A simple answer to complex questions: Caenorhabditis elegans as an experimental model for examining the DNA damage response and disease genes

Abstract

The genetic information is constantly challenged by genotoxic attacks. DNA repair mechanisms evolved early in evolution and recognize and remove the various lesions. A complex network of DNA damage responses (DDR) orchestrates a variety of physiological adaptations to the presence of genome instability. Erroneous repair or malfunctioning of the DDR causes cancer development and the accumulation of DNA lesions drives the aging process. For understanding the complex DNA repair and DDR mechanisms it is pivotal to employ simple metazoan as model systems. The nematode Caenorhabditis elegans has become a well-established and popular experimental organism that allows dissecting genome stability mechanisms in dynamic and differentiated tissues and under physiological conditions. We provide an overview of the distinct advantages of the nematode system for studying DDR and provide a range of currently applied methodologies.

Keywords: C. elegans; DNA damage response; DNA repair; aging; genome stability.

Figure 1. C. elegans life cycle and developmental stages.

(A) Scheme of the life cycle from fertilized embryo, through the four larval stages L1-L4 and the fertile adult. One life-cycle is completed in 2.5 days when animals are grown at 20°C on OP50 bacteria-seeded NGM agar plates. Adults lay 300 eggs that hatch within 9 hours ex utero development. (B) Stereoscopic bright field image showing mixed, distinguishable developmental stages on an agar plate. Scale bar is 100 µm.

Figure 2. Simplified summary of the NER pathway in C. elegans.

The scheme shows the NER factors that have been studied most extensively in the nematode. The damage is recognized either via GG-NER factors DDB-1 or XPC-1, or via TC-NER, in which CSB-1 and CSA-1 interact with the stalled RNA polymerase II. Both pathways employ a common core mechanism that is distinguished in DNA unwinding, damage excision and DNA synthesis to fill the gap.

Figure 3. Determination of developmental delay and embryonic survival upon UVB-exposure (see Methods).

(A) Wild type irradiated at various doses with two different types of UVB irradiation sources. (B) Embryonic survival assay in the wild type, the GG-NER mutant for XPC-1, the TC-NER mutant for CSB-1 and the DNA unwinding factor XPA-1. (C) Developmental assay of the TC-NER mutant CSB-1 compared to wild type. (D) Developmental delay determined in the highly UV-sensitive mutants for XPA-1 or ERCC-1, acting in the core NER machinery. Errors bars indicate standard deviations.

Figure 4. Commonly applied read-outs for somatic decline upon DNA damage induction (see Methods).

(A) Life span assay of wild type and xpa-1 mutants irradiated at day 1 of adulthood. (B) Pumping assay performed in adult-irradiated wild type and xpa-1 mutants 72 hours post-exposure. Error bars show SEM. (C) DAF-16::GFP nuclearization upon UVB-irradiation in xpa-1 mutant animals synchronized at L3 stage. The images are representative for the categories cytosolic, partially nuclear and fully nuclear. Size bars correspond to 100 µm. (D) Germline DNA damage induced stress resistance (GDISR) upon heat stress. Worms were treated either with 0 Gy (Control) or 90 Gy of IR at L4 larval stage before being shifted to 35°C 48 hours later. Experiment was done in biological triplicates. Error bars indicate standard error of the mean.

Figure 5. The C. elegans germline as model for cell cycle arrest and apoptosis upon IR-induced DSB-formation (see Methods).

(A) CED-1::GFP strain labeling somatic sheath cells during the engulfment of germ cells death. Day 1 adult worms were treated with IR at 90 Gy and 6 hours post-IR treatment pictures are acquired. Arrow shows a single corpse in 0 Gy and the square displays accumulation of corpses upon 90 Gy treatment in the gonad loop. Scale bars are 20 µm. (B) Representative images of the most distal (mitotic) zone to determine apoptosis. Scale bars are 20 µm. (C) Acridine Orange (AO) staining 6 h post-γ-irradiation (90 Gy). AO-stained apoptotic corpses (arrows) become visible under fluorescent microscope. Note that corpse clusters often result in diffuse staining signals thus exacerbating corpse scoring. Importantly, air bubbles (arrowhead) do not take up the dye. Scale bars: 20 µm. (C) RAD-51 foci indicating persistent DNA damage 16 h post-UV irradiation (120 mJ/cm²) in the mitotic zone of an adult C. elegans germline. Scale bars: 10 µm.