Marbles are a heterogeneous collection of sensory structures found in amphibians, reptiles, and fishes. The zebra fish develops two pairs of marbles, a smaller nasal pair, and a larger maxillary pair. These transparent appendages can be used to study the development, maintenance, and regeneration of several zebrafish cell types.
This video introduces maxillary marble development and demonstrates a surgical protocol to induce regeneration. The barbs are collected several days or weeks later, followed by dissection and embedding of regenerates and controls. Hi, I'm Elizabeth LeClair of the LeClair Lab at DePaul University And I'm Ya Chesky from Northwestern University Fried of Medicine and Children's Memorial Research Center.
Today we're gonna show you some procedures for studying the zebrafish maxillary marble. We use those procedures to study growth and development of zebrafish marble. So let's get started.
At approximately 30 to 40 days per post fertilization, the maxillary barbell appears as a transparent epithelial bud near the posterior ventral corner of the maxilla. As the zebrafish matures, the barbell extends into a narrow whisker leg appendage, several millimeters long. The mature barbell contains several important cell types, including pigment cells, gland cells, taste buds, circulatory vessels, and myelinated nerves.
Best of all, the marble is transparent at all stages of its development. Like other zebrafish organs, the maxillary barbell can regenerate following proximal amputation. In this section, we demonstrate a procedure to clip the barbell at the base of the maxilla.
This could also be called a unilateral ectomy. On the day of the surgery, prepare two small tanks of fish water, one for anesthesia and one for recovery. The anesthesia tank contains 0.015%trica buffered to pH 7.0.
In fish water, the recovery tank contains fish, water and a drop of methylene blue to control fungal infections. Anesthetize one to three adult zebra fish by immersion in the trica tank observe until swim movements stop and GI ventilation becomes slow and regular. Depending on fish size, complete anesthesia may take two to five minutes.
Use an aquarium fish net to transfer the fish to a piece of wet paper towel on a Petri dish. Using a blunt spatula, orient the fish so they are parallel to each other. With the left lateral side facing up, fold part of the paper towel over the fish to keep them moist.
Next, view the Petri plate under the stereo microscope using low angle incident light to illuminate the head. Focus on the base of the left maxillary marble using fine forceps. Tightly grasp the marble shaft slightly distal to the border of the maxilla.
Lift the marble and insert a fine tipped spring scissors just proximal to the forceps for stability. The scissors can touch the forceps or alternately the bottom of the petri plate. Slide the jaws of the scissors along the shaft of the marble until the cutting edge approaches the edge of the maxilla.
Cut through the marble shaft and remove the amputated marble. This tissue can be fixed and analyzed separately. After surgery, transfer the fish to the recovery tank.
Allow the fish to recover for several hours to overnight and return the fish to the rearing system. Wait one to two weeks for barb regeneration to occur. In the next section we'll show you how to prepare fixed marble tissue for analysis.
This procedure begins with marbles that have been collected and fixed days, weeks, or months after surgery. Rinse the fixed tissue in a buffered saline solution and dissect away the regenerated marble from the left side of the head. Don't forget the right barb, which serves as a control for temporary storage.
Pipette each matched pair of marbles into a number drop of saline on a Petri dish. Cover with a lid and leave on the bench while you go. Make your gel heat 150 milliliters of 2%agros until completely dissolved.
Equilibrate the solution in a 50 to 60 degrees Celsius water bath. Next, assemble a standard gel electrophoresis rig and add one to four small tooth to sample s. Pour the melted agros into the gel mold on a level surface, adding just enough to form some very shallow wells.
Immediately return the leftover aros to the warm water bath. Insert a long glass pasture pipette so that the pipette reaches the same temperature as the aros. After the gel cools, remove the combs and then the gel mold.
To secure the gel firmly tape both sides with your favorite color of laboratory tape. The tape should extend several millimeters above the surface of the aros so that additional agros can be poured in later position the gel mold on the stage of a a stereo microscope. Magnify an empty well and bring the edges into focus.
Pipette the marbles from the Petri dish onto the surface of the gel adjacent to the well Using handles fitted with bent insect pins. Drag the marbles into the well and push them to the bottom. Align the barbells parallel to each other and against the long edges of the well Before embedding, use a fine pipette or laboratory tissue to remove excess fluid.
Using the warmed glass pipette gently add fresh molten agros around the tissue. Make last minute adjustments before the agros hardens and don't overfill the well. Record the specimen number by adding a small numbered slip of paper nearby.
After all of the specimens are embedded, smooth out the upper surface of the gel by pouring a thin layer of aros over the top. Use as little as possible so the gel remains transparent for photography. Once the gel has hardened, it can be photographed under a stereo microscope to record the size and shape of each marble pair and collect morphometric measurements for further analysis.
Agar blocks containing matched pairs of marbles can be cut out of the gel with a scalpel or razor blade. These can then be processed for paraffin histology or cryosectioning by standard methods. Alternatively, individual marbles can be dissected out of the agros with fine needles rinsed in water, and then processed for other downstream applications such as whole mount immunohistochemistry or confocal microscopy.
Finally, the entire gel can be sealed in a bag and stored in the cold. The specimens will keep for several weeks. We've just shown you how to observe and analyze the developing and regenerating maxillary bar.
It's a new system in zebrafish, so there is a lot to be discovered. Thanks for watching and good luck with your experiments.
