Publication Date

9-2004

Degree Granted

Ph.D.

Institution Granting Degree

St. John's University

Keywords

Caspases, Apoptosis, Autophagy, Programmed cell death

Abstract

Embryogenesis takes place by cell proliferation, cell growth and Programmed Cell Death (PCD). Two pathways of PCD, apoptosis (or PCD-I) involving caspases, and autophagy (or PCD-II) involving lysosomes, have been well characterized in several species. Using the zebrafish ( Danio rerio ) model, we asked whether apoptosis or autophagy was a default program of death across organs and tissues with the other pathway playing a complementary role, or, whether they were parallel pathways independently active in different tissues.

To answer this, we used light and fluorescence microscopy techniques and investigated autophagy and apoptosis as functions of time and location during the first week of development of the zebrafish embryo. Lysosomal (autophagic) and caspase 3 (apoptosic) activities were tracked across organs and tissues by the acidotropic dye LysoTracker red ® and caspase 3 substrate PhiPhiLux ® , respectively.

We identified both autophagic and apoptosic activities in association with PCD that occurred during the morphogenesis of the olfactory placode, eyes, skin, tail, somites, and vent; but there were also several prominent differences; in the notochord we identified only caspase 3 activity, whereas in the myotome and developing fin buds, we observed only lysosomal activity. In the olfactory placode, apoptosis preceded autophagy.

When autophagy and apoptosis were inhibited individually by using 3 methyladenine and Z-DEVD-FMK, respectively, gross morphological abnormalities resulted (some resembling human congenital conditions) and these embryos died within a week. The phenotypic changes were found to be similar in organs and tissues where apoptotic and autophagic activity had overlapped and to vary in regions where one type of PCD had been exclusive.

Thus, we determined that in most organs and tissues, neither apoptosis nor autophagy is a default program of death, but that both activities are required for normal morphogenesis; while in some tissues they could be complementary, in most tissues they play essential, individual roles.

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