Long-Term High-Resolution Imaging of Developing C. elegans Larvae with Microfluidics.

Long-term studies of Caenorhabditis elegans larval development traditionally require tedious manual observations because larvae must move to develop, and existing immobilization techniques either perturb development or are unsuited for young larvae. Here, we present a simple microfluidic device to simultaneously follow development of ten C. elegans larvae at high spatiotemporal resolution from hatching to adulthood (∼3 days). Animals grown in microchambers are periodically immobilized by compression to allow high-quality imaging of even weak fluorescence signals. Using the device, we obtain cell-cycle statistics for C. elegans vulval development, a paradigm for organogenesis. We combine Nomarski and multichannel fluorescence microscopy to study processes such as cell-fate specification, cell death, and transdifferentiation throughout post-embryonic development. Finally, we generate time-lapse movies of complex neural arborization through automated image registration. Our technique opens the door to quantitative analysis of time-dependent phenomena governing cellular behavior during C. elegans larval development.

[1]  J. Sulston,et al.  Post-embryonic cell lineages of the nematode, Caenorhabditis elegans. , 1977, Developmental biology.

[2]  S. Franssila.  Introduction to microfabrication , 2004 .

[3]  James M. Rehg,et al.  Autonomous screening implicates new genes in synaptogenesis of C. elegans , 2012, Nature Methods.

[4]  M. Klass,et al.  Development of the reproductive system of Caenorhabditis elegans. , 1976, Developmental biology.

[5]  Hang Lu,et al.  Hydrogel-droplet microfluidic platform for high-resolution imaging and sorting of early larval Caenorhabditis elegans. , 2015, Lab on a chip.

[6]  V. Ambros,et al.  Efficient gene transfer in C.elegans: extrachromosomal maintenance and integration of transforming sequences. , 1991, The EMBO journal.

[7]  W. Wood.  The Nematode Caenorhabditis elegans , 1988 .

[8]  Nikos Chronis,et al.  An automated microfluidic platform for calcium imaging of chemosensory neurons in Caenorhabditis elegans. , 2010, Lab on a chip.

[9]  Hang Lu,et al.  Long-term high-resolution imaging and culture of C. elegans in chip-gel hybrid microfluidic device for developmental studies. , 2010, Lab on a chip.

[10]  Zhirong Bao,et al.  Control of cell cycle timing during C. elegans embryogenesis. , 2008, Developmental biology.

[11]  E. Jorgensen,et al.  EXP-1 is an excitatory GABA-gated cation channel , 2003, Nature Neuroscience.

[12]  R. Waterston,et al.  Automated cell lineage tracing in Caenorhabditis elegans. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[13]  N. Otsu.  A threshold selection method from gray level histograms , 1979 .

[14]  P. Sternberg,et al.  Functional transcriptomics of a migrating cell in Caenorhabditis elegans , 2012, Proceedings of the National Academy of Sciences.

[15]  Yun Lu,et al.  HSF-1 activates the ubiquitin proteasome system to promote non-apoptotic developmental cell death in C. elegans , 2016, eLife.

[16]  Matthew M. Crane,et al.  Automated on-chip rapid microscopy, phenotyping and sorting of C. elegans , 2008, Nature Methods.

[17]  P. Sternberg,et al.  Morphologically defined sub-stages of C. elegans vulval development in the fourth larval stage , 2015, BMC Developmental Biology.

[18]  Walter Fontana,et al.  Lifespan-on-a-chip: microfluidic chambers for performing lifelong observation of C. elegans. , 2010, Lab on a chip.

[19]  Marie-Anne Félix,et al.  Caenorhabditis elegans vulval cell fate patterning , 2012, Physical biology.

[20]  A. Hyman,et al.  Temperature Dependence of Cell Division Timing Accounts for a Shift in the Thermal Limits of C. elegans and C. briggsae. , 2015, Cell reports.

[21]  Hang Lu,et al.  Microfluidics for the analysis of behavior, nerve regeneration, and neural cell biology in C. elegans , 2009, Current Opinion in Neurobiology.

[22]  C. Kenyon,et al.  The nematode Caenorhabditis elegans. , 1988, Science.

[23]  Christopher V. Gabel,et al.  Long-Term Imaging of Caenorhabditis elegans Using Nanoparticle-Mediated Immobilization , 2013, PloS one.

[24]  S. Quake,et al.  Monolithic microfabricated valves and pumps by multilayer soft lithography. , 2000, Science.

