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 (Video) | JoVE Journal
Uploaded: 2017-10-03T14:00:00.000+00:00
Duration: 10 min 30 s
Description: Learn how to analyze unicellular tubulogenesis and polarized membrane biogenesis using the C. elegans excretory canal model. This method combines RNAi screening with live imaging to study intracellular lumen morphogenesis in vivo.

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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.

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The authors declare that they have no competing financial interests.

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><tbody><tr><td>&lt;strong&gt;Cloning&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Plasmid pPD95.75</td><td>Addgene</td><td>Cat. No. 37464</td><td></td></tr><tr><td>PCR Kit</td><td>Qiagen</td><td>Cat. No. 27106</td><td></td></tr><tr><td>Ligation kit</td><td>New England Biolabs</td><td>Cat. No. E2611L</td><td></td></tr><tr><td>DNA marker</td><td>Thermo Scientific</td><td>Cat. No. SM1331</td><td></td></tr><tr><td>Agarose DNA grade</td><td>Fisher Scientific</td><td>Cat. No. BP164-100</td><td></td></tr><tr><td>Competent cells</td><td>New England Biolabs</td><td>Cat. No. C2987H</td><td></td></tr><tr><td>Tris</td><td>Fisher Scientific</td><td>Cat. No. BP154-1</td><td></td></tr><tr><td>EDTA</td><td>Sigma</td><td>Cat. No. ED-1KG</td><td></td></tr><tr><td>Acetic acid</td><td>Fisher Scientific</td><td>Cat. No. A38S-500</td><td></td></tr><tr><td>Ethidium bromide</td><td>Fisher Scientific</td><td>Cat. No. BP1302-10</td><td></td></tr><tr><td>&lt;strong&gt;Equipments&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>PCR machine&amp;nbsp;</td><td>MJ Research</td><td>Cat. No. PTG-200&amp;nbsp;</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf&amp;nbsp;</td><td>Cat. No. 5415C</td><td></td></tr><tr><td>Water Bath</td><td>Precision Scientific&amp;nbsp;</td><td>Cat. No. 666A3&amp;nbsp;</td><td></td></tr><tr><td>Gel running instrument</td><td>Fisher Scientific</td><td>Cat. No. 09-528-165</td><td></td></tr><tr><td>Gel running power supply</td><td>Fisher Scientific</td><td>Cat. No. 45-000-465</td><td></td></tr><tr><td>Molecular Imager&amp;nbsp;Gel Doc XR System</td><td>Bio-Rad</td><td>Cat. No. 1708195EDU</td><td></td></tr><tr><td>Nanodrop Spectrophotometer</td><td>Thermo Scientific</td><td>Cat. No. ND1000</td><td></td></tr><tr><td>&lt;strong&gt;&lt;em&gt;C. elegans&lt;/em&gt; related&lt;sup&gt;1&lt;/sup&gt;&lt;/strong&gt;</td><td></td><td></td><td>&lt;sup&gt;1&lt;/sup&gt;see reference&lt;sup&gt;19&lt;/sup&gt; for standard C. elegans culture and maintenance procedures.</td></tr><tr><td>&lt;strong&gt;LB Medium and plates&lt;sup&gt;2&lt;/sup&gt;&lt;/strong&gt;</td><td></td><td></td><td>&lt;sup&gt;2&lt;/sup&gt;see reference&lt;sup&gt;19&lt;/sup&gt; for protocols.</td></tr><tr><td>Tryptone&amp;nbsp;</td><td>Acros Organics</td><td>Cat. no. 611845000</td><td></td></tr><tr><td>Yeast Extract</td><td>BD Biosciences</td><td>Cat. no. 212750</td><td></td></tr><tr><td>NaCl</td><td>Sigma</td><td>Cat. no. S7653</td><td></td></tr><tr><td>Bacto Agar&amp;nbsp;</td><td>BD Biosciences</td><td>Cat. no. 214040</td><td></td></tr><tr><td>Ampicillin</td><td>Sigma</td><td>Cat. no. A0116</td><td></td></tr><tr><td>Tetracycline</td><td>Fisher Scientific</td><td>Cat. no. BP912</td><td></td></tr><tr><td>&lt;strong&gt;M9 Medium&lt;sup&gt;2&lt;/sup&gt;&lt;/strong&gt;</td><td></td><td></td><td>&lt;sup&gt;2&lt;/sup&gt;see reference&lt;sup&gt;19&lt;/sup&gt; for protocols.</td></tr><tr><td>NaCl</td><td>Sigma</td><td>Cat. no. S7653</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>Cat. no. P0662</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>Cat. no. S7907</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>Cat. no. M2773</td><td></td></tr><tr><td>&lt;strong&gt;NGM plates &lt;sup&gt;2&lt;/sup&gt;&lt;/strong&gt;</td><td></td><td></td><td>&lt;sup&gt;2&lt;/sup&gt;see reference&lt;sup&gt;19&lt;/sup&gt; for protocols.