1Northwestern University Feinberg School of Medicine, Children's Hospital of Chicago Research Center, 2Departments of Pediatrics, Neurology and Physiology, Northwestern University Feinberg School of Medicine
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Yang, B., Geary, L. B., Ma, Y. C. In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development. J. Vis. Exp. (66), e4017, doi:10.3791/4017 (2012).
Dopaminergic neurons located in the ventral midbrain control movement, emotional behavior, and reward mechanisms1-3. The dysfunction of ventral midbrain dopaminergic neurons is implicated in Parkinson's disease, Schizophrenia, depression, and dementia1-5. Thus, studying the regulation of midbrain dopaminergic neuron differentiation could not only provide important insight into mechanisms regulating midbrain development and neural progenitor fate specification, but also help develop new therapeutic strategies for treating a variety of human neurological disorders.
Dopaminergic neurons differentiate from neural progenitors lining the ventricular zone of embryonic ventral midbrain. The development of neural progenitors is controlled by gene expression programs6,7. Here we report techniques utilizing electroporation to express genes specifically in the midbrain of Hamburger Hamilton (HH) stage 11 (thirteen somites, 42 hours) chick embryos8,9. The external development of chick embryos allows for convenient experimental manipulations at specific embryonic stages, with the effects determined at later developmental time points10-13. Chick embryonic neural tubes earlier than HH stage 13 (nineteen somites, 48 hours) consist of multipotent neural progenitors that are capable of differentiating into distinct cell types of the nervous system. The pCAG vector, which contains both a CMV promoter and a chick β-actin enhancer, allows for robust expression of Flag or other epitope-tagged constructs in embryonic chick neural tubes14. In this report, we emphasize special measures to achieve regionally restricted gene expression in embryonic midbrain dopaminergic neuron progenitors, including how to inject DNA constructs specifically into the embryonic midbrain region and how to pinpoint electroporation with small custom-made electrodes. Analyzing chick midbrain at later stages provides an excellent in vivo system for plasmid vector-mediated gain-of-function and loss-of-function studies of midbrain development. Modification of the experimental system may extend the assay to other parts of the nervous system for performing fate mapping analysis and for investigating the regulation of gene expression.
1. Supply Preparation (not in video)
2. Egg Preparation (in video)
3. Glass Injection Needle Preparation (in video)
4. Midbrain/Neural Tube Injection (in video)
5. Electroporation (in video)
|PULSE LENGTH:||25 ms|
6. Incubate and Harvest
7. Representative Results
A diagrammatic representation of electroporating and analyzing embryonic chick midbrain is shown in Figure 1. In successfully injected and electroporated chick embryos, mCherry expression should be visible in the midbrain region after further development (Figure 2A, B, C). Assuming that mCherry correlates with the expression of other co-electroporated genes, embryos exhibiting proper mCherry expression can then be fixed and embedded to prepare midbrain cryosections for further study (Figure 2D). Generally, about 50% of ventral midbrain neural progenitors can be efficiently transfected with the above protocol. The specification of dopaminergic fate in electroporated neural progenitors can be examined by the co-localization of mCherry, Flag-tagged gene, with dopaminergic neuron markers such as Tyrosine Hydroxylase (TH) (Figure 3). The patterning of progenitor domains in ventral midbrain can be investigated by co-immnostaining cryosections with different progenitor domain markers.
Insufficient mCherry expression suggests that ectopic genes are likely not efficiently expressed. Embryos under this condition should not be used for further studies. Similarly, embryos that did not survive to the end of the experimental procedure can't be used for data collection and analysis.
Figure 1. Illustration of the embryonic chick midbrain in ovo electroporation assay. HH stage 11 embryonic chick embryos are injected with pCAG-mCherry together with genes of interest mixed with fast green to visualize the injection process (A). After filled with DNA constructs, midbrains are electroporated with a square wave electroporator. Embryos are further incubated until desired HH stage 23 is reached. Then mCherry-positive embryos are harvested, fixed, and embedded (B). Midbrain cryosections are prepared with cutting planes indicated by the white line in Figure 1B, and analyzed by immunostaining with midbrain markers such as Tyrosine Hydroxylase (TH) (C). Click here to view larger figure.
