Fig. 2. Defective embryonic eyelid closure of the Mekk1^+/KDJnk1^-/- mice and impaired migration of the Mekk1^+/KDJnk1^-/- keratinocytes. (A) Photographs and histological analyses (H&E) of the eyes in Mekk1 heterozygous Jnk1-null (M1^+/KDJ1^-/-) and Mekk1 heterozygous Jnk2-null (M1^+/KDJ2^-/-) mice at E19, postnatal day (P)1 and P21. The M1^+/KDJ1^-/- mice have impaired developmental eyelid closure and an EOB phenotype. H&E staining of the eye tissue sections show developmental eyelid (arrowheads) and ocular tissue inflammation (asterisk). (B) H&E staining of the developing eye at E16. The Jnk1-null (J1^-/-), M1^+/KDJ1^+/- and M1^+/KDJ2^-/- fetuses had eyelid epithelium (arrowheads) covered ocular surface, whereas the M1^+/KDJ1^-/- fetus had opened eyelids and fully exposed ocular surface. Scale bar: 200 µm. (C) Immunofluorescence staining for BrdU (red) and keratins (green) of the developing eyes at E16. Similar levels of keratin 6 (K6) and keratin 10 (K10) expression and BrdU incorporation were detected in the developing eyelid epithelium of M1^+/KDJ1^-/- and M1^+/KDJ2^-/- fetuses. (D) An in vitro wound healing assay was performed on mouse primary epidermal keratinocytes isolated from wild-type, M1^+/KDJ1^-/- and M1^+/KDJ2^-/- mice, in the presence or absence of 10% fetal bovine serum (serum), individual growth factors, TGF (T) and activin B (Act B), or the JNK inhibitor (SP), as indicated. Photographs were taken immediately and 48 hours after wounding. The remaining wound areas at 48 hours were compared with the original wound area. The results represent the mean ± s.d. of at least four independent experiments. (E) Immunofluorescence staining showed PAI1 expression (green) was abundant in developing eyelid epithelium of the M1^+/KDJ2^-/-, but was almost undetectable in that of the M1^+/KDJ1^+/- fetuses. Nuclei were stained with DAPI (blue). Arrowheads indicate the developing eyelid tip. Scale bars: 50 µm.