Novel Metrics to Characterize Embryonic Elongation of the Nematode Caenorhabditis elegans

1. Characterization of Early Elongation Defects in WT and Mutant Animals

1. Mounting Embryos for Normarski DIC Microscopy
   1. Prepare the following culture media and material:
      1. M9 Buffer, dissolve 12.8 g/L Na₂HPO₄•7H₂O, 3 g/L KH₂PO₄, 5 g/L NaCl, 0.25 g/L MgSO₄•7H₂O in distilled water and autoclave to sterilize.
      2. NGM plates, dissolve 3 g/L NaCl, 16 g/L agar, 2.5 g/L bactopeptone in ddH₂O. Autoclave and add 1 mM MgSO₄, 1 mM CaCl₂, 1 mM phosphate buffer and 5 µg/ml cholesterol. Pour 6 ml of NGM per 60 mm dishes. Allow the medium to solidify for 24 hr at room temperature. Add a few drops of a saturated culture of E. coli OP50 bacteria grown in Luria broth (LB). Let the bacteria grow for 48 hr and store the plates at 4 °C.
      3. To generate the worm pick, mount a 0.01" diameter platinum wire on a short Pasteur pipette. Fasten the platinum wire to the extremity of the glass pipet by heating this extremity with a benzene burner. Flatten the extremity of the wire to generate a sort of shovel.
   2. Grow the worm strain on 60 mm NGM plates with OP50 at 20 °C.
      NOTE: Thermosensitive alleles may require growing the animals at non-permissive temperatures at up to 25.5 °C. Plates should contain many young adults and still plenty of food in order to pursue the protocols.
   3. Place a microscope slide between two spacer slides covered by two layers of masking tape (Figure 1A).
   4. Prepare a 3% agarose solution (weight/volume) in M9 buffer by dissolving 0.6 g of agarose powder in 20 ml M9 buffer by heating in the microwave during 30 sec. Allow the solution to cool down for 5 min.
      NOTE: The agarose solution can be used for a few days after melting in a microwave. It can also be aliquoted and stored at 4 °C for weeks.
   5. Place a drop of hot 3% agarose solution on the glass slide located between the two spacer-slides. Avoid forming bubbles.
   6. Rapidly cover the agarose with another slide as shown in Figure 1B and press down gently. The top slide will flatten the agarose drop and generate a pad with the thickness of two layers of masking tape.
   7. Allow the agarose to solidify for at least 1 min at room temperature or until the embryos are ready to be transferred to the pad.
   8. Using a worm pick, transfer 20 to 30 well fed young adults from the NGM-plate into a micro-centrifuge tube containing 400 µl of M9 buffer.
   9. Allow the worms to sediment by gravity for approximately 5 min and remove the supernatant using a micropipette. This step aims to remove the maximum of bacteria picked with the nematodes.
   10. Add 200 µl of M9 buffer to each tube.
   11. Using a Pasteur pipette, transfer the content of the tube (buffer and nematodes) to a watch glass.
   12. Use one or two 25G 5/8" needles to cut the animals at the mid-body section (between the spermatheca and the vulva).
       NOTE: A scalpel blade can also be used as an alternative to needle(s). Do this on swimming nematodes and may require some practice. The idea is to use needles as scissors or as a knife depending how many needles are used. Once the animals are cut open, hermaphrodites release their embryos into the buffer.
   13. Concentrate embryos at the center of the watch glass through generation of a circular vortex within the liquid.
   14. Remove most of the M9 from the watch glass using a micropipette. Leave approximately 30 µl of M9 with the embryos.
   15. Remove the slide covering the agarose pad (Figure 1B) by sliding it off the pad.
   16. Cut the pad with a razor blade in order to be able to completely cover it with a coverslip (Figure 1C).
   17. Using a Pasteur pipette, transfer all the embryos (and worm debris) onto the agarose pad.
   18. Group the embryos at the center of the pad using an eyelash glued at the extremity of a tip used for 200 µl micropipettes.
