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First published online 3 May 2006
doi: 10.1242/dev.02381
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1 Laboratory of Mammalian Genes and Development, National Institute of Child
Health and Human Development, Bethesda, MD 20892, USA.
2 German Cancer Research Center, Heidelberg, Germany.
3 Developmental Skin Biology Unit, National Institute of Arthritis and
Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD
20892, USA.
Author for correspondence (e-mail:
hw{at}mail.nih.gov)
Accepted 28 March 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Mouse, Dkk2, Cornea, Epidermis, Differentiation, Wnt/ß-catenin
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Cell
proliferation is commonly regulated by Wnt signaling, and Wnt knockout
phenotypes can often be explained by a loss of cell proliferation. For
example, tissue outgrowth is impaired in limb buds lacking Wnt5a
(Yamaguchi et al., 1999), and
expansion of the CNS fails in Wnt1 mutants
(Megason and McMahon, 2002).
Interestingly, the cell cycle regulators Myc and cyclin D1 are direct Wnt
signaling targets in colon cancer cells (reviewed by
Morin, 1999). Wnt signaling
also affects cell adhesion, where formation of an epithelial bud during hair
follicle development requires the repression of E-cadherin transcription.
Inputs from both Wnt, which stabilizes ß-catenin, and from the BMP
inhibitor Noggin, which induces Lef1 expression, directly repress the
E-cadherin promoter (Jamora et al.,
2003). Proper regulation of Wnt signaling is not only essential
for embryogenesis but also for postnatal development and tissue homeostasis.
This is illustrated by mutations in the Wnt co-receptor gene Lrp5
that lead to familial osteoporosis, high bone density syndromes and ocular
disorders (reviewed by He et al.,
2004). Wnt signaling also regulates stem-cell self-renewal and
differentiation in a range of epithelia, and the inappropriate activation of
these pathways contributes to epithelial cancers (reviewed by
Watt, 2004). Thus,
investigating Wnt signaling pathways is crucial for understanding development
and disease.
A variety of secreted Wnt inhibitors have been identified that regulate Wnt
signaling by binding either to Wnts or to their receptors. Inhibitors that
directly bind Wnts, such as sFRP (Rattner
et al., 1997), WIF (Hsieh et
al., 1999) and Cerberus
(Piccolo et al., 1999) are
structurally similar to the extracellular domains of Frizzled (Fz) family of
Wnt receptors. Cerberus is a multifunctional inhibitor of Bmp, Nodal and Wnt
signals (Piccolo et al.,
1999). These Wnt inhibitors have been implicated in repression of
the canonical Wnt/ß-catenin signaling pathway.
The Dickkopf (Dkk) family of secreted cysteine rich proteins
represent another class of Wnt inhibitors that regulate Wnt pathways by
interacting with the Wnt co-receptor LRP5/6 and with the Kremen class of
transmembrane proteins (He et al.,
2004; Mao and Niehrs,
2003). In Xenopus and mammals, Dkks are differentially
expressed in various neural, mesenchymal tissues
(Hashimoto et al., 2000;
Monaghan et al., 1999) and in
hindbrain-derived neural crest populations, suggesting that these ligand
proteins are involved in diverse inductive processes. Dkk1 is a Wnt
antagonist whose roles in head induction and limb development are well
established (Glinka et al.,
1998; Mukhopadhyay et al.,
2001). To investigate the function of the mammalian Dkk2
gene in development we have generated Dkk2 knockout mice through
homologous recombination. Our study uncovers an essential role for the Dkk2
gene in ocular surface epithelial fate determination.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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),
the targeting vector was constructed by replacing the entire first exon of
Dkk2 with a PGK-NEO cassette, preserving 2.9 kb (5') and 2.5 kb
(3') of flanking homologous sequences and using the thymidine kinase
gene for double selection. The targeting vector was linearized at a unique
NotI site before transfection of ES cells. Transfected ES cells were
subjected to double selection. Targeted disruption of the Dkk2 gene
via homologous recombination was confirmed by Southern blot analysis. Two
independently derived recombinant ES cell lines were injected into blastocysts
to generate chimeric mice. Chimeric males were mated with C57BL/6 females to
produce Dkk2+/– animals, which were intercrossed to
produce offspring for analysis.
