The overall goal of this group of experiments is to identify developmental defects in the nematode Caenorhabditis elegans during the early and late phases of embryonic elongation. This method can answer a key question in the developmental genetic field. It enables the characterization of genes controlling a late stage of the Caenorhabditis elegans embryonic development called embryonic elongation.
The main advantage of this group of techniques is that they identify genes controlling either early or late stage of the elongation and also those that control morphogenic processes at the anterior or posterior part of the embryo. I will demonstrate the procedure together with Olivier Rocheleau-Leclair, a former student of the laboratory. This procedure requires DIC lenses, prism, and a video capture system for time-lapse microscopy of Z stacks.
First, position the slide-mounted sample for viewing. Preparation details are in the text protocol. Beginning at low magnification, identify embryos in their pre-morphogenesis stages.
Then, add a drop of oil and use a more powerful objective. Now, identify the top and bottom points of the embryos to record and set the Z stack parameters. Set the time-lapse to take images every two minutes.
Make certain that the light shutter is closed between the image acquisitions. The time needed to capture the early elongation event can vary from one to eight hours, depending on the stage of the embryo. Now, run the image acquisition program and save the results.
Next, proceed with their analysis using Fiji ImageJ software. Once the images are loaded, choose the Hyperstick viewing mode. Therein, adjust the brightness and contrast by applying the Auto Mode from the Lookup Tables.
Next, go under Analyze Set Measurements and set the parameters. For each embryo, set the Z Scale bar to focus on the pharynx, which is a good landmark for the center point of the embryo. Now, adjust the Time Scale bar to the beginning of early elongation for a specific embryo.
Take note of this time. Then, calculate the length of the embro using the Analyze Measure tool to draw a line from the tip of the tail to the mouth along the midline. Take note of this length.
Next, adjust the time point to the end of the early elongation event and repeat the length measurement. Use these values to calculate the duration of early elongation and length increase during early elongation. The change in width can also be measured using a straight line tool to draw a line across the midline of the head.
This is a good location for consistent measurements. Do the same at the midline of the tail then calculate the head-to-tail ratio for any time point and use this to score changes in width. For this protocol, a flow cytometer must be prepared for large particle flow.
Such a machine essentially becomes a worm sorter. Before beginning start up the laser block, the compressor, the worm sorter, and the software. In the software, press Start and check the Argon Laser Control.
Select the Run mode and let the laser warm up to near 10 milliwatts of power then select Done. Let the pressure equilibrate for at least 15 minutes. Now, prepare the sample.
First, load 10 microliters of synchronized wild type L1 in a watch glass and estimate the fraction of dead eggs under a microscope. If the estimate is greater than 20%do not use the sample. Transfer the about 700 microliters of synchronized L1 in M9 solution to a 15-milliliter tube and add propidium iodide to 10 micrograms per milliliter.
Let the stain work for 30 minutes at room temperature. Now, prepare the system. It is vital to adjust the flow rate using the Sheath and Sample Flow Controllers to 5.1 and 5.51 respectively.
When pressures stabilize, click the Pressure Okay box. Next, use the Acquire command and manually flick the tubule to remove any bubbles from the channel between the sample cup and the analysis chamber. The Acquisition Window will eventually show that no bubbles are present.
This is also vital to the experiment. Now, set the parameters for the Fluorescents and TOF measurements. Set the scales for TOF, EXT, Green, Yellow, and Red to 256.
Then, set the Gain as noted. Then, set the PMT Control to 700 for both Green and Yellow and to 900 for Red. Now, calibrate the worm sorter.
Load 20 milliliters of general purpose high-fluorescence control particles into a sample cup and load it into the sorter. In the software, select Run Control Particles from the Tools and click Acquire. Check that the resulting coefficient of variation is greater or equal to 11 and the mean is equal to 21 plus or minus six.
If it is not the case, try to clean the tubules by clicking on the Clean button or by flicking the flow channel. Now, quit Control Particles mode by unchecking the command in Tools. Once the 30-minute staining of worms with propidium iodide is finished, add 10 milliliters of M9 to the stained worms and mix.
Incubate for at least 15 minutes at room temperature. Now, take five milliliters of the dilution and add it to a sample cup with 15 milliliters of M9.Run this dilution through the worm sorter. Press the Acquire button and observe the flow rate.
It may be necessary to adjust the concentration of worms to get a good flow, somewhere between 15 and 25 objects per second. Once the appropriate concentration of the objects is acheived, add control particles to the test to get a final concentration of about 0.5 to 1x control particles in solution. Now, adjust the Gating Parameter under the Gating menu to TOF versus Green.
Then adjust the Sorting parameters to TOF versus Red. This will sort the control particles from the dead animals and the living animals. Now, run the experiment by clicking Acquire.
Analyze about 10, 000 objects, which would correlate to about 8, 000 animals. Then stop the experiment and click Store to save the data as a text file for analysis. To establish the robustness of head width measurement, measurement was performed independently five times on 12 wild type embryos at the 1.2 fold stage.
There was no variances among the data when applying the Brown-Forsythe test. The reproducibility and batch effects associated with head width measurement was tested using 4D DIC imaging on three different days. Across these three measurement groups, no variance or batch effects were found.
Both described protocols were used to assess the length of arrested larvae in wild type and mutant backgrounds. Image analysis provided absolute measurements of length in micrometers. Mutants were smaller than wild types in this experiment.
Measurement of length by flow cytometry gave comparable results. However, these measurements were far more robust. Thus, this method should elucidate more subtle effects in mutant populations.
While attempting this procedure it is important to maintain a constant flow rate through the experiment to ensure a fair comparison between the analyzed strain. Once mastered, measurement of the larval length using flow cytometry enables the characterization of 18 to 24 condition or mutant strain per day, even considering the initial calibration of the equipment. Following this procedure, we are able to study the morphology of the hypodermal cell.
To do so we can use fluorescent marker and confocal microscopy to characterize the molecular function of the identified genes. Don't forget that propidium iodide can be extremely dangerous and precautions such as wearing gloves and protective glasses should always be taken while performing this procedure.
