Name: the c. elegans excretory canal as a model for intracellular lumen morphogenesis and in vivo polarized membrane biogenesis in a single cell: labeling by gfp-fusions, rnai interaction screen and imaging
Uploaded: 2017-10-03T14:00:00
Description: Watch this Scientific Journal Video about The C. elegans Excretory Canal as a Model for Intracellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis in a Single Cell: labeling by GFP-fu

We thank M. Buechner (University of Kansas, Kansas, USA), K. Nehrke (University of Rochester Medical Center, Rochester, New York, USA), and the Caenorhabditis Genetics Center, funded by National Institutes of Health, Office of Research Infrastructure Programs (P40 OD010440). This work was supported by grants NIH GM078653, MGH IS 224570 and SAA 223809 to V.G.

1. Labeling the C. elegans Excretory Canal by Fluorescent Fusion Proteins14
Note: See the accompanying paper on intestinal tubulogenesis3 for labeling by in situ antibody staining procedures adaptable to the excretory canal. See Table 1 for examples of molecules proven useful for visualizing C. elegans excretory canal endo- and plasma membranes, Table 2 for examples of promoters driving e...

<ol>
	<li>Lubarsky, B., Krasnow, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tube+morphogenesis:+making+and+shaping+biological+tubes.">Tube morphogenesis: making and shaping biological tubes.</a> Cell. 112 (1), (2003).</li><li>Sundaram, M. V., Cohen, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time+to+make+the+doughnuts:+Building+and+shaping+seamless+tubes.">Time to make the doughnuts: Building and shaping seamless tubes.</a> Semin. Cell Dev. Biol. S1084-9521, 30130-30136 (2016).</li><li>Zhang, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+C.+elegans+intestine+as+a+model+for+intercellular+lumen+morphogenesis+and+in+vivo+polarized+membrane+biogenesis+at+the+single-cell+level.">The C. elegans intestine as a model for intercellular lumen morphogenesis and in vivo polarized membrane biogenesis at the single-cell level.</a> JoVE. , (2017).</li><li>Andrew, D. J., Ewald, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphogenesis+of+epithelial+tubes:+Insights+into+tube+formation,+elongation,+and+elongation.">Morphogenesis of epithelial tubes: Insights into tube formation, elongation, and elongation.</a> Dev. Biol. 341 (1), 34-55 (2010).</li><li>Nelson, F. K., Albert, P. S., Riddle, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fine+structure+of+the+Caenorhabditis+elegans+secretory-excretory+system.">Fine structure of the Caenorhabditis elegans secretory-excretory system.</a> J. Ultrastruct. Res. 82 (2), 156-171 (1983).</li><li>Sundaram, M. V., Buechner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Caenorhabditis+elegans+Excretory+System:+A+Model+for+Tubulogenesis,+Cell+Fate+Specification,+and+Plasticity.">The Caenorhabditis elegans Excretory System: A Model for Tubulogenesis, Cell Fate Specification, and Plasticity.</a> Genetics. 203 (1), 35 (2016).</li><li>Altun, Z. F., Hall, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Excretory+system.">Excretory system.</a> WormAtlas. , (2009).</li><li>Buechner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tubes+and+the+single+C.+elegans+excretory+cell.">Tubes and the single C. elegans excretory cell.</a> Trends Cell Biol. 12 (10), 479-484 (2002).</li><li>Khan, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+lumen+extension+requires+ERM-1-dependent+apical+membrane+expansion+and+AQP-8-mediated+flux.">Intracellular lumen extension requires ERM-1-dependent apical membrane expansion and AQP-8-mediated flux.</a> Nat. Cell Biol. 15 (2), 143-156 (2013).</li><li>Kolotuev, I., Hyenne, V., Schwab, Y., Rodriguez, D., Labouesse, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+pathway+for+unicellular+tube+extension+depending+on+the+lymphatic+vessel+determinant+Prox1+and+on+osmoregulation.">A pathway for unicellular tube extension depending on the lymphatic vessel determinant Prox1 and on osmoregulation.</a> Nat. Cell Biol. 15 (2), 157-168 (2013).</li><li>Buechner, M., Hall, D. H., Bhatt, H., Hedgecock, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cystic+canal+mutants+in+Caenorhabditis+elegans+are+defective+in+the+apical+membrane+domain+of+the+renal+(excretory)+cell.">Cystic canal mutants in Caenorhabditis elegans are defective in the apical membrane domain of the renal (excretory) cell.</a> Dev. Biol. 214 (1), 227-241 (1999).