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

Summary

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.

Abstract

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.

Protocol

I. Electroporation Egg handling Place the eggs in an humidified incubator set to 37-38°C, preferable an incubator with a rocking trays. Embryo are electroporated after about 66 hours of the incubation, when they have reached Hamburger & 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 sho…

Discussion

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 neurons^1,3,5. 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…