Date of Award

2004

Document Type

Dissertation

Degree Name

Philosophy (Ph.D)

Department

Biological Sciences

First Advisor

Richard Lockshin

Second Advisor

Timothy Carter

Third Advisor

Pokay Ma

Abstract

The zebrafish early embryo acquires a general capability for apoptosis as a consequence of passage through the maternal-zygotic transition, the time that marks the beginning of zygotic transcription and the degradation of inherited maternal factors. We describe in detail the origin and regulation of the programmed cell death machinery found in the zebrafish early embryo. To elicit an apoptotic response from zebrafish early embryos, gastrulating germ-ring stage embryos were chronically exposed to the protein synthesis inhibitor cycloheximide. Chronic exposure of germ-ring stage embryos to cycloheximide resulted in the rapid activation of an apoptotic response by the embryos, which was evident by the presence of extensive DNA fragmentation, chromatin condensation, and caspase-3 activation. Zebrafish eggs synchronously die if attempted fertilization is unsuccessful. The lack of fertilization results in the elimination of the eggs through apoptosis within eight hours, a time equivalent to mid-gastrulation in fertilized control embryos. The presence of extensive cytoplasmic fragmentation and the activation of caspase-3 by the egg provides evidence that the death is apoptotic. In addition, if 64-cell stage embryos are chronically exposed to cycloheximide, the embryo enters cell cycle arrest, and fails to undergo classical apoptosis, yet dies via a caspase-3 mediated type of secondary necrosis within five hours, a time equivalent to mid-gastrulation in control embryos. The data reported here generate support for the hypothesis that the zebrafish egg is supplied with the machinery for apoptosis prior to fertilization, and that if fertilization occurs, the formation of the zygote state activates an inhibition of the apoptotic machinery that persists until mid-gastrulation. These maternal inhibitors are effective at preventing classical apoptosis until the embryo reaches the maternal-zygotic transition. Once the maternal-zygotic transition begins, the embryo regains the ability to undergo classical apoptosis as demonstrated by the apoptotic death of germ-ring stage embryos upon exposure to cycloheximide. These results lead us to believe that post-maternal-zygotic transition embryos have the capability to initiate activation of the apoptotic response without the need for de novo protein synthesis. Taken together, our data reveal an elaborate mechanism of cell suicide, which has its roots in the unfertilized egg.

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