This study was supported by: a National Natural Science Foundation of China grant (No. 32000697); the Science and Technology Program of Guangzhou (202102080139); the Guangdong Natural Science Foundation (2019A1515110625, 2021A1515010619); the Fundamental Research Funds for the Central Universities (11620324); a Research Grant of Key Laboratory of Regenerative Medicine, Ministry of Education, Jinan University (No. ZSYXM202107); the Fundamental Research Funds for the Central Universities of China (21621054); and the Medical Scientific Research Foundation of Guangdong Province of China (20191118142729581). We thank the medical experimental center of Jinan University. We thank Dr. Terra Bradley for careful editing of the manuscript.

Eggs and chicken embryos were handled with care and respect in accordance with the animal protocols approved by the Jinan University Animal Care and Use Committee.
1. Egg and plasmid preparation
<ol>
	<li>Egg preparation
	<ol>
		<li>Obtain fresh fertilized chicken eggs (Gallus gallus) from the South China Agricultural University and store at 16 &#176;C before incubation. For optimal viability, set all eggs for incubation within a week of arrival.</li>
		<li>Place eggs horizontally and incubate at 38 &#176;C for 46-48 h until Hamburger and Hamilton (HH) stage 1225 for neural tube transfection, or for 54-56 h until HH13 for otocyst transfection. Because the animal pole of the egg is lighter than the vegetal pole, horizontal placement positions the embryo on the top of the egg for easy manipulation. Use caution to keep the egg horizontal in this and all following steps.</li>
		<li>The location of the embryo appears as a dark area on the eggshell when casting light from the bottom of the egg using a flashlight. At the desired HH stages, use this flashlight method to mark the location of the embryo on the eggshell with pencil.</li>
		<li>Wipe the eggs with gauze containing 75% ethanol. Drill a hole in the pointy end of the eggs using the tip of scissors and remove 2 mL of albumin with an 18 G needle syringe. Ensure the hole is only large enough to allow needle insertion. Wipe away any leaking albumin with gauze and seal the hole with clear tape.</li>
		<li>To minimize cracks and to prevent falling shell debris during windowing, cover the top of eggs with clear tape, centering on the pencil-marked dark area.</li>
	</ol>
	</li>
	<li>Plasmid DNA extraction
	<ol>
		<li>Clone chicken Fmr1 shRNA using a transposon-based Tet-on system as described previously20. This system contains three plasmids: pCAGGS-T2TP, pT2K-CAGGS-rtTA-M2, and pT2K-BI-TRE-EGFP-Fmr1 shRNA, providing a drug-inducible vector system by which the expression of Fmr1 shRNA and EGFP is silenced after transfection and can be turned on by doxycycline (Dox) induction. 
		&#8203;NOTE: These plasmids are not currently available in a repository. The authors agree to provide the plasmids upon reasonable request.</li>
		<li>Extract plasmid DNAs using an endotoxin-free preparation kit according to manufacturer&#39;s instruction and precipitate by adding 1/15 volume of 7.5 M sodium acetate and 1 volume of 100% isopropanol.</li>
		<li>Precipitate plasmids at -20 &#176;C overnight and centrifuge at 13,000 x g for 10 min. Dissolve the pellet with the Tris-EDTA buffer provided in the kit to a final concentration of 4-5 &#181;g/&#181;L. Plasmids can be stored at -20 &#176;C for 12 months without reduced transfection efficiency.</li>
		<li>On the day of electroporation, thoroughly mix 2 &#181;L of each plasmid in a sterile centrifuge tube using a pipette tip. The resultant 6 &#181;L volume of the plasmid mixture is enough to electroporate three dozen eggs. To help visualize the plasmid during electroporation, add 1 &#181;L of 0.1% fast green (prepared in sterile ddH2O) for every 6 &#181;L of DNA mixture26, which turns the plasmid mixture blue.</li>
	</ol>
	</li>
</ol>
2. In ovo electroporation
<ol>
	<li>Egg windowing and embryo identification
	<ol>
		<li>On the day of electroporation, remove the eggs from the incubator, one dozen eggs at a time. Keeping eggs out of their incubator for more than 1 h introduces developmental variations and reduces viability.</li>
		<li>Wipe all surgery tools with gauze containing 75% ethanol.</li>
		<li>Place each egg in a custom-made foam holder. Wipe the tape-covered area with 75% ethanol and cut a window (1-2 cm2) around the circumference of the pencil mark using a small pair of scissors (Figure 1A). When cutting, hold the scissors flat to avoid damaging the embryo underneath.</li>
		<li>Place the windowed egg under a stereomicroscope with a 10x eyepiece and 2x zoom and identify the embryo. Adjust the angle and brightness of the light source for better visualization. 
		NOTE: It takes practice and experience to visualize the embryo and identify the neural tube at this stage.
		<ol>
			<li>For beginners, use the following optional ink injection to facilitate embryo identification. Prepare 10% Indian ink solution in 0.01 M phosphate-buffered saline (PBS) and autoclave in advance. Fill a 1 mL syringe with the ink solution and then fit a 27G needle. Bend the needle to a 45&#176; angle with forceps.</li>
			<li>Under the microscope, carefully poke from the edge of the area opaca and insert the needle beneath the embryo. Inject ~50 &#181;L of ink, which will diffuse below the area pellucida for embryo visualization. The ink will form a dark background for a clear visualization of the embryo. 
			NOTE: Expel the air from the syringe before insertion and injection. When skilled at visualizing, avoid the ink injection step as it reduces the survival rate.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Neural tube injection and electroporation 
	NOTE: This procedure is performed at HH12 for transfecting NM neurons in the brainstem.
	<ol>
		<li>Pull glass capillaries (~1 mm in diameter and 100 mm long) into pipettes using a pipette puller. Under a dissecting microscope, carefully open the tip of the capillary needle to 10-20 &#181;m in diameter with forceps. Store the pipettes (usually 5-10 at one time) in a storage box until use.</li>
		<li>Fill a capillary pipette with 0.5-1 &#181;L of the plasmid mixture by applying negative pressure through a rubber tube at the end of the pipette. This can be achieved by a syringe or air pump.</li>
		<li>Place the egg under the microscope so that the embryo is vertically oriented with the tail near to you (or horizontally for injecting capillary pipettes using a three-axis manipulator). Hold the capillary pipette with one hand or with the three-axis manipulator and drive the tip of the pipette to the r5/6 in a tail-to-head direction. 
		NOTE: The most anterior hindbrain area is r1 and each subsequent bulge along the neural tube is an individual rhombomere. R5/6 is at the same anteroposterior level as the otocyst, which is a readily recognizable cup-like structure (Figure 1B-C).</li>
		<li>Gently poke the tip through the vitelline 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 r5/6 and extends into r3 and r4. 
		NOTE: It is not necessary to make a slot on the vitelline membrane for injection.</li>
		<li>Check for a successful injection, which is achieved when the blue plasmid solution rapidly diffuses down the neural tube without leaking (Figure 1B-C). When leaking occurs, the blue quickly fades.</li>
		<li>Immediately after the injection, place a platinum bipolar electrode on either side of the neural tube (Figure 1D). Deliver two pulses of 12 V and 50 ms duration with an electroporator. Observe air bubbles at the ends of the bipolar electrode, with more on the negative side.</li>
		<li>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.</li>
		<li>Cover the window on the eggshell with a piece of transparent film that is pre-cut into 2 in squares and sprayed with 75% ethanol. Place the egg back into its incubator.</li>
		<li>Clean the bipolar electrode by delivering 10-20 pulses of 12 V and 50 ms duration in saline before proceeding to the next egg.</li>
	</ol>
	</li>
	<li>Otocyst injection and electroporation 
	NOTE: This procedure is performed at HH13 for transfecting hair cells and AG neurons in the inner ear.
	<ol>
		<li>At HH13 (which is ~embryonic day 2.5), the embryo has turned so the right side of the head faces up. Under the microscope, place the egg so that the embryo is vertical, with the tail near to you. Hold the capillary pipette and gently poke the right otocyst in a dorsolateral direction (Figure 2A). 
		NOTE: At this stage, the otocyst appears as a small circular structure on the top of the body at the same anteroposterior level as r5/6.</li>
		<li>Inject the plasmid mixture with air pressure until the otocyst is filled with blue solution27. Check for a successful injection, which is achieved when the blue plasmid mixture is confined within the otocyst and doesn&#39;t leak.</li>
