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

1. Labeling the C. elegans Excretory Canal by Fluorescent Fusion Proteins¹⁴

Note: See the accompanying paper on intestinal tubulogenesis³ for labeling by in situ antibody staining procedures adaptable to the excretory canal. See Table 1 for examples of molecules proven useful for visualizing C. elegans excretory canal endo- and plasma membranes, Table 2 for examples of promoters driving expression to the excretory canal, and Table 3 for resources for more comprehensive collections of markers and promoters, including references discussing the selection of different fluorophores.

1. Construction of tissue specific fluorescent marker plasmids by restriction enzyme based cloning¹⁵
   Note: See the Discussion for alternative techniques of constructing fluorescent fusion proteins.
   1. Identify the sequence of the promoter (for transcriptional fusions) or of the entire gene of interest with its promoter (for translational fusions) in WormBase⁴⁴.
      Note: For promoters, about 1–3 kilobase (kb) is sufficient for most C. elegans genes. Translational fusion proteins can also be constructed by placing a gene of interest under an excretory canal specific promoter (see Table 2).
   2. Design forward and reverse primers for amplification of the promoter (for transcriptional fusions) and/or a full gene with promoter (for translational fusions). Add restriction enzyme linkers at the 5’ and 3’ ends of the primers.
      Note: Choose restriction enzymes that are present in the vector plasmid (e.g., pPD95.79)¹⁶. For translational fusions, the 3’ linker should be designed so that after restriction enzyme digestion and ligation with the vector, the insert codon frame will be continuous with the codons of the fluorophore, e.g., GFP. One may need to add 1 or 2 more bases to the typical restriction enzyme linkers¹⁴; take care not to create a stop codon.
   3. Perform polymerase chain reaction (PCR) to amplify the promoter or full-length gene using live worms or wild-type genomic DNA or cDNA as template¹⁵.
      Note: When using worms as template, first lyse worms in lysis buffer (PCR buffer plus Proteinase K)¹⁷. Mixed stage worms can be used as template. Starved worms can be used to avoid contamination with bacterial DNA.
   4. Perform agarose (1%) gel electrophoresis on PCR products to identify the correct size of amplified product.
      Note: If band size is correct, proceed with next step. If multiple bands are generated, improve amplification conditions to produce single band. If this does not work, cut the correct band from the gel and purify DNA by standard methods¹⁵ and then proceed with next step.
   5. Perform restriction digest on the PCR product and the vector plasmid that contains the fluorophore (e.g., pPD95.79) in separate tubes by standard methods¹⁵.
   6. Separate the digested DNAs by gel electrophoresis and elute the PCR product and vector DNA bands in separate tubes.
   7. Purify the DNAs from gel slices by standard methods¹⁵. Measure DNA concentration by spectrophotometer.
   8. Ligate the PCR product and vector DNA by standard methods and transform the recombinant DNAs into competent cells by standard methods¹⁵.
   9. Spread 10 μL, 50 μL and 100 μL of the transformed cells on three individual Luria Broth (LB) plates supplemented with 50 μg/mL ampicillin.
      Note: Spread different amounts of cells onto different plates for a spectrum of transformation efficiencies. For instance, plating too densely may not permit the isolation of colonies if transformation is efficient.
   10. Incubate the plates at 37 °C overnight. Next morning, take out the plates from the incubator.
       Note: If the colonies are very small, incubate for several more hours.
   11. Prepare plasmid DNAs from single colonies by standard methods¹⁵. Mix template DNA and primers and send out for sequencing (typically performed in a core service center).
   12. Read the sequences and verify the integrity of the fusion construct.
       Note: CRITICAL: Confirm the correct codon frame between inserted gene and fluorescent marker gene for translation fusions. Ideally, sequence the whole gene to confirm that no mutation was introduced during PCR and ligation procedures.
   13. Generate more plasmid DNA (using step 1.1.11) for injection for step 1.2.
2. Generation of transgenic animals by microinjection of DNA for germline transformation¹⁸