</li><li>Fehon, R. G., McClatchey, A. I., Bretscher, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organizing+the+cell+cortex:+the+role+of+ERM+proteins.">Organizing the cell cortex: the role of ERM proteins.</a> Nat. Rev. Mol. Cell Biol. 11 (4), 276-287 (2010).</li><li>Gobel, V., Barrett, P. L., Hall, D. H., Fleming, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lumen+morphogenesis+in+C.+elegans+requires+the+membrane-cytoskeleton+linker+erm-1.">Lumen morphogenesis in C. elegans requires the membrane-cytoskeleton linker erm-1.</a> Dev. Cell. 6 (6), 865-873 (2004).</li><li>Sambrook, J., Sambrook, J., DW, R. u. s. s. e. l. l. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular cloning: a laboratory manual. , (2006).</li><li>Fire, A., Harrison, S. W., Dixon, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+modular+set+of+lacZ+fusion+vectors+for+studying+gene+expression+in+Caenorhabditis+elegans.">A modular set of lacZ fusion vectors for studying gene expression in Caenorhabditis elegans.</a> Gene. 93 (2), 189-198 (1990).</li><li>Kamath, R. S., Ahringer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+RNAi+screening+in+Caenorhabditis+elegans.">Genome-wide RNAi screening in Caenorhabditis elegans.</a> Methods. 30 (4), 313-321 (2003).</li><li>Timmons, L., Court, D. L., Fire, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ingestion+of+bacterially+expressed+dsRNAs+can+produce+specific+and+potent+genetic+interference+in+Caenorhabditis+elegans.">Ingestion of bacterially expressed dsRNAs can produce specific and potent genetic interference in Caenorhabditis elegans.</a> Gene. 263 (1-2), 103-112 (2001).</li><li>Pawley, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Handbook of Biological Confocal Microscopy. , (2006).</li><li>Hibbs, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Confocal Microscopy for Biologists. , (2004).</li><li>Bankhead, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Analyzing fluorescence microscopy images with ImageJ. , (2014).</li><li>Praitis, V., Casey, E., Collar, D., Austin, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Creation+of+low-copy+integrated+transgenic+lines+in+Caenorhabditis+elegans.">Creation of low-copy integrated transgenic lines in Caenorhabditis elegans.</a> Genetics. 157 (3), 1217-1226 (2001).</li><li>Fr&#248;kjaer-Jensen, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-copy+insertion+of+transgenes+in+Caenorhabditis+elegans.">Single-copy insertion of transgenes in Caenorhabditis elegans.</a> Nat. Genet. 40 (11), 1375-1383 (2008).</li><li>Mello, C. C., Kramer, J. M., Stinchcomb, D., Ambros, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+gene+transfer+in+C.+elegans:+extrachromosomal+maintenance+and+integration+of+transforming+sequences.">Efficient gene transfer in C. elegans: extrachromosomal maintenance and integration of transforming sequences.</a> EMBO J. 10 (12), 3959-3970 (1991).</li><li>Barrangou, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cas9+Targeting+and+the+CRISPR+Revolution.">Cas9 Targeting and the CRISPR Revolution.</a> Science. 344 (6185), 707-708 (2014).</li><li>Dickinson, D. J., Goldstein, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR-Based+Methods+for+Caenorhabditis+elegans+Genome+Engineering.">CRISPR-Based Methods for Caenorhabditis elegans Genome Engineering.</a> Genetics. 202 (3), 885-901 (2016).</li><li>Esposito, D., Garvey, L. A., Chakiath, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gateway+cloning+for+protein+expression.">Gateway cloning for protein expression.</a> Methods Mol. Biol. 498, 31-54 (2009).</li><li>Gibson, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzymatic+assembly+of+DNA+molecules+up+to+several+hundred+kilobases.">Enzymatic assembly of DNA molecules up to several hundred kilobases.</a> Nat. Methods. 6 (5), 343-345 (2009).</li><li>Hobert, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PCR+fusion-based+approach+to+create+reporter+gene+constructs+for+expression+analysis+in+transgenic+C.+elegans.">PCR fusion-based approach to create reporter gene constructs for expression analysis in transgenic C. elegans.</a> Biotechniques. 32 (4), 728-730 (2002).</li><li>Jorgensen, E. M., Mango, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+art+and+design+of+genetic+screens:+caenorhabditis+elegans.">The art and design of genetic screens: caenorhabditis elegans.</a> Nat. Rev. Genet. 3 (5), 356-369 (2002).