		<li>Immediately after the injection, place the bipolar electrode on the otocyst as depicted in Figure 2B. Position the positive and negative sides anterior and posterior to the otocyst, respectively. Deliver two pulses of 12 V and 50 ms duration with the electroporator.</li>
		<li>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. Cover the window on the eggshell with a transparent film and return the egg to the incubator.</li>
	</ol>
	</li>
</ol>
3. Administration of Dox to initiate and maintain plasmid transcription
<ol>
	<li>Preparation of the Dox solution
	<ol>
		<li>Measure 100 mg of Dox powder under a chemical hood and dissolve it in 100 mL of sterile PBS to make a 1 mg/mL of working solution. Filter the solution with a 0.22 &#181;m filter and store 1 mL aliquots at -20 &#176;C. Protect from light20,28.</li>
	</ol>
	</li>
	<li>Administration of Dox
	<ol>
		<li>For full-strength gene editing, at 24 h before the desired age, thaw a Dox aliquot on ice. Drop 50 &#181;L of Dox directly on the chorioallantoic membrane of the egg using a syringe penetrating the transparent film. Seal the needle hole with transparent film or tape after injection.</li>
		<li>To maintain the knockdown effect, administer Dox every other day until tissue harvest at the desired developmental stage.</li>
	</ol>
	</li>
</ol>
4. Tissue dissection and sectioning
<ol>
	<li>Brainstem
	<ol>
		<li>For embryos at embryonic stage 3 (E3) and E6, open the eggshell and cut the associated membrane with scissors. Spoon the embryo out and immerse it in 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer.</li>
		<li>For E9 embryos, open the eggshell, decapitate the embryo with scissors, and transfer the head to a silicon-bottomed plate filled with PBS. Pin the head down through the orbits and cut the top of the skull apart. Carefully place the forceps beneath the brain from both sides and elevate the entire brain from the skull with the shoulders of the forceps. Immerse the brain in 4% PFA.</li>
		<li>For E15 and E19 embryos, open the eggshell, decapitate the embryo with scissors, and place the head on a silicon-bottomed plate filled with PBS. Cut open the skin on top of the head to expose the skull.</li>
		<li>Incise through the skull vertically with a razor to separate the forebrain and the caudal brain. Immerse the caudal block in PBS, remove the top of the skull, and then take the brain off the skull.</li>
		<li>Pin the brain down through the optic lobe and separate the cerebellum and brainstem. Take care not to damage the auditory brainstem, which is located right below the cerebellum. Remove as much dura as possible from the brainstem and then immerse it in 4% PFA.</li>
		<li>Keep embryos and brain tissues in 4% PFA at 4 &#176;C overnight except for E19 brainstems, which should be kept under the same condition for 24 h.</li>
		<li>Following fixation, dehydrate the tissue in PBS containing 30% sucrose until it settles, which usually takes 1-3 days, depending on the age.</li>
		<li>Section the brainstem (E15 and E19) at 30 &#181;m at the coronal plane using a sliding microtome. Collect sections in PBS and store at 4 &#176;C before immunostaining.</li>
		<li>For E3 and E6 embryos and E9 brainstems, section at 50 &#181;m transversely. Collect sections in PBS and store at 4 &#176;C. Mount the sections on gelatin-coated microscope slides before immunostaining. Collecting thicker sections for these ages facilitates tissue handling.</li>
	</ol>
	</li>
	<li>Auditory duct
	<ol>
		<li>After removing the brainstems from E9, E15, and E19 embryos, the auditory duct, which is embedded in the temporal bone, is readily identifiable lying beneath the skull, and the temporal bone is easily separatable from the surrounding skull. For E9 embryos, fix the whole temporal bone in 4% PFA. For older embryos, remove the bony structure of the temporal bone to isolate the auditory duct and fix the auditory duct in 4% PFA.</li>
		<li>Keep all temporal bones and auditory ducts in 4% PFA at 4 &#176;C overnight before tissue embedding.</li>
		<li>Perform tissue embedding as described. Prepare the embedding solution as follows: Soak 10% gelatin (from bovine skin) in cold water until the gelatin granules swell and settle (about 30 min). Heat the mixture to ~50 &#176;C to dissolve the gelatin and add 20% sucrose into the gelatin solution and stir to dissolve. Store the gelatin-sucrose solution at 4 &#176;C for up to a month.</li>
		<li>For use, warm the gelatin-sucrose solution at 37 &#176;C. Soak the temporal bones/auditory ducts in the warm gelatin-sucrose solution on a 96-well plate at 37 &#176;C until they settle, usually 30-60 min.</li>
		<li>Line the bottom of the wells of a 12-well plate with a strip of transparent film. Add a layer of warm gelatin-sucrose solution and wait until it solidifies. Transfer a temporal bone/auditory duct to each well.</li>
		<li>Implant the tissue with a second layer of warm gelatin-sucrose solution. Adjust the tissue&#39;s position so that it is in the center of the gel and the auditory duct is oriented horizontally. Carefully move the plate into a 4 &#176;C refrigerator and wait until the gel is firm.</li>
		<li>Pull the transparent film strip to move the gel-tissue block out of the well. Trim extra gel into a square block and cut a corner of the block to identify the orientation. Wrap the gelatin block with a piece of foil, freeze on dry ice, and then store at -80 &#176;C until sectioning.</li>
		<li>Section the block at 20 &#181;m along the longitude of the auditory duct with a cryostat. Mount the sections directly on gelatin-coated microscope slides. Store the slides at -80 &#176;C before immunostaining.</li>
		<li>Prior to immunostaining, immerse the slides in pre-warmed PBS (45 &#176;C) for 5 min to dissolve the gelatin, and then wash the slides 3x with room temperature PBS to remove residual gelatin.</li>
	</ol>
	</li>
</ol>
5. Immunostaining and microscope imaging
NOTE: Two types of immunostaining are performed depending on whether sections are mounted on slides or free-floating in PBS.
<ol>
	<li>Immunostaining on slides
	<ol>
		<li>Wash the slides 3x for 10 min each with PBS. Circle the region containing the sections with an oil pen to prevent liquid leaking during staining. Cover the sections with a blocking solution containing 5% normal goat serum in PBS and incubate for 30 min at room temperature.</li>
		<li>Dilute the primary antibodies (for the final concentration used refer to Table of Materials) in PBS containing 0.3% TritonX-100 and 5% normal goat serum. Remove the blocking solution and then cover the sections with the primary antibody solution. Incubate the slides at 4 &#176;C overnight in a box containing a layer of distilled water at the bottom.</li>
		<li>Rinse the slides 3x for 10 min with PBS. Apply fluorescent secondary antibodies diluted at 1:500 in PBS containing 0.3% TritonX-100 on slides. Incubate the slides at room temperature for 4 h, shielded from light.</li>
		<li>Wash the slides 3x with PBS. Gently drop two drops of mounting medium on the slides and cover the sections with coverslips. Take care not to generate air bubbles between the coverslip and the slide.</li>
	</ol>
	</li>
	<li>Immunostaining for whole-mount embryos and free-floating sections
	<ol>
		<li>Wash the embryos/sections with PBS 3x for 10 min each in a well plate. Dilute the primary antibodies in PBS containing 0.3% TritonX-100 and 5% normal goat serum in centrifugal tubes. Transfer each embryo/section into a tube with a glass hook and incubate on a shaker at 60 rpm overnight at 4 &#176;C.</li>
		<li>Wash embryos/sections 3x with PBS for 10 min each in a well plate. Transfer each embryo/section into a dark centrifugal tube filled with fluorescent secondary antibody solution and incubate at room temperature for 4 h on the shaker.</li>
		<li>Wash the embryos/sections 3x with PBS in a well plate. Use a brush to mount the sections on gelatin-coated microscope slides in a 15 cm dish containing PBS. After the slides are dried slightly (~5 min), apply two drops of mounting medium and place a coverslip. Take care not to generate air bubbles between the coverslip and the slide. Keep the whole mount tissues in PBS for imaging.</li>
	</ol>
	</li>
	<li>Imaging
	<ol>
		<li>Image the whole mount tissues in PBS in a dish with a silicon bottom containing carbon powder, which provides a black background. Capture and process images using a commercial image processing Olympus software package, as described previously29.</li>
		<li>Image the sections on the slides with a confocal microscope as described previously20. Image the sections at a single focal plane with 10x, 20x, and 63x objectives. Capture all images from the same animal using the same parameters. Take care to adjust the laser level and imaging acquisition settings to avoid signal saturation.</li>
		<li>Perform image processing using the Fiji software. For illustration, adjust image brightness, contrast, and gamma using a professional image editing software.</li>
	</ol>
	</li>
</ol>

