Name: 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
Uploaded: 2017-10-03T13:00:00
Description: Watch this Scientific Journal Video about The C. elegans Intestine As a Model for Intercellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis at the Single-cell Level: Labeling by Anti

We thank Mario de Bono (MRC Laboratory of Molecular Biology, Cambridge, UK), Kenneth J. Kemphues (Cornell University, Ithaca, USA), Michel Labouesse (Institut de Biologie Paris Seine, Universite&#769; Pierre et Marie Curie, Paris, France), Gr&#233;goire Michaux (Universit&#233; deRennes 1, Rennes, France) and the CGC, funded by NIH Office of Research Infrastructure Programs (P40 OD010440), for strains and antibodies. This work was supported by grants NIH GM078653, MGH IS 224570 and SAA 223809 to V.G.

1 . Labeling the C. elegans intestine
Note: See the accompanying paper by the authors on the analysis of excretory canal tubulogenesis2 for the construction of tissue specific fluorescent marker plasmids and the generation of transgenic animals, including discussions on transcriptional and translational fusion proteins (the latter required for the subcellular localization of a molecule of interest). These procedures can be adapted by using specific promoters to d...

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	<li>Bryant, D. M., Mostov, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+cells+to+organs:+building+polarized+tissue.">From cells to organs: building polarized tissue.</a> Nat. Rev. Mol. Cell Biol. 9 (11), 887-901 (2008).</li><li>Zhang, N., Membreno, E., Raj, S., Zhang, H., Khan, L. A., Gobel, V. <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+excretory+canal+as+a+model+for+intracellular+lumen+morphogenesis+and+in+vivo+polarized+membrane+biogenesis+in+a+single+cell.">The C. elegans excretory canal as a model for intracellular lumen morphogenesis and in vivo polarized membrane biogenesis in a single cell.</a> JoVE. , (2017).</li><li>Rodriguez-Boulan, E., Macara, I. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organization+and+execution+of+the+epithelial+polarity+programme.">Organization and execution of the epithelial polarity programme.</a> Nat. Rev. Mol. Cell Biol. 15 (4), 225-242 (2014).</li><li>Mellman, I., Nelson, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coordinated+protein+sorting,+targeting+and+distribution+in+polarized+cells.">Coordinated protein sorting, targeting and distribution in polarized cells.</a> Nat. Rev. Mol. Cell Biol. 9 (11), 833-845 (2008).</li><li>Nelson, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptation+of+core+mechanisms+to+generate+cell+polarity.">Adaptation of core mechanisms to generate cell polarity.</a> Nature. 422, 766-774 (2003).</li><li>Goldstein, B., Macara, I. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+PAR+Proteins:+Fundamental+Players+in+Animal+Cell+Polarization.">The PAR Proteins: Fundamental Players in Animal Cell Polarization.</a> Dev. Cell. 13 (5), 609-622 (2007).</li><li>Rodriguez-Boulan, E., Kreitzer, G., Musch, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organization+of+vesicular+trafficking+in+epithelia.">Organization of vesicular trafficking in epithelia.</a> Nat. Rev. Mol. Cell Biol. 6 (3), 233-247 (2005).</li><li>Zegers, M. M., O'Brien, L. E., Yu, W., Datta, A., Mostov, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial+polarity+and+tubulogenesis+in+vitro.">Epithelial polarity and tubulogenesis in vitro.</a> Trends Cell Biol. 13 (4), 169-176 (2003).</li><li>Tepass, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+apical+polarity+protein+network+in+Drosophila+epithelial+cells:+regulation+of+polarity,+junctions,+morphogenesis,+cell+growth,+and+survival.">The apical polarity protein network in Drosophila epithelial cells: regulation of polarity, junctions, morphogenesis, cell growth, and survival.</a> Annu. Rev. Cell Dev. Biol. 28, 655-685 (2012).</li><li>Shivas, J. M., Morrison, H. A., Bilder, D., Skop, 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=Polarity+and+endocytosis:+reciprocal+regulation.">Polarity and endocytosis: reciprocal regulation.</a> Trends Cell Biol. 20 (8), 445-452 (2010).</li><li>Bryant, D. 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+molecular+network+for+de+novo+generation+of+the+apical+surface+and+lumen.">A molecular network for de novo generation of the apical surface and lumen.</a> Nat. Cell Biol. 12 (11), 1035-1045 (2010).</li><li>Zhang, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apicobasal+domain+identities+of+expanding+tubular+membranes+depend+on+glycosphingolipid+biosynthesis.">Apicobasal domain identities of expanding tubular membranes depend on glycosphingolipid biosynthesis.</a> Nat. Cell Biol. 13 (10), 1189-1201 (2011).</li><li>Zhang, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clathrin+and+AP-1+regulate+apical+polarity+and+lumen+formation+during+C.+elegans+tubulogenesis.">Clathrin and AP-1 regulate apical polarity and lumen formation during C. elegans tubulogenesis.</a> Development. 139 (11), 2071-2083 (2012).</li><li>Asan, A., Raiders, S. A., Priess, J. R. <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+the+C.+elegans+Intestine+Involves+Axon+Guidance+Genes.">Morphogenesis of the C. elegans Intestine Involves Axon Guidance Genes.</a> PLoS Genet. 12 (4), e1005950 (2016).</li><li>Leung, B., Hermann, G. J., Priess, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organogenesis+of+the+Caenorhabditis+elegans+intestine.">Organogenesis of the Caenorhabditis elegans intestine.</a> Dev. Biol. 216 (1), 114-134 (1999).</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=Alimentary+system,+intestine.">Alimentary system, intestine.</a> WormAtlas. , (2009).</li><li>Sulston, J. E., Horvitz, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Post-embryonic+cell+lineages+of+the+nematode,+Caenorhabditis+elegans.">Post-embryonic cell lineages of the nematode, Caenorhabditis elegans.</a> Dev. Biol. 56 (1), 110-156 (1977).</li><li>Rasmussen, J. P., English, K., Tenlen, J. R., Priess, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Notch+signaling+and+morphogenesis+of+single-cell+tubes+in+the+C.+elegans+digestive+tract.">Notch signaling and morphogenesis of single-cell tubes in the C. elegans digestive tract.</a> Dev. Cell. 14 (4), 559-569 (2008).</li><li>Maduro, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gut+development+in+C.+elegans.">Gut development in C. elegans.</a> Seminars in cell &amp; developmental biology. , (2017).</li><li>McGhee, 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=The+Caenorhabditis+elegans+intestine.">The Caenorhabditis elegans intestine.</a> Wiley Interdiscip. Rev. Dev. Biol. 2 (3), 347-367 (2013).</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>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>Brenner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genetics+of+Caenorhabditis+elegans.">The genetics of Caenorhabditis elegans.</a> Genetics. 77, 71-94 (1974).</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>Shakes, D. C., Miller, D. M., Nonet, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunofluorescence+microscopy.">Immunofluorescence microscopy.</a> Methods Cell Biol. 107, 35-66 (2012).</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>Simmer, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+the+Putative+RNA-Directed+RNA+Polymerase+RRF-3+Makes+C.+elegans+Hypersensitive+to+RNAi.">Loss of the Putative RNA-Directed RNA Polymerase RRF-3 Makes C. elegans Hypersensitive to RNAi.</a> Curr. Biol. 12 (15), 1317-1319 (2002).</li><li>Kennedy, S., Wang, D., Ruvkun, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+conserved+siRNA-degrading+RNase+negatively+regulates+RNA+interference+in+C.+elegans.">A conserved siRNA-degrading RNase negatively regulates RNA interference in C. elegans.</a> Nature. 427 (6975), 645-649 (2004).</li><li>Maddox, A. S., Maddox, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+imaging+of+cellular+processes+in+Caenorhabditis+elegans.">High-resolution imaging of cellular processes in Caenorhabditis elegans.</a> Methods Cell Biol. 107, 1-34 (2012).</li><li>Netherlands, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Nomarski Differential Interference Contrast Microscopy. , (2008).</li><li>Bates, M., Jones, S. A., 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=Stochastic+optical+reconstruction+microscopy+(STORM):+a+method+for+superresolution+fluorescence+imaging.">Stochastic optical reconstruction microscopy (STORM): a method for superresolution fluorescence imaging.</a> Cold Spring Harb. Protoc. 6, 498-520 (2013).</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, 356-369 (2002).</li><li>Lee, Y. U., Son, M., Kim, J., Shim, Y. H., Kawasaki, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CDC-25.2,+a+C.+elegans+ortholog+of+cdc25,+is+essential+for+the+progression+of+intestinal+divisions.">CDC-25.2, a C. elegans ortholog of cdc25, is essential for the progression of intestinal divisions.</a> Cell Cycle. 15 (5), 654-666 (2016).</li><li>Hall, D. H., Hartwieg, E., Nguyen, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modern+electron+microscopy+methods+for+C.+elegans.">Modern electron microscopy methods for C. elegans.</a> Methods Cell Biol. 107, 93-149 (2012).</li><li>Shi, A., Grant, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+analysis+of+recycling+endosomes+in+Caenorhabditis+elegans.">In vivo analysis of recycling endosomes in Caenorhabditis elegans.</a> Methods Cell Biol. 130, 181-198 (2015).</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>Kamath, R. <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>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 Res. 14 (10B), 2162-2168 (2004).</li></ol>

