Name: Deciphering Axonal Pathways of Genetically Defined Groups of Neurons in the Chick Neural Tube Utilizing in ovo Electroporation (Video) | JoVE Journal
Uploaded: 2010-05-02T20:17:00.000+00:00
Duration: 7 min 55 s
Description: Learn in ovo electroporation to visualize axonal pathways of genetically defined neurons in the embryonic chick spinal cord. This method enables precise study of neuronal development and connectivity.

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5 minutes

This work was supported by grants to AK from the Israel Science Foundation, The Israel ministry of health, and DFG (German Research Foundation).

I. Electroporation
Egg handling
<ol>
 <li>Place the eggs in an humidified incubator set to 37-38&deg;C, preferable an incubator with a rocking trays.</li>
 <li>Embryo are electroporated after about 66 hours of the incubation, when they have reached Hamburger &amp; Hamilton (HH) stage 18-20. At this stage the head lies at right angles to the trunk (condition called cervical flexure) and a vast set of extra-embryonic blood vessels, such as anterior, posterior, right and left vitelline veins should be seen. This is an optimal stage for enhancer-dependent specific expression 1,3,4. It is important to monitor temperature and humidity of the incubator, in order to obtain viable and synchronized embryos.</li>
 <li>After 66 hours of incubation, remove the eggs from the incubator. Place the eggs horizontally. Wait 5-10 minutes. The embryo now is located at the upper pole of the egg. </li>
</ol>
Preparations
<ol>
 <li>Prepare Hank's solution with penicillin/streptomycin (P/S) (1:100 dilution).</li>
 <li>Pull glass capillaries (0.5 mm diameter) to microcapillary. </li>
 <li>Place the electrodes (tungsten) into micromanipulator and connect electrodes to the pulse generator (ECM 830).</li>
 <li>Adjust the pulse generator with the following parameters: voltage 30v, number of pulses - 3, length of the pulse 50 ms.</li>
 <li>Prepare a mouth pipette, a syringe needle and a tape.</li>
 <li>Prepare a desired mixture of DNA (2-5&mu;g/&mu;l) and add a Fast Green dye.</li>
</ol>
Windowing and electroporation
<ol>
 <li>Using a syringe, remove 5-6 ml of albumin from each egg. </li>
 <li>With a small scissors open an oval window at the upper side of the egg.</li>
 <li>Moist the embryo with 0.5-1ml Hank's + P/S solution.</li>
 <li>Use a mouth pipette, to load the glass microcapillary with DNA mixture.</li>
 <li>Place the embryo with its tail toward you. At this stage, embryo's spinal cord is clearly seen. Hence, no contrasting techniques are required. The spinal cord is sealed at both ends, the head and the tail, but if your microcapillary is thin enough, you will be able to puncture a small hole and penetrate the neural tube at a shallow angle. A microcapillary of right diameter should be loaded easily with DNA, penetrate the tube without damaging it and release the DNA with a regular exhalation.</li>
 <li>Inject the DNA using a mouth pipette. The green dye should spread from the tip of the tail up to the vesicles of the developing brain.</li>
 <li>Place the electrodes in parallel to the neural tube, as near as you can, without touching the tube itself and pulse. At the time of the pulse, the electrodes must be covered with liquid for allowing electrical conductivity. Usually, the amount of Hank's added at the begging is sufficient. If not add one drop of Hank's just prior to the pulse.</li>
 <li>Carefully remove the electrodes and seal the window with a tape. It is important to seal the egg entirely to prevent drying of the embryo during the following incubation period.</li>
 <li>Place the egg back in the incubator.</li>
</ol>
II. Spinal cord open-book preparation
Detachment of the spinal cord from the embryo
<ol>
 <li>Remove the eletroporated embryo, at E6, from the egg and place it in a Petri dish coated with silicone containing PBS. </li>
 <li>Cut away the membranes and stretch the embryo on his ventral side using pins to hold it.</li>
 <li>Using a sharp tungsten microcapillary make a longitudinal incision along the roof plate, from the hindbrain down to the tail. </li>
 <li>Make an additional two longitudinal incisions at both sides of the spinal cord, detaching the dorsal root ganglia (DRGs) away from the spinal cord. </li>
 <li>Detach the floor plate from the tissue starting from the tail to the hindbrain, leaving the spinal cord intact.</li>
 <li>Cut the spinal cord in a transverse section at the hindbrain using fine scissors and separate it from the body.</li>
</ol>
Fixation
<ol>
 <li>Spread the isolated spinal cord in a new petri dish coated with silicone containing PBS, using pins to hold it, from the hindbrain to the tail producing a flat-mount preparation.</li>
 <li>Pour the PBS from the dish and replace it with 4% paraformaldehyde in phosphate-buffered saline.</li>
 <li>Incubate at room temperature for 1 hour.</li>
</ol>
Immunohistochemistry
<ol>
 <li>Transfer the fixed spinal cord into a vial containing primary antibody in PBS.</li>
 <li>Incubate with gentle agitations over night at 4&deg;C.</li>
 <li>Wash the antibody X5 times with PBS, each wash for 1 hour with gentle agitations.</li>
 <li>Add secondary antibody and incubate with gentle agitations over night at 4&deg;C.</li>
 <li>Wash the antibody X5 times with PBS, each wash for 1 hour with gentle agitations.</li>
</ol>
Mounting open book for imaging
<ol>
 <li>Smear grease or silicone in a rectangle shape on a slide and drip PBS within.</li>
 <li>Place the spinal cord stretched at the middle of the rectangle using tweezers and draw out the liquid around it with a Pasteur pipette.</li>
 <li>Place 1-2 drops of mounting media on the spinal cord and cover with cover slip. Squash it to avoid air bubbles.</li>
 <li>Keep the slides in dark in 4&deg;C.</li>
 <li>Inspect the spinal cord using confocal microscopy.</li>
</ol>

