This method addresses how to analyze developmental processes such as muscle contractions and rolling which occur in the Drosophila embryo. Some advantages of this technique are that it is noninvasive and quite detailed, yet it is relatively simple to perform. Our method can be developed further for high-content analysis-based screening to isolate and analyze new mutations that affect embryonic muscle contractions and other developmental processes.
When visualizing muscle contractions, it's important to properly time embryo collections because contractions begin only at a specific developmental stage. Step-by-step visual demonstration can greatly aid in the quantitative analysis of the developmental processes. For live imaging of mounted Drosophila embryos, place the embryos onto the stage of an epifluorescence microscope with a timelapse function and a digital camera with suitable emission filters and select a 10X water immersion objective lens.
Then live video record the embryos using the appropriate microscope recording software for about one to two hours with an acquisition rate of four frames per second. At the end of the experiment, export the recorded video directly into ImageJ. Select image and crop to draw a box around each individual embryo to crop the video recordings to the size of each embryo.
Click Image, Transform and Rotate to rotate the cropped images to achieve a vertical position of the embryo midline relative to the screen. To set distance measurements to micrometers, click Analyze and Set Scale, then enter the known pixel to micron ratio conversion factor for your microscope setup. All subsequent measurements will then be reported in micrometers.
To analyze embryo rolling, first mark the position of one or both trachea of an embryo in the first frame of the video midway between the posterior and anterior ends and click Analyze, Tools and Region of Interest Manager. Draw an approximate seven micrometer by seven micrometer box around the trachea and click the T key on the keyboard to record this position as an XY coordinate. Mark the position of the same area of the trachea after peristaltic contractions of interest and draw a line connecting the centers of each box clicking M on the keyboard to measure the distance from the pre-contraction position to the post-contraction position.
To analyze embryonic muscle contractions using embryos expressing fluorescent muscle markers, use the recording of the fluorescent readout to draw a region of interest centered around the fluorescing muscles of a particular body segment of interest. Select the Add T tab in the Region of Interest Manager to record the position of the region of interest. Click Region of Interest Manager and Measure to record the average fluorescent intensity of each region of interest selected for each frame of the video.
Move the box to the centers of other body segments of interest and click Add T to record their positions to obtain regions of interest of identical size in all of the body segments to be analyzed. In the Region of Interest Manager, hold the Control key while selecting all the regions of interest recorded as XY coordinates. Click More and Multi-Measure to measure the mean fluorescent intensity of each region of interest for all of the frames of the video.
This will report each measurement in a table with each region of interest as a column of the table and each frame as a row. Then transfer the table to a spreadsheet program for further analysis. Plot a graph with the frame number on the x-axis and the mean fluorescence intensity on the y-axis and convert the frame number to time using the four frames per second frame rate.
The muscle contractions increase the GFP intensity as they bring more GFP into the vicinity of the focal area as more muscles get pulled in during these contractions. To determine the muscle contraction amplitude, estimate the baseline GFP intensity as the average intensity of the regions between peaks. Then assess the increase in GFP fluorescence intensity relative to this baseline.
Then divide every region of interest intensity value by the baseline intensity to normalize the GFP intensity to the baseline. Compare the normalized GFP intensities at posterior, medial and anterior segments during muscle contraction wave to examine the changes in the extent of muscle contraction as the wave propagates and to determine the direction of the wave. To compare the muscle contractions on the left and right sides of the embryo, analyze the peak intensities for the same segments on both sides of the embryo.
Use the contraction amplitude and the timing of the peaks to reveal the differences, if any, and the extent and the timing of the peristaltic muscle contraction waves. Here normal peristaltic muscle contractions in a wild type Drosophila embryo are shown. In this video of rolling in a wild type embryo, note that the dorsal appendage does not move while the trachea does indicating that the embryo has rolled within its shell.
In this representative analysis, the muscle contraction peaks during the 165 to 178-second time period represent a single forward wave. For this embryo, no difference in the amplitude and the time of the muscle contractions was measured on the right and left sides of the embryo. A peristaltic contraction is designated as a forward type one wave if its profile has a peak that arises at the posterior region first followed by peaks at the middle and anterior regions.
A backward type one wave is a peak that first arises at anterior segments and then propagates toward posterior regions. Type two waves start at one end of the embryo and proceed toward the middle regions before returning to their origin as a sweeping wave re-initiated at the opposite end. Body posture mutant embryos demonstrate an abnormal relative frequency of type one to type two wave generation that results in a body posture abnormality designated as the body torsion or rotation phenotype.
To use fluorescence as a readout for the muscle contraction parameters, it is essential to use embryos with a reporter in the muscle tissue. This method can be used for the simultaneous recording of the muscle contractions of many embryos and for the assessment of the responses to various stimuli, drugs or environmental changes.