This protocol describes how to combine standard loss-of-function genetic/RNAi and imaging (labeling and microscopic) approaches to take advantage of the C. elegans intestinal epithelium as a model for the visual and molecular dissection of in vivo polarized membrane and lumen biogenesis.
Labeling
This protocol focuses on antibody staining. In situ labeling by antibodies is a highly specific alternative approach to labeling...

The generation of cellular and subcellular asymmetries, such as the formation of polarized membrane domains, is crucial for the morphogenesis and function of cells, tissues and organs1. Studies on polarized membrane biogenesis in epithelia remain a technical challenge, since directional changes in the allocation of subcellular components depend upon multiple consecutive and coincident extracellular and intracellular signals that are difficult to separate in most models and strongly depend on the model system. The model presented here - the single-layered Caenorhabditis elegans intestine - is a tissue of exquisite simplicity. Together with the single-cell C. elegans excretory canal (see accompanying paper on polarized membrane biogenesis in the C. elegans excretory canal)2, it provides several unique advantages for the identification and characterization of molecules required for polarized membrane biogenesis. The conservation of molecular polarity cues from yeast to man make this simple invertebrate organ an excellent &#34;in vivo tissue chamber&#34; to address questions on epithelial polarity that are of direct relevance to the human system, which is still far too complex to allow the visual dissection of these events at the single cell level in vivo.
Although multiple conserved polarity cues from (1) the extracelluar matrix, (2) the plasma membrane and its junctions, and (3) intracellular vesicular trafficking have been identified3, the underlying principles of their integration in the process of polarized epithelial membrane and tissue biogenesis is poorly understood4. The classical single-cell in vivo models (e.g.S. cerevisiae and the C. elegans zygote) have been instrumental in defining the principles of polarized cell division and anterior-posterior polarity and have identified critical membrane-associated polarity determinants (the small GTPases/CDC-42, the partitioning-defective PARs)5,6, but they depend upon unique symmetry breaking cues (bud scar, sperm entry) and lack junction-secured apicobasal membrane domains and, presumably, the corresponding intracellular apicobasal sorting machinery. Our current knowledge about the organization of polarized trafficking in epithelia, however, primarily relies on mammalian 2D monocultures7, which lack physiological extracellular and developmental cues that can change positions of membrane domains and directions of trafficking trajectories (a switch from 2D to 3D in vitro culture systems alone suffices to invert membrane polarity in MDCK (Madin-Darby canine kidney) cells)8. In vivo developmental studies on epithelial polarity in invertebrate model organisms were initially conducted in flat epithelia, for instance in the Drosophila melanogaster epidermis, where they identified the critical contribution of junction dynamics for polarized cell migration and cell sheet movement9, and of endocytic trafficking for polarity maintenance10. The 3D in vitro and in vivo analysis of lumen morphogenesis in tubular epithelia in MDCK cells and in the C. elegans intestine, respectively, have recently identified the requirement of intracellular trafficking for de novo (apical) domain and lumen biogenesis and positioning11,12,13. The thickness of tubular (versus flat) epithelial cells is an advantage for the 3D analysis of subcellular asymmetries since it permits a superior visual distinction of the apical-lumenal membrane, apico-lateral junctions, the lateral membrane, and the positions of intracellular organelles. To these visual advantages, the C. elegans model adds the in vivo setting, developmental axis, transparency, simplicity of body plan, invariant and defined cell lineage, analytical (genetic) and additional advantages described below.
C. elegans itself is a roundworm of tubular structure whose transparency and simple architecture make its likewise tubular internal organs directly accessible to the visual analysis of tube and lumen morphogenesis. The twenty cells of its intestine (21 or 22 cells on occasion)14 are derived from a single progenitor cell (E) and develop from a double-layered epithelium by one intercalation step into a bilaterally symmetrical tube of nine INT rings (four cells in the first ring; Figure 1 schematic)14,15,16. The intestine&#39;s lineage and tissue analysis, initially determined by Nomarski optics via nuclear identities17and subsequently by fluorescence microscopy via labeled membranes, has provided critical insights into its morphogenesis, in particular the cell-autonomous and cell-non-autonomous requirements for its directional cell divisions and movements (e.g., intercalation, right-left asymmetries, anterior and posterior tube rotation)14,18. Early endodermal cell specification and the gene regulatory network controlling the development of this clonal model organ are well characterized19,20. The focus here, however, is on the analysis of polarized membrane and lumen biogenesis in single tubular cells, and of the intracellular asymmetries of endomembranes, cytoskeletal structures and organelles that accompany this process. The analysis is facilitated by the simplicity of this tube, where all apical membranes (on the ultrastructural level distinguished by microvilli) face the lumen and all basal membranes face the outer tube surface, with lateral membranes contacting each other, separated from the apical membrane by junctions (Figure 1 schematic; see references (16,21) for the C. elegans-specific organization of tight and adherens junction components). Apical membrane biogenesis is thus coincident with lumen morphogenesis. Furthermore, the size of adult intestinal cells - the largest cells of this small animal (with exception of the excretory cell) - approximate the size of a mammalian cell, permitting the in vivo visual tracking of subcellular elements, e.g. vesicle trajectories, that is typically attempted in vitro in a culture dish.