<ol><li>Zisman, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Proteolysis+and+membrane+capture+of+F-spondin+generates+combinatorial+guidance+cues+from+a+single+molecule%5BTitle%5D)+AND+%22J+Cell+Biol%22%5BJournal%5D)">Proteolysis and membrane capture of F-spondin generates combinatorial guidance cues from a single molecule.</a> J Cell Biol. 178 (7), 1237-1249 (2007).</li>
<li>Arakawa, T., Iwashita, M., Matsuzaki, F., Suzuki, T., Yamamoto, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Paths%2C+elongation%2C+and+projections+of+ascending+chick+embryonic+spinal+commissural+neurons+after+crossing+the+floor+plate%5BTitle%5D)+AND+%22Brain+Res%22%5BJournal%5D)">Paths, elongation, and projections of ascending chick embryonic spinal commissural neurons after crossing the floor plate.</a> Brain Res. 1223, 25-33 (2008).</li>
<li>Avraham, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transcriptional+control+of+axonal+guidance+and+sorting+in+dorsal+interneurons+by+the+Lim-HD+proteins+Lhx9+and+Lhx1%5BTitle%5D)+AND+%22+Neural+Dev%22%5BJournal%5D)">Transcriptional control of axonal guidance and sorting in dorsal interneurons by the Lim-HD proteins Lhx9 and Lhx1.</a> Neural Dev. 4 (1), 21(2009).</li>
<li>Timmer, J., Johnson, J., Niswander, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+use+of+in+ovo+electroporation+for+the+rapid+analysis+of+neural-specific+murine+enhancers%5BTitle%5D)+AND+%22Genesis%22%5BJournal%5D)">The use of in ovo electroporation for the rapid analysis of neural-specific murine enhancers.</a> Genesis. 29 (3), 123-132 (2001).</li>
<li>Reeber, S. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Manipulating+Robo+expression+in+vivo+perturbs+commissural+axon+pathfinding+in+the+chick+spinal+cord%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">Manipulating Robo expression in vivo perturbs commissural axon pathfinding in the chick spinal cord.</a> J Neurosci. 28 (35), 8698-8708 (2008).</li>
</ol>

Electroporation of plasmid DNA into the chick embryo evolves as a powerful technique for in vivo ectopic expression. The combination of specific enhancers, and chick electroporation provides a quick and efficient tool for deciphering axonal pathways of a genetically defined group of neurons1,3,5. Utilizing the Cre/LoxP and the Gal4/UAS amplification systems can augment the levels and duration of expression. The emerging picture is of a complex divergence of axonal cues that arises from interneuron subpopulatio...

deciphering axonal pathways of genetically defined groups of neurons in the chick neural tube utilizing in ovo electroporation

Deciphering Axonal Pathways of Genetically Defined Groups of Neurons in the Chick Neural Tube Utilizing in ovo Electroporation

Employment of enhancer elements to drive expression of reporter genes in neurons is a widely used paradigm for tracking axonal projection. For tracking axonal projection of spinal interneurons in vertebrates, germ line-targeted reporter genes yield bilaterally symmetric labeling. Therefore, it is hard to distinguish between the ipsi- and contra-laterally projecting axons. Unilateral electroporation into the chick neural tube provides a useful means to restrict expression of a reporter gene to one side of the central nervous system, and to follow axonal projection on both sides 1 ,2-5. This video demonstrates first how to handle the eggs prior to injection. At HH stage 18-20, DNA is injected into the sacral level of the neural tube, then tungsten electrodes are placed parallel to the embryo and short electrical pulses are administered with a pulse generator. The egg is sealed with tape and placed back into an incubator for further development. Three days later (E6) the spinal cord is removed as an open book preparation from embryo, fixed, and processed for whole mount antibody staining. The stained spinal cord is mounted on slide and visualized using confocal microscopy.