   Note: See the discussion for alternative techniques for introducing transgenes. The outlined procedures can be used to generate transgenic animals that carry a fluorescence fusion protein or any other protein of interest. For instance, an exogenous protein can be newly introduced (e.g., a heterologous ortholog) or an endogenous protein can be reintroduced (e.g., into its corresponding germline mutant for rescue) or overexpressed to generate a phenotype (e.g., injection of erm-1 was used to generate the overexpression cystic canal phenotype that serves as the target for modification by the RNAi interaction screen described below).
   1. Mix construct DNA (1–50 ng/μL) with marker plasmid DNA (typically 100 ng/μL), for instance the dominant marker rol-6 (su1006) (see 1.2.3 for marker options).
      Note: CRITICAL: Concentration of injected DNA must be empirically determined to avoid introduction of artefactual phenotypes (cysts, extension defects, lethality) when expressing genes in the excretory canals that are particularly sensitive to the expression of transgenes. One can, for instance, make several mixtures of plasmids at concentrations of 1 ng/μL, 10 ng/μL, 50 ng/μL, and 100 ng/μL with 100 ng/μL rol-6(su1006) to test a range of concentrations for the generation of a viable strain with the desired expression or phenotype (high concentrations are likely to be non-specifically toxic for canal morphogenesis and may be lethal).
   2. Filter the DNA mixture through a 0.22-μm (micrometer) pore size spin-x centrifuge tube filter.
      Note: Do not leave the lid of the tube open to avoid dust that can block the microinjection needles.
   3. Microinject recombinant plasmids into the gonad of wild-type or mutant worms by standard methods for germline transformation (see reference¹⁸ for procedure details).
      Note: Standard marker plasmids are, for instance: rol-6(su1006), dpy-20, unc-119, pha-1. Dominant transgenes like rol-6(su1006) are introduced into wild type worms, whereas rescuing transgenes are introduced into their respective mutants. Marker plasmids are co-injected for easy maintenance of the transgenic lines since extrachromosomal transgenes are randomly lost during cell division (see 1.2.8). When injecting genes encoding for fluorophore fusions, one can also use the fluorophore, GFP, itself as marker. rol-6 induces worms to roll around themselves which is often advantageous for the evaluation of morphogenesis phenotypes.
   4. Transfer injected worms onto Escherichia coli seeded Nematode Growth Medium (NGM) plates (e.g., 5 worms/plate) (see reference¹⁹ for standard C. elegans culture and maintenance procedures and Table of Materials).
   5. Incubate the plates and let progeny develop at 20 °C for about 3 d.
   6. Examine the F1 progeny under the dissecting microscope for Rol (rolling) worms (or any other specific injection marker, e.g., GFP) and pick rollers to individual plates.
   7. Select plates with rolling F2 animals and confirm presence of fluorescence under a dissecting fluorescence microscope (usually all roller animals are GFP positive).
      Note: F2 rollers indicate the successful generation of a transgenic line. Individual lines may be different, e.g., with regard to transgene transmission rate. It is therefore useful to maintain and store several lines.
   8. Maintain the transgenic lines by enriching new plates for marker-positive animals.
      Note: The injected DNA is incorporated into the germline as an extrachromosomal array. Transmission rates for extrachromosomal arrays are variable but generally around ~50%. To not lose the strain, it is therefore critical to manually enrich lines with dominant markers (e.g., lines not secured by negative selection).
   9. Freeze transgenic lines by standard freezing techniques for long term storage at -80 °C¹⁹.
      Note: Transgenes on extrachromosomal arrays can also be integrated into the germline by UV irradiation in an additional step to yield homogenous lines¹⁸. For example, erm-1 was integrated into the germline by UV irradiation to obtain the ERM-1[++] strain fgIs2[erm-1p::erm-1;rol-6p::rol-6(su1006)] where every animal carries the transgene, a requirement for its use in the RNAi-based interaction screen described below. This strain was additionally labeled by cytoplasmic excretory canal GFP via crossing in a strain containing a vha-1p::GFP transgene (generated by the same procedures as outlined above; see reference²⁰ for basic genetic procedures such as crosses) and is referred to as ERM-1[++]; vha-1p::GFP strain below.

