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. 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.
Avraham
Oshri
O
Department of Medical Neurobiology, Institute for Medical Research Israel Canada, Hebrew University-Hadassah Medical School.
Zisman
Sophie
S
Hadas
Yoav
Y
Vald
Lilach
L
Klar
Avihu
A
D000818:Animals; D001369:Axons / Q000502:physiology*; D002642:Chick Embryo / Q000502:physiology*; D004566:Electrodes; D018274:Electroporation / Q000379:methods*; D017930:Genes, Reporter; D018613:Microscopy, Confocal / Q000379:methods; D009434:Neural Pathways / Q000502:physiology*; D054259:Neural Tube / Q000166:cytology / Q000502:physiology*; D009474:Neurons / Q000502:physiology*; D014414:Tungsten
D014414:Tungsten