Background: Lim-HD proteins control crucial aspects of neuronal differentiation, including subtype identity and axonal guidance. The Lim-HD proteins Lhx2/9 and Lhx1/5 are expressed in the dorsal spinal interneuron populations dI1 and dI2, respectively. While they are not required for cell fate acquisition, their role in patterning the axonal trajectory of dI1 and dI2 neurons remains incompletely understood.

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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.

The ventral midline of the embryonic neural tube, the floor plate, has a profound role in guiding axons during embryonic development. Floor plate-derived guidance cues attract or repel axons, depending on the neuronal subtype and developmental stage. Netrin-1 and its receptor, Deleted in Colon Carcinoma (DCC), are the key constituents of commissurral axons guidance cues toward the floor plate. Recent studies have implicated Down Syndrome Cell Adhesion Molecule (Dscam) as an additional Netrin-1 receptor. In this study, we examined the role of Dscam in guiding defined spinal dorsal interneuron populations. In vivo knockdown and ectopic expression of Dscam were performed in the dorsal dI1, dI2 and dI3 interneurons of chick embryos, by separately increasing or decreasing Dscam expression in each of these three specific interneuronal populations. Neuron-specific gain and loss of function of Dscam had no effect on the axonal trajectories of dI1-3 neurons. The commissural neurons, dI1c and dI2, crossed the midline, and the ipsilaterally projecting neurons, dI1i and dI3, projected ipsilaterally. However, the fasciculation of dI1 axons was diminished when Dscam expression was attenuated. Dscam is not required for either attraction to or repulsion from the floor plate. In contrast, Dscam is required for the fasciculation of axons, probably via homophilic interaction.

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