Abstract
Purpose: :
Evidence from mouse models implicated that FGF-signaling is essential for lens fiber differentiation and survival. However, the downstream signaling pathways of FGF receptor (FGFR) in the lens are still unclear. Previous studies using transgenic mice expressing a dominant-negative form of FGFR1 (tR1) in the lens have revealed defective fiber differentiation and cell death. The purpose of this work is to investigate the cell death mechanisms in tR1 transgenic mouse lenses.
Methods: :
To determine whether the classical mitochondrial apoptosis mechanism is involved in cell death in the tR1 lens, transgenic mice were crossed to either the Apaf1+/- or to the apoptosis-defective CytC-KA mutant mice. Animals were genotyped by PCR. The eyes were collected at E14.5, E16.5 and at birth. Lens histology (H&E staining), TUNEL assay, and Caspase-3 activity pattern were compared between the tR1 and tR1-Apaf1-/- or tR1-CytCKA/KA lenses.
Results: :
Histology analysis of the tR1 lens showed that differentiation of the fiber cells was perturbed with the presence of degenerating and dark pyknotic nuclei. The lens phenotype was neither delayed nor rescued when tR1 was expressed in the Apaf1-/- or CytCKA/KA background. High number of TUNEL-positive cells was detected in the fiber compartment of the tR1 lens, extending from the central to the cortical region. Cell death still occurred in the fiber cells of the tR1-Apaf1-/- and tR1-CytCKA/KA lens, however, the TUNEL-positive fiber cells were mostly localized in the central zone, suggesting that apoptosis in these cells is likely independent of the mitochondrial death pathway. A small number of TUNEL-positive cells were also found in the epithelial layer of the tR1 lens at E16.5, but not in the epithelium of the age-matched tR1-Apaf1-/- or tR1-CytCKA/KA lens, suggesting that cell death in the lens epithelial cells requires activation of the mitochondrial death pathway. Immunohistochemical staining showed that Caspase-3 activity was elevated in a few epithelial cells, but not in the degenerative fiber cells, in the tR1 lens. Such immunoreactivity was abolished in the epithelial layer of the tR1-Apaf1-/- and tR1-CytCKA/KA lenses.
Conclusions: :
Our results imply that different apoptosis machinery may operate in the tR1 transgenic lens. Cell death in the epithelial cells of the tR1 lens depends on the activation of the classical mitochondrial pathway, whereas cell death in the tR1 fiber cells is controlled by a mechanism which is independent of the CytC/Apaf1/Caspase-3 pathway.
Keywords: apoptosis/cell death • transgenics/knock-outs