2. Construction of a Targeted RNAi Library and Design of an RNAi Interaction Screen to Modify a Canal Phenotype

Note: A targeted RNAi-based genetic interaction screen is described that uses an overexpression cystic canal phenotype to search for interacting excretory canal morphogenesis genes. The ERM-1[++] strain (see step 1.2.9) serves as example⁹. This approach presents only one of many possible approaches for the genetic analysis of excretory canal lumen morphogenesis (see Introduction and Discussion for other genetic approaches). See the accompanying paper on intestinal tubulogenesis³ and references^17,21,22 for background on RNAi, details of RNAi procedures, modulation of RNAi strength (adjusted to the often lethal tubulogenesis genes) and discussion of technical problems connected to RNAi. See reference¹⁹ for standard worm culture and maintenance and Table of materials.

1. Search for ERM-1 (or other gene of interest) interacting molecules in databases and published articles.
   Note: Potential ERM-1 interactors would include all molecules that were experimentally shown to functionally, genetically, or physically interact with ERM proteins in any species and/or were predicted to do so by any in silico, high throughput or systems biology approach (see Table 3 for examples of databases and resources).
2. Generate a list of all genes and find C. elegans homologs where required.
   Note: Consider expanding the list of identified genes to gene classes, which takes into account the function of the gene of interest and widens the net for identifying interactors (e.g., for the membrane-actin linker ERM-1, select all actins and actin-related molecules).
3. Identify the corresponding RNAi bacterial feeding clones in commercially available genome-wide bacterial RNAi libraries for all genes (e.g., Ahringer genomic C. elegans RNAi feeding library²¹; Table of Materials)
4. Generate a spreadsheet for all genes and their corresponding RNAi plate and well number.
5. Pick and streak ~50 RNAi clones on LB/ampicillin/tetracycline plates (choice of antibiotic is determined by library construction) per day and continue until targeted RNAi library is generated.
   Note: Depending on library size and projected workflow, omit generating a full library and proceed directly with batch analysis. Plates can be stored at 4 °C, for no longer than approximately 2 weeks (if needed, re-streak onto new plates after that). Conversely, one can generate larger frozen libraries in replicate 96-well or 384-well formats for long term storage at -80 °C.
6. Incubate the plates at 37 °C overnight. Next morning, remove plates from incubator and store at 4 °C.
7. Pick RNAi bacteria from a plate by a sterile toothpick, mix bacteria with 600 μL LB/ampicillin (50 ng/μL) broth in 1.5 mL microtube, incubate the tubes at 37 °C, shake for 6 h.
   Note: Inoculate RNAi bacteria into the broth by rubbing the pick (toothpick or micropipette tip) along the side of the microtube.
8. Seed 70 μL cultured bacteria into each well of 6-well RNAi plates in duplicate or triplicate sets. Incubate the RNAi plates at 22 °C overnight.
   Note: RNAi plates are generated by standard procedures (Table of Materials and references^3,17,21) and here used in a 6-well tissue culture plate format for a higher throughput approach that still allows for the microscopic evaluation of canal morphogenesis in live animals on the plates.
9. Next morning, pick 3 L4 stage ERM-1[++]; vha-1p::GFP worms onto each well of the RNAi plates.
   1. To avoid contamination with OP50 bacteria (see reference¹⁹) that interfere with RNAi first seed the worms onto a NGM plate without bacteria and let the animals crawl for about 10 min. Only use non-starved healthy animals.
10. Incubate the plates at 22 °C for 3 d to allow animals to produce progeny.
11. Examine canal phenotypes in F1 progeny under the dissecting fluorescence microscope.