   19. Slowly place a coverslip on the pad avoiding bubble formation and seal it.
       NOTE: Several sealers can be used. We use drawing gum found in art and craft stores (usually used as masking gum for aquarelle painting). This gum is used because it is hydrophilic and solidifies reasonably fast and is not toxic for the worms. Slides can also be sealed with nail polish or VALAP (mixture made of Vaseline, Lanolin and Parafin wax).
   20. Allow the drawing gum dry for approximately 15 min.
2. Recording Early Elongation using Four-dimensional Normarski DIC Microscopy
   NOTE: The objective of this step is to record early elongation from comma to the beginning of late elongation — defined as the moment when the body-wall muscles start contracting. We also aim to record more than one embryo at the time. Eight hours of recording are usually required to record early elongation for a group of non-synchronized embryos.
   1. Place the mounted-slide on the stage of a microscope. Ensure that the microscope is equipped with 10X and 60X objectives as well as DIC lenses, prism, camera and capture software enabling time-lapse microscopy and generation of Z-stacks (automated Z-platform).
      NOTE: ensure that the temperature is constant between 20 and 23 °C in the microscopy room. A heating-cooling chamber may be required on the microscope stage, especially when using thermosensitive mutants.
   2. Identify a group of embryos at pre-morphogenesis stages using the 10X objective.
   3. Once located, slide off the 10X objective and add a drop of oil immersion on the slide.
   4. Using the 60X objective, identify the top and the bottom of embryos to setup the Z-stack imaging parameters.
      NOTE: Do not hesitate to set the Z-stack larger than the thickness of the embryos. Set the distance between two adjacent planes as 0.8 µm. This will enable to cover the total depth of the embryos in 35 to 50 Z-plans.
      NOTE: If the microscope contains an automated xy platform, embryos located at different locations of the pad could be recorded simultaneously. To do so, record xy coordinates of each location on the pad you wish to analyze during the course of the experiment. Make sure to select the xy when setting the recording parameter and follow the rest of the protocol as indicated. Thin Z-sectioning of the embryo during recording is not necessarily required for the measurements detailed in this protocol. Recording embryonic development of wt and mutant animals with the maximum resolution is however a good practice in order to build a library of recordings able to support additional analyses.
   5. Set up the time-lapse with 2 min intervals between two acquisitions lasting 8 hr.
      NOTE: The exposure time will depend on the light intensity set for the microscope. Cell movement during early elongation is very slow; exposure-time could be approximately one frame per second or less. If the embryos are at early morphogenesis stages (dorsal intercalation, ventral enclosure), early elongation would be recorded within 1 hr. Make sure light shutter is closed between each acquisition.
   6. Run the acquisition and save it.
3. Measurement of Early Elongation Defects using Image Analysis
   1. Open Fiji-ImageJ software (v1.48o – http://fiji.sc/Fiji).
   2. In the menu select Analyze/Set Measurements, select Perimeter. For each embryo, adjust the Z-scale bar to focus on the pharynx of the embryo as shown in Figure 2A. This will ensure that you focus on the center of the embryo.
   3. To measure the length of the embryo, adjust the time scale bar to have the embryo of interest at the start of the early elongation (comma stage; Figure 2A). Note the time ("Time-beginning").
   4. Choose the segmented line tool. Draw a segmented line from the tip of the mouth of the embryo up to the tip of its tail following the midline of the embryo (Figure 2A). Using the menu tab Analyze/Measure, obtain the length of the drawn line ("Length beginning").
   5. Repeat this measurement for the same embryo at the end of early elongation (moment when muscles start contracting). Note the time ("Time-end") and measure the length of the embryo ("Length-end") as detailed above (Figure 2B).