This video demonstrates how to visualize axonal pathways of genetically defined groups of neurons in the embryonic chick spinal cord utilizing in ovo electroporation of reporter genes under the control of specific enhancer elements.

Employment of enhancer elements to drive expression of reporter genes in neurons is a widely used paradigm for tracking axonal projection. For tracking ...

Hi, I am Ham from the laboratory of Dr.Review Klar at the medical neurobiology department in the Hebrew University of Jerusalem. Hi, I'm Sophie Esman and I'm also from a Lars lab. Today we are going to show you a procedure for Innovo, electroporation and spinal cord open book preparation.We use this procedure in our laboratory to study axonal trajectories of genetically defined neuronal population in the embryonic spinal cord. So let's get started. To begin this procedure, transfer fertilized white leghorn chicken eggs from room temperature to a humidified incubator, preferably with rocking trays at 37 to 38 degrees Celsius.It is important to monitor the temperature and humidity of the incubator in order to obtain viable and synchronized embryos. The chick embryos are electroporated when they have reached hamburger and Hamilton or HH stage 18 to 20, which is an optimal stage for enhancer dependence specific expression. At this stage, the head lies at right angles to the trunk.Also at HH stage 18 to 20, a vast set of extra embryonic blood vessels such as anterior, posterior right and left vital line vessels should also be seen. The chick embryos reach HH stage 19 to 20 after 66 hours of incubation, and at that time remove the eggs from the incubator After removing eggs, place them horizontally and wait for five to 10 minutes. The embryo is now located at the upper pole of the egg.Prior to electro pering, the embryos prepare Hank's solution with 100 units per milliliter of penicillin and 0.1 milligrams per milliliter of streptomycin. Prepare a desired mixture of DNA at two to five micrograms per microliter and add a few grains of the fast green dye for easy visualization of the injected DNA. Have a mouth pipette, a sterile syringe needle, small scissors, and a tape ready as well.Prepare micro capillaries by pulling 0.5 millimeter diameter glass capillaries with a pipette puller. Place the tungsten electrodes into the micro manipulators and connect the electrodes to the pulse generator. Set the voltage on the pulse generator to minus 30 volts the length of each pulse to 50 milliseconds and the number of pulses to three.Proceed to electroporation before exposing and handling the embryos. Insert a syringe at the narrow pole of the egg and remove five to six milliliters of albumin from each egg. The albumin is removed to avoid it from spilling.When cutting the in the next step, use the small scissors to cut. Open an oval window at the upper side of the egg after revealing the chick embryo, moisten it with 0.5 to one milliliters of hanks and penicillin streptomycin solution. Next, use a mouth perpet to load the glass micro capillary.With a DNA dye mixture, a micro capillary of the correct diameter should load easily with DNA. Under a binocular position the embryo for injecting the DNA into the neural tube. Place the embryo with its tail toward you.At this developmental stage, the spinal cord is clearly visible and therefore no contrasting techniques are required. The spinal cord is sealed at both the head and the tail end. However, with the micro capillary punctures more hole in the neural tube and penetrate it at a shallow angle, a micro capillary that is thin enough and of the correct diameter should penetrate the tube without damaging it.Inject the DNA using a mouth perpet. A micro capillary of the correct diameter should release the DNA with a regular exhalation. The green dye should spread from the tip of the tail up to the vesicles of the developing brain.Inject until the neural tube is filled with DNA electroporation is required for the injected DNA to enter the cells First. Place the electrodes in parallel to the neural tube and as close to it as possible without touching the tube itself. Make sure the electrodes are covered with liquid for allowing electrical conductivity during the pulse.Usually the Hank solution added immediately after exposing the embryo is sufficient. But if not a one drop of Hank's solution just prior to the pulse with the electrodes positioned and submerged in liquid pulse to electro operate, then carefully remove the electrodes and seal the window and the egg with tape. It is important to seal the egg entirely to prevent drying of the embryo during the following incubation period.Place the egg back in the incubator and incubate until the chick embryo reaches developmental embryonic. Day six or E six, when it is time for imaging when the electroporated embryo has reached developmental embryonic. Day six, E six three days after electroporation, remove it from the egg and place it into a silicone coated Petri dish containing PBS using fine scissors.Cut away the membranes and stretch the embryo on its ventral side using pins pushed into the silicone to hold it in place. Using a sharp tungsten micro capillary. Make a longitudal incision along the roof plate from the hind brain down to the tail.Make two additional longitudinal incisions on both sides of the spinal cord, detaching the dorsal root ganglia or DRGs from the spinal cord. Detach the floor plate from the tissue starting from the tail to the hind brain, leaving the spinal cord intact. Finally, use fine scissors to cut the spinal cord in a transverse section at the hind brain and separate the spinal cord from the body.Now that the spinal cord has been isolated, spread it in a new silicon coated Petri dish containing PBS using pins to hold it from the hind brain to the tail producing a flat mount preparation. Fix the spinal cord in 4%paraldehyde in PBS for one hour at room temperature immuno stain following the immunohistochemistry procedure, detailed and accompanying written protocol to mount the spinal cord open. Book preparation for imaging.Spread grease or silicone in a rectangle shape on a slide and drip PBS within the rectangle. Use tweezers to place the spinal cord, stretched the middle of the rectangle and draw out the liquid around it. With a pasta per pet place one to two drops of mounting media on the spinal cord and cover with a cover slip.Apply pressure to the cover slip. To avoid air bubbles. Keep the slides in dark in four degrees Celsius.Inspect the spinal cord using confocal microscopy. Here are some representative results of axonal rejections of chick spinal inter neurons in control experiments where RFP is expressed in all the neurons utilizing a ubiquitous enhancer element from CMV, most of the neurons and their axons are labeled. It is impossible to decipher the axonal traject of neuronal subpopulations.However, when applying specific enhance elements, a defined axonal pathway of a specific subpopulation DI two neurons in this example is revealed. So we've just shown you how to electro operate a cheek neural tube with a specific neuronal enhancer at E three and how to prepare an open book spinal cord at E six. When you're doing this procedure, you should remember to be careful and patient.So this is it. Thank you for watching and good luck with your experiments. Good luck.

