Requirement of the Rho GTPase Cdc42 during Optic Cup Morphogenesis in Mouse

Abstract

Purpose: Defects during eye morphogenesis result in congenital ocular malformations such as anophthalmia, microphthalmia and coloboma (MAC) that are prevalent in ~1 in 3-4,000 individuals and are the cause for over 25% of childhood blindness worldwide. The etiology of MAC in humans is complex and can result from disruption of multiple factors. However, more effort is required to define the molecular and cellular events underlying eye morphogenesis. During normal eye development, morphogenesis of the optic cup is a critical step that involves invagination of the distal optic vesicle and the overlying surface ectoderm. The ventral optic cup and stalk invaginate resulting in formation of the optic fissure. During these processes, neuroepithelial cells undergo significant changes in cell shape that require dynamic regulation of actin cytoskeleton, apicobasal polarity, and cell adhesion. The Rho GTPase Cdc42 is a key regulator of cell junction assembly and actin cytoskeleton; it is required for apical localization of adherens junction proteins and the PAR complex, filopodia formation, and cell fate decisions. We hypothesize that Cdc42 is critical during invagination of the optic cup and closure of the optic fissure.

Methods: For temporally controlled, tissue-specific inactivation, we used the tamoxifen-inducible Hes1^CreERT2. CreERT2 is inserted into the Hes1 locus and shows efficient activity in the optic vesicle and optic cup. We activated Hes1^CreERT2 at different stages to determine the effect of Cdc42 disruption on ocular development.

Results: Activation of Hes1^CreERT2 at E10.5 and analysis at E12.5 revealed that Cdc42 disruption caused a fully penetrant defect in optic fissure closure (coloboma), confirmed by persistent laminin expression. Cdc42 disruption induced 2 days earlier, at E8.5, resulted in microphthalmia and a severe reduction of the ventral optic cup. Apical enrichment of F-actin in optic cup progenitor cells of mutants can be in locally decreased suggesting defects in apicobasal organization of the tissue. Further experiments are underway to determine the precise temporal requirement of Cdc42, as well as the effect of Cdc42 inactivation on morphology, tissue regionalization, and cell polarity during optic cup morphogenesis.

Conclusions: These observations strongly suggest that Cdc42 is required for eye morphogenesis, particularly during closure of the optic fissure and formation of the ventral optic cup.