Genotyping
The genotyping was performed by polymerase chain reaction using the
following primers.
Dkk2 wild-type allele primer set: left primer, 5'-GGT CTC CTG GGT GAC CAA ACC TCT CCT AA-3'; right primer, 5'-GAG GCC TTG GCT AGC CTT TCA AGT CAG-3'.
Mutant allele primer set: left primer, 5'-GGT CTC CTG GGT GAC CAA ACC TCT CCT AA-3'; right primer, 5'-GTA GAA TTG ACC TGC AGG GGC CCT CGA-3'.
Histology and immunohistochemistry
Embryos were fixed in 4% paraformaldehyde, dehydrated, embedded in paraffin
and sectioned at 5 µm. Sections were either stained with Hematoxylin and
Eosin (H&E) or Toluidine Blue or periodic acid-Schiff base for
histological analyses. Immunohistochemistry was performed on paraffin sections
collected from E15, E17, P2, P5 and P10 eyes. Paraffin was removed by a series
of xylene and ethanol rinses. We quenched endogenous peroxidase activity by
incubating the slides in 2.5% hydrogen peroxide in methanol for 30 minutes.
Antigen retrieval was carried out by boiling slides in a 5 mM sodium citrate
buffer (pH 6.0). We incubated the sections with primary antibodies diluted
with PBS containing 2% normal serum. The antibodies used are as follows:
anti-K12 (Santa Cruz Biotechnology), anti-K1, anti-filaggrin and anti-Pax6
(Covance). After primary antibody incubation, sections were washed in PBS,
incubated with biotinylated secondary antibodies (Vector Laboratories) and
processed with a Vectastain ABC kit (Vector Laboratories). The sections were
developed by using AEC as chromogen (Zymed), counterstained with Hematoxylin,
and mounted with Aqua PolyMount (Polysciences). For alkaline
phosphatase-conjugated secondary antibodies, BM-purple blue was used as
chromogen (Roche). For immunofluorescence, P2, P10 and P15 eyes were embedded
in OCT compound (Tissue Tek) and 10 µm frozen sections were cut. Sections
were stained with commercially available anti-Dkk2 antibody (Santa Cruz
Biotechnology) and rhodamine-conjugated secondary antibodies (Molecular
Probes).
ß-Galactosidase expression analysis
ß-Galactosidase expression was visualized by X-gal staining of whole
eyes using a standard protocol (Hogan et
al., 1994). Stained eyes were embedded in paraffin, sectioned and
photographed.
RNA isolation and RT-PCR analysis
Total RNA was isolated from dissected corneas using the `S.N.A.P. total RNA
isolation kit' from Invitrogen (Carlsbad, CA). RNA quantification was
performed using RiboGreen RNA Quantification reagent and a kit from Molecular
Probes (Eugene, OR). For RT-PCR we used the `Cloned AMV first-strand synthesis
kit' from Invitrogen. The sequences of Shh primers used for RT-PCR
analysis are as follows: left primer, 5'-CCA TTA GCC TAC AAG CAG TTT ATT
CCC-3' and right primer, 5'-TTG GAT TCA TAG TAG ACC CAG TCG
AAA-3'. Lef 1 primers are: left primer, 5'-ACT CTG GCT
ACA TAA TGA TGC CCA ATA-3' and right primer, 5'-AGG TGT TAC AAT
AGC TGG ATG AGG GAT-3'. A ß-actin control primer-set from
Invitrogen was used for quantification.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
The outer surface of the eye is covered by the cornea, which consists of an
external stratified non-keratinizing epithelial cell layer, a neural
crest-derived collagenous stroma and a single cell layer endothelium
(Fig. 1E). By contrast, the
outer ocular surface of adult Dkk2–/– mice
appears opaque and we noticed hair growth. Histological analysis revealed the
presence of sebaceous glands as well as conjunctiva-specific goblet cells
(Fig. 1F-H). The goblet cells
localized preferentially to the periphery of the cornea, suggesting invasion
of conjunctival tissues as seen in several corneal diseases
(Puangsricharern and Tseng,
1995) and in Pax6+/– heterozygote mice
(Ramaesh et al., 2003). We
also observed presence of pigment granules in cells of the basal epithelial
layer of the mutant cornea (Fig.
