Dissecting Cell-Autonomous Function of Fragile X Mental Retardation Protein in an Auditory Circuit by In Ovo Electroporation

Our protocol is an in vivo method to investigate the cell-autonomous functions of FMRP during development. This can help to understand the cell type-specific pathophysiology of fragile X syndrome. This method employs stage, site, and direction-specific electroporation of a drug-inducible vector system containing Fmr1 small hairpin RNA.

With this method, we achieve the selective FMRP knockdown in the auditory circuit. This method can also be applied to investigate the function of other genes in the auditory circuit or the vestibular system. Begin by placing the eggs horizontally to position the embryo on the top of the egg for easy manipulation.

Incubate at 38 degrees Celsius for 46 to 48 hours until Hamburger and Hamilton, or HH stage 12, for neural tube transfection or for 54 to 56 hours until HH 13 for otocyst transfection. Remove the eggs from the incubator, one dozen eggs at a time. Keeping eggs out of their incubator for more than one hour introduces developmental variations and reduces viability.

Use a flashlight to cast light from the bottom of the egg. The location of the embryo appears as a dark area on the eggshell. Mark the location of the embryo on the eggshell with a pencil.

Wipe the eggs with gauze containing 75%ethanol and drill a hole in the pointy end of the eggs using the tip of scissors. Then, remove two milliliters of albumin with an 18-gauge needle syringe. Ensure the hole is only large enough to allow the needle insertion.

Wipe away any leaking albumin with gauze and seal the hole with clear tape. Cover the top of the eggs with clear tape, centering on the pencil-marked dark area to minimize cracks and prevent falling shell debris during windowing. Wipe all surgery tools with gauze containing 75%ethanol and wipe the tape-covered area with 75%ethanol.

Use a small pair of scissors to cut a one to two square-centimeter window around the circumference of the pencil mark. Hold the scissors flat to avoid damaging the embryo underneath. Place the windowed egg under a stereomicroscope with a 10X eye piece and 2X zoom.

Fill a one-milliliter syringe with the ink solution and then fit a 27-gauge needle. Bend the needle to a 45 degree angle with forceps. Under the microscope, carefully poke from the edge of the area opaqua and insert the needle beneath the embryo.

Inject around 50 microliters of ink, which will diffuse below the area pellucida, for embryo visualization. The ink will form a dark background for the clear visualization of the embryo. Pull glass capillaries into pipettes using a pipette puller.

Under a dissecting microscope, carefully open the tip of the capillary needle to 10 to 20 micrometers in diameter with forceps. Store the pipettes in a storage box until use. Next, fill a capillary pipette with 0.5 to 1 microliters of the plasmid mixture by applying negative pressure through a rubber tube at the end of the pipette with a syringe.

Place the egg under the microscope so that the embryo is vertically oriented with the tail near to you. Hold the capillary pipette with one hand or with the three-axis manipulator and drive the tip of the pipette to the rhombomere 5 to 6. in a tail-to-head direction.

Gently poke the tip through the vitellin membrane and into the dorsal neural tube, and then withdraw the pipette a bit so the tip is in the lumen of the neural tube. Inject the plasmid mixture by applying air pressure until the tinted plasmid diffuses fully into rhombomere 5 to 6 and extends into rhombomere 3 and rhombomere 4. Check for successful injection, which is achieved when the blue plasmid solution rapidly diffuses down the neural tube without leaking.

When leaking occurs, the blue quickly fades. Immediately after the injection, place a platinum bipolar electrode on either side of the neural tube. Deliver two pulses of 12 volts and 50 milliseconds duration with an electroporator.

Observe air bubbles at the ends of the bipolar electrode, with more on the negative side. Check for successful electroporation, which is achieved when the tinted plasmid mixture enters the neural tube tissue near the positive side of the electrode. After electroporation, carefully remove the bipolar electrode.

Cover the window on the the eggshell with a piece of transparent film precut to two-inch squares and sprayed with 75%ethanol. Place the egg back into its incubator. Clean the bipolar electrode by delivering 10 to 20 pulses of 12 volts and 50 milliseconds duration in saline before proceeding to the next egg.

Under the microscope, place the egg in such a way that the embryo is vertical with the tail near you. Hold the capillary pipette and gently poke the right otocyst in dorsolateral direction. Inject the plasmid mixture with air pressure until the otocyst is filled with blue solution.

Check for a successful injection, which is achieved when the blue plasmid mixture is confined within the otocyst and doesn't leak. Immediately after the injection, place the bipolar electrode on the otocyst and position the positive and negative sides anterior and posterior to the otocyst, respectively. Deliver two pulses of 12 volts and 50 milliseconds duration with the electroporator.

Check for successful electroporation, which is achieved when the blue plasmid mixture enters the tissue of the otocyst near the positive side of the electrode. After electroporation, carefully remove the bipolar electrode and cover the window on the eggshell with a transparent film. Finally, return the egg to the incubator.

The representative image shows an example of embryonic day three, or E3, following neuro tube transfection and dox treatment at embryonic day two, or E2.Transfected cells with Fmr1 shRNA and EGFP on the cross section are shown here. EGFP-expressing cells were confined to one side of the neural tube and the brainstem. A cross section at E15 with EGFP-expressing nucleus magnocellularis, or NM neurons, on the transfected side is shown here.

On the contralateral side, EGFP-containing axons were seen in the ventral portion of the nucleus laminaris, or NL.The knockdown effect of Fmr1 shRNA was validated by marked reductions in FMRP immunoreactivity in transfected NM neurons compared to neighboring non-transfected neurons. The auditory ganglion at E19 with FMRP immunoreactivity is shown here. Auditory ganglion neurons were not transfected.

Some glial cells derived from the cranial neural crest cells were transfected. The representative images show FMRP knock-down in the auditory duct following otocyst transfection. Auditory ducts at E9 exhibited extensive EGFP fluorescence.

On transverse sections, EGFP-expressing cells were located in the basilar papilla and the auditory ganglion. The knockdown effect of Fmr1 shRNA was validated by reduced FMRP immunoreactivity in transfected hair cells as compared to neighboring non-transfected hair cells. Similarly, FMRP immunoreactivity was largely diminished in transfected auditory ganglion neurons.

The central projection of the transfected auditory ganglion neurons was tracked to the brainstem via the auditory nerve with characterized endbulb terminals. While attempting this procedure, the most important thing is to control the site and direction of electroporation for selective transfection. Following this procedure, single cell type filling, immunostaining, cell sorting followed with mass spectrum, or single-cell sequencing can be performed to reveal the changes at morphological and biochemical levels.

This technique also enables selective editing of other genes with temporal control and component specificity in the auditory ear for stem circuits and can be modified to manipulate the vestibular system.