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

Summary

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.

Abstract

Electroporation of the chick embryonic neural tube has many advantages such as being quick and efficient for the expression of foreign genes into neuronal cells. In this manuscript we provide a method that demonstrates uniquely how to electroporate DNA into the avian hindbrain at E2.75 in order to specifically label a subset of neuronal progenitors, and how to follow their axonal projections and synaptic targets at much advanced stages of development, up to E14.5. We have utilized novel genetic tools including specific enhancer elements, Cre/Lox - based plasmids and the PiggyBac-mediated DNA transposition system to drive GFP expression in a subtype of hindbrain cells (the dorsal most subgroup of interneurons, dA1). Axonal trajectories and targets of dA1 axons are followed at early and late embryonic stages at various brainstem regions. This strategy contributes advanced techniques for targeting cells of interest in the embryonic hindbrain and for tracing circuit formation at multiple stages of development.

Introduction

The hindbrain represents a key relay hub of the nervous system by communicating between the central and peripheral nervous systems via ascending and descending neuronal networks. It regulates basic functions including respiration, consciousness, hearing, and motor coordination ^1-3. During early embryonic development, the vertebrate hindbrain is transiently subdivided along its anterior-posterior (AP) axis into repetitive rhombomeres, in which distinct neuronal cell types are formed and generate multiple brainstem nuclei centers ⁴. The hindbrain is also divided along its dorsal-ventral (DV) axis into a basal and alar plate, at which discrete neuronal progenitors become specified and differentiate in distinct DV locations ^3,5,6. How the early AP and DV-specific neuronal patterns are governing the establishment of functional brainstem circuitries is largely unknown.

To gain knowledge on this fundamental question, tools are required in order to label specific subsets of neurons in the early hindbrain and to trace their axonal trajectories and connectivity at more advanced stages. We have previously utilized specific enhancer elements, and a Cre/LoxP-based conditional expression system for tracking axonal trajectory of dorsal spinal interneurons in the early chick embryo ^7-9. In the current manuscript we have targeted the hindbrain and upgraded the experimental paradigm for labeling late embryonic hindbrain interneurons, axons and their synaptic targets, using a modified electroporation strategy and the PiggyBac - mediated DNA transposition. Our new strategy allows the tagging of distinct neuronal subtypes in one side of the hindbrain and the tracking of their axonal projections and synaptic sites at various embryonic stages, from 2 up to 12 days following electroporation. Based on this method, we labeled the dorsal-most subgroup of hindbrain interneurons (dA1/Atoh1⁺ cells) and revealed two contralateral ascending axonal projection patterns, each derives from a different AP location and elongates in a distinct funiculus. dA1 axons were found to project and form synapses in the auditory nuclei, midbrain and in multiple layers of the cerebellum ¹⁰.

The combination of chick electroporation, genetic tracing of neurons and analysis of projection sites at much advanced stages of development provides a unique platform to study the formation of neuronal networks in the brain and to elucidate molecular mechanisms that govern circuit formation.

Protocol

1. Hindbrain Electroporation

1.1 Egg handling

1. Place the eggs horizontally in a humidified incubator (37-38.5 °C). Embryos are electroporated after 65-70 hr of incubation, when they reach 16-17 (HH) stage (25-30 somites).
2. Remove the eggs from the incubator; they remain in the horizontal position.

1.2 Preparations

1. Pull glass capillaries (0.5 mm diameter).
2. Connect bent L-shaped gold Genetrodes electrodes (3 mm diameter) with an adaptor holder to the pulse generator. Electroporation parameters include 25 volts, 5 pulse numbers, 45 msec pulse length, 300 msec pulse intervals.
3. Prepare a mouth pipette, 10 ml syringe needle, parafilm strips, a soft brush, sharp dissecting scissors and 25 ml sterile PBS solution. This amount is usually sufficient to handle an egg tray (18 eggs).
4. Prepare an adequate mixture of DNA plasmids (5 μg/μl each) and add about 0.025% Fast Green dye to visualize DNA injection. Usually a volume of 4 μl is sufficient for the injection of a tray.

1.3 Windowing, Injection and Electroporation

1. Clean egg shells with 70% ethanol.
2. Handle one egg at a time to minimize embryonic exposure time to air.
3. Make a hole at the egg pole with the scissors ends and remove 3-4 ml of albumin with the syringe. Do not remove a larger amount of albumin as it reduces viability.
4. Open a small oval window (2.5 x 2 cm size) at the upper center of the egg shell using the dissecting scissors.
5. Drip few drops of PBS on top of the embryo to prevent dryness during the whole process.
6. Load a small amount of the DNA mixture into the glass capillary using a mouth pipette.
7. Place the embryo with its posterior end towards you. No ink injection is required at this stage as the embryo is clearly visible. Avoiding ink injection increases embryonic survival. Penetrate the caudal hindbrain by holding the capillary at ~45°. Do not remove any membrane from around the embryo. Inject DNA solution carefully into the hindbrain lumen and exhale without forming air bubbles. DNA solution spreads anteriorly.
8. Immediately after DNA injection, place the parallel electrodes at the precise AP and DV hindbrain position you aim to target. Push the electrodes slightly ventrally without touching the heart, as it may reduce viability. Pulse the electroporator. Electrodes should be covered with bubbles to indicate electrical conductivity (Figure 1A).
9. Carefully remove the electrodes and drip few drops of PBS to cool the embryo and to remove bubbles. Seal the egg opening properly with a parafilm strip to prevent drying during incubation. Wash electrodes in PBS with the soft brush.
10. Place egg in the incubator for the desired length of time. Survival of embryos is approximately 90% 2-3 days after electroporation, and reduces up to 50% 12 days following electroporation.