   6. Calculate the duration (D) of early elongation and the length increase (L) during this stage as follows:
          D = "Time-end"-"Time-beginning"
          L = "Length-end" -"Length-beginning"
   7. To measure the head width, adjust the time scale bar to have an embryo at the stage of interest (1.2-fold stage or end of early elongation). Choose the straight-line tool. Draw the transversal (dorso-ventral axis) midline of the head. This section is the thickest part of the embryo (Figure 2C). Using the menu Analyze/Measure, obtain the length of the line corresponding to the head width.
   8. At the same time point, repeat this step by drawing the transversal midline of the tail (Mid-line between the intestinal valve and the tip of the tail; Figure 2C) and measure it to obtain the tail width.
      NOTE: Use this measurement to compare embryos at the same stage across different phenotypes. To ensure the reproducibility of this measurement, make sure to find a location located around the mid-line of the tail of the animal that can easily be recognized from one animal to another.
   9. To calculate the head to tail width ratio, divide the head width by the tail width.

2. Characterization of Late Elongation Defects Using Image Analysis

1. Synchronization of L1 Larvae using Alkaline Hypochlorite Treatment
   1. From a non-starved 60 mm plate containing many gravid adults, collect all worms in a micro-centrifuge tube by washing the nematodes off the plate with 1 ml of M9 buffer using a Pasteur pipette.
   2. Sediment nematodes at 3,500 x g for 3 min at room-temperature and remove the supernatant using a micropipette without disturbing the worm pellet.
   3. Add 1 ml of hypochlorite solution to each tube (0.4 M hypochlorite, 0.5 M NaOH) and shake for 3 min.
      NOTE: Alkaline hypochlorite solution should be freshly prepared and embryos in this solution should be agitated gently but continuously to optimize dissolution of the adults and oxygenation of the embryos. After 3 min agitation, most adults should release their eggs into the solution.
   4. Spin at 3,500 x g for 3 min at room-temperature and quickly remove the supernatant using a micropipette without disturbing the pellet.
   5. Wash four times with 1 ml of M9 buffer followed by centrifugation at 3,500 x g for 3 min.
   6. After the fourth wash, remove the supernatant and resuspend eggs in 700 µl of M9 buffer without pipetting up the eggs (as the eggs would stick to the plastic of the tip).
   7. Tape the tube on a 3-mm orbital shaker placed in an incubator at the appropriate growth temperature and shake at 600 rpm overnight.
2. Length Measurement of Synchronized Larvae
   1. Centrifuge arrested L1 larvae at 3,500 x g for 3 min at room-temperature and remove the supernatant. Resuspend the larvae in 100 µl of M9 buffer and transfer them onto an agarose pad using a Pasteur pipette (agarose pads should be prepared as described at section 1.1.2 to 1.1.8). Place a coverslip on the pad.
      NOTE: The pad does not need to be sealed with gum for this experiment that involves short acquisition.
   2. Use a 10X objective and phase contrast illumination to measure the length of the larvae if a high resolution camera is used. Increase the intensity of the light to be able to capture images within a few milliseconds and consequently obtain a clear picture of the larvae (Figure 2D).
      NOTE: While the swimming trashes of larvae are reduced by the agarose pad, they are still moving. Therefore, fast recording is essential to obtain a clear image.
   3. Open Fiji-ImageJ and repeat step 1.3.1. To measure the length of larvae, choose the segmented line tool. Draw a segmented line from the tip of the head up to the tip of the tail following the midline of the larvae (Figure 2D, right panel).
   4. Using the menu Analyze/Measure, obtain the length of the drawn line corresponding to the length of the larvae in micrometer.

3. Characterization of Late Elongation Defects Using Flow Cytometry

1. Synchronize Larvae
   1. Purify embryos using the alkaline hypochlorite treatment as described in section 2.1 from full 100 mm plates of well-fed young adults and let the embryo hatch and the L1 arrest in M9 buffer for 16 to 24 hr at 20 or 25.5 °C depending on the strain analyzed.
