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

Summary

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.

Abstract

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´-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 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.

Introduction

Despite differences in morphology and cellular patterning between the mammalian and avian auditory sensory organ, the molecular factors and pathways responsible for sensory HC development are thought to be evolutionarily conserved ^1-3. The basilar papilla, which houses the auditory HCs and their surrounding SCs, develops as an outpocketing of the inner ear otocyst. Early on a pool of HC and SC progenitors is specified within the otic placode/otic cup neural-sensory competent domain (NSD). Fate-mapping data provide evidence that neuronal and sensory lineages are linked and arise from the NSD located in the antero-ventral region of the otic cup or otocyst in mice and chicken ^4,5. First, neuroblasts delaminate from the NSD to give rise to the neurons of the auditory-vestibular ganglion, which eventually split into auditory and vestibular ganglia. These neurons innervate the sensory HCs of the inner ear and nuclei in the brainstem. The cells that remain in the NSD are thought to give rise to various sensory patches, including the auditory sensory organ, consisting of the mechano-transducing sensory HCs and their associated SCs.

In both the chick and murine, auditory organ sensory progenitor cell-cycle exit and HC differentiation occur in opposing gradients. In chick, auditory progenitor cell-cycle exit starts around ~E5 and progresses from the base to the apex, and from the center to the periphery ⁶. One day later, HC differentiation starts in the apex and progresses to the base ⁷. Studying the development of the inner ear in chicken provides many technical advantages as functional mechanisms can be investigated with relative ease in ovo as opposed to manipulating embryos in utero in other model systems, which requires complex surgeries. The method described here uses in ovo micro-electroporation to target genes of interest in the presumptive basilar papilla area anterior-ventrally in the otic vesicle specifically at E4.

Electroporation in ovo is a technique that is well established and commonly used ^8-14. The principle of the electroporation technique is based on the fact that nucleotides (e.g., plasmid DNA, synthetic DNA, or RNA oligonucleotides) are negatively charged. The DNA is injected into the tissue of interest. When an electric current is placed across the tissue, the current opens up transient pores in the cell walls and allows for the uptake of the DNA, as the negatively charged DNA flows toward the positive electrode (anode). For gain-of-function experiments, genes of interest are commonly subcloned into expression plasmids that contain appropriate expression cassettes for green fluorescent protein (GFP) or red fluorescent protein (RFP). For loss-of-function experiments plasmid-based dominant negative repressor constructs, or RNAi, or morpholino constructs are commonly used ^15,16. To inhibit microRNA (miRNA) function miRNAs sponge constructs, which are typically plasmid based, can be used ¹⁷. The here described method allows for investigating the molecular mechanisms that control proliferation and differentiation in the auditory organ, as the in ovo micro-electroporation method can be easily combined with in ovo cell proliferation assay by adding EdU to the whole embryo.

The electroporation and addition of EdU is performed at E4 in the otic vesicle, which is one day before the onset of cell-cycle exit in the auditory organ. Analysis of the auditory organ is typically performed 18 - 96 hr after electroporation at E5 (onset of sensory progenitor cell-cycle exit), E6 (onset of HC differentiation), and E7 (during HC differentiation); and later up to ~E9 (end of HC differentiation). This electroporation method of gene transfer is transient lasting approximately ~4 days, because the genes of interest do not integrate into the genome, but the method is applicable for use with appropriate plasmid DNA-based expression vectors, which do have the ability to integrate into the genome, such as Tol2-mediated gene transfer ¹¹. With this method robust GFP or RFP expression lasts ~4 days in the cochlea, after which point the signals fade, yet provide an ample time window to study the intricate development of the auditory organ. This in ovo gene transfer method is novel and allows to specifically target the presumptive auditory organ at E4, which is optimal for investigations that focus on HC development in the auditory organ. It is a good addition to alternative methods, which electroporate at much younger developmental stages ^11,12 or compared to the use of in vitro basilar papillae explant cultures ¹⁸.

Protocol

The eggs and unhatched embryos are cared for and treated ethically and humanely. All protocols for unhatched embryo use were approved by the Animal Care and Use Committee at the Johns Hopkins School of Medicine, Baltimore, Maryland.

