Analysis of Skeletal Muscle Defects in Larval Zebrafish by Birefringence and Touch-evoke Escape Response Assays

The overall goal of this procedure is to evaluate the extent of myo fibrillar organization in translucent zebrafish larvae, and to quantify their swimming distance in response to tactile stimulation for both methods. This is accomplished first by mating the zebra fish and then collecting viable eggs in the second step of the assay for evaluating muscle structure. A coated anesthetized zebra fish embryo is positioned directly between two polarized lenses on a stereo microscope stage so that the fish is oriented along the lateral axis of its body and is in as flat an orientation as possible.

The birefringence is then visualized and the muscle integrity is quantified. In the second step of the touch evoked escape assay, a single coated larva is placed into a deep Petri dish and then centered in the field of view of the stereo microscope. A meno sensory stimulus is delivered to the larvae by touching the tail with an insect pin, and the animal's reaction is imaged until the embryo quit swimming or swims out of the field of view.

Ultimately, differences in muscle organization and locomotive responses can be quantified. The main advantages of these non-invasive techniques are that large number of fish can be analyzed quickly, and skeletal muscle defects in the same fish can be monitored in vivo over several days. Visual demonstrations of setting up polarized lenses and positioning larvae in the proper orientation are critical to ensure success.

With these assays, Clean out the inviable eggs from all clutches collected from mating adult fish. These generally appear white and opaque to the naked eye, or as a cluster of dark disorganized cells under the microscope, return the embryos to the incubator for further growth three to seven days post fertilization. If the embryo of interest remains in its corion, use a pair of sharp forceps to gently make a tear in the corion of the embryo of interest and then turn it upside down so that the embryo falls out.

Anesthetize the coated embryos with trica to aid in their correct positioning and then fit a dissecting microscope with a polarized lens that can be rotated to adjust the angle of polarized light. Once the embryos have straightened, position a second polarized lens on the microscope stage directly below the top lens and place embryos directly on top of the second lens. Each embryo of interest should then be positioned along the lateral axis of its body.

Rotate the top lens with the embryo of interest in view until the axis of polarization of the two lenses are oriented 90 degrees from one another and the background is completely dark. Move the fish around to make sure that they're lying as flat as possible. If a fish is curved, only the segments with a flat orientation will pass the polarized light and exhibit by frt.

Now, observe the bi frt phenotype of the wild type fish. Highly organized skeletal muscles show bright by frt as the refractive index for the light parallel to the myo filaments is higher than the polarized light perpendicular to these structures. Fish with disorganized skeletal muscle suggestive of a neurodegenerative myopathy or a developmental defect typically show an overall reduction in birefringence fish with disorganized skeletal muscle.

Characteristic of muscular dystrophy. Commonly exhibit a patch like pattern of birefringence with dark areas representing muscle degeneration among bright areas of normal muscle architecture. Collect images of the fish under polarized light for quantification of the birefringence.

Save the images as tiff files. Open the birefringence images in an image processing software program. If using image J as shown here for each image, first, use the polygon selections tool to draw a line around the body of the fish to set the area of zebrafish muscle for measurement.

Then under the analyze dropdown menu, choose set measurements to select the required statistics for the image. Check the boxes for area mean gray value and min and max gray value to set up the touch evoked escape behavior assay two to seven days post fertilization. Place the coated fish of interest individually into deep Petri dishes working with only one embryo at a time.

Center the fish in the field of view of a stereo microscope equipped with a digital camera system and begin sequential imaging using greater than or equal to 30 hertz frame rates. Then using an insect pin, touch the tail of the embryo to deliver the meno sensory stimulus, stopping the imaging when the embryo has stopped swimming or has swam out of the field of view. Finally, convert the sequential images into a video file or analyze the individual frames of time-lapse images.

Offline birefringence can be used as an efficient noninvasive assay to shed light on the state of Myo fibrillar organization in living zebrafish embryos, wild type zebrafish at five days post fertilization display highly frt skeletal muscles under polarized light due to their ordered array of myofilaments. In contrast, age-matched embryos homozygous for a pathogenic mutation in the distro glycan or DAG one gene display patchy birefringence indicative of areas of muscle degeneration. This patch like pattern and rapid progression of muscle degeneration throughout early development is consistent with other dystrophic fish models.

Quantifying the birefringence of five days post fertilization, dystrophic DAG one mutants identifies a significant reduction in brightness to 27.9 plus or minus 5.3%of the maximal birefringence seen in their wild type siblings. This reduction is more severe than the reduction to 52.4 plus or minus 5.5%observed in three days post fertilization myopathic mtm one morphines myopathic Fish models instead tend to show an overall reduction in birefringence compared to wild type, as there is often myo fibrillary disorganization in the major axial skeletal muscles, but no evidence of degenerative changes. Touchy focused escape behaviors can be used to demonstrate that zebrafish models of skeletal muscle disease display locomotive defects and swimming difficulties.

This first video shows a touch evoked escape behavior assay on wild type zebra fish. Note how at five days post fertilization, the normal fry swim away very rapidly. In response To touch in this second video, dag one fry, touch evoked escape behavior was captured.

Note how day five post fertilization mutant embryos exhibit an impaired response to touch and fail to exit the field of view after stimulation. The videos were then analyzed frame by frame to confirm that DAG one mutants exhibit impaired movement compared to wild type controls in response to tactile stimuli. The distances both types of embryos were able to move within a fixed time interval were averaged and indicate a diminished motor function in DAG one mutants.

The once mastered these techniques can be performed on large quantities of fish over a short period, which is critical when conducting high throughput chemical or mutagenesis screens. Following this procedure, more invasive approaches such as immunohistochemistry or electron microscopy can be performed to investigate defects in skeletal muscle at the molecular and structural levels.