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W., Fritzsch, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Auditory+system+development:+primary+auditory+neurons+and+their+targets.">Auditory system development: primary auditory neurons and their targets.</a> Annual Review of Neuroscience. 25, 51-101 (2002).</li><li>Cramer, K. S., Fraser, S. E., Rubel, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+origins+of+auditory+brain-stem+nuclei+in+the+chick+hindbrain.">Embryonic origins of auditory brain-stem nuclei in the chick hindbrain.</a> Developmental Biology. 224 (2), 138-151 (2000).</li><li>Chervenak, A. P., Hakim, I. S., Barald, K. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatiotemporal+expression+of+Zic+genes+during+vertebrate+inner+ear+development.">Spatiotemporal expression of Zic genes during vertebrate inner ear development.</a> Developmental Dynamics. 242 (7), 897-908 (2013).</li><li>Hamburger, V., Hamilton, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+series+of+normal+stages+in+the+development+of+the+chick+embryo.">A series of normal stages in the development of the chick embryo.</a> Journal of Morphology. 88 (1), 49-92 (1951).</li><li>Lu, T., Cohen, A. L., Sanchez, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+ovo+electroporation+in+the+chicken+auditory+brainstem.">In ovo electroporation in the chicken auditory brainstem.</a> Journal of Visualized Experiments. (124), e56628 (2017).</li><li>Evsen, L., Doetzlhofer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+transfer+into+the+chicken+auditory+organ+by+in+ovo+micro-electroporation.">Gene transfer into the chicken auditory organ by in ovo micro-electroporation.</a> Journal of Visualized Experiments. (110), e53864 (2016).</li><li>Schecterson, L. C., Sanchez, J. T., Rubel, E. W., Bothwell, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TrkB+downregulation+is+required+for+dendrite+retraction+in+developing+neurons+of+chicken+nucleus+magnocellularis.">TrkB downregulation is required for dendrite retraction in developing neurons of chicken nucleus magnocellularis.</a> Journal of Neuroscience. 32 (40), 14000-14009 (2012).</li><li>Wang, X. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+glucose+environment+inhibits+cranial+neural+crest+survival+by+activating+excessive+autophagy+in+the+chick+embryo.">High glucose environment inhibits cranial neural crest survival by activating excessive autophagy in the chick embryo.</a> Scientific Reports. 5, 18321 (2015).</li><li>Yu, X., Wang, X., Sakano, H., Zorio, D. A. R., Wang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+the+fragile+X+mental+retardation+protein+correlates+with+cellular+and+synaptic+properties+in+primary+auditory+neurons+following+afferent+deprivation.">Dynamics of the fragile X mental retardation protein correlates with cellular and synaptic properties in primary auditory neurons following afferent deprivation.</a> Journal of Comparative Neurology. 529 (3), 481-500 (2021).</li><li>Li, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Islet-1+expression+in+the+developing+chicken+inner+ear.">Islet-1 expression in the developing chicken inner ear.</a> Journal of Comparative Neurology. 477 (1), 1-10 (2004).</li><li>Carr, C. E., Boudreau, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Central+projections+of+auditory+nerve+fibers+in+the+barn+owl.">Central projections of auditory nerve fibers in the barn owl.</a> Journal of Comparative Neurology. 314 (2), 306-318 (1991).</li><li>Sandell, L. L., Butler Tjaden, N. E., Barlow, A. J., Trainor, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cochleovestibular+nerve+development+is+integrated+with+migratory+neural+crest+cells.">Cochleovestibular nerve development is integrated with migratory neural crest cells.</a> Developmental Biology. 385 (2), 200-210 (2014).</li><li>Cramer, K. S., Bermingham-McDonogh, O., Krull, C. E., Rubel, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EphA4+signaling+promotes+axon+segregation+in+the+developing+auditory+system.">EphA4 signaling promotes axon segregation in the developing auditory system.</a> Developmental Biology. 269 (1), 26-35 (2004).</li><li>Evsen, L., Sugahara, S., Uchikawa, M., Kondoh, H., Wu, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progression+of+neurogenesis+in+the+inner+ear+requires+inhibition+of+Sox2+transcription+by+neurogenin1+and+neurod1.">Progression of neurogenesis in the inner ear requires inhibition of Sox2 transcription by neurogenin1 and neurod1.</a> Journal of Neuroscience. 33 (9), 3879-3890 (2013).</li><li>Curnow, E., Wang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+animal+models+for+understanding+FMRP+functions+and+FXS+pathology.">New animal models for understanding FMRP functions and FXS pathology.</a> Cells. 11 (10), 1628 (2022).</li></ol>

