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.