Specific association of c-Jun-like immunoreactivity but not c-Jun p39 withnormal and induced programmed cell death in the chick embryo

We have examined c-Jun protein expression by immunocytochemistry in normal and pathologically induced cell death by focusing primarily on the developi ng neuromuscular system of the chick embryo. Several commercially available antibodies against c-Jun were used in combination with the TUNEL technique or propidium iodide staining for detection of cells undergoing programmed cell death (PCD). Among these, a rabbit polyclonal antibody raised against the amino acids 91-105 mapping to the amino terminal domain of mouse c-Jun p39 (c-Jun/sc45) transiently immunostained the cytoplasm of dying spinal co rd motoneurons at a time coincident with naturally occurring motoneuron dea th. Late apoptotic bodies were devoid of c-Jun/sc45 immunoreactivity. A mon oclonal antibody directed against a region corresponding to the amino acids 26-175 of c-Jun p39 (c-Jun/mAB) did not specifically immunostain dying neu rons, but, rather, showed nuclear immunolabeling in almost all healthy moto neurons. Experimentally induced programmed death of motoneurons by means of early limb bud ablation, axotomy, or in ovo injection of the neurotoxin P- bungarotoxin increased the number of dying cells showing positive c-Jun/sc4 5 immunoreactivity. Immunoelectron microscopy with c-Jun/sc45 antibody show ed that the signal was present in the cytoplasm without a specific associat ion with organelles, and was also present in large lysosome-like dense bodi es inside neuritic profiles. Similar findings were obtained in different ty pes of cells undergoing normal or experimentally induced PCD. These include dorsal root ganglion neurons, Schwann cells, muscle cells, neural tube and neural crest cells during the earliest stages of spinal cord development, and interdigital mesenchymal cells of hindlimbs. In all these cases, cells showed morphological and histochemical characteristics of apoptotic-like PC D. By contrast, motoneurons undergoing necrotic cell death induced by the e xcitotoxin N-methyl-D-aspartate did not show detectable c-Jun/sc45 immunore activity, although they displayed an increase in nuclear c-Jun/mAB immunost aining. In Western blot analysis of spinal cord extracts, c-Jun/sc45 antibo dy weakly detected a 39-kD band, corresponding to c-Jun, and more strongly detected two additional bands of 66 and 45 kD which followed developmental changes coincident with naturally occurring or experimentally stimulated ap optotic motoneuron death. By contrast, c-Jun/mAB only recognized a single p 39 band as expected for c-Jun, and did not display changes associated with neuronal apoptosis. From these data, we conclude that the c-Jun/sc45 antibo dy recognizes apoptosis-related proteins associated with the early stages o f morphological PCD in a variety of neuronal and nonneuronal cells, and tha t c-Jun/sc45 is a reliable marker for a variety of developing cells undergo ing programmed cell death. (C) 1999 John Wiley & Sons, Inc.