C. elegans is an excellent model system for studying neuroscience using genetics because of its relatively simple nervous system, sequenced genome, and the availability of a large number of transgenic and mutant strains. Recently, microfluidic devices have been used for high-throughput genetic screens, replacing traditional methods of manually handling C. elegans. However, the orientation of nematodes within microfluidic devices is random and often not conducive to inspection, hindering visual analysis and overall throughput. In addition, while previous studies have utilized methods to bias head and tail orientation, none of the existing techniques allow for orientation along the dorso-ventral body axis. Here, we present the design of a simple and robust method for passively orienting worms into lateral body positions in microfluidic devices to facilitate inspection of morphological features with specific dorso-ventral alignments. Using this technique, we can position animals into lateral orientations with up to 84% efficiency, compared to 21% using existing methods. We isolated six mutants with neuronal development or neurodegenerative defects, showing that our technology can be used for on-chip analysis and high-throughput visual screens.
References
[1]
Hilliard MA, Bargmann CI (2006) Wnt signals and frizzled activity orient anterior-posterior axon outgrowth in C-elegans. Developmental Cell 10: 379–390.
[2]
Antebi A, Norris CR, Hedgecock EM (1997) Cell and Growth Cone Migrations. In: Riddle DL, Blumenthal T, Meyer BJ, Priess JR, editors. C Elegans II. Cold Spring Harbor: Cold Spring Harbor Laboratory Press. pp. 583–610.
[3]
Arimura N, Kaibuchi K (2007) Neuronal polarity: from extracellular signals to intracellular mechanisms. Nature Reviews Neuroscience 8: 194–205.
[4]
Adler CE, Fetter RD, Bargmann CI (2006) UNC-6/Netrin induces neuronal asymmetry and defines the site of axon formation. Nature Neuroscience 9: 511–518.
[5]
Hao JC, Yu TW, Fujisawa K, Culotti JG, Gengyo-Ando K, et al. (2001) C-elegans slit acts in midline, dorsal-ventral, and anterior-posterior guidance via the SAX-3/Robo receptor. Neuron 32: 25–38.
[6]
Kaletta T, Hengartner MO (2006) Finding function in novel targets: C-elegans as a model organism. Nature Reviews Drug Discovery 5: 387–398.
[7]
Jorgensen EM, Mango SE (2002) The art and design of genetic screens: Caenorhabditis elegans. Nat Rev Genet 3: 356–369.
[8]
Samara C, Rohde CB, Gilleland CL, Norton S, Haggarty SJ, et al. (2010) Large-scale in vivo femtosecond laser neurosurgery screen reveals small-molecule enhancer of regeneration. Proceedings of the National Academy of Sciences of the United States of America 107: 18342–18347.
[9]
Chung KH, Crane MM, Lu H (2008) Automated on-chip rapid microscopy, phenotyping and sorting of C. elegans. Nature Methods 5: 637–643.
[10]
Crane MM, Chung K, Lu H (2009) Computer-enhanced high-throughput genetic screens of C. elegans in a microfluidic system. Lab on a Chip 9: 38–40.
[11]
Crane MM, Chung K, Stirman J, Lu H (2010) Microfluidics-enabled phenotyping, imaging, and screening of multicellular organisms. Lab on a Chip 10: 1509–1517.
[12]
Ben-Yakar A, Chronis N, Lu H (2009) Microfluidics for the analysis of behavior, nerve regeneration, and neural cell biology in C. elegans. Current Opinion in Neurobiology 19: 561–567.
[13]
Krajniak J, Lu H (2010) Long-term high-resolution imaging and culture of C. elegans in chip-gel hybrid microfluidic device for developmental studies. Lab on a Chip 10: 1862–1868.
[14]
Zeng F, Rohde CB, Yanik MF (2008) Sub-cellular precision on-chip small-animal immobilization, multi-photon imaging and femtosecond-laser manipulation. Lab on a Chip 8: 653–656.
[15]
Altun ZF, Hall DH (2008) Handbook of C. elegans Anatomy. In WormAtlas.
[16]
Hobert O, Community TCeR, editor (2005) Specification of the nervous system. WormBook: WormBook.
[17]
Chokshi TV, Bazopoulou D, Chronis N (2010) An automated microfluidic platform for calcium imaging of chemosensory neurons in Caenorhabditis elegans. Lab on a Chip 10: 2758–2763.
[18]
Huang X, Cheng HJ, Tessier-Lavigne M, Jin YS (2002) MAX-1, a novel PH/MyTH4/FERM domain cytoplasmic protein implicated in netrin-mediated axon repulsion. Neuron 34: 563–576.
[19]
Shaham S (2007) Counting Mutagenized Genomes and Optimizing Genetic Screens in Caenorhabditis elegans. PLoS One 2:
[20]
Unger MA, Chou HP, Thorsen T, Scherer A, Quake SR (2000) Monolithic microfabricated valves and pumps by multilayer soft lithography. Science 288: 113–116.
[21]
Stirman JN, Brauner M, Gottschalk A, Lu H (2010) High-throughput study of synaptic transmission at the neuromuscular junction enabled by optogenetics and microfluidics. Journal of Neuroscience Methods 191: 90–93.
[22]
Brenner S (1974) Genetics of Caenorhabditis-Elegans. Genetics 77: 71–94.
[23]
Jorgensen EM, Mango SE (2002) The art and design of genetic screens: Caenorhabditis elegans. Nature Reviews Genetics 3: 356–369.
[24]
Shaham S, Community TCeR, editor (2006) S Methods in cell biology. WormBook: WormBook.