</td></tr><tr><td>NaCl</td><td>Sigma</td><td>Cat. no. S7653</td><td></td></tr><tr><td>Peptone&amp;nbsp;</td><td>BD Biosciences</td><td>Cat. no. 211677</td><td></td></tr><tr><td>Tryptone&amp;nbsp;</td><td>Acros Organics</td><td>Cat. no. 611845000</td><td></td></tr><tr><td>Bacto Agar&amp;nbsp;</td><td>BD Biosciences</td><td>Cat. no. 214040</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>Cat. no. M2773</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td>Cat. no. C3881</td><td></td></tr><tr><td>Cholesterol&amp;nbsp;</td><td>Sigma</td><td>Cat. no. C8667</td><td></td></tr><tr><td>K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;&amp;nbsp;</td><td>Sigma</td><td>Cat. no. P3786</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>Cat. no. P0662</td><td></td></tr><tr><td>&lt;strong&gt;RNAi plates&lt;sup&gt;3&lt;/sup&gt;&lt;/strong&gt;</td><td></td><td></td><td>&lt;sup&gt;3&lt;/sup&gt;see reference&lt;sup&gt;21&lt;/sup&gt; for protocols.</td></tr><tr><td>NaCl</td><td>Sigma</td><td>Cat. no. S7653</td><td></td></tr><tr><td>Peptone&amp;nbsp;</td><td>BD Biosciences</td><td>Cat. no. 211677</td><td></td></tr><tr><td>Tryptone&amp;nbsp;</td><td>Acros Organics</td><td>Cat. no. 611845000</td><td></td></tr><tr><td>Bacto Agar&amp;nbsp;</td><td>BD Biosciences</td><td>Cat. no. 214040</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>Cat. no. M2773</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td>Cat. no. C3881</td><td></td></tr><tr><td>Cholesterol&amp;nbsp;</td><td>Sigma</td><td>Cat. no. C8667</td><td></td></tr><tr><td>K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;&amp;nbsp;</td><td>Sigma</td><td>Cat. no. P3786</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>Cat. no. P0662</td><td></td></tr><tr><td>IPTG&amp;nbsp;</td><td>US Biological</td><td>Cat. no. I8500</td><td></td></tr><tr><td>Carbenicillin</td><td>Fisher Scientific</td><td>Cat. no. BP2648</td><td></td></tr><tr><td>NaOH</td><td>Fisher Scientific</td><td>Cat. no. SS266-1</td><td></td></tr><tr><td>Sodium hypochlorite</td><td>Fisher Scientific</td><td>Cat. no. 50371500</td><td></td></tr><tr><td>&lt;strong&gt;Bacteria&lt;/strong&gt;</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>&lt;strong&gt;Genome-wide RNAi libraries&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Ahringer genome-wide RNAi feeding library (ref&lt;sup&gt;21,58,59&lt;/sup&gt;)</td><td>Source BioScience</td><td></td><td></td></tr><tr><td>C. elegans ORF-RNAi feeding library (ref&lt;sup&gt;60&lt;/sup&gt;)</td><td>Source BioScience</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Imaging related&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Lidocaine</td><td>MP Biomedicals,LLG</td><td>Cat. no. 193917</td><td></td></tr><tr><td>&lt;strong&gt;Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Vacuum Grease Silicone</td><td>Beckman</td><td>Cat. no. 335148</td><td></td></tr><tr><td>Microscope slides&amp;nbsp;</td><td>Fisher Scientific</td><td>Cat. no. 4448</td><td></td></tr><tr><td>Tissue culture plate, 6 well&amp;nbsp;</td><td>Corning Inc.</td><td>Cat. no. 08-772-33</td><td></td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>SMZ-U dissecting microscope (Nikon)</td><td></td><td></td><td></td></tr><tr><td>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><td>TCS SL Laser-scanning confocal microscope (Leica Microsystem)</td><td></td><td></td><td></td></tr><tr><td>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 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...

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 ...