1I), reminiscent of similar granules in epidermal keratinocytes.
These results suggest that the ocular surface epithelium of
Dkk2–/– eyes contain cell types normally found
in skin.
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).
Dkk2 expression in the cornea is maintained throughout development
(Ang et al., 2004).
Stratification of the corneal epithelia takes place during the first 2 weeks
after birth, and it is in corneal cells and in the limbus that Dkk2
is prominently expressed (Fig.
2). Its expression is localized to the epithelial layers of the
cornea and the limbus. The limbal epithelium at the border between cornea and
sclera is considered the site of the progenitors that are required for the
maintenance of the corneal epithelium
(Cotsarelis et al., 1989;
Pellegrini et al., 1999).
Thus, the expression pattern of Dkk2 is consistent with the development of the
observed phenotype in the null mice.
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;
Wolosin et al., 2004). In the
keratinizing epidermis, the intermediate filament-associated protein filaggrin
(FIL) is a marker for terminal differentiation and a precursor of the
cornified envelope (Candi et al.,
2005). Keratin and FIL immunostaining
(Fig. 3F-T), along with
Hematoxylin/Eosin staining (Fig.
3A-E) has generated useful information concerning the lineage
origin of the epithelial cells. Fig.
3 shows that the adult Dkk2–/–
cornea stains for K1 (Fig.
3L,N,O) and FIL (Fig.
3Q,S,T) but lacks the corneal epithelial marker K12
(Fig. 3G,I,J). Thus, the adult
mutant ocular surface expresses proteins characteristic of epidermis. As it
also contains hair follicles and sebaceous glands, i.e. typical epidermal
appendages, we conclude that the Dkk2 mutant cornea was transformed
into a stratified epidermis.
Molecular mechanism of Dkk2 gene function
Epithelial cells of the cornea derive from ectoderm that expresses
Pax6, a homeobox gene of profound importance for eye development and
maintenance of the cornea (Ramaesh et al.,
2003; Davis et al.,
2003). In the cornea of mice heterozygous for a Pax6 null
mutation, there is decreased expression of keratin K12 and, in addition,
conjunctival invasion (Ramaesh et al.,
2003; Davis et al.,
2003). We found that Pax6 gene expression is absent in
the ocular surface of the Dkk2 mutant
(Fig. 4B), whereas the nuclei
of corneal epithelial basal cells of wild-type corneas show Pax6
immunostaining (Fig. 4A).
Therefore, the observed cornea-to-skin fate change in the Dkk2-null
mutant eye occurs in the absence of Pax6 expression.
Wnt and sonic hedgehog (Shh) signaling pathways play key roles in the
complex series of events that determine the ectodermal appendages that appear
in the epidermis (Millar,
2003; St-Jacques et al.,
1998; Huelsken et al.,
2001). We examined ß-catenin activity in the
Dkk2-null mutant eye using the TOPGAL transgene in which
lacZ expression is controlled by a ß-catenin-responsive promoter
sequence (DasGupta and Fuchs,
1999). Upregulation of the Wnt/ß-catenin pathway was noticed
first shortly after birth (P2) in the form of ß-gal staining in the
limbal mesenchyme (stroma) (Fig.
4D,E). At later stages of development, we observed occasional
lacZ signals in hair follicles located in regions corresponding to
the stromal layer of the central cornea
(Fig. 4G). No ß-gal
staining was detected in the wild-type corneas
(Fig. 4C,F).
Additional evidence for a Wnt inhibitory role of Dkk2 in the mammalian eye
came from results of our RT-PCR analysis that shows upregulation of the Wnt
downstream gene Lef1 in the cornea of Dkk2 mutant mice
(Fig. 4H). Wnt upregulation in
the Dkk2 null mutant cornea also coincides with enhanced Shh
expression (Fig. 4I). Shh plays
a major role at discrete stages of hair follicle morphogenesis, and Wnt and
Shh pathways appear to interact during follicle development
(St-Jacques et al., 1998). The
appearance of hair follicles in the mutant cornea is consistent with these
findings.