The authors have nothing to disclose.

To determine the cell-autonomous function of FMRP, manipulating its expression in individual cell groups or cell types is necessary. Since one of the major functions of FMRP is to regulate synaptic formation and plasticity, selectively manipulating each synaptic component of a certain circuit is prerequisite for a full understanding of FMRP mechanism in synaptic communication. Using in ovo electroporation of chicken embryos, we demonstrated a method to target FMRP expression in t...

Fragile X syndrome (FXS) is a neurodevelopmental disorder characterized by intellectual disability, sensory abnormalities, and autistic behaviors. In most cases, FXS is caused by a global loss of fragile X mental retardation protein (FMRP; encoded by Fmr1 gene) starting at early embryonic stages1. FMRP is an RNA-binding protein that is normally expressed in most neurons and glial cells in the brain, as well as in sensory organs2,3,4. In mammalian brains, FMRP is likely associated with hundreds of mRNAs that encode proteins that are important for various neural activities5. Studies of conventional Fmr1 knockout animals demonstrated that FMRP expression is particularly important to the assembly and plasticity of synaptic neurotransmission6. Several conditional and mosaic knockout models have further demonstrated that FMRP actions and signals vary across brain regions, cell types, and synaptic sites&#160;during several developmental events including axonal projection, dendritic patterning, and synaptic plasticity7,8,9,10,11,12,13,14. Acute function of FMRP in regulating synaptic transmission was studied by intracellular delivery of inhibitory FMRP antibodies or FMRP itself in brain slices or cultured neurons15,16,17,18. These methods, however, do not offer the ability to track FMRP misexpression-induced consequences during development. Thus, developing in vivo methods to investigate the cell-autonomous functions of FMRP is in great need, and expected to help determine whether the reported anomalies in FXS patients are direct consequences of FMRP loss in the associated neurons and circuits, or secondary consequences derived from network-wide changes during development19.
The auditory brainstem of chicken embryos offers a uniquely advantageous model for in-depth functional analyses of FMRP regulation in circuit and synapse development. The easy access to embryonic chicken brains and the well-established in ovo electroporation technique for genetic manipulation have contributed greatly to our understanding of brain development at early embryonic stages. In a recently published study, this technique was combined with advanced molecular tools that allow temporal control of FMRP misexpression20,21. Here, the methodology is advanced to induce selective manipulations of presynaptic and postsynaptic neurons separately. This method was developed in the auditory brainstem circuit. Acoustic signal is detected by hair cells in the auditory inner ear and then conveyed to the auditory ganglion (AG; also called the spiral ganglion in mammals). Bipolar neurons in the AG innervate hair cells with their peripheral processes and in turn send a central projection (the auditory nerve) to the brainstem where they terminate in two primary cochlear nuclei, the nucleus magnocellularis (NM) and the nucleus angularis (NA). Neurons in the NM are structurally and functionally comparable to the spherical bushy cells of the mammalian anteroventral cochlear nucleus. Within the NM, auditory nerve fibers (ANFs) synapse on the somata of NM neurons via the large endbulb of Held terminals22. During development, NM neurons arise from rhombomeres 5 and 6 (r5/6) in the hindbrain23, while AG neurons are derived from neuroblasts residing in the otocyst24. Here, we describe the procedure to selectively knockdown FMRP expression in the presynaptic AG neurons and in the postsynaptic NM neurons separately.

