Fig. 6. Ectopic SDF1 expression disrupted migration of granule cells in vitro. GFP or SDF1 and GFP were electroporated into slice cultures and subsequently allowed to develop for 4 days. Cultures were then double immunostained for GFP and Prox1 to identify the extent of the electroporated tissue and location of granule cells, respectively, in the culture. (A) Schematic outlining the potential roles of SDF1 in dentate granule cell migration and the expected effect of ectopic expression (green shading) upon Prox1-positive cells (orange). (B) In order to express SDF1 ectopically in the hippocampus, we used focal electroporation. A schematic of the apparatus is shown. The slice culture on a membrane was placed on a 5% agarose section in contact with the positive electrode. Another agarose piece containing DNA was placed on the negative electrode and a current was applied. (C-F) Electroporation of GFP without SDF1 resulted in normal granule cell migration. Prox1 (red) is shown with GFP (green) staining to allow comparison of electroporation site with granule cell migration site. Additionally, to visualize clearly the location of all granule cells, Prox1 staining is shown alone. In both examples (C,E and D,F), granule cells migrate appropriately to the dentate gyrus despite GFP (green) expression throughout the hippocampus. (G-J) Electroporation of GFP and SDF1 resulted in disruption of granule cell migration. Again, GFP staining was used to identify the location of electroporated tissue. We show two distinct examples here, one with electroporation throughout the hippocampal formation, including the forming dentate gyrus (G,I) and the other throughout the hippocampus but not including the forming dentate gyrus (H,J). In both examples, Prox1-positive cells can be seen in the fimbrial region. Additionally, when ectopic expression included the middle of the developing dentate (G,I), many cells migrated to the position of the ectopically expressed SDF1 but failed to migrate any further into the dentate gyrus (arrows). When SDF1/GFP is electroporated into the hippocampus, excluding the dentate (H,J), very few cells leave the vicinity of the fimbria. For reference, the fimbria is labeled with an asterisk. Scale bar: 150 µm.