CDK1 is required for both lens epithelial cell proliferation and fiber cell denucleation

Abstract

Purpose: Since the ocular lens retains all its cells throughout life, and the cells undergo a well-documented differentiation program, the lens serves as an excellent model for studying the regulation of both cell proliferation and terminal differentiation. CDK1 plays a central role in cell proliferation by promoting the transition from the G2 to M phase of the cell cycle. We previously hypothesized that disassembly of the fiber cell nucleus requires CDK1 activity. However, a direct role for CDK1 in the promotion of terminal differentiation remains to be established. We used Cdk1 deficient lenses to test the hypothesis that Cdk1 activity is required for lens fiber cell denucleation.

Methods: Mice with exon 3 of Cdk1 flanked by LoxP sites (Cdk1^L/L) were bred to mice carrying either of two transgenes leading to the expression of Cre recombinase in the entire lens (MLR10) or exclusively in lens fiber cells (MLR39). Cdk1 deficient and control lenses were analyzed by BrdU incorporation, p57Kip2, CDK1 and TOMM20 expression, and TUNEL. Assays detecting the phosphorylation of CDK1 targets, lamin A/C and histone H1, assessed the level of CDK1activity in control and Cdk1 deficient lenses.

Results: The MLR10-Cdk1^L/L mice were microphthalmic while MLR39-Cdk1^L/L and Cre negative-Cdk1^L/L mice displayed eyes of normal size with clear lenses at birth. Immunohistochemistry and western blot determined that CDK1 protein remained in MLR39-Cdk1^L/L fiber cells whereas CDK1was largely depleted from the entire lens of MLR10-Cdk1^L/L eyes by E17.5. Cdk1 deficiency resulted in progressive loss of lens epithelial cells and the loss of both histone H1 and lamin A/C phosphorylation in lens fiber cell nuclei. Lens epithelial cell loss in the Cdk1 deficient lenses does not appear to involve increased apoptosis, but was associated with a dramatic reduction in proliferating cells. TUNEL positive fiber cells were dramatically reduced in Cdk1 deficient lenses suggesting an inhibition of denucleation. Both Cre negative and MLR10-Cdk1^L/L fiber cells underwent maturation-appropriate loss of mitochondria, but MLR10-Cdk1^L/Llens fibers contained nuclei through the center of the lens.

Conclusions: Loss of Cdk1 in the lens interfered with fiber cell denucleation in the presumptive organelle free zone without affecting the loss of mitochondria. Cdk1 deficiency also led to the apoptosis - independent depletion of the lens epithelial cell layer.

Keywords: 740 transgenics/knock-outs • 500 differentiation • 654 proliferation