<table><tbody><tr><td>&lt;strong&gt;Egg incubation&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>16 &amp;deg;C refrigerator</td><td>MAGAT</td><td></td><td>Used for fertilized egg storage.</td></tr><tr><td>Egg incubator</td><td>SHANGHAI BOXUN</td><td>GZX-9240MBE</td><td></td></tr><tr><td>Fertilized eggs</td><td>Farm of South China Agricultural University</td><td></td><td>Eggs must be used in one week for optimal viability.</td></tr><tr><td>&lt;strong&gt;Plasmid preparation&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Centrifuge</td><td>Sigma</td><td>10016</td><td></td></tr><tr><td>Fast green</td><td>Solarbio</td><td>G1661</td><td>Make 0.1% working solution in distilled water and autoclave.</td></tr><tr><td>Plasmid Maxi-prep kit</td><td>QIAGEN</td><td>12162</td><td>Dissolve plasmid DNA in Tris-EDTA (TE) buffer; endotoxin-free preparation kit</td></tr><tr><td>Sodium Acetate</td><td>Sigma-Aldrich</td><td>S2889</td><td>Make 7.5M working solution in nuclase-free water.</td></tr><tr><td>&lt;strong&gt;Electroporation and Doxycycline Administration&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Electroporator</td><td>BTX</td><td>ECM399</td><td></td></tr><tr><td>1 mL / 5 mL Syringe</td><td>GUANGZHOU KANGFULAI</td><td></td><td></td></tr><tr><td>Dissecting microscope</td><td>CNOPTEC</td><td>SZM-42</td><td></td></tr><tr><td>Doxcycline</td><td>Sigma-Aldrich</td><td>D9891</td><td>Use fresh aliquots for each dose and store at -20 &amp;deg;C.</td></tr><tr><td>Glass capillary</td><td>BEIBOBOMEI</td><td>RD0910</td><td>0.9-1.1 mm*100 mm</td></tr><tr><td>Laboratory parafilm</td><td>PARAFILM</td><td>PM996</td><td>transparent film</td></tr><tr><td>Pipette puller</td><td>CHENGDU INSTRUMENT FACTORY</td><td>WD-2</td><td>Pulling condition: 500 &amp;deg;C for 15 s</td></tr><tr><td>Platinum elctrodes</td><td>Home made</td><td></td><td>0.5 mm diameter, 1.5 mm interval.</td></tr><tr><td>Rubber tube</td><td>Sigma-Aldrich</td><td>A5177</td><td></td></tr><tr><td>&lt;strong&gt;Tissue Dissection and Fixation&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Forceps</td><td>RWD</td><td>F11020-11</td><td>Tip size: 0.05*0.01 mm</td></tr><tr><td>Other surgery tools</td><td>RWD</td><td></td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma-Aldrich</td><td>158127</td><td>Freshly made 4% PFA solution in phosphate-buffered saline can be stored in 4 &amp;deg;C for up to 1 week.</td></tr><tr><td>SYLGARD 184 Silicone Elastomer Kit</td><td>DOW</td><td>01673921</td><td>For black background plates, food-grade carbon powder is applied.</td></tr><tr><td>&lt;strong&gt;Sectioning&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Cryostat</td><td>LEICA</td><td>CM1850</td><td></td></tr><tr><td>Gelatin</td><td>Sigma-Aldrich</td><td>G9391</td><td>From bovine skin.</td></tr><tr><td>Sliding microtome</td><td>LEICA</td><td>SM2010</td><td></td></tr><tr><td>&lt;strong&gt;Immunostaining&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Alexa Fluor 488 goat anti-Mouse</td><td>Abcam</td><td>ab150113</td><td>1:500 dilution, RRID: AB_2576208</td></tr><tr><td>Alexa Fluor 555 goat anti-rabbit</td><td>Abcam</td><td>ab150078</td><td>1:500 dilution, RRID: AB_2722519</td></tr><tr><td>DAPI</td><td>Abcam</td><td>ab285390</td><td>1: 1000 dilution</td></tr><tr><td>Fluoromount-G mounting medium</td><td>Southern&amp;nbsp;Biotech</td><td>Sb-0100-01</td><td></td></tr><tr><td>FMRP antibody</td><td>Y. Wang, Florida State University</td><td>#8263</td><td>1:1000 dilution, RRID: AB_2861242</td></tr><tr><td>Islet-1 antibody</td><td>DSHB</td><td>39.3F7</td><td>1:100 dilution, RRID: AB_1157901</td></tr><tr><td>Netwell plate</td><td>Corning</td><td>3478</td><td></td></tr><tr><td>Neurofilament antibody</td><td>Sigma-Aldrich</td><td>N4142</td><td>1:1000 dilution, RRID: AB_477272</td></tr><tr><td>Parvalbumin antibody</td><td>Sigma-Aldrich</td><td>P3088</td><td>1:10000 dilution, RRID: AB_477329</td></tr><tr><td>SNAP25 antibody</td><td>Abcam</td><td>ab66066</td><td>1:1000 dilution, RRID: AB_2192052</td></tr><tr><td>&lt;strong&gt;Imaging&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Adobe photoshop</td><td>ADOBE</td><td></td><td>image editing software</td></tr><tr><td>Confocal microscope</td><td>LEICA</td><td>SP8</td><td></td></tr><tr><td>Fluorescent stereomicroscope</td><td>OLYMPUS</td><td>MVX10</td><td></td></tr><tr><td>Olympus Image-Pro Plus 7.0</td><td>OlYMPUS</td><td></td><td>commercial image processing software package</td></tr></tbody></table>

dissecting cell-autonomous function of fragile x mental retardation protein in an auditory circuit by in ovo electroporation

Fragile X mental retardation protein (FMRP) is an mRNA-binding protein that regulates local protein translation. FMRP loss or dysfunction leads to aberrant neuronal and synaptic activities in fragile X syndrome (FXS), which is characterized by intellectual disability, sensory abnormalities, and social communication problems. Studies of FMRP function and FXS pathogenesis have primarily been conducted with Fmr1 (the gene encoding FMRP) knockout in transgenic animals. Here we report an in vivo method for determining the cell-autonomous function of FMRP during the period of circuit assembly and synaptic formation using chicken embryos. This method employs stage-, site-, and direction-specific electroporation of a drug-inducible vector system containing Fmr1 small hairpin RNA (shRNA) and an EGFP reporter. With this method, we achieved selective FMRP knockdown in the auditory ganglion (AG) and in one of its brainstem targets, the nucleus magnocellularis (NM), thus providing a component-specific manipulation within the AG-NM circuit. Additionally, the mosaic pattern of the transfection allows within-animal controls and neighboring neuron/fiber comparisons for enhanced reliability and sensitivity in data analyzing. The inducible vector system provides temporal control of gene editing onset to minimize accumulating developmental effects. The combination of these strategies provides an innovative tool to dissect the cell-autonomous function of FMRP in synaptic and circuit development.

By performing in ovo electroporation at different sites and at different developmental stages, we achieved selective FMRP knockdown either in the auditory periphery or in the auditory brainstem.
FMRP knockdown in NM 
Small hairpin RNA (shRNA) against the chicken Fmr1 was designed and cloned into the Tet-On vector system as described previously20. The setup for in ovo electroporation is shown in Figure...