</li><li>Berry, K. L., Bulow, H. E., Hall, D. H., Hobert, O. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=C.+elegans+CLIC-like+protein+required+for+intracellular+tube+formation+and+maintenance.">C. elegans CLIC-like protein required for intracellular tube formation and maintenance.</a> Science. 302 (5653), 2134-2137 (2003).</li><li>Mattingly, B. C., Buechner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+FGD+homologue+EXC-5+regulates+apical+trafficking+in+C.+elegans+tubules.">The FGD homologue EXC-5 regulates apical trafficking in C. elegans tubules.</a> Dev. Biol. 359 (1), 59-72 (2011).</li><li>Shaye, D. D., Greenwald, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+disease-associated+formin+INF2/EXC-6+organizes+lumen+and+cell+outgrowth+during+tubulogenesis+by+regulating+F-actin+and+microtubule+cytoskeletons.">The disease-associated formin INF2/EXC-6 organizes lumen and cell outgrowth during tubulogenesis by regulating F-actin and microtubule cytoskeletons.</a> Dev. Cell. 32 (6), 743-755 (2015).</li><li>Lant, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CCM-3/STRIPAK+promotes+seamless+tube+extension+through+endocytic+recycling.">CCM-3/STRIPAK promotes seamless tube extension through endocytic recycling.</a> Nat. Commun. 6 (6), 6449 (2015).</li><li>Paupard, M. C., Miller, A., Grant, B., Hirsh, D., Hall, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immuno-EM+localization+of+GFP-tagged+yolk+proteins+in+C.+elegans+using+microwave+fixation.">Immuno-EM localization of GFP-tagged yolk proteins in C. elegans using microwave fixation.</a> J. Histochem. Cytochem. 49 (8), 949-956 (2001).</li><li>Lukyanov, K. A., Chudakov, D. M., Lukyanov, S., Verkhusha, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoactivatable+fluorescent+proteins.">Photoactivatable fluorescent proteins.</a> Nat. Rev. Mol. Cell Biol. 6 (11), 885-891 (2005).</li><li>Bates, M., Huang, B., Dempsey, G. T., Zhuang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multicolor+super-resolution+imaging+with+photo-switchable+fluorescent+probes.">Multicolor super-resolution imaging with photo-switchable fluorescent probes.</a> Science. 317 (5845), 1749-1753 (2007).</li><li>Sherman, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+abts+and+sulp+families+of+anion+transporters+from+Caenorhabditis+elegans.">The abts and sulp families of anion transporters from Caenorhabditis elegans.</a> Am. J. Physiol. Cell Physiol. 289 (2), C341-C351 (2005).</li><li>. Transgeneome website Available from: <a target="_blank" href="https://transgeneome.mpi-cbg.de/transgeneomics/index.html">https://transgeneome.mpi-cbg.de/transgeneomics/index.html</a> (2017)</li><li>. National BioResource Project (NBRP)::C. elegans Available from: <a target="_blank" href="https://shigen.nig.ac.jp/c.elegans/">https://shigen.nig.ac.jp/c.elegans/</a> (2017)</li><li>Heppert, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+assessment+of+fluorescent+proteins+for+in+vivo+imaging+in+an+animal+model+system.">Comparative assessment of fluorescent proteins for in vivo imaging in an animal model system.</a> Mol. Biol. Cell. 27 (22), 3385-3394 (2016).</li><li>Shaner, N. C., Steinbach, P. A., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+guide+to+choosing+fluorescent+proteins.">A guide to choosing fluorescent proteins.</a> Nat Methods. 2 (12), 905-909 (2005).</li><li>. BLAST Available from: <a target="_blank" href="https://blast.ncbi.nlm.nih.gov/Blast.cgi">https://blast.ncbi.nlm.nih.gov/Blast.cgi</a> (2017)</li><li>Kamath, R. S., Martinezcampos, M., Zipperlen, P., Fraser, A. G., Ahringer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effectiveness+of+specific+RNA-mediated+interference+through+ingested+double-stranded+RNA+in+Caenorhabditis+elegans.">Effectiveness of specific RNA-mediated interference through ingested double-stranded RNA in Caenorhabditis elegans.</a> Genome Biol. 2 (1), RESEARCH0002 (2001).</li><li>Kamath, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+functional+analysis+of+the+Caenorhabditis+elegans+genome+using+RNAi.">Systematic functional analysis of the Caenorhabditis elegans genome using RNAi.</a> Nature. 421 (6920), 231-237 (2003).</li><li>Rual, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+improving+Caenorhabditis+elegans+phenome+mapping+with+an+ORFeome-based+RNAi+library.">Toward improving Caenorhabditis elegans phenome mapping with an ORFeome-based RNAi library.</a> Genome research. 14 (10B), 2162-2168 (2004).</li></ol>