The overall goal of this combination of standard procedures is to demonstrate how the C.elegans excretory canal can be used for the in vivo analysis of unicellular tube, intracellular lumen, and de novo polarized membrane biogenesis. This method can help answer key questions in the fields of tubulogenesis and polarity, such as how an extended intracellular lumen is established or which molecules are required to expand an apical membrane. The main advantage of this model system is that a huge 2000-fold expansion of an apical versus a basal lateral membrane can be visually checked in vivo in the single cell.Generally, individuals new to this model will struggle because the C.elegans excretory canal is very thin and sensitive to osmotic stress and to any manipulation required for imaging. This video on excretory canal tubulogenesis is accompanied by a video on intestinal tubulogenesis, mutually informative models for the in vivo analysis of polarized membrane biogenesis. With regard to labeling, the accompanying video on intestinal tubulogenesis describes the alternative labeling procedure, staining cellular components with fluorescently tagged antibodies.This technique is also applicable to the excretory canal. This video describes live imaging analysis by using animals with fluorescently labeled fusion proteins. How to construct a plasmid containing a gene of interest attached to GFP, and how to generate transgenic animals expressing a fusion protein is described in the text.The protocol also has examples of excretory canal-specific membrane markers, organelle markers, and promoters. Other resources for both labeling procedures are also provided. In this video a transgenic strain that labels the canal cytoplasm with GFP and has a cystic canal phenotype is screened for modifiers using RNAi.The phenotype is caused by over-expression of ERM-1, a lumen morphogenesis gene. From an overnight growth of RNAi expressing bacteria on ampicillin plus tetracycline plates, pick colonies and inoculate them into 600 microliters of liquid LB medium with ampicillin. Incubate these tubes at 37 degrees Celsius on a shaker for six hours.From the liquid cultures, seed 70 microliter aliquots onto the wells of six well RNAi plates in duplicate or triplicate sets. Then, incubate the RNAi plates overnight at 20 to 22 degrees Celsius. The next morning, pick three L4 stage worms and load them into each well of the RNAi plates.Each should look healthy, well-fed, and should express the appropriate marker in the excretory canal. Then, incubate the plates at 20 to 22 degrees Celsius for three days. After three days of consuming RNAi bacteria, examine the canal phenotypes of the F1 progeny under a dissecting fluorescence microscope.To score extension of the canal, examine L3 L4 larval stage worms. For this procedure, have a scoring sheet for the screen that includes all possible phenotypes. Several canal phenotypes are described in the text protocol.Scan the wells systematically, beginning with a control such as worms fed an empty vector RNAi bacteria. First, use bright light at lower magnification and then at higher magnification to find general phenotypes such as embryonic lethality, sterility, clear appearance, and dumpy appearance. On the scoring sheet, tally these observations.Second, under fluorescent light, examine excretory canal phenotypes, first at lower magnification, and then at higher magnification. Zoom in and score quantifiable phenotypes such as the length of the canal, the width of the lumen, or the size of the cysts. Marker plasmids such as role, can be useful for imaging by keeping animals on the spot rather than moving forward.And may also provide an improved view on the phenotype with the animal rolling around itself. Score at least 100 animals per plate for a quantitative analysis that takes into account phenotypes with lower penetrance. Perform these experiments in triplicate.During this process, acquire images of the predominant phenotypes from at least three different animals using a CCD camera and image capture software. Then acquire images at both lower and higher magnifications. To analyze the worms of interest by 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 not be thicker than 0.1 millimeters. Next, add about six microliters of five percent lidocaine solution 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 place a cover slip onto the grease circle and allow it to settle under its own weight. Check for immobility under a dissecting microscope.The worms should be undamaged and sticking to the slide, not floating in solution. Now, place a drop of oil on the coverslip and focus on the worms under bright light with a 10x objective on a confocal microscope. Then, carefully switch to an oil-immersion objective and switch to using fluorescence light to view the labeled excretory canals.Now, examine the excretory canals cellular and subcellular phenotypes to find a specimen suitable for imaging. Work swiftly to avoid photobleaching. Look at the lumen shape, lumen diameter, and the size and shape of the cysts.Also, examine the subcellular components labeled for analysis, such as the apical membrane, the basal membrane, cytoplasm, vesicles, or organelles. Using the software control, adjust the laser intensity and acquire images. For the best resolution, use a low gain, do not open the pinhole too much, and use about four averages per image.Also, do not over saturate the color intensity. First, acquire an image of a single section, and then acquire an image of multiple sections along the z axis. 15 to 20 sections generally suffice.By making a bracket on a specific region of the excretory canal in the scan area in the control panel an area of the canal can be magnified. For double or multiply labeled canals it is critical to use sequential scanning to avoid bleed-through between channels. Otherwise co-localization studies of the signals will not be accurate.Concomitantly, acquire corresponding DIC Nomarski images to quantify the canal length or lumen diameters in relation to the worm's body length and diameter. Later, overlay the fluorescence and Nomarski images to demonstrate landmarks. 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.Subcellular components such as apical membranes, the cytoplasm, and endosomal versus canalicular vesicles can be visualized and distinguished in the same cell, using component-specific fluorescent fusion proteins. A worm over-expressing an apical membrane associated molecule called ERM-1 was fed RNAi bacteria to screen for modifications of its unique excretory canal phenotype. Suppressors and enhancers were identified using a fluorescent dissecting microscope.Many subcellular aspects of the modified phenotypes could be examined in detail at higher magnification by confocal microscopy. They included abnormal membranes, lumenal cysts rather than cytoplasmic vacuoles, localization of intracellular canaliculi, discontinuous lumen with abnormal canal tip morphology, lumen versus canal extension defects, and recruitment of subcellular components to the lumen. Many of the effects from the suppressors and enhancers of the ERM-1 induced phenotype could also be quantified, such as cyst size, canal length, and fluorescently labeled subcellular components.After watching this video you should have a good understanding of how to combine standard RNAi and ERM procedures to characterize C.Elegans excretory canal phenotypes under fluorescent dissecting and confocal microscopes. Using these techniques, you should be able to investigate de novo polarized membrane biogenesis, and tubulogenesis in a single cell in living animals.