Watch this Scientific Journal Video about Dissecting Cell-Autonomous Function of Fragile X Mental Retardation Protein in an Auditory Circuit by In Ovo Electroporation at JoVE.com

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

Using in ovo electroporation, we devised a method to selectively transfect the auditory inner ear and cochlear nucleus in chicken embryos to achieve a cell-group-specific knockdown of fragile X mental retardation protein during discrete periods of circuit assembly.

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

Fragile X mental retardation protein (FMRP) is an mRNA-binding protein that regulates local protein translation. FMRP loss or dysfunction leads to ...

dissecting-cell-autonomous-function-fragile-x-mental-retardation

Research

JoVE Journal

Neuroscience

2.2K Views.  Jinan University. Ons protocol is een in vivo methode om de cel-autonome functies van FMRP tijdens de ontwikkeling te onderzoeken. Dit kan helpen om de celtype-specifieke pathofysiologie van het fragiele X-syndroom te begrijpen. Deze methode maakt gebruik van fase-, plaats- en richtingsspecifieke elektroporatie van een geneesmiddelinduceerbaar vectorsysteem dat Fmr1 klein haarspeld-RNA bevat.Met deze methode bereiken we de selectieve FMRP-knockdown in het auditieve circuit. Deze methode kan ook worden t...

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

Induction of Protein Deletion Through In Utero Electroporation to Define Deficits in Neuronal Migration in Transgenic Models

Genetic deletion using the Cre-Lox system in transgenic mouse lines is a powerful tool used to study protein function. However, except in very specific Cre models, deletion of a protein throughout a tissue or cell population often leads to complex phenotypes resulting from multiple interacting mechanisms. Determining whether a phenotype results from disruption of a cell autonomous mechanism, which is intrinsic to the cell in question, or from a non-cell autonomous mechanism, which would result from impairment of that cell&#8217;s environment, can be difficult to discern. To gain insight into protein function in an in vivo context, in utero electroporation (IUE) enables gene deletion in a small subset of cells within the developing cortex or some other selected brain region. IUE can be used to target specific brain areas, including the dorsal telencephalon, medial telencephalon, hippocampus, or ganglionic eminence. This facilitates observation of the consequences of cell autonomous gene deletion in the context of a healthy environment. The goal of this protocol is to show how IUE can be used to analyze a defect in radial migration in a floxed transgenic mouse line, with an emphasis on distinguishing between the cell autonomous and non-cell autonomous effects of protein deletion. By comparing the phenotype resulting from gene deletion within the entire cortex versus IUE-mediated gene deletion in a limited cell population, greater insight into protein function in brain development can be obtained than by using either technique in isolation.

Induction of Protein Deletion Through In Utero Electroporation to Define Deficits in Neuronal Migration in Transgenic Models

Neuron migration is regulated by numerous cell autonomous and non-cell autonomous factors. This protocol shows how in utero electroporation can be used to determine whether a phenotype in a transgenic mouse is due to disruption of a cell intrinsic mechanism or impairment of interaction between the neuron and its environment.

Genetic deletion using the Cre-Lox system in transgenic mouse lines is a powerful tool used to study protein function. However, except in very specific ...

Developmental Biology

Culture of Embryonic Mouse Cochlear Explants and Gene Transfer by Electroporation

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair cells are aligned in one row of inner hair cells and three rows of outer hair cells that extend along the basal to apical axis of the cochlea. The explant culture technique described here provides an efficient method to isolate and maintain cochlear explants from the embryonic mouse inner ear. Also, the morphology and molecular characteristics of sensory hair cells and nonsensory supporting cells within the cochlear explant cultures resemble those observed in vivo and can be studied within its intrinsic cellular environment. The cochlear explants can serve as important experimental tools for the identification and characterization of molecular and genetic pathways that are involved in cellular specification and patterning. Although transgenic mouse models provide an effective approach for gene expression studies, a considerable number of mouse mutants die during embryonic development thereby hindering the analysis and interpretation of developmental phenotypes. The organ of Corti from mutant mice that die before birth can be cultured so that their in vitro development and responses to different factors can be analyzed. Additionally, we describe a technique for electroporating embryonic cochlear explants ex vivo which can be used to downregulate or overexpress specific gene(s) and analyze their potential endogenous function and test whether specific gene product is necessary or sufficient in a given context to influence mammalian cochlear development1-8.

We present a method that describes isolation and culture of cochlear explants from embryonic mouse inner ear. We also demonstrate a method for gene transfer into cochlear explants via square-wave electroporation. The in vitro explant culture coupled with gene transfer technique enables researchers to study the effects of altering gene expression during development.

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair ...

Gene Transfer into the Chicken Auditory Organ by In Ovo Micro-electroporation

Chicken embryos are ideal model systems for studying embryonic development as manipulations of gene function can be conducted with relative ease in ovo. The inner ear auditory sensory organ is critical for our ability to hear. It houses a highly specialized sensory epithelium that consists of mechano-transducing hair cells (HCs) and surrounding glial-like supporting cells (SCs). Despite structural differences in the auditory organs, molecular mechanisms regulating the development of the auditory organ are evolutionarily conserved between mammals and aves. In ovo electroporation is largely limited to early stages at E1 - E3. Due to the relative late development of the auditory organ at E5, manipulations of the auditory organ by in ovo electroporation past E3 are difficult due to the advanced development of the chicken embryo at later stages. The method presented here is a transient gene transfer method for targeting genes of interest at stage E4 - E4.5 in the developing chicken auditory sensory organ via in ovo micro-electroporation. This method is applicable for gain- and loss-of-functions with conventional plasmid DNA-based expression vectors and can be combined with in ovo cell proliferation assay by adding EdU (5-ethynyl-2&#180;-deoxyuridine) to the whole embryo at the time of electroporation. The use of green or red fluorescent protein (GFP or RFP) expression plasmids allows the experimenter to quickly determine whether the electroporation successfully targeted the auditory portion of the developing inner ear. In this method paper, representative examples of GFP electroporated specimens are illustrated; embryos were harvested 18 - 96 hr&#160;after electroporation and targeting of GFP to the pro-sensory area of the auditory organ was confirmed by RNA in situ hybridization. The method paper also provides an optimized protocol for the use of the thymidine analog EdU to analyze cell proliferation; an example of an EdU based cell proliferation assay that combines immuno-labeling and click EdU chemistry is provided.