C. elegans&#8217; genetic versatility, transparency, simple body plan and invariant cell lineage all make it an excellent model for the analysis of morphogenesis. This protocol describes how to combine standard genetic manipulations and imaging studies to take advantage of the 2 micron thin C. elegans excretory canals to study polarized membrane and intracellular lumen biogenesis in a single cell tube.
Labeling 
The C. elegans excretory canals c...

All internal organs are composed of tubes, crucial for their many different functions, such as the transport and exchange of gases, liquids and nutrients and the excretion of metabolic waste. Their polarized character, with distinct apical and lumenal membranes, is adapted to these specific functions, and defects in the biogenesis of their endo- and plasma membrane systems are a frequent cause of human disease1,2. The majority of tubes of the vasculature and of internal organs are multicellular and form a lumen intercellularly; however, unicellular tubes, which form the lumen intracellularly, can, for example, represent as much as 30&#8211;50% of human capillary beds2. The polarized membranes of multi- and unicellular tubes are similar in composition, although their microdomains may differ based on the tube&#8217;s specific function (e.g., excretory canal canaliculi versus intestinal microvilli in Caenorhabditis elegans; see accompanying paper on C. elegans intestinal tubulogenesis)3. The principles of polarized membrane biogenesis and tubulogenesis are conserved among metazoans, and a similar molecular machinery directs them1,2,4.
The C. elegans excretory system consists of five cells: the excretory cell (EC), duct cell (DC), pore cell (PC) and two gland cells. Ablation of the EC, DC or PC causes fluid accumulation in the body cavity and the animals die at an early larval stage5. Intriguingly, these three unicellular tubes create their lumens in three different ways: by cell hollowing (EC); cell wrapping coupled with autocellular junction formation (PC); and by cell wrapping coupled with autofusion (DC); different mechanisms of lumen morphogenesis that are all phylogenetically conserved6,7. The EC, located at the left lateral side of the posterior pharyngeal bulb, sends out two lateral extensions from which the four canals branch out to extend anteriorly and posteriorly (on both the right and left side) to the tip of the worm&#8217;s nose and tail, respectively (Figure 1)5,6,8. The EC extends from approximately 1 &#181;m to 2 x 1,000 &#181;m, making it the largest cell in the animal. On a subcellular level, the excretory canal is a simple tube, generated from a basal membrane directed towards the pseudocoelom, and tunneled by a lumenal membrane (endotube). The canal lumenal membrane connects to the duct lumenal membrane at its only intercellular junction; the canals are otherwise junctionless along their lengths (Figure 1). The excretory canal lumenal membrane and its submembraneous cytoskeleton are apical, defined by their molecular composition that resembles the composition of the apical membrane and submembraneous cytoskeleton of multicellular tubes, such as the intestine, and of other (e.g., flat) epithelia. Cytoplasmic organelles, including endosomal vesicular and other (e.g., Golgi) endomembranes are distributed along the length of the canal. In addition, multiple canalicular vesicles - either connected to the lumenal membrane, and/or interconnected, or isolated - are threaded through the canal cytoplasm7,8,9,10. This dynamic plasma-membrane/canalicular connection further expands the canal&#8217;s membrane system and contributes to both lumen morphogenesis and osmoregulation10. The excretory canal thus consists almost entirely of endo- and plasma membranes, providing an excellent model for the analysis of polarized membrane biogenesis and the regulation of the endo- to plasma membrane interface. The dramatic expansion of the apical membrane during canal morphogenesis - in this single-cell system coincident with lumen extension &#8211; also allows to analyze the architectural problems arising by the need to stabilize and center an intracellular lumenal membrane. This protocol focuses on the analysis of the canal tube&#8217;s and lumen&#8217;s structural morphogenesis and the intracellular membrane dynamics required for this process rather than on the signals that direct the cell movements that generate the EC&#8217;s position in the excretory system and construct its intricate connections to other cellular elements (reviewed in6).
A further advantage of the C. elegans single-cell canal system for the analysis of polarized membrane and intracellular lumen biogenesis is its ability to separate, through developmental time, the generation of different components of its membranes and junctions. The EC is born at the time of ventral closure and settles ventro-laterally of the pharynx during mid embryogenesis5,6,8, during which time lateral canal extension and branching occur. This is followed by anterior-posterior canal extension during late embryogenesis, a process that continues into the L1-larval stage (Figure 1). In a newly hatched larva, the posterior canal tip reaches approximately the middle of the worm, fully extending to the tail at the end of the L1 stage, after which time the canal elongates along with the worm8. Active canal growth at a speed exceeding that