1. Eggs and Preparation of Expression Constructs

1. Eggs:
   1. Incubate 3 - 4 dozen fertilized chicken eggs (Gallus gallus) lying flat on their sides at 37 °C.
      1. After 24 hr of incubation, pull 3 cc of albumen with a syringe from the round back end of the egg and seal the hole with clear tape. This creates an air chamber between the embryo and the eggshell, allowing for ease of access to the embryo without the embryo sticking to the shell when cutting the window for access on top of the egg ¹⁹.
      2. At 117 hr of incubation time, stage the embryos according to Hamburger and Hamilton ²⁰ developmental stages at E4 (HH24-25) (See Figure 1D).
2. Expression Constructs:
   1. Prepare the plasmid DNA using reagents and the protocol provided by the manufacturer of a commercially available preparation kit. At the final step of preparation, elute the DNA into 500 µl TE (Tris-EDTA) buffer provided in the kit into a 1.5 ml tube.
   2. Ethanol precipitate the DNA to concentrate it by adding 50 µl of 3 M Sodium Acetate and 1 ml of 100% Ethanol to the tube. Place the tube at -20 °C O/N.
   3. Centrifuge the tube for 30 min at 14,000 rpm at 4 °C. Remove the supernatant and air-dry the pellet for 10 min at RT. Dissolve the pellet in 15 µl TE buffer, which should yield a concentration of ~4 µg/µl.
      NOTE: The control expression constructs used here are pMES-IRES-GFP¹⁰, which is driven by a chicken β-actin promoter, or pCI-H2B-IRES-RFP¹⁰, which is driven by the CMV promoter.

2. Chicken In Ovo Micro-electroporation and In Ovo Cell Proliferation Assay

1. In Ovo Micro-injection of Expression Construct and Micro-electroporation:
   1. Pull the glass capillary tubes into fine needles using a micro-pipette puller with the following optimized parameters for a 2.5 x 2.5 mm Box Platinum Heating Filament: Pressure = 500, Heat = 600 °C, Pull = 64, Velocity = 105, and Time = 150 msec (see Table 1).
   2. Set up the Left- and Right-handed micro-manipulator stages flanking the microscope (see Figure 1A). The Right-handed micro-manipulator holds the glass capillary needle and the Left-handed micro-manipulator holds the electrodes (Figure 1A).
   3. Prepare the finely pulled glass capillary needle for micro-injection by cutting the tip of the needle with a forceps while working under the microscope.
   4. Fill the needle manually by hand using the micro-manipulator with 2.5 µl of plasmid DNA tinted with 0.1% Fast Green while working under the microscope.
   5. Place the egg lying on its side within a mold/egg holding device (see Figure 1A). Cut a round window on top of the egg using scissors ¹⁹ (Figure 1A).
   6. While working under the microscope, carefully open the two membranes overlaying the embryo using forceps.
   7. While working under the microscope, deliver approximately ~0.5 µl of plasmid DNA to the right otic vesicle lumen by micro-injection manually using the right hand by using the micro-manipulator and by micro-electroporation.
      1. To do so, micro-inject the right otic vesicle lumen with the DNA with the Right-hand micro-manipulator (Figure 1B-D) while at the same time using the left hand holding a forcep to steady the head of the embryo, because the head of the embryo dips at stage E4. Do not micro-inject past the otic vesicle lumen, because this will damage the otic vesicle.
      2. Immediately after micro-injection, while working under the microscope, use the Left-hand micro-manipulator to place the positive (anode) 2 mm platinum electrode anterior-ventral to the right otic vesicle and the negative 2 mm electrode (cathode) in parallel 1 mm apart (see Figure 1 C). Deliver 4 pulses at 12 V with 100 msec duration and 200 msec spacing. The left otic vesicle serves as an internal untreated control.
         NOTE: Clean the electrodes in between electroporations with a cotton-tipped swab dipped in 1x PBS.
2. In Ovo Cell Proliferation Assay:
   1. Immediately after electroporation add 50 μl of 0.25 mg/ml EdU in 1x PBS by manually dropping the 50 μl EdU solution onto the whole embryo in ovo using a pipette. Seal the eggs with tape and return to the incubator for 18 - 96 hr.
   2. Carefully remove the tape and check the embryos for fluorescent GFP or RFP signal within the otic vesicle after 18 - 24 hr using a standard fluorescent microscope (see Figure 1 E-E'). If fluorescent signal is present, at this point harvest the embryos for analyses or reseal with tape and return it to the incubator for harvesting and analyses at later stages (see Figure 1 F-P'').
      NOTE: Expression of GFP signal with the pMES-IRES-GFP construct is evident 6 - 7 hr after electroporation ¹⁰.