Gene Transfer into the Chicken Auditory Organ by In Ovo Micro-electroporation

The auditory organ mediates hearing. Here we present a modified in ovo micro-electroporation method optimized for studying auditory progenitor cell proliferation and differentiation in the developing chicken auditory organ.

Chicken embryos are ideal model systems for studying embryonic development as manipulations of gene function can be conducted with relative ease in ovo. ...

In Ovo Electroporation in the Chicken Auditory Brainstem

Electroporation is a method that introduces genes of interest into biologically relevant organisms like the chicken embryo. It is long established that the chicken embryo is an effective research model for studying basic biological functions of auditory system development. More recently, the chicken embryo has become particularly valuable in studying gene expression, regulation and function associated with hearing. In ovo electroporation can be used to target auditory brainstem regions responsible for highly specialized auditory functions. These regions include the chicken nucleus magnocellularis (NM) and nucleus laminaris (NL). NM and NL neurons arise from distinct precursors of rhombomeres 5 and 6 (R5/R6). Here, we present in ovo electroporation of plasmid-encoded genes to study gene-related properties in these regions. We show a method for spatial and temporal control of gene expression that promote either gain or loss of functional phenotypes. By targeting auditory neural progenitor regions associated with R5/R6, we show plasmid transfection in NM and NL. Temporal regulation of gene expression can be achieved by adopting a tet-on vector system. This is a drug inducible procedure that expresses the genes of interest in the presence of doxycycline (Dox). The in ovo electroporation technique &#8211; together with either biochemical, pharmacological, and or in vivo functional assays &#8211; provides an innovative approach to study auditory neuron development and associated pathophysiological phenomena.

In Ovo Electroporation in the Chicken Auditory Brainstem

Auditory brainstem neurons of avians and mammals are specialized for fast neural encoding, a fundamental process for normal hearing functions. These neurons arise from distinct precursors of embryonic hindbrain. We present techniques utilizing electroporation to express genes in the hindbrain of chicken embryos to study gene function during auditory development.

Electroporation is a method that introduces genes of interest into biologically relevant organisms like the chicken embryo. It is long established that ...

CRISPR-mediated Loss of Function Analysis in Cerebellar Granule Cells Using In Utero Electroporation-based Gene Transfer

Brain malformation is often caused by genetic mutations. Deciphering the mutations in patient-derived tissues has identified potential causative factors of the diseases. To validate the contribution of a dysfunction of the mutated genes to disease development, the generation of animal models carrying the mutations is one obvious approach. While germline genetically engineered mouse models (GEMMs) are popular biological tools and exhibit reproducible results, it is restricted by time and costs. Meanwhile, non-germline GEMMs often enable exploring gene function in a more feasible manner. Since some brain diseases (e.g., brain tumors) appear to result from somatic but not germline mutations, non-germline chimeric mouse models, in which normal and abnormal cells coexist, could be helpful for disease-relevant analysis. In this study, we report a method for the induction of CRISPR-mediated somatic mutations in the cerebellum. Specifically, we utilized conditional knock-in mice, in which Cas9 and GFP are chronically activated by the CAG (CMV enhancer/chicken &#223;-actin) promoter after Cre-mediated recombination of the genome. The self-designed single-guide RNAs (sgRNAs) and the Cre recombinase sequence, both encoded in a single plasmid construct, were delivered into cerebellar stem/progenitor cells at an embryonic stage using in utero electroporation. Consequently, transfected cells and their daughter cells were labeled with green fluorescent protein (GFP), thus facilitating further phenotypic analyses. Hence, this method is not only showing electroporation-based gene delivery into embryonic cerebellar cells but also proposing a novel quantitative approach to assess CRISPR-mediated loss-of-function phenotypes.

CRISPR-mediated Loss of Function Analysis in Cerebellar Granule Cells Using In Utero Electroporation-based Gene Transfer

Conventional loss-of-function studies of genes using knockout animals have often been costly and time-consuming. Electroporation-based CRISPR-mediated somatic mutagenesis is a powerful tool to understand gene functions in vivo. Here, we report a method to analyze knockout phenotypes in proliferating cells of the cerebellum.

Brain malformation is often caused by genetic mutations. Deciphering the mutations in patient-derived tissues has identified potential causative factors ...

In Ovo and Ex Ovo Methods to Study Avian Inner Ear Development

The inner ear perceives sound and maintains balance using the cochlea and vestibule. It does this by using a dedicated mechanosensory cell type known as the hair cell. Basic research in the inner ear has led to a deep understanding of how the hair cell functions, and how dysregulation can lead to hearing loss and vertigo. For this research, the mouse has been the pre-eminent model system. However, mice, like all mammals, have lost the ability to replace hair cells. Thus, when trying to understand cellular therapies for restoring inner ear function, complementary studies in other vertebrate species could provide further insights. The auditory epithelium of birds, the basilar papilla (BP), is a sheet of epithelium composed of mechanosensory hair cells (HCs) intercalated by supporting cells (SCs). Although the anatomical architecture of the basilar papilla and the mammalian cochlea differ, the molecular mechanisms of inner ear development and hearing are similar. This makes the basilar papilla a useful system for not only comparative studies but also to understand regeneration. Here, we describe dissection and manipulation techniques for the chicken inner ear. The technique shows genetic and small molecule inhibition methods, which offer a potent tool for studying the molecular mechanisms of inner ear development. In this paper, we discuss in ovo electroporation techniques to genetically perturb the basilar papilla using CRIPSR-Cas9 deletions, followed by dissection of the basilar papilla. We also demonstrate the BP organ culture and optimal use of culture matrices, to observe the development of the epithelium and the hair cells.

In Ovo and Ex Ovo Methods to Study Avian Inner Ear Development

The chick is a cost-effective, accessible, and widely available model organism for a variety of studies. Here, a series of protocols is detailed to understand the molecular mechanisms underlying avian inner ear development and regeneration.

The inner ear perceives sound and maintains balance using the cochlea and vestibule. It does this by using a dedicated mechanosensory cell type known as ...