      NOTE: One full plate of well-fed adults per strain is sufficient for the analysis described below.
2. Calibration of the Worm Sorter
   NOTE: The flow cytometer used here is a large particle flow cytometer (hereafter referred to as the worm sorter). The worm sorter includes a 670 nm red diode laser, which is located in front of an extinction detector. The worm sorter also contains a multi-line argon laser for fluorescent excitation. The standard instruments have three photomultiplier tubes (PMT) fluorescence detectors used to detect fluorescence emissions in the green, yellow, and red regions of the spectrum. In the protocol described here, TOF will be used to measure the size of the larvae, the red channel will be used to identify dead worms that will be stained with Propidium Iodide (PI). GP (General Purpose) High Fluorescence Control Particles used to calibrate the instrument and as an internal control in our experiment display high fluorescence in green, yellow and red. They will be the only objects in the sample analyzed with high emission detected in the green channel and will subsequently be identified based on this characteristic.
   NOTE: Living animals are identified based on the absence of fluorescence in the Green and Red channel as well as on the TOF. Autofluorescent emission from the gut of the larvae is not detectable at L1 stage.
   1. Switch on the laser block, the computer, the compressor and the worm sorter instrument. Press the START button on the opened worm sorter software as indicated in the instruction manual of the instrument.
   2. Observe the argon laser control pop-up window. Select RUN mode and wait as the laser powers reach 10 ± 1 mW. Select DONE on the laser control window.
   3. Set the sheath and the sample pressures to 5.10 ± 0.02 and 5.51 ± 0.01 respectively. Allow the pressure to equilibrate for at least 15 min and click the PRESSURE OK check box.
      NOTE: The flow rate depends on the sheath and the sample pressures.
   4. Make sure to eliminate all bubbles present in the flow channel tubules between the sample cup and the analyzing chamber. To do so, click ACQUIRE and gently flick the tubule until no bubble are acquired (as seen in the acquisition window).
   5. Setup of all parameters for fluorescent and TOF measurement and detection as follows:
      1. Set the scales for TOF, Ext, Green, Yellow and Red to 256. Set gain as described in Table 1. Set PMT Control at 700 for Green and Yellow and to 900 for Red.
         NOTE: Two excitation filters are available (488 nm and 514 nm) and can be used to excite either Green fluorescent protein (GFP) or Yellow fluorescent protein (YFP) or any fluorochromes excited at these wavelengths with the multiline argon laser. Appropriate filters need to be inserted in the filter chamber of the equipment. We used the 488 nm filter for the following experiment.
   6. To calibrate the worm sorter, select "run control particles" in tool menu. Put 20 ml of 1x GP (General Purpose) High Fluorescence Control Particles in the cup (these particles are fluorescent particles of precise size, sold by the cytometry manufacturer to calibrate their instrument).
   7. Click on ACQUIRE button to begin sheath and sample flow.
      NOTE: Expect a mean related to the control particle distribution of 21 ± 6 and a coefficient of variation around that mean (C.V.) ≤11. If the C.V. is >11 and the mean is >27 try to clean the tubules by clicking several times on the clean button or by flicking the flow channel.
3. Acquisition of Animal TOF
   NOTE: Light scatters when an object passes in the flow cell between the laser source and the extinction detector. The time it takes for the light to be scattered by the flying object (time of flight, TOF) is used to measure the axial length of the object. The extent of light masked by the object (extinction, EXT) is used as a measure of its opacity/optical density.
   1. Place 10 µl of synchronized wild-type (wt) L1 in a watch glass and estimate the percentage of dead-eggs using a dissecting microscope.
      NOTE: Synchronized L1 displaying high Emb (higher than 20%) in wt should not be used for further analysis.
   2. Transfer synchronized L1 from the microcentrifuge (approximately 700 µl of M9 containing L1) to a 15 ml conical tube. Add propidium iodide (PI) at a final concentration of 10 µg/ml (dilution 1/100^th from a 1 mg/ml stock solution) and incubate for 30 min at room temperature.
