Chapter 1
Neuroendocrine Regulation in the Genetic Model C. elegans

Charline Borghgraef1* , Pieter Van de Walle2* , Sven Van Bael1, Liliane Schoofs1, Wouter De Haes1,2§ and Isabel Beets1,3§

1Functional Genomics and Proteomics, Department of Biology, KU Leuven, Leuven, Belgium

2Molecular and Functional Neurobiology, Department of Biology, KU Leuven, Leuven, Belgium

3Cell Biology Division, MRC Laboratory of Molecular Biology, Cambridge, UK

1.1 A brief history on the model organism C. elegans

Research on the neurobiology of Caenorhabditis elegans has its roots in the 1960s, when Sydney Brenner proposed to use the nematode as a model organism for studying development and functioning of the nervous system. Brenner (Nobel Prize 2002) pioneered C. elegans genetics, by isolating and genetically mapping hundreds of mutant strains. Two decades later, John White and colleagues reconstructed the anatomy and synaptic connections (connectome) of all 302 C. elegans neurons in the adult hermaphrodite from electron micrographs. More recently, the wiring diagram of the posterior mating circuit in the adult male was mapped. Because C. elegans has a fixed number of somatic cells, researchers were able to construct a complete cell lineage by tracking the fate of each cell from fertilization to adulthood. This work was achieved by John Sulston and Robert Horvitz (Nobel Prize 2002), Judith Kimble, David Hirsh and Einhard Schierenberg. The neuronal connectome and cell lineage map allowed unprecedented insight into the worm's anatomy, development and neuronal makeup. These resources provided the basis for several key discoveries, including the characterization of genes regulating programmed cell death and axon guidance.

At the start of the genomic era in the 1990s, C. elegans was one of the simplest and best-studied animals available for undertaking whole-genome sequencing. The nematode was the first multicellular eukaryote to have its genome sequenced, a project completed in 1998. In the same year, RNA-interference (RNAi) was first demonstrated by Andrew Fire and Craig Mello (Nobel Prize 2006) using C. elegans. It has since been widely adopted as a tool for gene silencing in many organisms. C. elegans' transparent body facilitated another breakthrough that revolutionized the analysis of gene function. In 1994, Martin Chalfie (Nobel Prize 2008) showed that DNA encoding green fluorescent protein (GFP) could be used to mark gene expression in vivo in C. elegans. These landmark discoveries have been fundamental for establishing C. elegans as a versatile, genetic model system which is used today for studying questions on diverse research topics ranging from aging to metabolism, behavior, innate immunity and neuroendocrinology.

1.2 C. elegans genetics and anatomy

C. elegans is a small, free-living nematode that has two sexual forms: hermaphrodites and males (Figure 1.1). Both sexes have five autosomal chromosomes. Males have one X chromosome resulting from a spontaneous non-disjunction during meiosis, which occurs at low frequency (0.1%). After mating, the proportion of male progeny rises to 50%. Self-fertilizing hermaphrodites have two X chromosomes. They are easily cultivated and ensure transfer of homozygous mutations to the next generation. Therefore, they are studied far more commonly than males and used to maintain strain collections. C. elegans strains can be stored long-term by freezing them in a glycerol-rich solution at -80°C or in liquid nitrogen. The C. elegans research community has generated an extensive resource of mutants for most genes, which are summarized in the online database 'Wormbase', together with manually curated functional descriptions of all genes (www.wormbase.org). Over 21,000 protein-coding genes are annotated in the C. elegans genome (~100 Mb), over 30% of which have human orthologs.

Figure 1.1 Schematic body plans of adult C. elegans hermaphrodite and male, showing the pharynx in orange, intestine in yellow, gonads in green and cuticle in grey. In hermaphrodites, the gonads are connected to the spermatheca (dark green), followed by the uterus with eggs (blue). Males have a single gonad, which is connected to the vas deferens (dark green) and male-specific copulatory apparatus (blue), consisting of a fanned tail with copulatory spicules.