For the purpose of this cellular and subcellular analysis, appropriate labeling is critical. Intestinal endo- or plasma-membrane domains, junctions, cytoskeletal structures, nuclei and other subcellular organelles can be visualized by labeling their specific molecular components. Many such components have been characterized and continue to be discovered (Table 1 gives a few examples and refers to resources). For instance, various molecules distinguishing the tubular and/or vesicular compartments of the intestinal endomembrane system, from the ER to the Golgi via post-Golgi vesicles to the plasma membrane, have been identified22. The specific proteins (as well as lipids and sugars) can either be labeled directly, or indirectly via binding proteins. This protocol focuses on in situ antibody staining of fixed specimens, one of two standard labeling techniques (see the accompanying paper on excretory canal tubulogenesis for a description of the other technique2 - in vivo labeling via fluorescent protein fusions - which is directly applicable to the intestine; Table 2 provides examples of intestine-specific promoters that can be used to drive expression of such fusion proteins to the intestine). Double- or multiple labeling with either approach, or with a combination of both plus additional chemical staining, allows greater in-depth visual resolution and the examination of spatial and temporal changes in co-localization and recruitment of specific molecules or of subcellular components (Figure 2). The fixation and staining procedures described in this protocol support preservation of green fluorescent protein (GFP) labeling during immunostaining procedures. For imaging, key points of the detection and characterization of tubulogenesis phenotypes via standard microscopic procedures (fluorescence dissecting and confocal microscopy) are described (Figure 3, 4). These can be extended to higher resolution imaging approaches, for instance superresolution microscopy and transmission electron microscopy (not described here).
A key strength of this system is the ability to analyze polarity in individual cells at different developmental stages, from embryogenesis through adulthood. For instance, apical membrane domain and lumen biogenesis can be tracked throughout development at the single-cell level via labeling with ERM-1, a highly conserved membrane-actin linker of the Ezrin-Radixin-Moesin family23,24. ERM-1 visualizes apical membrane biogenesis (1) during embryonic tube morphogenesis, when it occurs concomitantly with polarized cell division and migration (cells move apically around the lumen during intercalation)15; (2) during late embryonic and larval tube extension that proceeds in the absence of cell division or migration; and (3) in the adult intestine, where polarized membrane domains are maintained (Figure 1). In the expanding post-mitotic larval epithelium, de novo polarized membrane biogenesis can thus be separated from polarized tissue morphogenesis, which is not possible in most in vivo and in vitro epithelial polarity models, including those with single-cell resolution (e.g. the 3D MDCK cyst model8). With labeling for other components, this setting provides the opportunity (particularly at the L1 larval stage when cells have a higher cytoplasm/nucleus ratio) to distinguish those intracellular changes that are specific to polarized membrane biogenesis (e.g. the reorientation of trafficking trajectories) from those concomitantly required for polarized cell division and migration.
The genetic versatility of C. elegans is well known25and makes it a powerful model system for the molecular analysis of any biological question. A study on morphogenesis, for instance, can start with a wild-type strain, a transgenic strain where the structure of interest (e.g. a membrane) is labeled with a fluorescent marker, or with a loss- or gain-of-function mutant with a defect in this structure. A typical reverse genetic study may generate a mutant where the gene of interest is deleted in the germline (e.g. by a targeted deletion), modified by mutagenesis (typically producing point mutations with consequent loss, reduction, or gain in function of the gene), or where its transcript is reduced by RNAi. The ease of RNAi by feeding in C. elegans26 also lends itself to the design of targeted screens that examine a larger group of genes of interest. A genetic model organism&#39;s arguably greatest strength is the ability to conduct in vivo forward screens (e.g. mutagenesis, systematic or genome-wide RNAi screens) that permit an unbiased inquiry into the molecular cause for a phenotype of interest. For instance, an unbiased visual C. elegans RNAi tubulogenesis screen, starting with a transgenic animal with ERM-1-labeled apical membranes, discovered an intriguing reversible intestinal polarity conversion and ectopic lumen phenotype, used here as an example for this type of analysis. This screen identified the depletion of glycosphingolipids (GSLs; obligate membrane lipids, identified via their GLS-biosynthetic enzymes) and components of the vesicle coat clathrin and its AP-1 adaptor as the specific molecular defects causing this polarity conversion phenotype, thereby characterizing these trafficking molecules as in vivo cues for apical membrane polarity and lumen positioning12,13. When starting with a specific genetic mutation/morphogenesis phenotype, such screens (or single genetic/RNAi interaction experiments) can also examine functional interactions between two or multiple genes of interest (see accompanying paper on the excretory canal for an example of such an analysis)2. This protocol focuses on RNAi which, in addition to its ability to directly identify the gene whose loss causes the phenotype in forward screens, provides specific advantages for the analysis of morphogenesis. Since gene products directing morphogenesis often work in a dose-dependent fashion, RNAi is usually successful in generating a spectrum of phenotypes. The ability to generate informative partial-loss-of-function phenotypes also helps to address the problem that the majority of important tubulogenesis genes are essential and that their losses cause sterility and early embryonic lethality. This protocol includes conditional RNAi strategies to overcome this difficulty and suggests ways to optimize the generation of a broader spectrum of phenotypes, such as an allelic series produced by mutagenesis.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td>Antibody staining</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td>poly-L-lysine</td>
			<td>Sigma</td>
			<td>P5899</td>
			<td></td>
		</tr>
		<tr >
			<td>Methanol</td>
			<td>Fisher Scientific</td>
			<td>A452-4</td>
			<td></td>
		</tr>
		<tr >
			<td>Acetone</td>
			<td>Fisher Scientific</td>
			<td>A949SK-4</td>
			<td></td>
		</tr>
		<tr >
			<td>Tween</td>
			<td>Fisher Scientific</td>
			<td>50-213-612</td>
			<td></td>
		</tr>
		<tr >
			<td>Permount</td>
			<td>Fisher Scientific</td>
			<td>SP15-100</td>
			<td></td>
		</tr>
		<tr >
			<td>Powdered milk</td>
			<td>Sigma</td>
			<td>MT409-1BTL</td>
