Ablation of the Pax6-Expressing Cells in the Mouse Eye Alters the Corneal Differentiation Program

Abstract

Purpose:: Studies performed in chicks and mice show that the lens is critical for proper differentiation of surrounding ocular tissues. The purpose of this study was to genetically ablate the lens precursor cells in the surface ectoderm in mice and assess the developmental consequences.

Methods:: In order to ablate the lens precursors, we have generated transgenic mice that express an attenuated version of diphtheria toxin (Tox176) linked to a modified Pax6 promoter that is active in the lens and corneal precursors. Ocular development of wild type and transgenic mice was analyzed by standard histological techniques, in situ hybridization and immunohistochemistry.

Results:: Five transgenic families (GOV1-5) that express the Pax6-Tox176 transgene were generated. Adult transgenic mice in families GOV1, GOV4 and GOV5 are microphthalmic and GOV2 and GOV3 families do not show any ocular abnormalities. Similar alterations in ocular morphology are seen during embryonic development in transgenic families GOV1 and GOV5 and post natally in family GOV4. In these mice, lens precursors in the surface ectoderm undergo apoptosis and do not form a placode with concomitant loss of lens induction markers, Sox2 and Prox1. The retina is folded with improper placement of the pigmented epithelium. Unexpectedly, dramatic alterations were seen at birth in the corneal differentiation program. The corneal epithelium displays histological features of the skin including a distinctive granular layer and a stratum corneum. The corneal epithelial cells fail to upregulate keratin 12, a marker of corneal epithelial differentiation. Instead, these cells express markers of skin differentiation such as Keratin 1 and 10.

Conclusions:: Expression of an attenuated version of diphtheria toxin in the pax6-expressing cells of the surface ectoderm results in complete loss of the lens and profound alterations in corneal differentiation. Though corneal abnormalities have been reported in Pax6 null mice, our results provide the first in vivo demonstration that the Pax6-expressing cells in the surface ectoderm are critical for the proper differentiation of the corneal epithelium and loss of these cells will result in the induction of the skin differentiation program. These results support the hypothesis that the skin differentiation program may be the ‘default’ mode of development for the surface ectoderm.

Keywords: cornea: basic science • differentiation • transgenics/knock-outs