We next examined corneas from embryonic stages to determine the temporal
requirement for Dkk2 gene function in cornea development. Expression
of the cornea-specific keratinocyte marker K12 is first induced in the
developing cornea at embryonic day 15.5. We observed a punctate staining
pattern of the K12 gene in wild-type corneal epithelium at this stage
(Fig. 4J), while mutant corneas
were devoid of any K12 staining (Fig.
4K). In the developing cornea, Pax6 acts as the upstream regulator
of cyokeratin-K12 gene expression (Ramaesh
et al., 2003). In the mutant corneal epithelium Pax6 expression
was absent at E15.5 (compare Fig. 4L with
4M). Moreover, its expression was already affected at E14.5 (data
not shown), a day before K12 expression is normally induced in wild-type
corneas. The absence of Pax6 expression in mutant corneas may well be a
consequence of increased Wnt signaling. In support of this notion, we observed
an induction of TOPGAL reporter expression in the limbal stroma region of the
mutant cornea but not in the wild-type cornea at E15.5 (compare
Fig. 4N with 4O). These data
suggest that proper development of the corneal epithelium requires suppression
of Wnt pathways in the limbus rather than in the central cornea. As the Dkk2
protein is expressed in the outer epithelial layer of the cornea, it stands to
reason that this Wnt regulator acts non-autonomously by inhibiting signaling
in the adjacent stroma.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Furthermore, our experiments indicate that suppression of cornea-specific gene
expression is preceded by downregulation of Pax6 expression in the developing
corneal epithelium. This agrees with recent findings that transdifferentiation
of corneal epithelium into epidermis involves downregulation of Pax6
expression in basal corneal epithelia
(Pearton et al., 2005).
The development of epidermal appendages, such as hair follicles, requires
reciprocal interactions between the surface epithelium and the mesenchyme
(reviewed by Millar, 2003).
Wnt signaling has been implicated in the initiation of hair follicle
development. In the mouse, ectopic expression of Dkk1 in the skin
completely blocks development of skin appendages
(Andl et al., 2002). Using an
activated form of ß-catenin, Silva-Vargas et al. showed that adult
epidermis can be reprogrammed to form interfollicular epidermis, sebaceous
glands or new hair follicles, depending on the strength of the ß-catenin
signaling (Silva-Vargas et al.,
2005). In the present study, we show that the activation of
ß-catenin in the mutant limbal mesenchyme, owing to lack of Dkk2
expression, is concomitant with the formation of a stratified epidermis and
derived ectodermal appendages. All together, these data suggest that Dkk2
expression in the corneal/limbal epithelium is required to modulate Wnt
activity in the limbal stroma region, and that this is a required step in the
pathway that leads to corneal epithelium formation and prevents the
development of a stratified epidermis and skin appendages.
At present, we know little about the nature of cells that are the initial
targets of Dkk2-mediated epithelial transformation. It is very likely, that
they reside in the limbus because it is here, in the transitional zone between
the transparent cornea and the white conjunctiva that we observe strong Wnt
signaling in the mutant. Some of the steps in the profound change of
differentiation programs that bring about the epidermal components of the
Dkk2-null mutant ocular surface are suggested by two recent reports.
Nicolas et al. (Nicolas et al.,
2003) report that conditional ablation of the Notch1 gene
function leads to epidermal hyperplasia and extensive hyperplasia and
keratinization of the corneal epithelial cells through activation of Wnt
pathways, though hair follicles were not observed. The second study by Pearton
et al. (Pearton et al., 2005),
described transdifferentiation of corneal epithelium into epidermis in a
setting where central rabbit cornea is abutting embryonic mouse dermis. The
authors show data that are compatible with a model whereby the change is
brought about by a stepwise process involving a dedifferentiation of basal
epithelial cells in the cornea, followed by induction of hair follicles and
subsequent formation of interfollicular epidermis. In this process, Wnts are
thought to be responsible for the downregulation of Pax6 and to interact with
Noggin to induce hair. Although limbus tissue was excluded from this in vitro
experiment, we may nonetheless hypothesize that a similar series of events is
triggered by Wnt signals emanating from the limbus of the newborn
Dkk2-null mutant eye.
Our findings provide direct genetic evidence that Dkk2 controls the integrity of the cornea. In its absence, an increase of Wnt signaling in the limbus triggers epidermal and suppresses corneal pathways of differentiation.
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ACKNOWLEDGMENTS |
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Footnotes |
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