2. Analysis of Embryos

2.1 Flat-mount preparation and immunofluorescence

1. Flat-mount preparations of the hindbrain can be performed up to E6.5 to easily visualize axons under a microscope or a stereo-microscope.
2. Dissect the embryo carefully by separating it from surrounding membranes and blood vessels. Place the embryo in a small silicone-coated Petri dish containing PBS. Attach the embryo to the dish with tungsten pins facing dorsally. Perform a dorsal slit in the hindbrain roof-plate using sharp glass capillaries.
3. Using sharp tweezers and micro-scissors hold the upper part of the head and pull the hindbrain tissue very carefully from rostral to caudal. After separation of the hindbrain remove remaining ventral tissues to reach a clean preparation.
4. Incubate the hindbrain with 4% paraformaldehyde (PFA) solution for 1-2 hr at room-temperature (RT). Wash the hindbrain with PBT (PBS/0.1% Triton X-100) to remove PFA.
5. For Immunofluorescence in flat-mounted hindbrains add 500 μl of blocking solution (5% of goat serum in PBT and incubate for 2 hr at RT Incubate with 1^st antibody ON at 4 °C. Wash with PBT (15 min x 3). Incubate with 2^nd antibody for 2 hr at RT. Wash with PBT (15 min x 3).
6. Place the hindbrain facing dorsally on a glass slide. Add fluorescent mounting media onto the hindbrain. Use glass capillaries or tungsten needles to flatten the hindbrain on the slide.
7. Add small amount of grease at each corner to prevent squeezing of the tissue by the cover slip. Carefully place a cover slip on top of the hindbrain and avoid air bubbles. For adherence add nail polish to cover glass edges.

2.2 Cryo-sections and Immunofluorescence

1. Cryo-sections of the brainstem can be performed at any stage to visualize axonal trajectories. Yet, after E6.5 this should be the preferred method due to the difficulty to visualize axons in the flat-mounted embryo, which becomes too thick. Moreover, demonstration of synapses requires sectioning of the tissue and a high-magnification view under a microscope.
2. Dissect the embryo and remove all surrounding tissues. Rinse with PBS. Cut the embryo at the caudal part of the medulla using micro-scissors and sharp tweezers. Make a wide incision between the cerebellum and the midbrain and remove the entire brainstem. The tissue should contain the medulla, pons and cerebellum.
3. Fix the brainstem in 4% PFA for overnight (ON) at 4 °C, rinse with PBS (10 min x 3), and incubate with 30% sucrose for ON at 4 °C until sinking.
4. Embed the hindbrain in a cryo-mold with OCT (Optimal Cutting Temperature) compound, and place it at -20 °C until freezing, as described elsewhere ¹¹.
5. Place the OCT block in the cryostat at the desired sectional axis. Section width is 12 μm.
6. Dry the slides. Add 100 μl blocking solution on each slide for 2 hr at RT. Add 100 μl of antibodies (diluted in the blocking solution to the desired concentration). Incubation period for 1^st and 2^nd antibodies is ON at 4 °C or 2 hr at RT, respectively. For each incubation time, gently add parafilm strip on top of the slide to prevent dryness. Wash with PBS (10 min x 3) between antibodies. If required, add 1:1,000 Dapi (4'-6-Diamidino-2-phenylindole), diluted in PBS for 20 min at RT. Rinse with PBS.
7. Mount the slide with fluorescent mounting media. Let dry for 1 hr before analysis.

Results

This protocol was recently used to uncover the axonal patterns and projection sites of dA1 subgroup of interneurons in the chick hindbrain ¹⁰. To specifically label these axons, an enhancer element (Atoh1), that has previously been characterized as specific for spinal dI1 neurons ^8,12,13, was confirmed to be expressed in hindbrain dA1 cells ¹⁰. The element was cloned upstream to Cre recombinase and co-electroporated at E2.75 along with a Cre dependent cytoplasmic GFP reporter plasmid (pCA...

Discussion

In ovo electroporation is a feasible, reliable, and effective tool to examine cell specification and axonal guidance during chick nervous system development ²⁰. In this protocol we describe a mode of electroporation in the chick hindbrain at E2.75 using enhancer elements which enable the conditional labeling of specific interneurons. This strategy is combined with the PiggyBac-mediated transposition system to insert foreign genes into the chick genome, which enables tracking of axonal routes, projecti...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
L-shaped gold Genetrodes 3 mm electrodes	BTX, Harvard Apparatus	45-0162
pulse generator, ECM 830	BTX, Harvard Apparatus	45-0002
OCT (Optimal Cutting Temperature) Compound	Tissue-Tek Sakura	4583 O.C.T. Compound
Nail Polish	From Any Commercial Supplier