of the animal&#8217;s growth thus ends at the first larval stage, however, further growth occurs in parallel with the growth of the whole animal during the additional larval stages (L2&#8211;4). This setting provides the opportunity to analyze different steps of de novo polarized membrane biogenesis independent of polarized cell division or migration. Moreover, it permits the separation of this process from the assembly of junctions (which occur in the embryo before lumen initiation); their exact requirement in membrane polarization is still an open question in the polarity field. Finally, it uniquely separates apical from basal membrane expansion, the latter process preceding the former in the excretory canals10. The C. elegans excretory canal model is therefore a particularly informative complement to the intestinal model which shares a number of these advantages for the analysis of polarized membrane biogenesis but executes it in a multicellular setting (see the accompanying paper on intestinal tubulogenesis3).
Although wild-type canals are ultrathin tubules in this tiny worm, their lumens can be visualized directly by Nomarski optics in this transparent animal. In fact, mutant cystic canal morphologies can be characterized in unlabeled animals using low magnification dissecting microscopy, which has been used to great effect in forward genetic screens to identify genes involved in tubulogenesis11. Improved visualization of the morphology of canals and distinction of their polarized membranes, cytoskeletal components, different intracellular organelles and other subcellular structures, however, requires labeling and higher power fluorescent dissecting and confocal microscopy. Although the canals&#8217; fine structure poses a number of difficulties for labeling and microscopy, membranes and subcellular components can be distinguished via the specific molecules unique to each compartment, and animals can be safely mounted for microscopy if certain precautions are taken to avoid introducing artifacts (see Protocol and Discussion). Labeling can be done by immunohistochemistry in fixed specimens, or by generating transgenic worms expressing fluorescent fusion proteins under the control of their own or excretory canal-specific promoters for in vivo imaging. This protocol describes the latter labeling technique (see the accompanying paper on intestinal tubulogenesis for antibody staining3).
The ability to combine in vivo loss- or gain-of-function studies with in vivo imaging analysis at the single cell level throughout development makes the C. elegans excretory canal a particularly strong model for the molecular and cellular analysis of unicellular tubulogenesis. Forward or reverse genetic screens can be performed starting with a wild-type or labeled transgenic animal to identify canal morphogenesis phenotypes (for instance, cysts) and their underlying gene defects. Alternatively, such screens can start with a mutant phenotype (e.g., a cystic canal) and identify suppressors or enhancers of this phenotype to identify genes that functionally interact with the gene causing the mutant phenotype. The genetic defect causing the mutant phenotype can induce a loss (e.g., via gene deletion) or a gain (e.g., via an activating mutation or via the introduction of excess gene copies) of the investigated function. Forward mutagenesis or systematic RNAi screens are without preconceptions on gene function and permit the unbiased identification of genes involved in the function of interest. Given the availability of genome-wide RNAi feeding libraries, almost every gene can be easily knocked down by RNAi in C. elegans, such that any single gene of interest or any group of genes (e.g., in targeted screens) can also be rapidly probed for their effect in a reverse genetics approach. To demonstrate a possible combination of approaches, we here describe a targeted RNAi interaction screen, starting with a gain-of-function cystic excretory canal mutant, labeled with cytoplasmic canal green fluorescent protein (GFP). The mutant phenotype was generated by overexpression of erm-1, a highly conserved C. elegans ortholog of the membrane-actin linker family Ezrin-Radixin-Moesin (ERM), which has been implicated in lumen morphogenesis and membrane organization in many species12. C. elegans ERM-1 localizes to lumenal membranes of internal organs, such as the excretory canal and intestine, and is required for lumen formation in both13. ERM-1 overexpression recruits excess actin and vesicles to the canal lumenal membrane, increasing flux into the lumen and generating a short cystic canal and a crimped lumenal membrane with thickened actin undercoat9. The protocol describes how to generate transgenic strains with excretory-canal-expressed labeled fusion proteins (or other proteins); how to perform targeted RNAi screens starting with such strains, to identify modifiers of a canal phenotype; and how to visually analyze the results of such screens by fluorescence dissecting and confocal microscopy, including simple ways to quantify informative tubulogenesis phenotypes. Alternative labeling techniques and the details of RNAi, adjusted to the often lethal tubulogenesis genes, can be found in the accompanying paper on intestinal tubulogenesis3. All methods can be used in various combinations for the investigation of other questions on canal tubulogenesis.