			<td></td>
		</tr>
		<tr >
			<td>Primary antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td>MH27 (mouse)</td>
			<td></td>
			<td></td>
			<td>Concentration: 1:20 Resources: Developmental Studies Hybridoma Bank.</td>
		</tr>
		<tr >
			<td>MH33 (mouse)</td>
			<td></td>
			<td></td>
			<td>Concentration: 1:10 Resources: Developmental Studies Hybridoma Bank.</td>
		</tr>
		<tr >
			<td>anti-ICB4 (rabbit)</td>
			<td></td>
			<td></td>
			<td>Concentration: 1:5 Resources: A gift from MariodeBono (Medical Research Council, England)</td>
		</tr>
		<tr >
			<td>anti-PAR-3 (rabbit)</td>
			<td></td>
			<td></td>
			<td>Concentration: 1:50 Resources: A gift from Kenneth J. Kemphues (Cornell University)</td>
		</tr>
		<tr >
			<td>Secondary antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td>Alexa Floor 568 (anti-rabbit)</td>
			<td>ABCam</td>
			<td>AB175471</td>
			<td>Concentration: 1:200</td>
		</tr>
		<tr >
			<td>Cy5 (anti-mouse)</td>
			<td>Life technologies</td>
			<td>A10524</td>
			<td>Concentration: 1:200</td>
		</tr>
		<tr >
			<td>TRITC (anti-rabbit)</td>
			<td>Invitrogen</td>
			<td>T2769</td>
			<td>Concentration: 1:200</td>
		</tr>
		<tr >
			<td>FITC (anti-mouse)</td>
			<td>Sigma</td>
			<td>F9006</td>
			<td>Concentration: 1:100</td>
		</tr>
		<tr >
			<td>Labeled chemicals</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td>Texas Red-Phalloidin</td>
			<td></td>
			<td></td>
			<td>Concentration: 1:100 Resources: Molecular Probes-T7471</td>
		</tr>
		<tr >
			<td>Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td>Vacuum Grease Silicone</td>
			<td>Beckman</td>
			<td>335148</td>
			<td></td>
		</tr>
		<tr >
			<td>Microscope slides</td>
			<td>Fisher Scientific</td>
			<td>4448</td>
			<td></td>
		</tr>
		<tr >
			<td>Microscope coverslips (22x22-1)</td>
			<td>Fisher Scientific</td>
			<td>12-542-B</td>
			<td></td>
		</tr>
		<tr >
			<td>C. elegans related</td>
			<td></td>
			<td></td>
			<td>see reference29 for standardC. elegans culture and maintenance procedures.</td>
		</tr>
		<tr >
			<td>LB Medium and plates</td>
			<td></td>
			<td></td>
			<td>see reference29 for protocols.</td>
		</tr>
		<tr >
			<td>Tryptone</td>
			<td>Acros Organics</td>
			<td>611845000</td>
			<td></td>
		</tr>
		<tr >
			<td>Yeast Extract</td>
			<td>BD Biosciences</td>
			<td>212750</td>
			<td></td>
		</tr>
		<tr >
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S7653</td>
			<td></td>
		</tr>
		<tr >
			<td>Bacto Agar</td>
			<td>BD Biosciences</td>
			<td>214040</td>
			<td></td>
		</tr>
		<tr >
			<td>Ampicillin</td>
			<td>Sigma</td>
			<td>A0116</td>
			<td></td>
		</tr>
		<tr >
			<td>Tetracycline</td>
			<td>Fisher Scientific</td>
			<td>BP912</td>
			<td></td>
		</tr>
		<tr >
			<td>M9 Medium</td>
			<td></td>
			<td></td>
			<td>see reference29 for protocols.</td>
		</tr>
		<tr >
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S7653</td>
			<td></td>
		</tr>
		<tr >
			<td>KH2PO4</td>
			<td>Sigma</td>
			<td>P0662</td>
			<td></td>
		</tr>
		<tr >
			<td>Na2HPO4</td>
			<td>Sigma</td>
			<td>S7907</td>
			<td></td>
		</tr>
		<tr >
			<td>MgSO4</td>
			<td>Sigma</td>
			<td>M2773</td>
			<td></td>
		</tr>
		<tr >
			<td>NGM plates</td>
			<td></td>
			<td></td>
			<td>see reference29 for protocols.</td>
		</tr>
		<tr >
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S7653</td>
			<td></td>
		</tr>
		<tr >
			<td>Peptone</td>
			<td>BD Biosciences</td>
			<td>211677</td>
			<td></td>
		</tr>
		<tr >
			<td>Tryptone</td>
			<td>Acros Organics</td>
			<td>611845000</td>
			<td></td>
		</tr>
		<tr >
			<td>Bacto Agar</td>
			<td>BD Biosciences</td>
			<td>214040</td>
			<td></td>
		</tr>
		<tr >
			<td>MgSO4</td>
			<td>Sigma</td>
			<td>M2773</td>
			<td></td>
		</tr>
		<tr >
			<td>CaCl2</td>
			<td>Sigma</td>
			<td>C3881</td>
			<td></td>
		</tr>
		<tr >
			<td>Cholesterol</td>
			<td>Sigma</td>
			<td>C8667</td>
			<td></td>
		</tr>
		<tr >
			<td>K2HPO4</td>
			<td>Sigma</td>
			<td>P3786</td>
			<td></td>
		</tr>
		<tr >
			<td>KH2PO4</td>
			<td>Sigma</td>
			<td>P0662</td>
			<td></td>
		</tr>
		<tr >
			<td>RNAi plates</td>
			<td></td>
			<td></td>
			<td>see reference30 for protocols.</td>
		</tr>
		<tr >
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S7653</td>
			<td></td>
		</tr>
		<tr >
			<td>Peptone</td>
			<td>BD Biosciences</td>
			<td>211677</td>
			<td></td>
		</tr>
		<tr >
			<td>Tryptone</td>
			<td>Acros Organics</td>
			<td>611845000</td>
			<td></td>
		</tr>
		<tr >
			<td>Bacto Agar</td>
			<td>BD Biosciences</td>
			<td>214040</td>
			<td></td>
		</tr>
		<tr >
			<td>MgSO4</td>
			<td>Sigma</td>
			<td>M2773</td>
			<td></td>
		</tr>
		<tr >
			<td>CaCl2</td>
			<td>Sigma</td>
			<td>C3881</td>
			<td></td>
		</tr>
		<tr >
			<td>Cholesterol</td>
			<td>Sigma</td>
			<td>C8667</td>
			<td></td>
		</tr>
		<tr >
			<td>K2HPO4</td>
			<td>Sigma</td>
			<td>P3786</td>
			<td></td>
		</tr>
		<tr >
			<td>KH2PO4</td>
			<td>Sigma</td>
			<td>P0662</td>
			<td></td>
		</tr>
		<tr >
			<td>IPTG</td>
			<td>US Biological</td>
			<td>I8500</td>
			<td></td>
		</tr>
		<tr >
			<td>Carbenicillin</td>
			<td>Fisher Scientific</td>
			<td>BP2648</td>
			<td></td>
		</tr>
		<tr >
			<td>NaOH</td>
			<td>Fisher Scientific</td>
			<td>SS266-1</td>
			<td></td>
		</tr>
		<tr >
			<td>Sodium hypochlorite</td>
			<td>Fisher Scientific</td>
			<td>50371500</td>
			<td></td>
		</tr>
		<tr >
			<td>Bacteria</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td>OP50 bacteria</td>
			<td>CGC</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td>HT115 bacteria</td>
			<td>CGC</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td>Genome-wide RNAi libraries Ahringer genome-wide RNAi feeding library (ref 30,49,50)</td>
			<td>Source BioScience</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td>C. elegans ORF-RNAi feeding library (ref51)</td>
			<td>Source BioScience</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td>Imaging related</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td>Sodium azide</td>
			<td>Fisher Scientific</td>
			<td>BP9221-500</td>
			<td></td>
		</tr>
		<tr >
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td>dissecting microscope</td>
			<td>Nikon</td>
			<td>SMZ-U</td>
			<td></td>
		</tr>
		<tr >
			<td>dissecting microscope 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>Olympus</td>
			<td>SZX12</td>
			<td></td>
		</tr>
		<tr >
			<td>Laser-scanning confocal microscope</td>
			<td>Leica Microsystem</td>
			<td>TCS SL</td>
			<td></td>
		</tr>
		<tr >
			<td>laser-scanning confocal mounted on an ECLIPSE Ti-E inverted microscope</td>
			<td>Nikon</td>
			<td>C2</td>
			<td></td>
		</tr>
	</tbody>
</table>

