The overall goals of this procedure are to use the embryos of transgenic Medaka bone reporter lines for drug testing and for live imaging of bone-cell behavior after the induction of osteoporosis like conditions. This method can help answer key questions in skeletal research such as how do different type of bone cells interact with each other in vivo? The main advantage of this technique is that the optical clarity of Medaka larvae allows live imaging of bone-cell behavior in an intact specimen.
The implications of this technique extends towards the development of novel therapeutics for common bone diseases as it allows the visualization of long term effects of drug treatment in C2.All procedures described in this video were approved by IACUC of the National University of Singapore. To harvest the embryos during the first few hours of the light cycle, first use a fine meshed net to capture female adults carrying an egg cluster. Let the fish briefly rest in the net, and then gently massage the fish's abdomen to carefully strip the fertilized egg cluster from the abdomen of the female.
Transfer 20 to 30 eggs into individual plastic 60 millimeter petri dishes, and use a plastic pipette to rinse the embryos in each plate with 5 to 10 milliliters of daneous solution. Gently roll egg cluster to form a knot of attachment filaments. Using forceps, carefully remove the attachment filaments from the fertilized egg clusters to obtain individual embryos.
Then, culture the embryos in a 28 degrees Celsius incubator with daily medium changes to ensure normal embryonic development. For transgenic embryo screening, at two days post fertilization or later, heat shock the larvae for 20 minutes at 39 degrees Celsius. Then, use a stereo microscope equipped with a mercury lamp for fluorescence imaging to identify ubiquitous CFP expression.
On day 5 post fertilization, mCherry reporter expression can be observed in osteoblasts of the early forming cranial bones on both sides of the posterior head and at a central position in the ventral cranium. Starting from day 6 post fertilization, robust nlGFP expression can be visualized in cathepsin positive cells of the head and tail. For the induction of an osteoporosis-like phenotype, subject the embryos to a 1.5 to 2 hour heat shock treatment at 9 days post fertilization or later to induce large numbers of ectopic osteoclasts in the trunk region.
For bisphosphonate treatment, first transfer 6 Medaka larvae per well into a 6-well plate. Then use a clean, plastic pipette to carefully remove the fish medium. Add approximately 0.5 milliliters of bisphosphonate to each well to dilute any traces of medium.
Then replace the wash solution with 4 milliliters of fresh bisphosphonate solution, and return the larvae to the incubator with daily medium changes for 2 to 6 days. At the appropriate experimental time point, filter freshly prepared bone staining solution through a single use 0.2 micron strainer. And incubate the larvae in a 1 and 10 dilution of the stain in fresh fish medium, for the appropriate staining period in a 28 degree Celsius incubator in the dark.
At the end of the incubation, use a clean plastic pipette to transfer the larvae into fresh fish medium, and replace the stain contaminated medium with another aliquot of fresh medium. When there is no visible stain left in the medium, allow the larvae to rest in the fish medium for 30 to 60 minutes before imaging. For live fluorescence imaging of the Medaka larvae, use a plastic microloader to orient the anesthetized larvae according to the region of interest.
And place them under a stereo microscope with fluorescence illumination. Select the appropriate magnification and image individual sections of each larvae using a suitable image processing software to stitch together the images at the appropriate overlapping regions. After each larvae has been imaged, return the animal to its well in the 6-well plate in fresh fish medium for recovery.
For live confocal imaging, add 0.5 to 1 milliliter of 30 degree Celsius 1.5%liquid low melting agarose in fish medium to a glass bottom petri dish. Before the agarose solidifies, transfer an anesthetized larvae to the agarose and use a plastic microloader to push the larvae to the bottom of the dish. And use the yoke extension to orient the larvae according to the region of interest.
After the agar has solidified, image the larvae by confocal microscopy. Then, use a pair of fine syringe needles to release the larvae from the agarose. And transfer each larvae and residually attached agarose into a new petri dish containing fresh fish medium for recovery.
In these images, triple transgenic larvae were used for the simultaneous detection of premature osteoblasts and differentiated osteoclasts. Overview images were taken with a stereo microscope, while confocal microscopy was used to visualize processes at the cellular level. ALC stained bone matrix along the neural arches and sentry, was used as a reference for determining the position of the fluorescently labeled bone cells.
RankL induction resulted in extensive formation of ectopic osteoclasts around the vertebral bodies. These excessive osteoclasts cause an osteoporosis-like phenotype which are observed as cavities in the mineralized centra, and as absent mineralization in the neural arches. After incubation in alendronate, osteoclasts formed normally but vertebral columns are intact, with no lesions and fully mineralized arches suggesting an efficient inhibition of osteoclast activity.
Further, successive staining of the bone matrix with ALC followed by calcein, reveals a de novo mineralized bone matrix, with a simultaneous visualization of the osteoclasts and osteoblasts in the same intact larvae. Once mastered, this technique can be completed in 3 days from the induction of the osteoporotic lesions, until the imaging of the drug effect if it is performed properly. While attempting this procedure, it is important to remember to handle the living larvae with extreme care and to mound the larvae properly so that the region of interest will be in one focal plane.
Following this procedure, other methods like noninvasive sulfate checking of individual bone precursor sites can be performed to answer additional questions such as bone-cell origin, activation, and attraction to the lesion sites for subsequent bone repair. After the development of this technique, it pave the way for scientists in the field of bone research to explore the effects of various chemical compounds, or genetic mutations on the dynamic behavior of cells during bone homeostasis. After watching this video, you should now have a good understanding of how to use Medaka bone transgenic lines for drug screening and live imaging of bone-cell behavior.
