Activation of apoptosis and caspase-3 in zebrafish early gastrulae

Nonmammalian vertebrate embryos do not manifest apoptosis before gastrulation, and it has been suggested that their cells are inhibited from undergoing apoptosis. To study this interesting possibility, the zebrafish (Danio rerio) embryo is an excellent model. However, the appearance of apoptosis varies among species, and many components of cell death are not highly conserved. To undertake the larger investigation, we first need to document by several criteria that cell death in the zebrafish embryo is apoptotic. Exposure of gastrulating germ-ring stage embryos to cycloheximide or staurosporine elicits an arrest in development and cell death within 8 hr. Caspase-3 activity increases, followed by translocation of phosphatidylserine, loss of cell-cell adhesion, cleavage of poly (ADP-ribose) polymerase, terminal deoxynucleotidyl transferase-mediated dNTP-fluorescein nick end labeling (TUNEL)-positive nuclei, internucleosomal DNA fragmentation, chromatin condensation and margination, and blebbing of the nuclear membrane. Thus, by many criteria, cell death in zebrafish is apoptotic; many of the markers of apoptosis found in mammals are conserved in zebrafish; and post-midblastula transition embryos have the capacity to activate a caspase-dependent apoptotic response well before naturally occurring programmed cell death is seen.