<table border="0" cellpadding="0" cellspacing="0" width="983">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:811px;">Cloning</td>
			<td style="width:173px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Plasmid pPD95.75</td>
			<td>Addgene</td>
			<td>Cat. No. 37464</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PCR Kit</td>
			<td>Qiagen</td>
			<td>Cat. No. 27106</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ligation kit</td>
			<td>New England Biolabs</td>
			<td>Cat. No. E2611L</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DNA marker</td>
			<td>Thermo Scientific</td>
			<td>Cat. No. SM1331</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Agarose DNA grade</td>
			<td>Fisher Scientific</td>
			<td>Cat. No. BP164-100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Competent cells</td>
			<td>New England Biolabs</td>
			<td>Cat. No. C2987H</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tris</td>
			<td>Fisher Scientific</td>
			<td>Cat. No. BP154-1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EDTA</td>
			<td>Sigma</td>
			<td>Cat. No. ED-1KG</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Acetic acid</td>
			<td>Fisher Scientific</td>
			<td>Cat. No. A38S-500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ethidium bromide</td>
			<td>Fisher Scientific</td>
			<td>Cat. No. BP1302-10</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Equipments</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PCR machine&#160;</td>
			<td>MJ Research</td>
			<td>Cat. No. PTG-200&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Centrifuge</td>
			<td>Eppendorf&#160;</td>
			<td>Cat. No. 5415C</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Water Bath</td>
			<td>Precision Scientific&#160;</td>
			<td>Cat. No. 666A3&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gel running instrument</td>
			<td>Fisher Scientific</td>
			<td>Cat. No. 09-528-165</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gel running power supply</td>
			<td>Fisher Scientific</td>
			<td>Cat. No. 45-000-465</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Molecular Imager&#160;Gel Doc XR System</td>
			<td>Bio-Rad</td>
			<td>Cat. No. 1708195EDU</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Nanodrop Spectrophotometer</td>
			<td>Thermo Scientific</td>
			<td>Cat. No. ND1000</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">C. elegans related1</td>
			<td></td>
			<td></td>
			<td>1see reference27 for standard C. elegans culture and maintenance procedures.</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">LB Medium and plates2</td>
			<td></td>
			<td></td>
			<td>2see reference24 for protocols.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tryptone&#160;</td>
			<td>Acros Organics</td>
			<td>Cat. no. 611845000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Yeast Extract</td>
			<td>BD Biosciences</td>
			<td>Cat. no. 212750</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NaCl</td>
			<td>Sigma</td>
			<td>Cat. no. S7653</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bacto Agar&#160;</td>
			<td>BD Biosciences</td>
			<td>Cat. no. 214040</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ampicillin</td>
			<td>Sigma</td>
			<td>Cat. no. A0116</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tetracycline</td>
			<td>Fisher Scientific</td>
			<td>Cat. no. BP912</td>
			<td></td>
		</tr>
		<tr height="23">
			<td colspan="3" height="23" style="height:23px;">M9 Medium2</td>
			<td></td>
			<td></td>
			<td>2see reference24 for protocols.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NaCl</td>
			<td>Sigma</td>
			<td>Cat. no. S7653</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">KH2PO4</td>
			<td>Sigma</td>
			<td>Cat. no. P0662</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Na2HPO4</td>
			<td>Sigma</td>
			<td>Cat. no. S7907</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">MgSO4</td>
			<td>Sigma</td>
			<td>Cat. no. M2773</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">NGM plates 2</td>
			<td></td>
			<td></td>
			<td>2see reference24 for protocols.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NaCl</td>
			<td>Sigma</td>
			<td>Cat. no. S7653</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Peptone&#160;</td>
			<td>BD Biosciences</td>
			<td>Cat. no. 211677</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tryptone&#160;</td>
			<td>Acros Organics</td>
			<td>Cat. no. 611845000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bacto Agar&#160;</td>
			<td>BD Biosciences</td>
			<td>Cat. no. 214040</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">MgSO4</td>
			<td>Sigma</td>
			<td>Cat. no. M2773</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">CaCl2</td>
			<td>Sigma</td>
			<td>Cat. no. C3881</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cholesterol&#160;</td>
			<td>Sigma</td>
			<td>Cat. no. C8667</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">K2HPO4&#160;</td>
			<td>Sigma</td>
			<td>Cat. no. P3786</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">KH2PO4</td>
			<td>Sigma</td>
			<td>Cat. no. P0662</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">RNAi plates3</td>
			<td></td>
			<td></td>
			<td>3see reference60 for protocols.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NaCl</td>
			<td>Sigma</td>
			<td>Cat. no. S7653</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Peptone&#160;</td>
			<td>BD Biosciences</td>
			<td>Cat. no. 211677</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tryptone&#160;</td>
			<td>Acros Organics</td>
			<td>Cat. no. 611845000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bacto Agar&#160;</td>
			<td>BD Biosciences</td>
			<td>Cat. no. 214040</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">MgSO4</td>
			<td>Sigma</td>
			<td>Cat. no. M2773</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">CaCl2</td>
			<td>Sigma</td>
			<td>Cat. no. C3881</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cholesterol&#160;</td>
			<td>Sigma</td>
			<td>Cat. no. C8667</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">K2HPO4&#160;</td>
			<td>Sigma</td>
			<td>Cat. no. P3786</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">KH2PO4</td>
			<td>Sigma</td>
			<td>Cat. no. P0662</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">IPTG&#160;</td>
			<td>US Biological</td>
			<td>Cat. no. I8500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Carbenicillin</td>
			<td>Fisher Scientific</td>
			<td>Cat. no. BP2648</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NaOH</td>
			<td>Fisher Scientific</td>
			<td>Cat. no. SS266-1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium hypochlorite</td>
			<td>Fisher Scientific</td>
			<td>Cat. no. 50371500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bacteria</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">OP50 bacteria</td>
			<td colspan="2">CGC</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HT115 bacteria</td>
			<td colspan="2">CGC</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Genome-wide RNAi libraries</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Ahringer genome-wide RNAi feeding library (ref29,49)</td>
			<td>Source BioScience</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">C. elegans ORF-RNAi feeding library (ref50)</td>
			<td>Source BioScience</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Imaging related</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lidocaine</td>
			<td>MP Biomedicals,LLG</td>
			<td>Cat. no. 193917</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vacuum Grease Silicone</td>
			<td>Beckman</td>
			<td>Cat. no. 335148</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microscope slides&#160;</td>
			<td>Fisher Scientific</td>
			<td>Cat. no. 4448</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microscope coverslips (22&#215;22-1&#65289;</td>
			<td>Fisher Scientific</td>
			<td>Cat. no. 12-542-B</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tissue culture plate, 6 well&#160;</td>
			<td>Corning Inc.</td>
			<td>Cat. no. 08-772-33</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SMZ-U dissecting microscope (Nikon)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:811px;">SZX12 dissecting microscope (Olympus), equipped with a high-power stereo fluorescence attachment (Kramer Scientific), CCD camera with Q capture software and X-Cite fluorescent lamp (Photonic Solutions).</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TCS SL Laser-scanning confocal microscope (Leica Microsystem)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">C2 laser-scanning confocal mounted on an ECLIPSE Ti-E inverted microscope (Nikon)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

