Advantages of Invertebrate Model Organisms

Simple model organisms are becoming increasingly important for investigating principal biochemical and molecular mechanisms. Caenorhabditis elegans (C. elegans) and Drosophila melanogaster (Drosophila) have been instrumental in deciphering the molecular underpinnings of cell death. Both animals are ideal for genetic and molecular studies and additionally they have proved to be highly relevant models for studying human disorders, such as neurodegenerative diseases (Driscoll and Gerstbrein 2003; Celotto and Palladino 2005).

Both are multi cellular organisms with a relatively simple anatomy. In the case of C. elegans, the total number of cells of the animal is 959, including 302 neurons that form a simple nervous system. The cell lineage in the development of the nematode is fully deciphered and a complete lineage tree is available (see http://; Sulston et al. 1983). During development, 131 cells undergo programmed cell death (Ellis and Horvitz 1986). This makes C. elegans a powerful tool for investigating developmental biology (Bargmann and Avery 1995).

An important advantage of the nematode is its transparency, which makes microscopy far easier, permitting every cell division throughout development to be tracked. The simple nervous system is well documented, all neurons are mapped, and an almost complete wiring diagram has been created (see; White et al. 1983; Hall and Russell 1991). Specific behaviors, such as locomotion, chemo- or thermotaxis, as well as learning and memory, can be experimentally associated with the relevant neuron(s) (Thomas and Lockery 2005). The detailed characterization of its nervous system renders C. elegans particularly suited for the study of neurodegeneration and aging (Murakami 2007). The nervous system of Drosophila is far more complex and includes an intricate brain structure. The fly has been utilized with resounding success to study programmed cell death, neurodevelopment, as well as neurodegenerative diseases (Tabata and Takei 2004; Carthew 2007; Leyssen and Hassan 2007; Li and Baker 2007).

Both organisms go through a short life cycle and likewise, have a short mean life span. C. elegans develops from the fertilized egg to a self-fertilizing adult hermaphrodite within 3.5 days by undergoing four larval stages (L1 to L4). Due to food starvation or harsh environmental conditions the developing larva can enter the so-called dauer stage before completing the L1 stage, which increases the mean life span for more than 5 months. Favorable food conditions allow the animal to reenter the normal life cycle as an L4 larva. After entering the adult stage an approximately 3 day reproductive period follows, during which the animal lays about 300 eggs. C. elegans lives around 20 days, of which the last 2 weeks are characterized by a decline in locomotion, food pumping, and recognizable tissue degeneration, revealing typical symptoms of aging. A low percentage of male animals (about 0.1 % of the progeny) is generated by hermaphrodites during self fertilization. These males enable genetic crosses that allow easy construction of double or multiple mutants (Riddle et al. 1997).

Drosophila needs about 8.5 days to develop from the zygote to the adult stage. After hatching, the animal undergoes three instar larval stages (first to third), followed by a prepupa and pupa stage, finally giving rise to the reproductive animal, which is either male or female. Females store the sperm of the male after mating and thereafter lay about 400 eggs (Lawrence 1992). Due to their short life span, both the nematode and the fruit fly are particularly popular for studying the mechanisms of aging and senescent decline (Lim et al. 2006).

Another important advantage of both animals is the easy maintenance in the laboratory. C. elegans feeds on bacteria (usually Escherichia coli strain OP50), which are grown either on solid agar plates or in liquid culture medium, and grows best at a temperature of 20°C. Drosophila is simply cultured at room temperature (25°C) and can be fed on different media containing a sugar source, like malt medium (Lakovaara 1969; Brenner 1974). The culturing temperature affects development timing of both animals. For example, C. elegans grows about 30% slower at 16°C compared to 20°C, while Drosophila needs about twice the time to complete a life cycle when grown at 18°C instead of 25°C, making it convenient to time experimental procedures. Both organisms can be cultured on a large scale.

Both the C. elegans and Drosophila genomes have been fully sequenced and annotated (Waterston and Sulston 1995; Kornberg and Krasnow 2000). Physical maps of the genome for both organisms based on the use of cosmids and yeast artificial chromosomes (YACs) have been created (Coulson et al. 1988; Hartl et al. 1992). The C. elegans genome is organized in five autosomes plus the sex chromosome X (sequence database: Drosophila only carries three autosomes plus the sex chromosome (sequence database: Approximately 20,000 open reading frames (ORFs) for the nematode and about 14,000 ORFs for the fruit fly have been predicted (Blumenthal et al. 2002; Halligan and Keightley 2006). Additionally detailed protein interaction networks have been modeled for both organisms (Walhout et al. 2000; Lin et al. 2006).

The availability of fully-charted genomes allows the implementation of large-scale, genome-wide genetic and molecular methodologies such as double-stranded RNA-mediated interference (dsRNAi; Mello and Conte 2004). In C. elegans high-throughput RNAi screens against all 20,000 ORFs have been published (Simmer et al. 2003). The use of RNAi in the nervous system of the nematode has been less successful so far, but can be offset by the use of special hypersensitive mutants or the introduction of double-stranded hairpin RNAs (dshRNAs) through microinjection (Tavernarakis et al. 2000; Schmitz et al. 2007).

Both organisms are genetically malleable (Lee et al. 2004; Venken and Bellen 2005). The most straightforward method of creating mutants in both cases is random mutagenesis through the use of the chemical ethyl methanesulfonate (EMS).

Mutants for almost every gene are available or can be ordered. Animals carrying multiple mutations can be constructed and efficient genetic mapping is possible, by utilizing precise single nucleotide polymorphism (SNP) maps available for both model organisms (Jakubowski and Kornfeld 1999; Berger et al. 2001).

In the case of Drosophila loss of function mutants can also be generated by the use of P transposable elements or introducing dshRNAs through the GAL4/ upstream activating sequence (GAL4/UAS) expression system, which is broadly used for gene overexpression (Brand and Perrimon 1993; Spradling et al. 1995; Cauchi and van den Heuvel 2006). In the fruit fly, the flippase (Flp)/flippase recom-binase target (FRT) genetic mosaic system is also used (Golic 1991; Cauchi and van den Heuvel 2006). Other genetic manipulation methods are additionally available in Drosophila (Greenspan 1997).

In C. elegans, transgenic animals can be obtained by microinjection of engineered DNA samples into the gonad, where they generate inherited extrachromosomal arrays. This extrachromosomal array can further be integrated and stabilized in the genome through mutagenesis-induced integration (Mello and Fire 1995; Jin 2005; Rieckher et al. 2009).

In conclusion, both C. elegans and Drosophila are exceptionally powerful and convenient model organisms for investigating diverse biological phenomena, including cell death.

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