Figure 2. Preparation of cryosections from embryonic chick midbrain. After 41 hours incubation, HH stage 23 chick embryos expressing mCherry and genes of interest are harvested (A, B and C), fixed with PFA, treated with sucrose, and embedded in OCT for cryosectioning. Chick embryos are carefully oriented to ensure the preparation of midbrain sections. A typical embryo morphology and the cutting plane for obtaining midbrain sections are shown (D). Click here to view larger figure.
Figure 3. Analysis of electroporated midbrain sections. Cryosections from embryos with high levels of mCherry expression (A) are prepared and stained with antibodies that recognize the Flag-tagged gene of interest (B) and midbrain dopaminergic neuron marker TH (C). Embryonic midbrains electroporated with pCAG-mCherry and pCAG-Flag empty vector serve as control. Click here to view larger figure.
The in ovo electroporation of embryonic chick midbrain offers a low cost and rapid alternative to the generation of transgenic or knockout animals to perform in vivo study of gene functions in midbrain dopaminergic neuron development. Using short 2 mm long L-shaped platinum electrodes together with embryonic midbrain-specific DNA injection is the key for achieving efficient expression of the gene of interest in midbrain dopaminergic neuron progenitors. In addition, using correct HH stage 11 chick embryos is critical. This is because first, at HH stage 11, the development of chick midbrain is advanced enough such that the forebrain, the midbrain, and the hindbrain are morphologically distinguishable. This allows for the accurate positioning of the injection needle and electrodes at the midbrain region, and thus efficient DNA injection and transfection. Second, at HH stage 11, the anterior neuropore of the chick embryo is not yet completely closed, allowing the injected plasmid DNA to easily enter specifically into the midbrain region. The narrow junctions between embryonic forebrain, midbrain and hindbrain also help to prevent fast diffusion of DNA constructs injected specifically into the midbrain. Lastly, embryos at later stages tend to curl their heads to the side of the neural axis, making it very difficult to target the electrode specifically at the midbrain. The incubation time necessary to obtain HH stage 11 embryos may vary slightly in response to deviations in incubation temperature, as well as natural differences between eggs. Under our experimental conditions, it takes about 41 hours incubation at 100 °F.
To obtain embryonic chick midbrain sections for analysis, embedding and cutting midbrains at the correct orientation is very critical. Forty-one hours after electroporation, chick embryos often display a morphology similar to Figure 2D. Preparing cryosections with a cutting plane parallel to the ones shown in Figure 2D maximizes the chance of getting correct midbrain sections for further immunostaining and quantification.
The fact that midbrain-specific ectopic gene expression can be achieved by electroporation opens up the possibility that this approach can be applied to other regions of the central nervous system. The location of the injection, as well as positioning of the electrodes, could significantly affect the brain regions that are efficiently transfected with DNA constructs7,15. This technique may also prove useful in reducing specific gene expression in midbrain by RNAi-mediated knockdown16. Similarly, slightly modified techniques may be implemented to study gene regulation by expressing enhancers linked to reporter genes to identify, map, and characterize new regulatory regions from genes in the chick. When applied to mouse embryos, this technique would be much quicker and easier to use than classic transgenic approaches.
The authors have no potential conflicting interests to disclose.
We thank Dr. Takahiko Matsuda for providing the pCAG-mCherry construct. YCM is supported by a Career Development Award from the Schweppe Foundation and a grant from the Whitehall Foundation.
|Fertilized chicken eggs||Charles River Laboratories|
|Egg incubator||GQF Manufacturing||1502 Sportsman|
|BTX Electroporator||BTX Harvard Apparatus||ECM830|
|Electrodes||BTX Harvard Apparatus||45-0162 L-shaped genetrodes||For use with ECM830 electroporator|
|Platinum iridium wire||Alfa Aesar||#10056||0.5 mm diameter
For making the 2 mm long electrodes
|Glass capillary tube||World Precision Instrument||TW100F-4|
|Microloader pipette tip||Eppendorf||5242 956.003|
|India ink||Staples||Filter 20% before use|
Parker Hannifin Cooperation
|Fluorescent dissection microscope||Leica||MZ16F|
|Micropipette puller||Sutter Instrument||D-97|