[25]  M. Yanik,et al.  Microfluidic immobilization of physiologically active Caenorhabditis elegans , 2010, Nature Protocols.

[26]  I. Greenwald,et al.  Control of cell-fate plasticity and maintenance of multipotency by DAF-16/FoxO in quiescent Caenorhabditis elegans , 2013, Proceedings of the National Academy of Sciences.

[27]  N. Munakata.  [Genetics of Caenorhabditis elegans]. , 1989, Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme.

[28]  Yun Lu,et al.  Control of Nonapoptotic Developmental Cell Death in Caenorhabditis elegans by a Polyglutamine-Repeat Protein , 2012, Science.

[29]  Ze Cheng,et al.  The auxin-inducible degradation (AID) system enables versatile conditional protein depletion in C. elegans , 2015, Development.

[30]  I. Greenwald,et al.  A Caenorhabditis elegans model for epithelial–neuronal transdifferentiation , 2008, Proceedings of the National Academy of Sciences.

[31]  Henry Pinkard,et al.  Advanced methods of microscope control using μManager software. , 2014, Journal of biological methods.

[32]  Mehmet Fatih Yanik,et al.  Microfluidic system for on-chip high-throughput whole-animal sorting and screening at subcellular resolution , 2007, Proceedings of the National Academy of Sciences.

[33]  Paul W. Sternberg,et al.  Pattern formation during vulval development in C. elegans , 1986, Cell.

[34]  D. Sherwood,et al.  The unfolded protein response is required for dendrite morphogenesis , 2015, eLife.

[35]  Maxwell G. Heiman,et al.  Duplication of a Single Neuron in C. elegans Reveals a Pathway for Dendrite Tiling by Mutual Repulsion. , 2016, Cell reports.

[36]  J. Fisher,et al.  Cell-cycle regulation of NOTCH signaling during C. elegans vulval development , 2012, Molecular systems biology.

[37]  Iva Greenwald,et al.  LIN-14 Inhibition of LIN-12 Contributes to Precision and Timing of C. elegans Vulval Fate Patterning , 2010, Current Biology.

[38]  P. Shaw.  Comparison of Widefield/Deconvolution and Confocal Microscopy for Three-Dimensional Imaging , 2006 .

[39]  H. Horvitz,et al.  The lin-12 locus specifies cell fates in caenorhabditis elegans , 1983, Cell.

[40]  A. Yoo,et al.  LIN-12/Notch Activation Leads to MicroRNA-Mediated Down-Regulation of Vav in C. elegans , 2005, Science.

[41]  Z. Bao,et al.  The Regulatory Landscape of Lineage Differentiation in a Metazoan Embryo. , 2015, Developmental cell.

[42]  Z. Bao,et al.  De Novo Inference of Systems-Level Mechanistic Models of Development from Live-Imaging-Based Phenotype Analysis , 2014, Cell.

[43]  Eugene W. Myers,et al.  Analysis of Cell Fate from Single-Cell Gene Expression Profiles in C. elegans , 2009, Cell.

[44]  Cori Bargmann,et al.  Microfluidics for in vivo imaging of neuronal and behavioral activity in Caenorhabditis elegans , 2007, Nature Methods.

[45]  J. V. van Zon,et al.  Long-term time-lapse microscopy of C. elegans post-embryonic development , 2016, Nature Communications.

[46]  Oliver Hobert,et al.  Neurogenesis in the nematode Caenorhabditis elegans. , 2010, WormBook : the online review of C. elegans biology.

[47]  Yun Lu,et al.  A morphologically conserved nonapoptotic program promotes linker cell death in Caenorhabditis elegans. , 2007, Developmental cell.

[48]  Millet Treinin,et al.  Time-lapse imaging and cell-specific expression profiling reveal dynamic branching and molecular determinants of a multi-dendritic nociceptor in C. elegans. , 2010, Developmental biology.

[49]  S. Shaham,et al.  Temporal Control of Cell-Specific Transgene Expression in Caenorhabditis elegans , 2007, Genetics.

[50]  Xiao Liu,et al.  Straightening Caenorhabditis elegans images , 2007, Bioinform..

[51]  Wei Li,et al.  Live imaging of cellular dynamics during Caenorhabditis elegans postembryonic development , 2012, Nature Protocols.

[52]  Adela Ben-Yakar,et al.  CO2 and compressive immobilization of C. elegans on-chip. , 2009, Lab on a chip.