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

Multicellular tubes, fundamental units of all internal organs, are composed of polarized epithelial or endothelial cells, with apical membranes lining the lumen and basolateral membranes contacting each other and/or the extracellular matrix. How this distinctive membrane asymmetry is established and maintained during organ morphogenesis is still an unresolved question of cell biology. This protocol describes the C. elegans intestine as a&#160;model for the analysis of polarized membrane biogenesis during tube morphogenesis, with emphasis on apical membrane and lumen biogenesis. The C. elegans twenty-cell single-layered intestinal epithelium is arranged into a simple bilaterally symmetrical tube, permitting analysis on a single-cell level. Membrane polarization occurs concomitantly with polarized cell division and migration during early embryogenesis, but de novo polarized membrane biogenesis continues throughout larval growth, when cells no longer proliferate and move. The latter setting allows one to separate subcellular changes that simultaneously mediate these different polarizing processes, difficult to distinguish in most polarity models. Apical-, basolateral membrane-, junctional-, cytoskeletal- and endomembrane components can be labeled and tracked throughout development by GFP fusion proteins, or assessed by in situ antibody staining. Together with the organism&#39;s genetic versatility, the C. elegans intestine thus provides a unique in vivo model for the visual, developmental, and molecular genetic analysis of polarized membrane and tube biogenesis. The specific methods (all standard) described here include how to: label intestinal subcellular components by antibody staining; analyze genes involved in polarized membrane biogenesis by loss-of-function studies adapted to the typically essential tubulogenesis genes; assess polarity defects during different developmental stages; interpret phenotypes by epifluorescence, differential interference contrast (DIC) and confocal microscopy; quantify visual defects. This protocol can be adapted to analyze any of the often highly conserved molecules involved in epithelial polarity, membrane biogenesis, tube and lumen morphogenesis.

This protocol describes how to molecularly analyze and visualize polarized membrane biogenesis and lumen morphogenesis in the C. elegans intestine, at the single cell and subcellular level. The twenty-cell single-layered C. elegans intestine is formed by directed cell division and migration during mid embryogenesis. At this time, polarized membrane domains become established, yet de novo polarized membrane biogenesis continues in the mature but expanding epithel...

Watch this Scientific Journal Video about The C. elegans Intestine As a Model for Intercellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis at the Single-cell Level: Labeling by Anti

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

The transparent C. elegans intestine can serve as an "in vivo tissue chamber" for studying apicobasal membrane and lumen biogenesis at the single-cell and subcellular level during multicellular tubulogenesis. This protocol describes how to combine standard labeling, loss-of-function genetic/RNAi and microscopic approaches to dissect these processes on a molecular level.

