Purpose. To determine the mechanism by which lens fiber cells eliminate their nuclei during differentiation. Methods. We used confocal microscopy to follow the process of nuclear loss in embryonic chicken lenses. The state of the chromatin was examined by a modified TUNEL assay and in situ electrophoresis. Results. At the border of the organelle-free zone (OFZ), fiber cell nuclei became irregular in shape, with marginalized chromatin. Holes appeared in the nuclear envelope and underlying laminae and the nuclei collapsed into condensed, spherical structures. Nuclear remnants, containing DNA, histones, lamin B2, and fragments of nuclear membrane were detected deep in the OFZ. We used m situ electrophoresis to demonstrate that fragmented DNA was present only in cells bordering the OFZ. Confocal microscopy of TdT-labeled lens slices confirmed that DNA fragmentation was a late event in differentiation, occurring after the loss of the nuclear membrane. DNA fragments with 3'-OH or 3'-PO4 ends were not observed elsewhere in the lens under normal conditions, although they could be produced by pretreatment with DNase I or micrococcal nuclease, respectively. Dual-labeling with TdT and an antibody against protein disulfide isomerase, an ER-resident protein, revealed a distinct spatial and temporal gap between the disappearance of ER and nuclear membranes and the onset of DNA degradation. Conclusions. Fiber cell chromatin disassembly differs from classical apoptosis, in both the sequence of events and the time course of the process. DNA degradation occurs only after the loss of mitochondria!. ER and nuclear membranes, sueoestinp lhat damace tn intrazellular membranes may be an initiating event in nuclear breakdown.
|Journal||Investigative Ophthalmology and Visual Science|
|State||Published - Dec 1 1997|