36 Analysis of Cell Death in Zebrafish

1. INTRODUCTION

Although the fruit fly Drosophila melanogaster and the small nematode worm Caenorhabditis elegans have been crucial for advancing our understanding of the molecular mechanisms of cell death, there is a need for vertebrate model organisms to accelerate studies of human disease biology. A valuable vertebrate model system is the mouse Mus musculus, but this system suffers from the drawbacks of small litter sizes and in utero development. With these limitations in mind, zebrafish can provide unique advantages for in vivo experiments in a vertebrate model system to dissect conserved signal transduction pathways. In this chapter, we describe the zebrafish Danio rerio as a very useful vertebrate model system for studying cell death processes in both embryos and adults. First, we detail the advantages of the zebrafish animal model for discovering and analyzing cell death regulators. Second, we summarize a few studies in the field that have delineated distinct cell death mechanisms. Next, we highlight studies of developmental cell death and the very cancer-relevant p53 pathway in zebrafish, and we finish with some perspectives on the future of this exciting field of research. We hope to convey both a basic understanding of the strengths of this system and an overview of the important studies to date, which have enhanced our knowledge of the mechanisms that cells use to self-destruct when triggered by either developmental or environmental cues.

2. WHY USE ZEBRAFISH TO STUDY CELL DEATH?

Zebrafish offer a number of advantages for studying the cellular, molecular, and genetic processes of cell death. In this section, we highlight some of the major strengths of the zebrafish as a vertebrate model system for

studying cell death, and we also describe recent technologies that should have a profound impact on how researchers address the molecular genetics of cellular destruction in this model system.

2.1. The zebraf ish life cycle

Zebrafish have a generation time similar to that of the mouse, reaching sexual maturity in 3 months (Figure 36-1). Unlike the mouse, however, embryonic development occurs ex utero, or outside the mother, and most developmental patterning is achieved within the first 24 hours after birth. One group of embryos (called a clutch) obtained from a single mating pair often contains hundreds of individuals that can be used to perform a variety of experiments, such as those detailed in the following pages. Zebrafish embryos are completely transparent, allowing easy visualization of such diverse developmental processes as muscle formation, brain patterning, heart tube folding, vasculogenesis, and blood flow. They develop pigment beginning at approximately 30 hours postfertilization (hpf), and they begin to freely swim at approximately 4 days postfertilization (dpf). Feeding also starts at 4 dpf, and the juvenile zebrafish grows rapidly from this stage until it reaches adulthood.

2.2. Molecular techniques to rapidly assess gene function in embryos

2.2.1. Studies of gene function using microinjections into early embryos

Both transient gain-of-function and loss-of-function experiments can be performed for any gene of interest by injections into the one-cell stage zebrafish embryo