the c. elegans excretory canal as a model for intracellular lumen morphogenesis and in vivo polarized membrane biogenesis in a single cell: labeling by gfp-fusions, rnai interaction screen and imaging

The four C. elegans excretory canals are narrow tubes extended through the length of the animal from a single cell, with almost equally far extended intracellular endotubes that build and stabilize the lumen with a membrane and submembraneous cytoskeleton of apical character. The excretory cell expands its length approximately 2,000 times to generate these canals, making this model unique for the in vivo assessment of de novo polarized membrane biogenesis, intracellular lumen morphogenesis and unicellular tubulogenesis. The protocol presented here shows how to combine standard labeling, gain- and loss-of-function genetic or RNA interference (RNAi)-, and microscopic approaches to use this model to visually dissect and functionally analyze these processes on a molecular level. As an example of a labeling approach, the protocol outlines the generation of transgenic animals with fluorescent fusion proteins for live analysis of tubulogenesis. As an example of a genetic approach, it highlights key points of a visual RNAi-based interaction screen designed to modify a gain-of-function cystic canal phenotype. The specific methods described are how to: label and visualize the canals by expressing fluorescent proteins; construct a targeted RNAi library and strategize RNAi screening for the molecular analysis of canal morphogenesis; visually assess modifications of canal phenotypes; score them by dissecting fluorescence microscopy; characterize subcellular canal components at higher resolution by confocal microscopy; and quantify visual parameters. The approach is useful for the investigator who is interested in taking advantage of the C. elegans excretory canal for identifying and characterizing genes involved in the phylogenetically conserved processes of intracellular lumen and unicellular tube morphogenesis.

