Anthony Graham 1 Introduction

Cell death occurs by two processes that are cytologically and biochemically distinct. Pathological cell death, or necrosis, is characterized by early cell swelling and the lysis of intracellular organelles with nuclear breakdown following at a later stage. In contrast, nonpathological cell death or apoptosis is associated with early nuclear fragmentation, cytoplasmic shrinkage, and preservation of intracellular organelles (1). Necrosis is usually associated with cell injury; ATP levels fall early, and consequently, protein synthesis is inhibited. On the other hand, in apoptosis, ATP levels remain near normal, and new protein synthesis is required to initiate the process. One result that stems from the early nuclear fragmentation that is associated with apoptosis is that DNA degradation, resulting in the formation of a DNA ladder, is frequently observed.

During development, programmed cell death plays a vital role in eliminating unwanted cells, and it is invariably apoptotic in nature with the dying cells displaying the hallmarks of this process (2). Programmed cell death will act to prune cells that have been produced in excess, such as neurons. It is also involved in the eradication of cells of structures that are no longer necessary, such as the tail of tadpoles or the Mullerian duct in males (2). The apoptotic elimination of cells is also important in morphogenesis, and in both the limb and the cranial neural crest, it functions to ablate populations of cells locally and, as such, spatially separates the surviving cell populations from each other (2,3).

In developing systems, one can often not obtain enough material to analyze programmed cell death by biochemical means, such as assaying for DNA

laddering, and therefore one must use alternative methods. The most definitive method of determining whether or not apoptosis, as opposed to necrosis, is occurring in a group of cells is to analyze the cells ultrastruc-turally using electron microscopy (EM). This is a powerful tool because it will allow one to identify unequivocally a cell as being apoptotic or necrotic. The problem with using EM to analyze cell death in developing embryos is that it is a cumbersome technique that cannot be used practically to detect apoptosis or to gain an impression of a spatiotemporal profile of apoptosis. This chapter will describe two methods for detecting apoptosis in whole embryos, both of which will give a complete picture of apoptosis in any system. Both of these techniques are based on detection of the DNA fragmentation that occurs early in the apoptotic program. One point that should be noted is that although both of these techniques will pick out apoptotic sites in the embryo, the fact that the cells are actually dying by apoptosis should also then be confirmed independently by EM analysis.

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