The Expression of Pax6 in Lens and Corneal Epithelium Is Required for Development of Trabecular Meshwork and Schlemm’s Canal

Abstract

Purpose: : In previous work we have shown that development of trabecular meshwork (TM) and Schlemm’s canal (SC) is affected in heterozygous Pax6-deficient mice. Here, we employed Cre/loxP-mediated inactivation of a single Pax6 allele in either lens/cornea or distal optic cup to further dissect the sensitivity of TM and SC to Pax6 haploinsufficiency and to identify those tissues, in which Pax6 needs to be expressed to control the morphogenetic processes of chamber angle development.

Methods: : Pax6^flox/+;Le-Cre and Pax6^flox/+;alpha-Cre mutant mice were obtained by crossing either Le-Cre or alpha-Cre animals with Pax6^flox/flox mice. TM, SC, and optic nerve (ON) head of the animals were investigated by light and electron microscopy. Intraocular pressure (IOP) was measured by invasive cannulation and correlated with non-invasive measurements (TonoLab rebound tonometer). The total number of axons was counted on semithin, cross sections of the ON.

Results: : In Pax6^flox/+;Le-Cre mice with conditional inactivation of Pax6 in lens and corneal epithelium, the chamber angle was filled with an undifferentiated mass of mesenchymal cells, and SC and TM were completely absent. IOP measured invasively at 12 weeks of age was at 14.21 ± 0.61 mmHg (mean ± SEM) in wild-type animals and significantly increased in Pax6^flox/+;Le-Cre mice (21.00 ± 1.57, p < 0.002). At 3 months of age, ON heads of Pax6^flox/+;Le-Cre mice showed a substantial excavation which was not seen in wild-type littermates. The total number of axons in the ON was 10,533.80 ± 910.77 (mean ± SD) in 3 month old wild-type animals and significantly lower in Pax6^flox/+;Le-Cre littermates (5,650.25 ± 1,540.72, p < 0.001). In 6 month old animals, the number of ON axons was 10,653.25 ± 1,953.38 in wild-type animals and 6,353 ± 594.30 in Pax6^flox/+;Le-Cre littermates (p < 0.001). The different number of ON axons might not entirely result from glaucomatous damage, as the eyes of Pax6^flox/+;Le-Cre mice are smaller than that of wild-type littermates. In Pax6^flox/+;alpha-Cre animals with inactivation of Pax6 in retina, and ciliary body/iris epithelia development of TM and SC was essentially normal.

Conclusions: : Signaling factors that are under control of Pax6 and are secreted from lens and/or corneal epithelium during development are required for formation of TM and SC. Lack of chamber angle differentiation is the likely cause for juvenile glaucoma associated with anirida.

Keywords: genetics • transcription factors • transgenics/knock-outs