The overall goal of the following experiment is to deliver antisense, oligonucleotide, morphos, or other development perturbing molecules directly into the zebrafish otics at the one day or 24 hour post fertilization stage. In this example, we use morph morphos to the earliest neurotrophin responsible for static acoustic ganglion development, an immune system cytokine called macrophage migration, inhibitory factor, or MIF, to determine if morphos to this molecule affect inner ear development or neurogenesis. This is achieved by first introducing Morphos into the lumen of the otic vesicle using microinjection, then electroporation is performed, which facilitates the penetration of morphos into the cells of the otics from the lumen.
Next, the embryos are raised at 28.5 degrees Celsius in order to observe the effects of the morphos on inner ear development and neurogenesis. Over the next few days, results are obtained that show a decrease in inner ear sensory hair cells, as well as a decrease in innervation by the stato acoustic ganglion based on immunohistochemistry and foid and staining, which label acetylated tubulin in Kinocilia and the acton in Stereocilia on the hair cell surface respectively. We originally developed this method of injection and electroporation of morphos in the chick embryo and a very talented undergraduate student in the lab.
Katie Holmes successfully scaled it down to zebrafish dimensions. The present research team, including Yuchi, she a postdoc, and Matt Wyatt, a graduate student, and a former postdoc in the lab, Deb Thompson refined this technique for zebrafish. This method hopefully can answer key, key questions in the fields of developmental biology and auditory research, including whether specific morph eno effects on an organ system like the ear can be distinguished from generalized developmental effects that affect many organ systems and are secondary on the ear, for example, effects on the brain that secondarily affect the ear.
The main advantage of this technique over existing methods like micro injections at one to four cell stages is that this method directly introduces morphos into the ear at later stages. So in direct effects of morphoses on the brand, that secondary effect the inner ear can be avoided To make electrodes begin by cutting 75 micrometer diameter tungsten wire about three to five centimeters in length. Put the tungsten wire through a Molex cable connector.
Next, wrap the tungsten wires and Molex cable connector with shrink tubing and apply heat with a bunsen burner or a heat gun to seal the shrinking tubing to the wire. Then to sharpen the tip of the tungsten wire, dip the wire into one normal sodium hydroxide and using a paperclip as the other electrode electrolyze the wire with a square wave electro. The conditions we use are five 50 volt pulses, each lasting 100 milliseconds with 50 milliseconds in between pulses.
Perform three repetitions with the tip of the electrode submerged in the one normal sodium hydroxide. Insert the electrode slightly further into the sodium hydroxide and perform one more repetition. Insert the electrode further, still advancing the electrode approximately the same distance both times and perform one last repetition.
This graduated sharpening produces a very fine point on the electrode, allowing for more accurate, less damaging insertion of the electrodes into the developing zebrafish embryo. Keep the wires pressed into modeling clay in a tray prior to use for electroporation for micro injecting morphos into zebrafish otic vesicles. First set up breeding tanks with a plastic divider to separate males from females in the evening.
Place four males and four females into the tank and leave the tank in an incubation room with a 14 to 10 light dark cycle overnight. The next morning after the lights are turned on, pull the divider and leave the fish undisturbed for about 20 to 30 minutes. Separate males from females and remove the breeders from the breeding tank.
Put them in tanks containing fresh fish water. Harvest the embryos from the breeding tank by pouring the egg containing water through a strainer. Rinse eggs in the strainer several times with fresh egg water before draining them into a Petri dish.
Remove any unfertilized or opaque eggs and incubate the embryos in embryo raising medium with 0.3 PP m methylene blue at 28.5 degrees Celsius overnight while the embryos incubate. Pull glass needles with a Sutter P 97 electrode puller just before injection. Warm up an AROS gel base consisting of 1%agros in fish water in a 10 millimeter Petri dish to 37 degrees Celsius in a water bath heat, 1%low melting point AGROS or LMPA in fish water until melted and keep it warm in the 37 degrees Celsius water bath.
Fill a glass needle with a morpho solution and cut the tip to the appropriate diameter. Approximately 10 to 15 microns. Mount the injection needle onto a micro manipulator that is attached to a PV eight 20 pneumatic pico pump with nitrogen tank dec coate the embryos at 24 hours post fertilization and anesthetize them with trica in 0.3 PPM methylene blue fish water.
Next, align three to five anesthetized embryos onto the AROS gel base with the right side up for convenient uniform analysis, put one to two drops of 1%LMPA over each embryo and let it solidify to fix the embryo in place. Rotate the Petri dish so that the otic vesicle is on the right side. Then place the needle into the lumen of the otic vesicle and inject the morpho solution using the foot pedal.
Immediately after microinjection, move the mounted embryos to the electroporation station to set up the electroporation station. First tape the sharpened 75 micrometer tungsten electrodes to the micro manipulators. Then place the microm manipulators on either side of the dissecting microscope stage.
Turn on the electro and set up the parameters for electroporation. The most effective parameters. For this experiment were found to be three 13 volt pulses at two milliseconds, each with 100 milliseconds in between each pulse.
Insert the electrodes so that the otic vesicle is evenly flanked with one electrode superficial to the otic vesicle and the other deep to the otic vesicle. Next, apply a current using a foot pedal or by pushing a switch when the electroporation is finished. Pour fish water onto the aros and remove the embryos using a glass pipette and by gently swirling the plate.
Use caution not to damage the embryos for embryos that are difficult to remove. Use a needle to gently break away any LMPA surrounding them. Transfer the embryos into a plate with methylene blue fish water and raise the embryos to the desired stages for morphological immunohistochemical.
In C two hybridization and microscopic analysis, representative results of ear injection and electroporation of morphos are shown here. The posterior or auditory macular labeled PM is one of the hearing organs in the fish when it is labeled with Phin red. In this photograph, the acton bundles in the stereocilia on the sensory hair cells appear as red dots on the ping pong.
Paddle shaped macula as seen by fluorescent labeling with Phin a three days post fertilization embryo injected with MIF morphs only shows more actin filaments within the stereocilia on the hair cells when compared with embryos of the same age that were electro as well as injected. However, injection and electroporation with the standard control morpho did not cause reduced F and staining compared to injection of the morpho only. The results are similar in the posterior macula, though less dramatic after five days in larvae that were injected and electroporated when compared with the embryo that was injected with MIF Morphoses only.
There was no difference between injection only and injection with electroporation when the standard control morph was used. This embryo, which is stained with an antibody against acetylated tubulin did not receive any injection or electroporation of MIF Morphoses. This embryo was electroporated but received no injection.
Both embryos show normal acetylated tubulin staining. These embryos were both injected and electroporated. They had visibly diminished tubulin staining compared to controls and far fewer neuronal processes associated with the ear associated as well as the surrounding ganglia.
When the control morpho was used for injection and electroporation, we did not see a reduction in the extent of a ated tubulin staining, nor a decrease in innervation by the acoustic ganglion when compared with injection of the morpho only Once mastered. This technique can be done in about two hours if it is performed properly. The most difficult part of the technique to master is the electroporation particular care should be taken not to damage the brain while sharpened electrodes will help to alleviate this problem.