deciphering-axonal-pathways-genetically-defined-groups-neurons-chick

Research

JoVE Journal

Biology

In utero electroporation is a valuable method for transfecting neuronal progenitor cells in vivo. Depending upon the placement of the electrodes and the developmental timepoint of electroporation, certain subsets of cortical cells can be targeted. Targeted cells can then be analyzed in vivo or in vitro for effects of genetic alteration.

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In utero electroporation allows for rapid gene delivery in a spatially- and temporally-controlled manner in the developing central nervous system (CNS). Here we describe a highly adaptable in utero electroporation protocol that can be used to deliver expression constructs into multiple embryonic CNS domains, including the telencephalon, diencephalon and retina.

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

To assess the function and the regulation of genes during the development of midbrain dopaminergic neurons, we describe a method that involves in ovo electroporation of plasmid DNA constructs into embryonic chick ventral midbrain dopaminergic neuron progenitors. This technique can be used to achieve efficient expression of genes of interest to study different aspects of midbrain development and dopaminergic neuron differentiation.

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

A method by which gene expression in the neural tube can be downregulated in a cell type-specific, traceable manner is described. We demonstrate how in ovo electroporation of microRNA-based plasmids that elicit spatiotemporally controlled RNA interference can be used to investigate commissural axon guidance in the developing neural tube.

In ovo Electroporation of miRNA-based Plasmids in the Developing Neural Tube and Assessment of Phenotypes by DiI Injection in Open-book Preparations

How neuronal networks are established in the embryonic brain is a fundamental question in developmental neurobiology. Here we combined an electroporation technique with novel genetic tools, such as Cre/Lox–plasmids and PiggyBac-mediated DNA transposition system in the avian hindbrain to label dorsal interneurons and track their axonal projections and synaptic targets at various developmental stages.

Electroporation of the Hindbrain to Trace Axonal Trajectories and Synaptic Targets in the Chick Embryo

This video shows the basic steps for performing whole transcriptome analysis on dissected chick embryonic spinal cord samples after transfection with in ovo electroporation.

RNA-Seq Analysis of Differential Gene Expression in Electroporated Chick Embryonic Spinal Cord

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.

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

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.

In Ovo Electroporation in the Chicken Auditory Brainstem

We describe the methods for fluorescent labeling of tangentially migrating cells by electroporation, and for time-lapse imaging of the labeled cell movement in a flat-mount culture in order to visualize migrating cell behavior in the developing chick optic tectum.

Visualization of Tangential Cell Migration in the Developing Chick Optic Tectum

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.

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

Learn in ovo electroporation to visualize axonal pathways of genetically defined neurons in the embryonic chick spinal cord. This method enables precise study of neuronal development and connectivity.

Deciphering Axonal Pathways of Genetically Defined Groups of Neurons in the Chick Neural Tube Utilizing in ovo Electroporation

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