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.