3. Embryo Harvesting and Tissue Processing for RNA In Situ hybridization and Immunohistochemistry

1. Harvest the embryos using forceps. Rinse the embryo in cold 1x PBS. Remove the heads of the embryos, by cutting the head by its neck using forceps, and place the heads into 4% paraformaldehyde O/N at 4 °C. Puncture the brain using a forcep to allow penetration of the 4% paraformaldehyde inside the head.
2. Dehydrate the heads in 30% sucrose (in 1x PBS) O/N at 4 °C.
3. Mount the heads in cryoprotective medium by rapid freezing in a slurry of dry ice and methylbutane (2-Methylbutane). Alternatively, rapid freeze the heads in cryoprotective medium with liquid nitrogen. Store the mounted heads at -80 °C.
4. Cryosection the heads into 12 µm thick tissue sections and collect all the inner ear-containing tissue sections onto superfrosted microscope glass slides.
5. Perform standard RNA in situ hybridization and immunohistochemistry as previously described ^4,21, and analyze for EdU cell proliferation following the protocol provided by the manufacturer of the kit.
   NOTE: Hair cells can be identified using rabbit polyclonal α-MyosinVIIa (Myo7a) antibody (1:1,000, Proteus Biosciences) and fluorescently-labeled secondary antibody (1:250, Goat anti-Rabbit AlexaFluor 488; Invitrogen).

4. Image Capture and Processing

1. Take images with digital imaging equipment. Image immunohistochemistry results with a standard fluorescent microscope and results of the in situ using a standard wide-field microscope (ranging from 5x to 40x in magnification).
2. Process the images using image processing software.

Results

In this method paper plasmid DNA consisting of green fluorescent protein (GFP) expression cassettes was targeted into the developing chicken basilar papilla (BP) with optimized parameters of 12 V and 4 pulses with 100 msec pulse duration and intervals of 200 msec, yielding a ~50% embryo survival rate and efficiency of plasmid-DNA targeting into the BP. Fluorescent imaging of native GFP expression in developing embryos showed this method of electroporation preferentially targets the anteri...

Discussion

The here described method of in ovo micro-electroporation is optimized for gene transfer into the developing auditory organ. It is compatible with plasmid DNA-based expression vectors typically used to manipulate gene function/expression. The timing of electroporation at E4 is optimal for investigations that focus on HC development in the auditory organ. The most critical steps are micro-injecting the DNA into the otic vesicle lumen without going too deep with the needle and damaging the otic vesicle (see

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Sterile 1x PBS pH 7.4	gibco	10010-023
Fast Green FCF Powder	Sigma	F7252-5G
EdU Powder	Invitrogen	E10187
Click-IT EdU Alexa Fluor 555 Imaging Kit	Invitrogen	C10338
HiSpeed Plasmid Midi Kit (25)	Qiagen	12643
ECM 830 ElectroSquarePorator	BTX Harvard Apparatus
Banana to Micrograbber Cable Kit	BTX Harvard Apparatus	45-0216
Right Handed & Left Handed Micromanipulators	World Precision Instruments Inc.	M3301R & M3301L
Two 12 mm Magnetic Holding Device Stages with 7 inch vertical posts	World Precision Instruments Inc.	M10
Metal Steel Base Plate 12x24 inch	World Precision Instruments Inc.	5479
Scissors for Eggs 12 cm long curved, 12 mm extrafine blades; Spring Scissors	World Precision Instruments Inc.	14120
Micropippette Puller for pulling needles	Sutter Instrument Co.	P-97
2.5x2.5 mm Box Platinum Heating Filament	Sutter Instrument Co.
Glass Capillary Tubes/Needles/No Fiber/Borosil 1 mm	FHC	27-30-0
Hamilton Glass Syringe 100 μl	Hamilton	80601 Model 710LT
Mineral Oil (Heavy) for Hamilton Glass Syringe	Fisher Scientific	O122-1
Polyethylene Tubing for connecting the Glass Syringe and Glass Cappillary Needles/ Non Toxic	Becton Dickinson and Company (BD)	427420 Intramedic Clay Adams Brand
3 cc Disposable Syringes	Becton Dickinson and Company (BD)	309657
Disposable 21 Gauge Needles	Becton Dickinson and Company (BD)	305122
One pair of 2 mm Platinum Electrodes	Bulldog Bio. / Nepagene	CUY611P3-2
Electrode Holder	Bulldog Bio. / Nepagene	CUY580
One pair of Dumont fine forceps number 5	Fine Science Tools (FST)
Matte finish invisible tape for sealing eggs	Office Depot	520-928
Cotton-Tipped Swabs	Fisher Scientific	23-400-101
Sterile filter tips