Figure 4

Using electroporation to study retino-tectal projections in vivo: a-b: Regions of the brain can be differentially targeted by sliding the embryo in the main channel (compare upper and lower panels in a). When the caudal part of the head was exposed, most of the optic pathway was electroporated (b). c-e: The transfected area can be restricted by reducing the amount of embryo area directly facing the electrodes. The modified chamber used to restrict electroporation is depicted in c (note the narrowing of the transverse channel in the inset), and a representative example of GFP expression 12 h post electroporation in a live embryo is shown in d. GFP expression in the tectum is shown on a wholemount dissected brain (e). Axons emanating from these neurons can be clearly observed (arrow). The dashed line delineates the diencephalon/mesencephalon boundary. The transfected area is restricted to the OT (dorsal mesencephalon). f-g: Electrodes can be placed dorsal and ventral to the embryo to target the ventral or dorsal part of the brain. A frontal section through the midbrain (g) demonstrating that ventral populations can be targeted by placing the embryo on its side in the specifically designed chamber represented in f. h-r: Retinas can be electroporated without affecting eye development. 48 h post electroporation, GAP-GFP was detected in all the retinal layers and outlined different retinal cell types with their characteristic morphologies (h-i). Eye microanatomy appeared normal (h). Eye-targeted electroporation can be performed by placing the embryo ventral side up, so that the eye but not the brain faces the electrodes (j). Eye-specific electroporation can be performed with limited brain transfection. Insert: side view of a transfected embryo 24 h after eye-targeted electroporation. GFP signal was detected in the eye and the RGC axons navigating to the tectum (arrow) but not in the brain on frontal sections (k). l-n: Co-electroporation of pCS2GAP-RFP with pEGFP. Most of the GAP-RFP positive cells (m) are also EGFP positive (n). Double positive cells are marked with white dots and the arrows point to axons leaving the retina. Outlines of the retina and lens were drawn from the corresponding DAPI counterstainings. After GAP-GFP electroporation, axons can be monitored using time-lapse microscopy (o-q) and growth cone morphology can be analyzed (r) in wholemount brain preparations. Axons were monitored as they entered the tectum. Initial positions of the two growth cones are indicated (white dot and rectangle). Time is in hours. Epi., epiphysis. Scale bars: 400 μm in a, d and insert j; 200 μm in b and e; 100 μm in k; 50 μm in h, i and l; 25 μm in o-q; 10 μm in m and n; 5 μm in r.