This protocol describes how to use the C. elegans excretory canals to visually and molecularly analyze unicellular tubulogenesis and intracellular lumen morphogenesis in a single cell. During their extension from the time of mid-embryogenesis to adulthood, the four excretory canals continue to expand their basolateral and apical/lumenal membranes together with their canalicular and endosomal endomembrane system, providing a unique model for the in vivo analysis of de...

Watch this Scientific Journal Video about The C. elegans Excretory Canal as a Model for Intracellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis in a Single Cell: labeling by GFP-fu

The C. elegans Excretory Canal as a Model for Intracellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis in a Single Cell: labeling by GFP-fusions, RNAi Interaction Screen and Imaging

The C. elegans excretory canal is a unique single-cell model for the visual in vivo analysis of de novo polarized membrane biogenesis. This protocol describes a combination of standard genetic/RNAi and imaging approaches, adaptable for the identification and characterization of molecules directing unicellular tubulogenesis, and apical membrane and lumen biogenesis.

The C. elegans Excretory Canal as a Model for Intracellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis in a Single Cell: labeling by GFP-fusions, RNAi Interaction Screen and Imaging

The four C. elegans excretory canals are narrow tubes extended through the length of the animal from a single cell, with almost equally far extended ...

Research

JoVE Journal

Developmental Biology

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration ...

Spatial and Temporal Analysis of Active ERK in the C. elegans Germline

Spatial and Temporal Analysis of Active ERK in the C. elegans Germline

The evolutionarily conserved&#160;extracellular signal transducing RTK-RAS-ERK pathway is an important kinase-signaling cascade that controls multiple ...

Drug Treatment and In Vivo Imaging of Osteoblast-Osteoclast Interactions in a Medaka Fish Osteoporosis Model

Drug Treatment and In Vivo Imaging of Osteoblast-Osteoclast Interactions in a Medaka Fish Osteoporosis Model

Bone-forming osteoblasts interact with bone-resorbing osteoclasts to coordinate the turnover of bone matrix and to control skeletal homeostasis. Medaka ...

A Novel Mammary Fat Pad Transplantation Technique to Visualize the Vessel Generation of Vascular Endothelial Stem Cells

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper ...

The C. elegans Intestine As a Model for Intercellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis at the Single-cell Level: Labeling by Antibody Staining, RNAi Loss-of-function Analysis and Imaging

The C. elegans Intestine As a Model for Intercellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis at the Single-cell Level: Labeling by Antibody Staining, RNAi Loss-of-function Analy

Multicellular tubes, fundamental units of all internal organs, are composed of polarized epithelial or endothelial cells, with apical membranes lining the ...

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain ...

In Vivo Imaging of Muscle-tendon Morphogenesis in Drosophila Pupae

In Vivo Imaging of Muscle-tendon Morphogenesis in Drosophila Pupae

Muscles together with tendons and the skeleton enable animals including humans to move their body parts. Muscle morphogenesis is highly conserved from ...

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C (UV-C) Damage

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C UV-C Damage

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational ...

A Semi-high-throughput Imaging Method and Data Visualization Toolkit to Analyze C. elegans Embryonic Development

A Semi-high-throughput Imaging Method and Data Visualization Toolkit to Analyze C. elegans Embryonic Development

C. elegans is the premier system for the systematic analysis of cell fate specification and morphogenetic events during embryonic development. One ...

Visualizing Ocular Morphogenesis by Lightsheet Microscopy Using rx3:GFP Transgenic Zebrafish

Vertebrate eye development is a complex process that begins near the end of embryo gastrulation and requires the precise coordination of cell migration, ...