3. In Vivo Imaging of the C. elegans Excretory Canal by Fluorescence Dissecting Microscopy and Scoring of Tubulogenesis Phenotypes

1. Prepare a phenotype scoring sheet (example shown in Table 4 and Figure 5).
2. Place the agar plate with worms directly under the fluorescence dissecting microscope, open lid of the plate for evaluation, use lower magnification to focus.
   Note: This protocol describes the use of a scope with 1.5X and 10X objectives and a zoom range from 3.5 to 45 (Table of Materials).
3. Evaluate animals by focusing on each well separately, starting with well 1, and work down the plate.
   Note: Always start with the evaluation of controls. For instance, a mock (empty vector) negative control (HT115 RNAi bacteria (see references^17,21) without or with an unrelated gene insert) and appropriate positive controls, e.g., in this interaction screen, erm-1 RNAi (suppresses the ERM-1[++] phenotype) and sma-1/spectrin RNAi (enhances the ERM-1[++] canal phenotype).
4. First, examine general phenotypes visible under bright light (for example: Let/lethal, Clr/clear, Emb/embryonic lethal, Ste/sterile, Unc/uncoordinated, Dpy/dumpy, etc.), quantify the phenotype by counting total number of animals and number of animals with the phenotype, record the numbers (see Table 4).
   Note: For knockdowns of genes causing defects that may affect evaluation of canal phenotypes (e.g., Emb, Ste), consider repeating the experiment with conditional, post embryonic RNAi (see accompanying paper for procedures³).
5. Second, examine excretory canal phenotypes under fluorescent light, score quantifiable phenotypes (e.g., length of canal, width of the lumen, cysts), record the numbers and describe phenotypes on a scoring sheet (see Table 4, Figure 5).
   Note: Higher magnification with zoom range is required to evaluate more subtle canal phenotypes. Move back and forth between low and high magnification to carefully evaluate the canal’s length and width and any other canal morphogenesis phenotype. For quantification or semi-quantification of simple phenotypes, count 100 animals (e.g., L4s in this targeted RNAi screen; phenotypes: canal length of 1/4, 1/2, 3/4 and full extension of posterior canals, and lumen diameter of posterior canals, small cysts (< 1/3 of animal width), large cysts (> 1/3 of animal width); see Table 4).
6. Acquire images of the predominant phenotypes from at least 3 different animals by a microscope mounted digital charge-coupled device (CCD) camera and corresponding imaging software (see Table of Materials)
   1. To acquire images, first turn on camera, turn on attached computer, double click the image capture software icon, focus an area of the worm plate manually under low magnification, and open the camera shutter.
   2. Click on the “live preview” icon of image capture software on the computer screen to visualize the worms on the computer screen, adjust the focus manually to clearly visualize the worms on the screen, then click the “snap” icon, then click the “save” icon.
      Note: Animals will move faster under fluorescent light, therefore keep one hand on computer mouse ready while moving the plate into the area of interest with the other hand. Then promptly click the “snap” icon to acquire the image. It is usually possible to acquire a good image with several tries.
   3. Save the acquired images with a proper file name (include strain name, RNAi clone name and date).
      Note: Faster moving wild-type worms with thin and long canals are more difficult to image than mutants. Mutants with cystic canals and/or other phenotypes are likely to move slowly, thus facilitating imaging. Marker plasmids such as Rol 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.

