REACTIVE OXYGEN SPECIES PARTICIPATE IN THE CONTROL OF MOUSE EMBRYONIC-CELL DEATH

Programmed cell death or apoptosis is an essential process during the morphogenesis of a large number of structures. Evidence obtained over the past few years indicates that, in some cases, the generation of re active oxygen species (ROS) is an important event during the course of apoptosis, Using an in vitro culture system in which digit individual ization of developing limbs normally occurs, we assayed the effect of different antioxidants on the cell death that takes place at interdigi ts, The addition of phenol, dimethyl sulfoxide, or 2',7'-dichlorodihyd rofluorescein diacetate (DCDHF-DA) to murine developing limbs in cultu re prevented digit individualization as well as the typical interdigit al cell death. Two ROS-sensitive dyes, 3-(4,5-dimethylthiazol)-2,5-dip henyl tetrazolium bromide and DCDHF-DA, stained interdigits and the so -called ''necrotic zones,'' implying that they contain cells under oxi dative stress. Very few interdigital cells were doubly stained with th e ROS probes and two cell death indicators (i.e., acridine orange and propidium iodide), suggesting that they detect a different stage durin g the course of apoptosis. Furthermore, we found cells stained for ROS that did not express a specific macrophage marker and in a few cases were seen surrounded by a macrophage. Surprisingly, many regions of th e midgestation mouse embryo that are undergoing cell death correlated with those that have a markedly higher level of ROS. Our data suggest that the generation of oxidative stress is a common requirement for ce ll death that occurs during mouse embryonic development. (C) 1998 Acad emic Press.