Direct Delivery of MIF Morpholinos Into the Zebrafish Otocyst by Injection and Electroporation Affects Inner Ear Development

In recent years, electroporation has become a popular technique for in vivo transfection of DNA, RNA, and morpholinos into various tissues, including the eye, brain, and somites of zebrafish. The advantage of electroporation over other methods of genetic manipulation is that specific tissues can be targeted, both spatially and temporally, for the introduction of macromolecules by the application of electrical current. Here we describe the use of electroporation for transfecting mif and mif-like morpholinos into the tissues of the developing inner ear of the zebrafish. In past studies, mif morpholino injected into embryos at the 1- to 8-cell stage resulted in widespread morphological changes in the nervous system and eye, as well as the ear. By targeting the tissues of the inner ear at later stages in development, we can determine the primary effects of MIF in the developing inner ear, as opposed to secondary effects that may result from the influence of other tissues. By using phalloidin and acetylated tubulin staining to study the morphology of neurons, neuronal processes, and hair cells associated with the posterior macula, we were able to assess the efficacy of electroporation as a method for targeted transfection in the zebrafish inner ear. The otic vesicles of 24hpf embryos were injected with morpholinos and electroporated and were then compared to embryos that had received no treatment or had been only injected or electroporated. Embryos that were injected and electroporated showed a decrease in hair cell numbers, decreased innervation by the statoacoustic ganglion (SAG) and fewer SAG neurons compared with control groups. Our results showed that direct delivery of morpholinos into otocysts at later stages avoids the non-specific nervous system and neural crest effects of morpholinos delivered at the 1-8 cell stage. It also allows examination of effects that are directed to the inner ear and not secondary effects on the ear from primary effects on the brain, neural crest or periotic mesenchyme.

A method to deliver morpholinos directly into the zebrafish otocyst at 24hpf has been developed. Using microinjection of morpholinos into the lumen of otic vesicle and electroporation to effect penetration, we were able to bypass the effect of morpholinos on the brain and obtain effects specific to the inner ear.

In recent years, electroporation has become a popular technique for in vivo transfection of DNA, RNA, and morpholinos into various tissues, including the ...

Biology

A Novel Strategy Combining Array-CGH, Whole-exome Sequencing and In Utero Electroporation in Rodents to Identify Causative Genes for Brain Malformations

Birth defects that involve the cerebral cortex &#8212; also known as malformations of cortical development (MCD) &#8212; are important causes of intellectual disability and account for 20-40% of drug-resistant epilepsy in childhood. High-resolution brain imaging has facilitated in vivo identification of a large group of MCD phenotypes. Despite the advances in brain imaging, genomic analysis and generation of animal models, a straightforward workflow to systematically prioritize candidate genes and to test functional effects of putative mutations is missing. To overcome this problem, an experimental strategy enabling the identification of novel causative genes for MCD was developed and validated. This strategy is based on identifying candidate genomic regions or genes via array-CGH or whole-exome sequencing and characterizing the effects of their inactivation or of overexpression of specific mutations in developing rodent brains via in utero electroporation. This approach led to the identification of the C6orf70 gene, encoding for a putative vesicular protein, to the pathogenesis of periventricular nodular heterotopia, a MCD caused by defective neuronal migration.

A Novel Strategy Combining Array-CGH, Whole-exome Sequencing and In Utero Electroporation in Rodents to Identify Causative Genes for Brain Malformations

Periventricular nodular heterotopia (PNH) is the most common form of malformation of cortical development (MCD) in adulthood but its genetic basis remains unknown in most sporadic cases. We have recently developed a strategy to identify novel candidate genes for MCDs and to directly confirm their causative role in vivo.

Birth defects that involve the cerebral cortex &#8212; also known as malformations of cortical development (MCD) &#8212; are important causes of ...

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

The nervous system is composed of an enormous range of distinct neuronal types. These neuronal subpopulations are characterized by, among other features, their distinct dendritic morphologies, their specific patterns of axonal connectivity, and their selective firing responses. The molecular and cellular mechanisms responsible for these aspects of differentiation during development are still poorly understood.
Here, we describe combined protocols for labeling and characterizing the structural connectivity and excitability of cortical neurons. Modification of the in utero electroporation (IUE) protocol allows the labeling of a sparse population of neurons. This, in turn, enables the identification and tracking of the dendrites and axons of individual neurons, the precise characterization of the laminar location of axonal projections, and morphometric analysis. IUE can also be used to investigate changes in the excitability of wild-type (WT) or genetically modified neurons by combining it with whole-cell recording from acute slices of electroporated brains. These two techniques contribute to a better understanding of the coupling of structural and functional connectivity and of the molecular mechanisms controlling neuronal diversity during development. These developmental processes have important implications on axonal wiring, the functional diversity of neurons, and the biology of cognitive disorders.

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

This manuscript provides protocols that use in utero electroporation (IUE) to describe the structural connectivity of neurons at the single-cell level and the excitability of fluorescently labeled neurons. Histology is used to characterize dendritic and axonal projections. Whole-cell recording in acute slices is used to investigate excitability.

The nervous system is composed of an enormous range of distinct neuronal types. These neuronal subpopulations are characterized by, among other features, ...

Inducing Cre-lox Recombination in Mouse Cerebral Cortex Through In Utero Electroporation

Cell-autonomous neuronal functions of genes can be revealed by causing loss or gain of function of a gene in a small and sparse population of neurons. To do so requires generating a mosaic in which neurons with loss or gain of function of a gene are surrounded by genetically unperturbed tissue. Here, we combine the Cre-lox recombination system with in utero electroporation in order to generate mosaic brain tissue that can be used to study the cell-autonomous function of genes in neurons. DNA constructs (available through repositories), coding for a fluorescent label and Cre recombinase, are introduced into developing cortical neurons containing genes flanked with loxP sites in the brains of mouse embryos using in utero electroporation. Additionally, we describe various adaptations to the in utero electroporation method that increase survivability and reproducibility. This method also involves establishing a titer for Cre-mediated recombination in a sparse or dense population of neurons. Histological preparations of labeled brain tissue do not require (but can be adapted to) immunohistochemistry. The constructs used guarantee that fluorescently labeled neurons carry the gene for Cre recombinase. Histological preparations allow morphological analysis of neurons through confocal imaging of dendritic and axonal arbors and dendritic spines. Because loss or gain of function is achieved in sparse mosaic tissue, this method permits the study of cell-autonomous necessity and sufficiency of gene products in vivo.

Inducing Cre-lox Recombination in Mouse Cerebral Cortex Through In Utero Electroporation

Cell-autonomous functions of genes in the brain can be studied by inducing loss or gain of function in sparse populations of cells. Here, we describe in utero electroporation to deliver Cre recombinase into sparse populations of developing cortical neurons with floxed genes to cause loss of function in vivo.

Cell-autonomous neuronal functions of genes can be revealed by causing loss or gain of function of a gene in a small and sparse population of neurons. To ...

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

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