IN-SITU DETECTION OF APOPTOSIS DURING EMBRYOGENESIS WITH ANNEXIN-V - FROM WHOLE-MOUNT TO ULTRASTRUCTURE

Apoptosis is of paramount importance during embryonic development, Thi s insight stems from early studies which correlated cell death to norm al developmental processes and now has been confirmed by linking aberr ant cell death patterns to aberrant development, Linking apoptosis to the phenotype of a developing organism requires spatial information on the localization of the dying cells, making in situ detection essenti al, This prerequisite limits the tools available for such studies (1) to vital dyes, which can be detected at the whole mount level only; (2 ) to detection based upon apoptotic morphology by routine light micros copy and electron microscopy; and (3) to staining for apoptosis associ ated DNA fragmentation via, e.g., the TUNEL procedure, which marks cel ls in a relative late phase of apoptosis, New apoptosis markers need t o be specific and should preferebly detect cells early during this pro cess. In the present study we show that the recently discovered in vit ro marker of apoptosis, Annexin V meets these requirements for in vivo detection, Through intracardiac injections of biotin labeled Annexin V, a Ca2+ dependent phosphatidylserine binding protein, we were able t o visualize apoptotic cells derived from each germ layer in the develo ping mouse embryo from the whole mount level up to the ultrastructural level, Double-labeling on paraffin sections for both this method and TUNEL revealed that cells become Annexin V-biotin labeled early during the process of apoptosis. (C) 1997 Wiley-Liss, Inc.