Abstract
Purpose: :
Despite great advances in lens developmental genetics, there is incomplete understanding of the molecular mechanisms leading to denucleation of lens fiber cells. The ocular lens is composed oflens epithelial cells which line the anterior hemisphere of the lens and lens fiber cells which comprise the bulk of lens tissue. Primary fiber cells derive from the posterior cells of the embryonic lens vesicle while secondary fiber cells differentiate from lens epithelial cells displaced posterior to the equator by cell proliferation in the germinative zone. Differentiation of both primary and secondary fiber cells involves elongation, interdigitation with neighboring fiber cells, and the loss of intracellular organelles including the nucleus. Cyclin Dependent Kinase 1Cdk1, formerly known as Cdc2) is a highly conserved protein which is commonly known to be the major kinase activated to stimulate mitotic entry. One of its main roles in mitotic entry is breaking down the nuclear envelop by depolymerizing the nuclear lamina through the phosphorylation of lamin B receptor. We hypothesize that Cdk1 is an important component in lens cell denucleation. Once activated, Cdk1 phosphorylates the nuclear lamin on the nuclear envelope promoting the nuclear envelope breakdown and allows entry of DNAse IIB into the nucleus to degrade DNA.
Methods: :
Mice in which exon 3 of Cdk1 is flanked by LoxP sites were crossed with MLR10 Cre transgenic mice to achieve conditional deletion of Cdk1 in the lens epithelium and lens fiber cells. Mouse lens cells at E15.5, and P0 were tested for proliferation with BrDU incorporation assay. Morphology and nuclear retention were examined using hematoxylin and eosin staining.
Results: :
The Cdk1 knockout showed a 9% reduction in the S-phase fraction at E15.5. There was a 65% reduction in total epithelial cells. At P0, few lens epithelial cells remained in the Cdk1 knockout, yet the 54% of these remaining cells incorporated BrDU. The overall size of the Cdk1 knockout lens was reduced, and the lenses were flatter across the anterior surface and more rounded at theposterior. The fiber cells appear to be less tightly packed. Within this fiber cell mass, cellular debris appears to accumulate, and vacuoles form. There was retention of nuclei in the organelle free zone of P0 lenses, suggesting a role of Cdk1 in the denucleation process.
Conclusions: :
Cdk1 is required for normal lens development. It is essential for the maintenance of the epithelial cell layer through proliferation. Cdk1 may play a role in lens cell denucleation through thephosphorylation of the nuclear lamin, and nuclear envelope breakdown.
Keywords: differentiation • development • proliferation