Adult C. elegans have an invariant number of somatic cells (eutely). Adult hermaphrodites measure around 1 mm in length and consist of 959 somatic nuclei, including 302 neurons. The adult male comprises 1031 somatic nuclei with 381 neurons. Most male-specific neurons are located in the copulatory circuits of the male tail. Similar to other nematodes, C. elegans has a simple body plan (Figure 1.1) that consists of an unsegmented inner and outer tube, separated by the pseudocoelomic body cavity. The outer tube contains the cuticle, the hypodermis, the muscles, the neurons and the excretory system; the inner tube comprises the pharynx, the intestine and the gonads. The most important endocrine sites in C. elegans are the nervous system, the intestinal and the gonadal tissues.

The small nervous system of C. elegans and its fully mapped connectome make it a prime model for studying the neuroendocrine control of physiology and behavior. The C. elegans neural network consists of two distinct systems: the large somatic nervous system (282 neurons) and a smaller pharyngeal nervous system (20 neurons) (Figure 1.2). The pharyngeal nervous system drives pumping of the pharynx and operates largely autonomously. The majority of neurons in the somatic nervous system have cell bodies in the head. Their processes are organized in a nerve ring surrounding the pharynx. A smaller number of somatic neurons are located in the lateral and tail ganglia, with processes that often project into the nerve ring. Sensory perception primarily relies on two symmetrically placed multicellular sensory organs, called amphids, which are located in the head. They can detect a wide range of sensory cues including olfactory, mechanical and water-soluble chemical stimuli. Smaller sensory organs, termed phasmids, are laterally located in the tail and are involved in the integration of stimuli sensed at the anterior and posterior parts of the body. For example, the phasmid neurons PHA and PHB, together with the polymodal amphid neuron ASH, mediate behavioral responses to chemical repellants.

Figure 1.2 Schematic wiring diagram of the C. elegans hermaphrodite nervous system, which includes 20 pharyngeal neurons (blue) and 282 neurons of the somatic nervous system. Cell bodies of neurons in the somatic nervous system are primarily located in ganglia in the head and tail, and along the ventral nerve cord (VNC). Most head neurons are organized around a ring-shaped bundle of neuron processes, called the nerve ring. Over 60% of all somatic neurons project axons or processes into the nerve ring. The detection of sensory stimuli relies largely on the amphid neurons (green) in the head and phasmid neurons (red) in the tail.

The worm's alimentary system - comprising the pharynx, intestine and anus - is involved in feeding and digestion. Since C. elegans consumes microorganisms, the intestine is also involved in immune and stress responses. In addition, the intestine and pharynx play important roles in the regulation of metabolic and endocrine processes, and in the storage of macromolecules. For example, the intestine is a main target site for insulin-like peptides. The somatic gonad also expresses several bioactive peptides and is thought to be the main site of synthesis of steroid hormones, termed dafachronic acids, which are involved in the regulation of development and lifespan. Males have only one gonadal arm for spermatogenesis (Figure 1.1). Hermaphrodites have two gonadal arms (Figure 1.1), in which oogenesis occurs in the distal tips. Hermaphrodite spermatogenesis takes place during development in the distal gonad, and sperm is stored in the spermatheca near the uterus.

1.3 C. elegans life-history

C. elegans is found worldwide, predominantly in humid and temperate environments. The nematode is commonly present in composting plant material, on plant stems, in rotting fruit and other bacteria-rich substrates. Its life-cycle consists of an embryonic stage, four larval stages (L1 to L4) and an adult stage. The timing of transitions between each stage depends on ambient temperature, but usually takes between three to four days from egg to adulthood (Figure 1.3). One of C. elegans' appealing features is its short generation time. The embryogenesis of hermaphrodites mainly occurs ex utero and lasts ~11 hours (at 20°C). The transition through the four larval stages typically requires ~65 hours. The end of each larval stage is characterized by a phase of lethargy and molting of the cuticle. In hermaphrodites, spermatogenesis takes place only during the fourth larval stage, after which oocytes are exclusively produced. Adults can lay eggs for up to 5 or 6 days and live for up to 3 weeks. C. elegans is easy to cultivate in the laboratory as the only requirements are...