Ultrastructural and cytochemical identification of apoptotic cell death accompanying development of the fetal rat olfactory nerve layer

It has been previously shown that the embryonic olfactory nerve contains, in addition to glial ensheathing cells, a large population of differentiated neurons that migrate from the developing olfactory epithelium, in close association with the olfactory axon fascicles. The purpose of our study was to verify the hypothesis according to which a process of physiological cell death might be involved in the progressive disappearance of these migrating neurons that has been reported during late embryonic stages in several immunocytochemical studies. To do so, we have investigated the development of the olfactory nerve layer in rat embryos by using light and electron microscopy, with special reference to the presence of cell death processes within this structure. We have also applied the histochemical TUNEL method allowing in situ visualization of cells degenerating by apoptosis. In order to determine if neurons were present among dying cells, a procedure of double-labeling was performed by combining the DNA-specific bisbenzimide with two neuronal markers, the protein B-50/GAP-43 and the lectin Ulex europaeus I. Results brought out the precise temporal and spatial patterns of programmed cell death accompanying the morphogenesis of the olfactory nerve layer. A cell death process was observed within the olfactory nerve layer from its onset at embryonic day 13 (E13). While only few pycnotic cells were observed in E13 and E14 embryos, their number increased from E15 to reach a maximum at E16 and then diminished. Few dying cells were also observed along the olfactory axon fascicles when they penetrated the olfactory nerve layer. Degenerating cells appeared strongly TUNEL-labeled and exhibited morphological features of cell death by apoptosis. Double-labeling experiments revealed that some of the apoptotic cells were neurons. These observations indicate that apoptosis may account for the progressive decrease in the number of migrating neurons present within the embryonic olfactory nerve layer. Otherwise, a zone of massive cell death by apoptosis was observed at E14 within the nasal mesenchyme located ventrally and caudally to the olfactory nerve layer. Double-labeling experiments showed that apoptotic cells present within this zone were not neurons. Our findings strongly suggest that apoptotic cell death of migrating neurons may allow the elimination of non-functional cells whereas that of mesenchymal cells may facilitate outgrowth of the newly formed olfactory axon fascicles by pathway formation.