Abstract
Whole-organism phenotypic assays are central to the assessment of neuromuscular function and health in model organisms such as the nematode C. elegans. In this study, we report a new assay format for engaging C. elegans in burrowing that enables rapid assessment of nematode neuromuscular health. In contrast to agar environments that pose specific drawbacks for characterization of C. elegans burrowing ability, here we use the optically transparent and biocompatible Pluronic F-127 gel that transitions from liquid to gel at room temperature, enabling convenient and safe handling of animals. The burrowing assay methodology involves loading animals at the bottom of well plates, casting a liquid-phase of Pluronic on top that solidifies via a modest temperature upshift, enticing animals to reach the surface via chemotaxis to food, and quantifying the relative success animals have in reaching the chemoattractant. We study the influence of Pluronic concentration, gel height and chemoattractant choice to optimize assay performance. To demonstrate the simplicity of the assay workflow and versatility, we show its novel application in multiple areas including (i) evaluating muscle mutants with defects in dense bodies and/or M-lines (pfn-3, atn-1, uig-1, dyc-1, zyx-1, unc-95 and tln-1), (ii) tuning assay conditions to reveal changes in the mutant gei-8, (iii) sorting of fast burrowers in a genetically-uniform wild-type population for later quantitation of their distinct muscle gene expression, and (iv) testing proteotoxic animal models of Huntington and Parkinson’s disease. Results from our studies show that stimulating animals to navigate in a dense environment that offers mechanical resistance to three-dimensional locomotion challenges the neuromuscular system in a manner distinct from standard crawling and thrashing assays. Our simple and high throughput burrowing assay can provide insight into molecular mechanisms for maintenance of neuromuscular health and facilitate screening for therapeutic targets.
Highlights
Whole-organism phenotypic assays are central to the assessment of neuromuscular function and health in model organisms such as the nematode C. elegans
It is a standard practice to culture C. elegans on agar (in the form of Nematode Growth Media (NGM)) plates, and studies so far have focused on using agar as the burrowing medium[16,18,20]
To compare the locomotory characteristics in Pluronic F-127 (PF-127) gel with swimming and crawling, we examined C. elegans locomotion in a 26% w/w Pluronic gel with a layer thickness of approximately 1 mm
Summary
Whole-organism phenotypic assays are central to the assessment of neuromuscular function and health in model organisms such as the nematode C. elegans. Results from our studies show that stimulating animals to navigate in a dense environment that offers mechanical resistance to three-dimensional locomotion challenges the neuromuscular system in a manner distinct from standard crawling and thrashing assays. The millimeter-long round worm Caenorhabditis elegans is an excellent model for investigating conserved molecular mechanisms regulating neuromuscular health and its decline with age[1]. Additional benefits of this model organism include 60% homology with human genes, a short life cycle, 95 body wall muscle cells similar to vertebrate muscle, and 302 mapped neurons[1,2,3]. Additional challenges include injection of tens of animals into a localized spot (which may result in animal loss or damage), and visualization difficulty due to the translucency of agar and refraction at cylindrical surfaces
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