Modeling molecular and cellular aspects of human disease using the nematode Caenorhabditis elegans

Abstract

As an experimental system, Caenorhabditis elegans offers a unique opportunity to interrogate in vivo the genetic and molecular functions of human disease-related genes. For example, C. elegans has provided crucial insights into fundamental biologic processes, such as cell death and cell fate determinations, as well as pathologic processes such as neurodegeneration and microbial susceptibility. The C. elegans model has several distinct advantages, including a completely sequenced genome that shares extensive homology with that of mammals, ease of cultivation and storage, a relatively short lifespan and techniques for generating null and transgenic animals. However, the ability to conduct unbiased forward and reverse genetic screens in C. elegans remains one of the most powerful experimental paradigms for discovering the biochemical pathways underlying human disease phenotypes. The identification of these pathways leads to a better understanding of the molecular interactions that perturb cellular physiology, and forms the foundation for designing mechanism-based therapies. To this end, the ability to process large numbers of isogenic animals through automated work stations suggests that C. elegans, manifesting different aspects of human disease phenotypes, will become the platform of choice for in vivo drug discovery and target validation using high-throughput/content screening technologies.

Figure 1

C. elegans cell death pathways. A. The now classical core apoptosis pathway occurs in development and the germ line, but rarely in the adult soma. Upstream regulators block activation of the caspase, CED-3. B. The neurodegenerin pathway was first described in mechanosensory neurons and stimulated by mutations that elicit excitotoxic injury. A rise in [Ca²⁺]_i triggers peptidase activity that kills the cells over several hours. Although not pictured, lysosomal injury and acidification of the cytoplasm may contribute to injury. The necrosis pathway is triggered after different types of stress (hypotonic, thermal, oxidative, hypoxic) and is most evident within the intestinal cytoplasm. C. An intracellular serpin (SRP-6) protects the lysosome from stress-induced injury and also protects the cell itself from lysosome-induced damage. There is strong genetic, biochemical and morphological evidence for the conservation of these pathways in higher eukaryotes, including humans.

Figure 2

Intestinal cell necrosis in C. elegans. (Center) Three dimensional image showing hypotonic shock-induced intestinal cell necrosis in srp-6 null C. elegans stained with Lysotracker Red and the cysteine peptidase substrate, (Z-FR)₂-R110. Animals undergo extensive lysosome disintegration (red) and propagate a wave of cysteine peptidase activity (blue) across the cytoplasm. As an agonal event, adult worms often extrude their uteri and necrotic intestine through the vulva. Images were collected using an Olympus Fluoview1000 confocal microscope with 488 and 568 lasers. The micrograph is an overlay of fluorescent images taken over a z-series and merged. Volocity (Improvision) was used to psuedocolor and render the images. Background subtraction was performed using Canvas (Deneba). Scale bar = 50 μm. (Perimeter) Four-dimensional, time-lapsed images of intestinal cell necrosis occurring in live srp-6 null C. elegans stained with Lysotracker Red. Merged DIC and fluorescent images start at upper left-hand corner and progress clockwise. Images were captured at 20-second intervals (total time ~10 minutes) after the initiation of hypotonic shock. Intestinal cells undergo lysosomal rupture (red) and large vacuoles appear (purple) as intestinal cell necrosis progresses over time. Images were collected using a Zeiss Axioskop II Mot microscope using DIC optics at 250 × magnification. Fluorescent images were captured using excitation and emission wavelengths of 572 nm and 630 nm, respectively. Volocity and Canvas was used to pseudocolor and render the images. Scale bar (first frame) = 5 μm.

Figure 3

Global strategy for high-throughput/high-content screening of lead compounds using C. elegans. A, compounds, culture media and E. coli are dispensed into multi-well (96- or 384- well) plates using a high-throughput liquid handler (Evolution-P3 ©2001–2008 Perkin Elmer Inc., all rights reserved, printed with permission). B, Animals of the desired size and/or fluorescence profiles are sorted into multi-well plates using a COPAS BIOSORT (Union Biometrica Inc., image used with permission). C, An automated high-throughput imaging system, ArrayScan R VTi HCS Reader (Thermo Fisher Scientific, images used with permission) captures images and identifies changes in phenotype of interest. D, Lead compounds are identified and further scrutinized to confirm positives and eliminate nuisance compounds. E, New compounds are synthesized to develop potential therapeutics.