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High-Throughput Visual Screens in Neurodegeneration and Neuronal Development Studies
Positioning and orienting C. Elegans for high throughput neuronal screening
Achievement/Results
The model organism Caenorhabditis elegans is a commonly used in neuroscience and genetics studies because of its comparatively simple nervous system, consisting of 302 neurons of known connectivity, and for its ease of use in genetic research. The animal is a soil dwelling nematode measuring 1mm in length when fully grown, is capable of producing hundreds of genetically identical offspring within three days because of its hermaphroditic nature and fast generation time, and has a large number of transgenic and mutant strains available. In recent years, scientists have begun to use microfluidics for C. elegans research to take advantage of high-throughput applications such as imaging, sorting, long-term culture, laser ablation, and drug screening. These methodologies, however, are not capable of orientating an animal within the device, hindering visual inspection of transgenic markers requiring specific orientations.
With NSF support we provide, for the first time, a device capable of laterally orienting C. elegans on-chip by using curved channel geometries (Fig. 1A). Using this device we are capable of positioning animals into lateral orientations up to 84% of instances as compared to 21% using existing and conventional straight channel methods (Fig. 1B). Altering the device radius of curvature (RoC) had no significant effect on the ability of the device to laterally orient C. elegans (Fig. 1B). Figure 1C demonstrates a wild-type animal positioned laterally within the curved channel of our device, scored by ensuring that both the ventral and dorsal nerve cords, VNC and DNC respectively, are fully visible throughout the animal body length. Figure 1E demonstrates a wild-type animal in a conventional straight channel design that is not positioned laterally. This is noted by the obstruction of the DNC from the focal plane. Figures 1D and 1F show a diagram of the animal body cross section and dorsal ventral axis in order to judge the difference in orientation between the two example images.
The advantages of our system our threefold: first, the system is passive, facilitating inspection of animals requiring specific orientation for analysis in a simple manner. Second, our curved channel area increases the amount of animal body that is within the microscope field of view. Lastly, operation of our device is simple, and can be used by non-engineers to perform visual screens. Using our device, we performed a phenotypic screen and inspected over 10,000 animals on-chip searching for animals with morphological defects involved in neurodegeneration and neural development. We successfully isolated six mutants as a result of our screen whose phenotypes are described further in our publication: Cáceres et. al, PLoS One, 2012. Of particular interest is the fact that one mutant discussed in our publication was isolated by a member of our collaborator’s laboratory, a non-engineer, that had been trained on site in Queensland, Australia as a result of the international travel benefits of the IGERT Hybrid Neural Microsystems fellowship.
Address Goals
Discovery: This technology was developed to accelerate the discovery process. As described above, it enables the process of screening mutants to occur much faster than with manual screening by hand. In the example above, over 10,000 animals were screened, identifying six novel mutants. While trained humans were still involved, this greatly accelerated the discovery process.
Infrastructure: Using this technology, we plan to implement custom image analysis software to recognize mutant phenotypes in order to fully automate our system, effectively eliminating laborious standard techniques involving manual manipulations of animals on glass slides. This would provide a single platform and location to perform all screening operations, versus current methods involving three separate steps of slide preparation, phenotype analysis, and rescue of animals of interest. Ultimately, our technology provides a tool for researchers to potentially saturate screens within months (versus years using current methods), and to potentially discover new therapies to treat devastating neural pathologies such as Alzheimer’s, Parkinson’s, and Huntington’s disease.