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

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

The overall goal of this combination of standard methods is to demonstrate how the C.elegans intestine can be used for the in vivo analysis of polarized membrane biogenesis and lumen morphogenesis at the single-cell and sub-cellular level. This method can help answer key questions in the polarity and biogenesis fields such as how apical versus basolateral membrane domains and other sub-cellular asymmetries are established and maintained during and after morphogenesis. The main advantage of this model system is that this transparent single layer of only 20 cells can survive in vivo tissue chamber to dissect single cell polarized membrane and lumen biogenesis.The techniques can be extended to the analysis of other C.Elegans morphogenesis phenotypes the focus here is on the analysis of membrane biogenesis, specifically on apical membrane and lumen biogenesis. The procedures include refined imaging techniques in fixed and alive animals to distinguish membrane domains and sub-cellular components and to identify lumen morphogenesis phenotypes in a C.elegans intestine. This video on intestinal tubulogenesis is accompanied by a video on excretory canal tubulogenesis mutually informative models where the in vivo analysis of polarized membrane biogenesis.With regard to labeling the generation of transgenic animals with fluorescently labeled fusion proteins for live imaging is described in the accompanying video on the analysis of excretory canal tubulogenesis. Such animals can be directly used in RNAi experiments. Third generation is typically the first step in this combination of procedures.This video starts with the alternative labeling method antibody staining for which animals must be fixed. The different labeling procedures can be combined with immunohistochemistry typically being used for the final imaging analysis. This protocol also has examples of intestine specific membrane markers promoters and other resources useful for both labeling procedures.After demonstrating immunohistochemistry this video uses a transgenic strain that labels the intestinal apical membrane with ERM-1 GFP to search for polarity and lumen morphogenesis phenotypes using RNAi. First prepare a slide to fix the worms. Pipette 30 microliters of poly-L-lysine onto a slide and then set a second slide onto the other and drop them together to spread the solution evenly onto both.Now peel apart the two slides and let them air dry for 30 minutes. Next place a flat metal block into a container filled with liquid nitrogen. Wash the plates with M9 solution and transfer about 10 microliters of M9 containing the worms.Alternatively pick the worms and add M9 to the slide. Once the worms are transferred gently drop a cover slip cross ways onto them such that the cover slip over hangs one edge. Then gently press the cover slip without making any lateral movements.Now immediately transfer the slide to the metal block in liquid nitrogen and let it freeze. After five minutes flick off the cover slip using the overhanging edge. Do this swiftly to get the proper crack of the cuticle.Next immerse the slide in a methanol filled glass Coplin jar at minus 20 degrees Celsius. After five minutes transfer the slide to an acetone filled glass Coplin jar for another five minutes at the same tempature. The slides can be stained directly or stored at minus 20 degrees Celsius.Remove the slide from acetone and let it air dry at room temperature. Next mark the location for a circular wall of petroleum jelly on the underside of the slide then build up the jelly wall. Do not rub off the jelly during this procedure.Next prepare a wet chamber in a bin with wet paper towels and place the slide onto a rack within. There should be no contact between the towels and slide or between adjacent slides. Now pipette enough PBS into the jelly circle to cover the area.Gently place a pipette tip at the edge of the circle and allow fluid to smoothly disperse over the worms. Then close the lid and wait five minutes. After five minutes tilt the slide and slowly aspirate the PBS taking care not to lose worms from the slide.Next carefully fill the jelly circle with freshly prepared blocking solution. And let the slide incubate for 15 minutes at room tempature. After 15 minutes replace the blocking solution with blocking solution containing the primary antibody.And incubate the slide at four degrees Celsius overnight. The next day remove the primary antibody and wash the slide by applying and removing blocking solution three times. Allow each block application to incubate for 10 minutes.Next add the secondary antibody diluted in blocking solution and incubate the slide for an hour at room temperature. Thereafter wash the slide two times with blocking solution and then once with PBS. Then remove as much of the PBS as possible without permitting the specimen to dry out and carefully remove the jelly around the specimen.Apply a drop of mounting medium onto the specimen followed by a cover slip and a nail polish seal. Then store the slides in the dark at four degrees Celsius. Transfer the RNAi library plate from minus 80 degrees Celsius to dry ice.Remove the sealing tape. And transfer the clone of interest onto selective auger. Then streak out the clone and grow the bacteria over night.Before returning the library plate to the minus 80 degrees Celsius freezer apply new sealing tape. The next day inoculate colonies from the plate into one milliliter aliquots of liquid medium containing ampicillin. Then shake them for about 14 hours at 37 degrees Celsius.The following day seed 200 microliters per clone onto separate RNAi plates. Let the plates dry and induce the RNAi production with an overnight incubation at room tempature. A day later transfer five L4 stage worms to each RNAi plate labeled with the specific gene that is targeted.Then transfer the plates to an incubator typically at 20 degrees Celsius. First examine control versus RNAi animals under bright light at low and high magnification. Next examine control versus RNAi animals under fluorescent light under the appropriate filters.Systematically scan a plate with RNAi animals from the upper left to the lower right of the entire plate searching for phenotypes. Look for animals of interest. For a closer examination of the selected RNAi phenotypes focus on the intestine and use the the zoom to assess tubulogenesis and lumen morphogenesis phenotypes.To examine the worms of interest under confocal microscopy first mount and immobilize them by hand make a thin circle of vacuum grease or petroleum jelly on a glass slide. The grease layer should be thin not thicker than 0.1 millimeter. Next add about 3.5 microliters of 10 millimolar sodium azide into the middle of the circle.Then swiftly pick worms of interest into the solution under a dissecting microscope before the solution dries. Then gently apply a cover slip and image the animals within 30 minutes. Use mild pressure and under the microscope verify that the worms are not floating in solution.Under the confocal microscope find the worms under low power and focus. Then use a 60 or 100X oil immersion objective to view and image the intestine. Next examine the animal under fluorescent light with the appropriate channel to select areas that display the cellular defect of interest.Move swiftly to avoid photo bleaching. Then switch to laser scanning and restrict the image to the intestine by setting scanning boundaries at its dorsal and ventral side. Capture an image series and later merge the images into a single projection image.This protocol describes how to molecularly analyze and visually dissect polarized membrane biogenesis in the developing C.elegans intestine where apical membrane and lumen morphogenesis coincide. Apical and basolateral membrane domains, membrane associated junctions and the intestinal lumen could be resolved and their relation to each other could be examined throughout development. These sub-cellular components could be labeled by fluorescently tagged antibodies or fluorescent fusion proteins for double or triple labeling as described in the accompanying paper.Gross lumen defects could be quickly scanned by using a dissecting fluorescence microscope. The apical membranes cytoskeleton linker ERM-1 coupled to GFP is a robustly expressed marker useful for a RNAi screen. An array of novel intestinal lumen morphogenesis and polarity phenotypes along with their underlying gene defects were uncovered when using this marker.Then higher magnification images acquired by a confocal laser scanning were used to more carefully examine the phenotypes to better understand the underlying defects. Some phenotypes could be quantified. Quantification was used to compare such defects and track their progression through development.After watching this video you should have a good understanding of how to label intestinal membrane and endo membrane compartments by immuno staining and how to analyze function RNAi phenotypes of polarized membrane and lumen biogenesis.

