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First published online 3 May 2006
doi: 10.1242/dev.02381

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Dkk2 plays an essential role in the corneal fate of the ocular surface epithelium

Mahua Mukhopadhyay¹, Marat Gorivodsky¹, Svetlana Shtrom¹, Alexander Grinberg¹, Christoph Niehrs², Maria I. Morasso^3,* and Heiner Westphal^1,*,

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.

View larger version (46K): [in a new window]   Fig. 1. Gene targeting at the Dkk2 locus and the cornea phenotype of Dkk2–/– mutant mice. (A) Partial restriction maps of the wild-type Dkk2 locus, the targeting vector and the disrupted Dkk2 allele. Exon 1 and part of the intron 1 of the Dkk2 allele were replaced with the neomycin (neo) cassette. The expected size of EcoRI-generated fragments are 7 and 5.2 kb. RI, EcoRI; RV, EcoRV; H3, HindIII; Ap, ApaI. (B) Southern blot analysis: EcoRI-digested genomic DNA isolated from wild-type and three targeted ES cell lines (lanes 1-3) generated 7 kb and 5.2 kb bands, respectively, when detected with a 5' external genomic probe. ApaI-digested genomic DNA generated 7 kb and 5 kb bands for wild-type and mutant alleles, respectively, when detected with a 3' external genomic probe. (C,D) Corneal phenotype. An opaque plaque and hairs (arrow) are visible on the ocular surface of a Dkk2–/– mutant (D), while an age-matched wild-type control eye contains a transparent cornea (C). (E-G) Paraffin sections of eyes stained with Hematoxylin/Eosin, indicating epithelial layer (ep) and the stroma (st). (E) Wild type. Corneal epithelial hyperplasia is evident in the Dkk2–/– eye (F). (G) Higher magnification view of F; skin appendages such as hair follicles (arrow) and sebaceous glands (arrowhead) are indicated. (H) Periodic acid-Schiff base staining of the Dkk2–/– cornea demonstrates presence of goblet cells (arrow). (I) Ectopic pigment-granules are present in the epithelial basal cells of the mutant cornea. The border between the epithelial (ep) and stromal (st) layers of a mutant cornea is indicated (broken line). Scale bars: in C, 1 mm for C,D; in E, 200 µm for E,F; 50 µm in H; 35 µm in I.

View larger version (103K): [in a new window]   Fig. 2. Expression of the Dkk2 protein in adult cornea. (A,B) Paraffin sections from wild-type cornea are stained with anti-Dkk2 antibody. Expression of the Dkk2 protein is localized to the epithelial layers in the cornea (A, arrow) and the limbus (B, arrowhead). The epithelial and stromal border of the cornea and the limbus are indicated (broken lines). Scale bars: 100 µm.

View larger version (83K): [in a new window]   Fig. 3. Expression of epithelial keratinocyte markers in wild-type and Dkk2–/– corneas. Hematoxylin/Eosin staining (A-E) and expression of epithelial markers (F-J,K-O,P-T) in wild-type and mutant corneas from P5 (A,B,F,G,K,L,P,Q), P10 (C,D,H,I,M,N,R,S) and 6-month-old (E,J,O,T) mice. (A-E) Hyperplasia of the corneal epithelial layer (ep) is apparent in the mutant corneas at all stages. The more superficial cells of the mutant cornea form interwoven and partially desquamated scaly layers (arrows). (F-J) K12 expression is present in wild-type corneas (F,H), but not in mutant corneas (G,I,J) at all postnatal stages. Conversely, K1 (K-O) and filaggrin (P-T) expression is induced in mutant corneas (L,N,O,Q,S,T) but not in wild-type corneas (K,M,P,R). Scale bars: 50 µm.

View larger version (67K): [in a new window]   Fig. 4. Molecular mechanism of Dkk2 gene function. (A,B) At P10, Pax6 protein is detected in the nuclei of the epithelial basal cells of the wild-type cornea (A) but is absent or excluded from the nuclei of the mutant cornea (B). (C-G) X-gal staining on paraffin sections taken from wild-type and Dkk2–/– corneas of mice carrying a ß-galactosidase (lacZ) gene under the control of ß-catenin-responsive elements. At P2, significant lacZ expression is detected in the limbus of the mutant cornea (D), but not in the wild-type cornea (C). (E) Higher magnification view of D, showing localization of the X-gal staining in the stroma of the limbus. The limbal epithelial-stromal border is indicated. At P15, lacZ expression is absent in the central region of a wild-type cornea (F), but detected in a few hair follicles located in the central cornea of a mutant eye (G). (H,I) RT-PCR analysis. Lef1 mRNA expression is significantly upregulated in mutant corneas compared with wild-type corneas; Shh mRNA expression is induced in mutant corneas and absent in wild-type corneas. (J-M) Immunostaining and (N,O) X-gal staining on paraffin-embedded sections from E15.5 corneas. K12 expression is present (J) in wild-type cornea but absent (K) in a Dkk2–/– cornea. Pax6 expression is present in a wild-type cornea (L), but absent in a mutant cornea (M). TOPGAL expression is induced in the limbal stroma region of a Dkk2–/– cornea (O), but not in wild-type cornea (N). Scale bars: in A, 50 µm for A,B; in C, 100 µm for C,D; in F, 100 µm for F,G; in J, 50 µm for J-M; in N, 150 µm for N,O.