Antebi, A. | bioRxiv Science

bioRxiv2019

Pluronic gel-based burrowing assay for rapid assessment of neuromuscular health in C. elegans

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 Parkinsons 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.

neuroscience↗

bioRxiv2019

Cell cycle dynamics during diapause entry and exit in an annual killifish revealed by FUCCI technology

BackgroundAnnual killifishes are adapted to surviving and reproducing over alternating dry and wet seasons. During the dry season, all adults die and desiccation-resistant embryos remain encased in dry mud for months or years in a state of quiescence, delaying hatching until their habitats are flooded again. Embryonic development of annual killifishes deviates from canonical teleost development. Epiblast cells disperse during epiboly, and a \"dispersed phase\" precedes gastrulation. In addition, annual fish have the ability to enter diapause and block embryonic development at the dispersed phase (diapause I), mid-somitogenesis (diapause II) and the final phase of development (diapause III).\n\nDevelopmental transitions associated with diapause entry and exit can be linked with cell cycle events. Here we set to image this transitions in living embryos.\n\nResultsTo visibly explore cell cycle dynamics during killifish development in depth, we created a stable transgenic line in Nothobranchius furzeri that expresses two fluorescent reporters, one for the G1 phase and one for the S/G2 phases of the cell cycle, respectively (fluorescent ubiquitination based cell cycle indicator, FUCCI). Using this tool, we observed that, during epiboly, epiblast cells progressively become quiescent and exit the cell cycle. All embryos transit through a phase where dispersed cells migrate, without showing any mitotic activity, possibly blocked in the M phase (diapause I).\n\nThereafter, exit from diapause I is synchronous and cells enter directly into the S phase without transiting through G1. The developmental trajectories of embryos entering diapause and of those that continue to develop are different. In particular, embryos entering diapause have reduced growth along the medio-lateral axis. Finally, exit from diapause II is synchronous for all cells and is characterized by a burst of mitotic activity and growth along the medio-lateral axis such that, by the end of this phase, the morphology of the embryos is identical to that of direct-developing embryos.\n\nConclusionsOur study reveals surprising levels of coordination of cellular dynamics during diapause and provides a reference framework for further developmental analyses of this remarkable developmental quiescent state.\n\nList of AbbreviationsIn this paper, we will refer to several developmental stages or morphological structures using abbreviations. To make the reading easier, we resume here a list of all the abbreviations, to which the reader can refer at any time.

developmental biology↗