the-c-elegans-intestine-as-model-for-intercellular-lumen

Research

JoVE Journal

Developmental Biology

Imaging Cleared Embryonic and Postnatal Hearts at Single-cell Resolution

Single clonal tracing and analysis at the whole-heart level can determine cardiac progenitor cell behavior and differentiation during cardiac development, and allow for the study of the cellular and molecular basis of normal and abnormal cardiac morphogenesis. Recent emerging technologies of retrospective single clonal analyses make the study of cardiac morphogenesis at single cell resolution feasible. However, tissue opacity and light scattering of the heart as imaging depth is increased hinder whole-heart imaging at single cell resolution. To overcome these obstacles, a whole-embryo clearing system that can render the heart highly transparent for both illumination and detection must be developed. Fortunately, in the last several years, many methodologies for whole-organism clearing systems such as CLARITY, Scale, SeeDB, ClearT, 3DISCO, CUBIC, DBE, BABB and PACT have been reported. This lab is interested in the cellular and molecular mechanisms of cardiac morphogenesis. Recently, we established single cell lineage tracing via the ROSA26-CreERT2; ROSA26-Confetti system to sparsely label cells during cardiac development. We adapted several whole embryo-clearing methodologies including Scale and CUBIC (clear, unobstructed brain imaging cocktails and computational analysis) to clear the embryo in combination with whole mount staining to image single clones inside the heart. The heart was successfully imaged at single cell resolution. We found that Scale can clear the embryonic heart, but cannot effectively clear the postnatal heart, while CUBIC can clear the postnatal heart, but damages the embryonic heart by dissolving the tissue. The methods described here will permit the study of gene function at a single clone resolution during cardiac morphogenesis, which, in turn, can reveal the cellular and molecular basis of congenital heart defects.

We describe a protocol to volumetrically image fluorescent protein labeled cells deep inside intact embryonic and postnatal hearts. Utilizing tissue-clearing methods in combination with whole mount staining, single fluorescent protein-labeled cells inside an embryonic or postnatal heart can be imaged clearly and accurately.

Single clonal tracing and analysis at the whole-heart level can determine cardiac progenitor cell behavior and differentiation during cardiac development, ...

Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae

Many parallels exist between the Drosophila and mammalian hematopoietic systems, even though Drosophila lack the lymphoid lineage that characterize mammalian adaptive immunity. Drosophila and mammalian hematopoiesis occur in spatially and temporally distinct phases to produce several blood cell lineages. Both systems maintain reservoirs of blood cell progenitors with which to expand or replace mature lineages. The hematopoietic system allows Drosophila and mammals to respond to and to adapt to immune challenges. Importantly, the transcriptional regulators and signaling pathways that control the generation, maintenance, and function of the hematopoietic system are conserved from flies to mammals. These similarities allow Drosophila to be used to genetically model hematopoietic development and disease.
Here we detail assays to examine the hematopoietic system of Drosophila larvae. In particular, we outline methods to measure blood cell numbers and concentration, visualize a specific mature lineage in vivo, and perform immunohistochemistry on blood cells in circulation and in the hematopoietic organ. These assays can reveal changes in gene expression and cellular processes including signaling, survival, proliferation, and differentiation and can be used to investigate a variety of questions concerning hematopoiesis. Combined with the genetic tools available in Drosophila, these assays can be used to evaluate the hematopoietic system upon defined genetic alterations. While not specifically outlined here, these assays can also be used to examine the effect of environmental alterations, such as infection or diet, on the hematopoietic system.

Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae

Drosophila and mammalian hematopoietic systems share many common features, making Drosophila an attractive genetic model to study hematopoiesis. Here we demonstrate dissection and mounting of the major larval hematopoietic organ for immunohistochemistry. We also describe methods to assay various larval hematopoietic compartments including circulating hemocytes and sessile crystal cells.

Many parallels exist between the Drosophila and mammalian hematopoietic systems, even though Drosophila lack the lymphoid lineage that characterize ...

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 cellular and developmental processes principally via activation of ERK, the terminal kinase of the pathway. Tight regulation of ERK activity is essential for normal development and homeostasis; overly active ERK results in excessive cellular proliferation, while underactive ERK causes cell death. C. elegans is a powerful model system that has helped characterize the function and regulation of RTK-RAS-ERK signaling pathway during development. In particular, the RTK-RAS-ERK pathway is essential for C. elegans germline development, which is the focus of this method. Using antibodies specific to the active, diphosphorylated form of ERK (dpERK), the stereotypical localization pattern can be visualized within the germline. Because this pattern is both spatially and temporally controlled, the ability to reproducibly assay dpERK is useful to identify regulators of the pathway that affect dpERK signal duration and amplitude and thus germline development. Here we demonstrate how to successfully dissect, stain, and image dpERK within the C. elegans gonad. This method can be adapted for spatial localization of any signaling or structural protein in the C. elegans gonad, provided an antibody compatible with immunofluorescence is available.

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

We present an immunofluorescence&#160;imaging-based method for spatial and temporal localization of active ERK in the dissected C. elegans gonad. The protocol described here can be adapted for visualization of any signaling or structural protein in the C. elegans gonad, provided a suitable antibody reagent is available.

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

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.

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

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

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 and protect cellular identities. The conversion of germ cells into specific neurons in the nematode Caenorhabditis elegans (C. elegans) is a powerful tool for performing genetic screens in order to dissect regulatory pathways that safeguard established cell identities. Reprogramming of germ cells to a specific type of neurons termed ASE requires transgenic animals that allow broad over-expression of the Zn-finger transcription factor (TF) CHE-1. Endogenous CHE-1 is expressed exclusively in two head neurons and is required to specify the glutamatergic ASE neurons fate, which can easily be visualized by the gcy-5prom::gfp reporter. A trans gene containing the heat-shock promoter-driven che-1 gene expression construct allows broad mis-expression of CHE-1 in the entire animal upon heat-shock treatment. The combination of RNAi against the chromatin-regulating factor LIN-53 and heat-shock-induced che-1 over-expression leads to reprogramming of germ cell into ASE neuron-like cells. We describe here the specific RNAi procedure and appropriate conditions for heat-shock treatment of transgenic animals in order to successfully induce germ cell to neuron conversion.

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

This protocol describes how to study cellular processes during cell fate conversion in Caenorhabditis elegans in vivo. Using transgenic animals, allowing heat-shock promoter-driven overexpression of the neuron fate-inducing transcription factor CHE-1 and RNAi-mediated depletion of the chromatin-regulating factor LIN-53 germ cell to neuron reprogramming can be observed in vivo.

