The overall goal of this protocol is to examine and quantify muscle regeneration in a zebrafish model of muscle disease. This method provides a high-throughput pipeline that is not only cost-effective, but also highly reproducible in scoring the regenerative potential of a diseased muscle in an in vivo setting. Demonstrating the procedure with me will be Avnika Ruparelia, a research fellow at the Australian Regenerative Medicine Institute.
For live embryo genotyping, peel the clear protective film from the top surface of a 24-chamber DNA extraction chip and use a 20 microliter filter tip with a cut tip to transfer a single anesthetized embryo in 13 microliters of embryo medium into each chamber of the chip. When all of the embryos have been loaded, gently mount the chip onto the zebrafish embryo genotyping platform by placing one side in first, followed by the rest of the chip. Affix the magnetic platform lid over the chip to prevent the evaporation of embryo medium during the DNA extraction protocol and close the lid.
Set the base unit to 2.4 volts, 0.051 amps, and 0.12 watts and start the DNA extraction protocol. The platform should start vibrating, which can be assessed by gently touching the lid. While the program is running, prepare a 24-well plate by adding one milliliter of embryo medium to each well and label eight-well strip tubes to be used for the collection of DNA material from each embryo.
After eight minutes of extraction, press the on/off button to stop the vibration of the platform, gently remove the magnetic lid and lift the chip from the platform. Transfer 10 microliters of embryo medium from one chamber into the appropriate well of the eight-well strip tube and immediately add two drops of fresh embryo medium to the chambers. Transfer the embryo from the chamber to an appropriate well of the previously prepared 24-well plate.
When all of the embryos have been transferred, place the plate in a 28 degree Celsius incubator. Perform the appropriate downstream genotyping assays on the genetic material collected in the eight-well strip tubes to determine the genotype of each embryo. Once the genotype of each embryo has been identified, transfer the embryos to 90 millimeter Petri dishes containing 25 milliliters of medium water and incubate the plates at 28 degrees Celsius until muscle injury.
At four days post-fertilization, use a pipette to transfer one anesthetized larva into a new Petri dish and carefully remove any excess medium from the dish under a dissecting microscope. Orient the fish such that the head is on the left, the tail is on the right, the dorsal region is up, and the ventral region is down, and use a 30 gauge needle to make a quick precise stab into one to two somites in the epaxial muscle above the anal pore and horizontal to the myoseptum. Apply a drop of embryo medium to the injured zebrafish and carefully transfer the larva into one well of a 24-well plate containing one milliliter of fresh embryo medium per well.
When all of the larvae have been injured, place the plate in the 28 degree Celsius incubator until the zebrafish are imaged. To quantify the muscle regeneration, open a one-day post-injury image in an appropriate image analysis software program and use the polygon tool to draw a shape around the wound site. Use the software to measure the area and mean birefringence intensity of the region and copy these values to cells D3 and E3 of the provided template.
Draw two additional regions each spanning one to two uninjured somites and measure the area and mean birefringence intensities of each of these regions. Copy these values to cells D4 and D5 and E4 and E5 in the template. Then repeat the measurement for same regions in the three days post-injury image.
When the birefringence has been measured in the same manner in all of the images, calculate the normalized birefringence for each region by dividing the mean birefringence intensity of each region by its area. For each time point, calculate the average normalized birefringence of the two uninjured regions to provide a reference point for the uninjured muscle. To determine the extent of muscle injury at day one post-injury, divide the normalized birefringence of the injury region by the average normalized birefringence of the uninjured regions.
To determine the extent of muscle regeneration, divide the normalized birefringence of the injury region in the three days post-injury image by the average normalized intensity of the uninjured regions at this stage. Finally, calculate the regenerative index by dividing the value for the extent of the muscle regeneration at day three post-injury by the value for the extent of the muscle injury at day one post-injury. In this representative analysis, a clutch of embryos from a cross between two Lama2 heterozygous zebrafish was transferred to a DNA extraction chip and subsequently genotyped.
While muscle injury results in a reduction in birefringence intensity at day one post-injury, the successful regeneration of muscle results in an increased birefringence within the same region. It is also noteworthy that while wild type larvae display a uniform birefringence intensity due to a normal muscle patterning, the birefringence intensity in the muscle of Lama2 knockout larvae is uneven and highly sporadic likely due to a reduced muscle integrity. As observed, both wild type and Lama2-deficient larvae demonstrate a significantly increased birefringence intensity in the wound site at three days post-injury compared to at one day post-injury, indicating that the muscle has regenerated.
Determination of the regenerative index reveals that Lama2-deficient larvae display a striking increase in muscle regeneration compared to wild type animals. While this protocol will allow us to identify any alterations in the ability of muscle to regenerate in models of muscle disease, downstream cellular and molecular analysis need to be performed to identify the mechanisms that are responsible for the observed changes. This method has allowed us to identify how impaired muscle stem cell function and an impairment in associated niche elements contributes to muscle disease pathogenesis, therefore allowing us to identify novel disease mechanisms.
