A role for Spred in lens development and growth.

Abstract

Purpose: The maintenance of the defined architecture and growth of the ocular lens is dependent on epithelial cell proliferation and subsequent fiber differentiation. These cellular processes are known to require MAPK/ERK1/2 signaling that in turn is regulated by inhibitory molecules, including members of the Spred family of proteins. As Spreds are expressed in the lens throughout its morphogenesis and growth, we hypothesized that these proteins play a role in regulating lens cell proliferation and fiber differentiation.

Methods: We examined lens development in murine embryos deficient for both Spred1 and Spred2 in the lens (Spred1/2^null) at embryonic days E12.5-E16.5. Embryos were processed for histology and examined with PAS staining, immunolabeling and in situ hybridization. The lens ocular phenotype was characterized with particular attention to changes in lens cell proliferation and fiber differentiation. Lens fibre cell length and epithelial cell numbers were calculated and Mann-Whitney tests were used for statistical analysis.

Results: Spred1/2^null embryos exhibited microphthalmia and severe lens epithelial defects when compared to their Spred1/2^het counterparts, or wildtype tissue. Spred1/2 deficiencies were shown to result in a small lens phenotype, with compromised primary fibre cell length, significantly at E12.5 (p=0.020) and E14.5 (p=0.031), but no significant difference at E16.5 (p=0.56). Furthermore, an increase in lens epithelial cell number in Spred1/2^null embryos was observed at E12.5 (p=0.027), E14.5 (p=0.016) and E16.5 (p=0.029), with this increase resulting in abnormal multilayering of the lens epithelium. Interestingly, BrdU assays revealed no significant correlation between increased epithelial cell number and an increase in rate of proliferative activity in Spred1/2^null embryos. Immunolabeling detected higher levels of phosphorylated ERK1/2 in Spred1/2^null embryos, in areas of lens cell proliferation and differentiation, when compared to control tissues.

Conclusions: Spred proteins appear to play an important role in tightly managing lens cell behavior at earlier stages of lens morphogenesis, and may be required for the continuous fetal growth phase of the lens. This study provides a greater understanding of the molecules that help drive lens development and growth, potentially developing strategies to preserve and regenerate normal lens cell biology as an alternative therapeutic treatment.