      NOTE: Dead eggs and larvae will be stained using PI and detected as highly fluorescent objects using the red channel.
   3. Add 10 ml of M9 buffer to dilute stained populations. Take 5 ml of the diluted populations and dilute them four times by adding 15 ml of M9. Place this dilution in the sample cup.
   4. Run the sample flow by clicking ACQUIRE and observe the flow rate.
      NOTE: In order to have accurate measurement, the flow rate should stay between 15 and 25 objects/sec.
   5. If necessary, adjust the amount of animals in the cup to reach the desired flow rate.
      NOTE: The flow rate will depends on the pressures (sheath and sample) that are constant as indicated in 3.2.3 and on the concentration of larvae in the cup. If the flow rate is higher than 25 objects/sec add some buffer in the cup. If it is lower than 15 objects/sec add concentrated L1 (from step 3.3.3) in the cup.
   6. Once the appropriate concentration of object is reached, evaluate the volume in the cup and add 1/4^th of this volume in High Fluorescent GP Control Particles 4x solution.
   7. Adjust the gating and sorting parameter. To do so, click 'Gating' on the menu and select ' TOF vs.' and 'Green'. Click on 'Sorting' on the menu and select 'TOF vs. ' and 'Red'. Gating and sorting graphics will then appear as shown in Figure 3A.
      NOTE: This will allow the visualization of the control particles (emission in Green and Red), dead animals stained by PI and bubbles (emission in Red and not Green) and living animals (no emission in neither Green nor Red channels) (Figure 3A).
   8. Run the experiment by clicking ACQUIRE.
      NOTE: To identify small size differences between larvae, analyze around 10,000 objects, so approximately 8,000 animals. Stop the experiment and export the data as .txt format by clicking on STORE.
4. Measurement of the Relative Size of Living Animals in Synchronized Populations
   1. Open the .txt file using a software able to manage large data tables.
   2. From the analyzed objects extract the TOF values associated with control particles that are characterized by emission in the green channel higher than 100 arbitrary units (this value depends on the parameters set in section 3.2.5). Create a new column and call it 'control particles'. Create a column containing all the other objects except the control particles called 'sample'. NOTE: the fluorescence emission of new particle batch needs to be verified before starting the acquisitions. This will set the fluorescent threshold enabling the identification of control particles over L1s.
   3. For each analyzed strain identify the portion of the experiment where the flow rate has been altered (due to the presence of a plug of eggs for example). To do so:
      1. Plot the TOF of 'control particles' for each sample as a factor of time (Figure 3B).
      2. Draw the linear trendline using the trendline tool and display the equation on the chart.
         NOTE: The TOF of control particles should be constant over time (horizontal line, Figure 3B). Considering the equation of the linear trendline drawn on the data y = ax+b, ensure that the 'a' value is lower that 10^-4. All measurements made within time sections displaying a rupture in the alignment of control particle should be removed from the analysis.
   4. Remove the dead embryos and bubbles (emission higher than 15 in the red) as well as small debris and large egg plugs (TOF lower than 10 and higher than 70 respectively) from the 'sample' measurement.
   5. Plot the 'control particles' distribution for each strain analyzed. As seen in Figure 3C these distributions should overlay almost perfectly. This ensures that TOF measurements obtained for different strains can be compared. NOTE: normalization of sample elements TOF over control particles can be used if the distributions of control particles in a sample do not overlay with that of control samples. Normalized TOF are computed as followed:
      1. Normalize TOF for genotype G = (sample TOF for G) / (average 'control particle' TOF for G) x (average 'control particle' TOF for wt) where wt is the control sample.
   6. Plot Larvae TOF distribution for each strain including control sample, in our case wt animals (Figure 3D). Measure the mean and the standard error of the mean (SEM) for each population (Figure 5A and B).