Live Imaging and Analysis of Muscle Contractions in Drosophila Embryo

Abstract

Coordinated muscle contractions are a form of rhythmic behavior seen early during development in Drosophila embryos. Neuronal sensory feedback circuits are required to control this behavior. Failure to produce the rhythmic pattern of contractions can be indicative of neurological abnormalities. We previously found that defects in protein O-mannosylation, a posttranslational protein modification, affect the axon morphology of sensory neurons and result in abnormal coordinated muscle contractions in embryos. Here, we present a relatively simple method for recording and analyzing the pattern of peristaltic muscle contractions by live imaging of late stage embryos up to the point of hatching, which we used to characterize the muscle contraction phenotype of protein O-mannosyltransferase mutants. Data obtained from these recordings can be used to analyze muscle contraction waves, including frequency, direction of propagation and relative amplitude of muscle contractions at different body segments. We have also examined body posture and taken advantage of a fluorescent marker expressed specifically in muscles to accurately determine the position of the embryo midline. A similar approach can also be utilized to study various other behaviors during development, such as embryo rolling and hatching.

Figure 1.. Muscle contraction amplitude.

A, GFP intensity is plotted against (Y- axis) time (X-axis) for different body segments of the embryo. B, GFP intensity (Y- axis) plotted against time for left and right sides of the same segment of a contracting embryo. Frame rate is 4 frames/sec for both graphs.

Figure 2.. Type 1 and Type 2 peristaltic muscle contraction wave profiles.

A, Type 1 wave profile in which individual lines represent normalized GFP intensities of particular body segments over time, while the peaks indicate contraction events. B, Type 2 wave profile that shows an example of a biphasic contraction wave, plotted in the same way as in A.

Figure 3.. Fixed and stained POMT mutant (rt^–) and WT embryos.

Note the rotation in body segments of the POMT mutant embryo, highlighted by a dashed line tracing the position of midline. Muscles are visualized using staining with fluorescein-conjugated phalloidin. Anterior is to the left, dorsal is up. Scale bar is 100 μm.

Figure 4.. Series of contraction waves generated by wild-type and POMT mutant embryos.

Blue and red bars depict Type 1 and Type 2 waves, respectively. Note that POMT mutant generates increased proportion of Type 2 waves, as compared to WT. The WT graph represents contractions shown in Movie 1.