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 contai ns, in addition to glial ensheathing cells, a large population of diff erentiated neurons that migrate from the developing olfactory epitheli um, in close association with the olfactory axon fascicles. The purpos e of our study was to verify the hypothesis according to which a proce ss of physiological cell death might be involved in the progressive di sappearance of these migrating neurons that has been reported during l ate embryonic stages in several immunocytochemical studies. To do so, we have investigated the development of the olfactory nerve layer in r at embryos by using light and electron microscopy, with special refere nce to the presence of cell death processes within this structure. We have also applied the histochemical TUNEL method allowing in situ visu alization of cells degenerating by apoptosis. In order to determine if neurons were present among dying cells, a procedure of double-labelin g was performed by combining the DNA-specific bisbenzimide with two ne uronal markers, the protein B-50/GAP-43 and the lectin Ulex europaeus I. Results brought out the precise temporal and spatial patterns of pr ogrammed cell death accompanying the morphogenesis of the olfactory ne rve layer. A cell death process was observed within the olfactory nerv e layer from its onset at embryonic day 13 (E13). While only few pycno tic cells were observed in E13 and E14 embryos, their number increased from E15 to reach a maximum at E16 and then diminished. Few dying cel ls were also observed along the olfactory axon fascicles when they pen etrated the olfactory nerve layer. Degenerating cells appeared strongl y TUNEL-labeled and exhibited morphological features of cell death by apoptosis. Double-labeling experiments revealed that some of the apopt otic cells were neurons. These observations indicate that apoptosis ma y account for the progressive decrease in the number of migrating neur ons present within the embryonic olfactory nerve layer. Otherwise, a z one 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 presen t within this zone were not neurons. Our findings strongly suggest tha t apoptotic cell death of migrating neurons may allow the elimination of non-functional cells whereas that of mesenchymal cells may facilita te outgrowth of the newly formed olfactory axon fascicles by pathway f ormation.