the-c-elegans-excretory-canal-as-model-for-intracellular-lumen

Research

JoVE Journal

Developmental Biology

The C. elegans embryo is a powerful system for studying cell biology and development.  We present a protocol for live imaging of C. elegans embryos utilizing DIC optics or fluorescence using readily available epifluorescent microscopes and open-source software.

Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

This article describes a technique for the visualization of the early events of embryogenesis in the nematode Caenorhabditis elegans.

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

This protocol describes how to image dividing cells within a tissue in Caenorhabditis elegans embryos. While several protocols describe how to image cell division in the early embryo, this protocol describes how to image cell division within a developing tissue during mid late embryogenesis.

Visualizing Neuroblast Cytokinesis During C. elegans Embryogenesis

Here it is shown how to track and quantify developmental processes in C. elegans. The methods presented are based on open-source tools that can be easily implemented. It is demonstrated how to reconstruct 3D cell-shape models, how to manually track subcellular structures, and how to analyze cortical contractile flow.

Tracking and Quantifying Developmental Processes in C. elegans Using Open-source Tools

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

This protocol demonstrates how to track a protein nuclear translocation under heat stress by using a green fluorescence protein (GFP) fusion protein as a marker and 4',6-diamidino-2-phenylindole (DAPI) staining. The DAPI staining protocol is fast and preserves the GFP and protein subcellular localization signals.

Detecting Protein Subcellular Localization by Green Fluorescence Protein Tagging and 4',6-Diamidino-2-phenylindole Staining in Caenorhabditis elegans

Here, we present a combinatorial approach using high-resolution microscopy, computational tools, and single-cell labeling in living C. elegans embryos to understand single cell dynamics during neurodevelopment.

Isotropic Light-Sheet Microscopy and Automated Cell Lineage Analyses to Catalogue Caenorhabditis elegans Embryogenesis with Subcellular Resolution

This work describes a semi-high-throughput protocol that allows simultaneous 3D time-lapse imaging of embryogenesis in 80–100 C. elegans embryos in a single overnight run. Additionally, image processing and visualization tools are included to streamline data analysis. The combination of these methods with custom reporter strains enables detailed monitoring of embryogenesis.

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

A protocol for imaging the dynamic microtubules in vivo using fluorescently labeled End binding protein has been presented. We described the methods to label, image, and analyze the dynamic microtubules in the Posterior lateral microtubule (PLM) neuron of C. elegans.

In vivo Assessment of Microtubule Dynamics and Orientation in Caenorhabditis elegans Neurons

The protocol describes a simple microfluidic chip design and microfabrication methodology used to grow C. elegans in presence of a continuous food supply for up to 36 h. The growth and imaging device also enables intermittent long-term high-resolution imaging of cellular and sub-cellular processes during development for several days.

A Simple Microfluidic Chip for Long-Term Growth and Imaging of Caenorhabditis elegans

This protocol describes microfluidic-based C. elegans time-lapse imaging across the entire post-embryonic development.

High-Resolution C. elegans Imaging Across All Larval Stages

Learn how to analyze unicellular tubulogenesis and polarized membrane biogenesis using the C. elegans excretory canal model. This method combines RNAi screening with live imaging to study intracellular lumen morphogenesis in vivo.

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 (Video) | JoVE Journal

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

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