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

CRISPR-mediated Loss of Function Analysis in Cerebellar Granule Cells Using In Utero Electroporation-based Gene Transfer

Brain malformation is often caused by genetic mutations. Deciphering the mutations in patient-derived tissues has identified potential causative factors of the diseases. To validate the contribution of a dysfunction of the mutated genes to disease development, the generation of animal models carrying the mutations is one obvious approach. While germline genetically engineered mouse models (GEMMs) are popular biological tools and exhibit reproducible results, it is restricted by time and costs. Meanwhile, non-germline GEMMs often enable exploring gene function in a more feasible manner. Since some brain diseases (e.g., brain tumors) appear to result from somatic but not germline mutations, non-germline chimeric mouse models, in which normal and abnormal cells coexist, could be helpful for disease-relevant analysis. In this study, we report a method for the induction of CRISPR-mediated somatic mutations in the cerebellum. Specifically, we utilized conditional knock-in mice, in which Cas9 and GFP are chronically activated by the CAG (CMV enhancer/chicken &#223;-actin) promoter after Cre-mediated recombination of the genome. The self-designed single-guide RNAs (sgRNAs) and the Cre recombinase sequence, both encoded in a single plasmid construct, were delivered into cerebellar stem/progenitor cells at an embryonic stage using in utero electroporation. Consequently, transfected cells and their daughter cells were labeled with green fluorescent protein (GFP), thus facilitating further phenotypic analyses. Hence, this method is not only showing electroporation-based gene delivery into embryonic cerebellar cells but also proposing a novel quantitative approach to assess CRISPR-mediated loss-of-function phenotypes.

CRISPR-mediated Loss of Function Analysis in Cerebellar Granule Cells Using In Utero Electroporation-based Gene Transfer

Conventional loss-of-function studies of genes using knockout animals have often been costly and time-consuming. Electroporation-based CRISPR-mediated somatic mutagenesis is a powerful tool to understand gene functions in vivo. Here, we report a method to analyze knockout phenotypes in proliferating cells of the cerebellum.

Brain malformation is often caused by genetic mutations. Deciphering the mutations in patient-derived tissues has identified potential causative factors ...

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 animals to humans. Therefore, the powerful Drosophila model system can be used to study concepts of muscle-tendon development that can also be applied to human muscle biology. Here, we describe in detail how morphogenesis of the adult muscle-tendon system can be easily imaged in living, developing Drosophila pupae. Hence, the method allows investigating proteins, cells and tissues in their physiological environment. In addition to a step-by-step protocol with helpful tips, we provide a comprehensive overview of fluorescently tagged marker proteins that are suitable for studying the muscle-tendon system. To highlight the versatile applications of the protocol, we show example movies ranging from visualization of long-term morphogenetic events &#8211; occurring on the time scale of hours and days &#8211; to visualization of short-term dynamic processes like muscle twitching occurring on time scale of seconds. Taken together, this protocol should enable the reader to design and perform live-imaging experiments for investigating muscle-tendon morphogenesis in the intact organism.

In Vivo Imaging of Muscle-tendon Morphogenesis in Drosophila Pupae

Here, we present an easy-to-use and versatile method to perform live imaging of developmental processes in general and muscle-tendon morphogenesis in particular in living Drosophila pupae.

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

Assessing Lysosomal Alkalinization in the Intestine of Live Caenorhabditis elegans

The nematode Caenorhabditis elegans (C. elegans) is a model system that is widely used to study longevity and developmental pathways. Such studies are facilitated by the transparency of the animal, the ability to do forward and reverse genetic assays, the relative ease of generating fluorescently labeled proteins, and the use of fluorescent dyes that can either be microinjected into the early embryo or incorporated into its food (E. coli strain OP50) to label cellular organelles (e.g. 9-diethylamino-5H-benzo(a)phenoxazine-5-one and (3-{2-[(1H,1&#39;H-2,2&#39;-bipyrrol-5-yl-kappaN(1))methylidene]-2H-pyrrol-5-yl-kappaN}-N-[2-(dimethylamino)ethyl]propanamidato)(difluoro)boron). Here, we present the use of a fluorescent pH-sensitive dye that stains intestinal lysosomes, providing a visual readout of dynamic, physiological changes in lysosomal acidity in live worms. This protocol does not measure lysosomal pH, but rather aims to establish a reliable method of assessing physiological relevant variations in lysosomal acidity. cDCFDA is a cell-permeant compound that is converted to the fluorescent fluorophore 5-(and-6)-carboxy-2&#39;,7&#39;-dichlorofluorescein (cDCF) upon hydrolysis by intracellular esterases. Protonation inside lysosomes traps cDCF in these organelles, where it accumulates. Due to its low pKa of 4.8, this dye has been used as a pH sensor in yeast. Here we describe the use of cDCFDA as a food supplement to assess the acidity of intestinal lysosomes in C. elegans. This technique allows for the detection of alkalinizing lysosomes in live animals, and has a broad range of experimental applications including studies on aging, autophagy, and lysosomal biogenesis.

Assessing Lysosomal Alkalinization in the Intestine of Live Caenorhabditis elegans

A step-by-step guide to probe loss of lysosomal acidity in the intestine of C. elegans using the pH-sensitive vital dye 5(6)-carboxy-2&#39;,7&#39;-dichlorofluorescein diacetate (cDCFDA)

The nematode Caenorhabditis elegans (C. elegans) is a model system that is widely used to study longevity and developmental pathways. Such studies are ...

Cell Cycle Analysis in the C. elegans Germline with the Thymidine Analog EdU

Cell cycle analysis in eukaryotes frequently utilizes chromosome morphology, expression and/or localization of gene products required for various phases of the cell cycle, or the incorporation of nucleoside analogs. During S-phase, DNA polymerases incorporate thymidine analogs such as EdU or BrdU into chromosomal DNA, marking the cells for analysis. For C. elegans, the nucleoside analog EdU is fed to the worms during regular culture and is compatible with immunofluorescent techniques. The germline of C. elegans is a powerful model system for the studies of signaling pathways, stem cells, meiosis, and cell cycle because it is transparent, genetically facile, and meiotic prophase and cellular differentiation/gametogenesis occur in a linear assembly-like fashion. These features make EdU a great tool to study dynamic aspects of mitotically cycling cells and germline development. This protocol describes how to successfully prepare EdU bacteria, feed them to wild-type C. elegans hermaphrodites, dissect the hermaphrodite gonad, stain for EdU incorporation into DNA, stain with antibodies to detect various cell cycle and developmental markers, image the gonad and analyze the results. The protocol describes the variations in the method and analysis for the measurement of S-phase index, M-phase index, G2 duration, cell cycle duration, rate of meiotic entry, and rate of meiotic prophase progression. This method can be adapted to study the cell cycle or cell history in other tissues, stages, genetic backgrounds, and physiological conditions.

Cell Cycle Analysis in the C. elegans Germline with the Thymidine Analog EdU

An imaging-based method is described that can be used to identify S-phase and analyze cell cycle dynamics in the C. elegans hermaphrodite germline using the thymidine analog EdU. This method requires no transgenes and is compatible with immunofluorescent staining.

Cell cycle analysis in eukaryotes frequently utilizes chromosome morphology, expression and/or localization of gene products required for various phases ...