In situ detection of apoptosis during embryogenesis with annexin V: from whole mount to ultrastructure.

Apoptosis is of paramount importance during embryonic development. This insight stems from early studies which correlated cell death to normal developmental processes and now has been confirmed by linking aberrant 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 essential 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 microscopy and electron microscopy; and (3) to staining for apoptosis associated DNA fragmentation via, e.g., the TUNEL procedure, which marks cells in a relative late phase of apoptosis. New apoptosis markers need to be specific and should preferably detect cells early during this process. In the present study we show that the recently discovered in vitro 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 to visualize apoptotic cells derived from each germ layer in the developing 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.