4. Imaging of the C. elegans Excretory Canal at High Resolution by Laser Scanning Confocal Microscopy

1. Mounting live animals
   1. Place a tiny amount of grease or petroleum jelly at the tip of a cotton swab or at the tip of the index finger, and spread the grease to generate an ultrathin circle with a diameter of ~ 6–8 mm in the middle of a clean glass slide.
   2. Place 6 μL 5% lidocaine solution (anesthetic) into the circle by a micropipette.
      Note: A lidocaine stock solution can be made by dissolving lidocaine powder in water. Dilute to 5% with M9 buffer¹⁹ (Table of Materials). It is critical for the analysis of the excretory canal to avoid the common immobilization solutions (such as sodium azide) that cause cysts to rupture and that induce canal phenotypes.
   3. Pick several animals from an RNAi plate, and place them into the lidocaine solution by immerging the worm pick¹⁹ into the solution.
      Note: Preferably pick stage-specific animals that will facilitate even-mounting by uniform thickness. Animals can be preselected on the dissecting fluorescence microscope.
   4. Place a 22 mm x 22 mm coverslip onto the glass slide; let it gently settle onto the grease circle.
      Note: Do not apply any physical pressure on the coverslip which may damage canal morphology, especially in mutants or RNAi treated worms with canal and possibly other phenotypes. It is important therefore to avoid a thick grease circle; ideally animals are gently sandwiched between glass slide and cover slip.
   5. Write the name of the sample on the frosted side of the glass slide. Immediately take the slide to the confocal microscope for analysis of canal phenotype and to acquire images.
      Note: Delays may result in damage of canal cysts or change in canal lumen morphology.
2. Acquiring confocal images
   1. Place the slide on the sample stage of the confocal microscope, focus the worms at low magnification (10X).
   2. View and select animals under 60X and/or 100X objectives, examine the excretory canal’s cellular and subcellular phenotypes, e.g., lumen shape and diameter, size and shape of cysts; or the subcellular components labeled for analysis, e.g., apical/lumenal membrane, basal membrane, cytoplasm, endosomal versus canalicular vesicles, other organelles (see Discussion, Figure 2, and Figure 4).
   3. Acquire images of the specific phenotype of interest.
      Note: This protocol describes the use of a laser scanning confocal microscope (Table of Materials). To resolve subcellular components in the thin C. elegans excretory canals, higher magnification objectives (60X to 100X) are required. A spinning-disk confocal microscope can be used to acquire time lapse images but provides less confocality (see Discussion).
      1. To acquire confocal images, turn on the computer, double click on the confocal microscope software, and select the laser by clicking on a specific laser icon.
      2. Click the “scan” icon to visualize the focused worm on the computer screen, adjust laser intensity through the software, and click again on “scan” icon to stop scanning, then click on “capture” icon to capture an image, then click on “save” icon.
      3. Save images with proper file name and include RNAi clone name, strain name and date.
         Note: Images can be acquired as single and multiple sections (e.g., 10–15 sections along the z-axis). Sectioning allows 3D visualization. Acquire projection images and save separately if required (depending on microscope). For optimal resolution, use laser settings with low gain, do not open pinhole too much, and add several averaging per image (see references^23,24 for general discussion of confocal imaging). Take care to acquire images at a brightness below saturation level to allow for modification by imaging software if required (preferably use unmodified images).
      4. Acquire images of double- or multiply labeled canals (e.g. green, red, and blue) in the same fashion, by clicking on multiple laser icons, but use sequential scanning to avoid bleed-through between channels (critical for co-localization studies).
         Note: One may need to take into consideration the time required for sequential scanning (which also results in a corresponding increase in photo bleaching) and modify scanner settings. Do not scan animals from one slide for more than 30 min maximum to avoid unspecific effects on canal morphology. Mount new slide if longer scanning is required.
      5. Acquire corresponding differential interference optics (DIC)/Nomarski images, particularly if quantifying canal length and lumen diameter in relation to the worm’s body length and diameter. Overlay fluorescence and Nomarski images by clicking on “overlay” icon to demonstrate landmarks (Figure 1D and Figure 4A–D).
   4. For quantification, measure fluorescence intensity of a labeled component of interest by ImageJ software²⁵ (Figure 5C).