Abstract
Abstract: :
Purpose: As lens cells differentiate they lose their nuclei and organelles through an apoptotic–like process, yet these cells survive. We examined apoptosis and survival factors that signal organelle loss and fiber cell survival, respectively, during lens cell differentiation. Methods: Lenses removed from embryonic day 10 chicken embryos were microdissected into four developmentally distinct regions; the central epithelium (EC), the equatorial epithelium (EQ), the cortical fiber zone (FP), and the core nuclear fiber region (FC). Expression and phosphorylation of Bcl–2 family members were determined by immunoblot analysis. Protease activation was examined using immunoblot analysis and commercially available activity assays. Cytochrome c release from mitochondria was determined by immunoblot analysis of membrane and cytosolic fractions. Results: Organelle loss during lens cell differentiation is likely controlled by a balance between proteins that regulate apoptosis and survival. Pro–apoptotic Bcl–2 family members control the release of cytochrome c into the cytosol to facilitate the initiation of the caspase cascade. Although an organelle free zone is not apparent until embryonic day 12 of chicken development we found that the molecular factors that control the process of organelle loss are active at least 2 days earlier. In the E10 lens, increased expression of the pro–apoptotic Bcl–2 family members occurred in the EQ region and persisted into the FP zone. Similarly, cytochrome c was released from the mitochondria into the cytosol in both the EQ and FP zones. The release of cytochrome c from mitochondria activates the caspase cascade culminating in the activation of "executioner caspases", such as caspase 3. In the E10 lens caspase 3–like activity was most pronounced in the FP region, coincident with the release of cytochrome c into the cytosol. The survival of lens cells during induction of apoptotic signaling cascades to rid the cells of organelles is likely to be mediated by the expression of pro–survival proteins. Anti–apoptotic Bcl–2 family members were highly expressed in the EQ and the FP zones. This was accompanied by phosphorylation of Bad, a survival signal, and the expression of inhibitor of apoptosis proteins (IAPs). Conclusions: We have shown that the apoptotic–related molecular machinery that signals organelle loss during lens cell differentiation was activated 2 days prior to the appearance of an organelle free zone. Anti–apoptotic factors were expressed concurrently, likely signaling the survival of the lens fiber cells.
Keywords: apoptosis/cell death • signal transduction • phosphorylation