfr2
in the surface ectoderm by Fgf10 from the underlying mesenchyme
(Tao et al., 2005) is required
for the localized expression of Bmp4 in the palpebral mesenchyme. The
signal from the epithelium that promotes Bmp4 expression in the
mesenchyme is likely to be Shh, as Shh is expressed in the eyelid
margin from E13.5 and active Shh signaling (indicated by Ptch1
expression) has an expression pattern similar to that of Bmp4 in the
wild-type eyelid. Moreover, the pattern of residual Shh signaling in the
Fgfr2CKO eyelid is similar to the pattern of residual
Bmp4 expression. These observations, together with the fact that
preventing BMP signaling did not alter Shh function, suggested that Shh
mediates the Fgfr2-dependent cross-talk between epithelium and mesenchyme.
|
The signaling pathways involved in eyelid closure
In addition to the functions of the FGF, BMP, Shh and Wnt pathways examined
in this study, germline deletion showed that activin β-B, TGF,
HB-EGF and the EGF receptor are each required for eyelid closure
(Luetteke et al., 1994;
Luetteke et al., 1993;
Miettinen et al., 1995;
Vassalli et al., 1994). Notch
signaling might also be involved (see Fig. S1 in the supplementary material).
The pathways activated by these morphogens interact in a remarkably complex
web to assure the proper migration and fusion of a small population of
periderm cells (Fig. 10B).
Further studies are needed to fully define the functions and interactions of
these pathways. Such studies will provide a more complete understanding of
eyelid fusion and, perhaps, other morphogenetic events that depend on
epithelial fusion, such as closure of the neural tube, the optic fissure, the
lens vesicle and the palatal shelves.
|
Footnotes |
|---|
Supplementary material available online at http://dev.biologists.org/cgi/content/full/136/10/1741/DC1
The authors are indebted to Drs Zhen Mahoney and Jeff Miner for generously sharing reagents, for many suggestions on the technical aspects of this work and for assistance in editing. Belinda McMahan and Jean Jones prepared the histological sections and Dr Claudia Garcia provided guidance with the Fgfr2 conditional knockouts, which were generously provided by Dr David Ornitz, Washington University, St Louis. The Psen1 and Psen2 floxed and mutant mice were generously provided by Dr J. Shen, Brigham and Women's Hospital, Boston, MA and Dr Raphael Kopan, Washington University, St Louis. Drs Peter Gruss and Ruth Ashery-Padan generated and provided the Le-Cre mice. Research was supported by NIH grant EY04853 (D.C.B.) and NIH Core Grant P30 EY02687 and an unrestricted grant from Research to Prevent Blindness to the Department of Ophthalmology and Visual Sciences. Deposited in PMC for release after 12 months.
|
REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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