Abstract
Purpose:
Lens development requires Fgf receptor (Fgfr) activation. Fgfr stimulation activates a signal transduction cascade leading to the phosphorylation of Akt (pAkt) and Erk (pErk). Loss of Fgfr2 in the lens placode reduces both pAkt and pErk and leads to lens degeneration resulting from substantial lens cell apoptosis and impairment of lens fiber cell differentiation. Since the tumor suppressor Pten normally antagonizes Akt phosphorylation, Pten was removed in Fgfr2 deficient lenses in an attempt increase pAkt and to prevent apoptosis associated with Fgfr2 loss. This experiment tested what aspects of the Fgfr2 deficient lens phenotype, if any, could be rescued by simultaneous deletion of Pten.
Methods:
Conditional (loxP-flanked) mutations in Pten (PtenL/L) and Fgfr2 (Fgfr2L/L) were used to remove these genes in the ocular surface ectoderm using Le-Cre transgenic mice. Two different genotypes were analyzed: Le-Cre; PtenL/L Fgfr2L/L and Le-Cre; Fgfr2L/L. Littermates lacking the Le-Cre transgene, and homozygous for the corresponding loxP flanked alleles were used as controls. Tissues were examined by hematoxylin and eosin for morphology, BrdU incorporation for cell cycle, TUNEL for apoptosis, and immunohistochemistry for γ-crystallin and pAkt.
Results:
As shown previously, Le-Cre; Fgfr2L/L lenses exhibit decreased pAkt and increased apoptosis relative to control lenses. However, both Akt phosphorylation and apoptosis demonstrated substantial rescue with the simultaneous deletion of Pten and Fgfr2 (Le-Cre; PtenL/L Fgfr2L/L). At birth, Le-Cre; Fgfr2L/L lens sections exhibited a 63.3% (+/- 13.2) reduction in planar area compared to the Cre negative control lenses. However, deletion of both Pten and Fgfr2 (Le-Cre; PtenL/L Fgfr2L/L) resulted in a much reduced 18.5% decrease increase in lens section planar area relative to control lenses. Despite alleviating reduced pAkt, apoptosis and decreased lens size, Le-Cre; PtenL/L FGFR2L/L lenses displayed an impaired differentiation response marked by marked nuclear retention in the center of the lens, as well as reduced γ-crystallin expression.
Conclusions:
Loss of Pten in Fgfr2 deficient lenses restored pAkt, cell survival and lens size but was unable to restore normal levels of γ-crystallin expression or fiber cell denucleation. These findings support a survival-independent role for Fgfr signaling in lens fiber differentiation.
Keywords: 497 development •
500 differentiation •
449 cell survival