Figure 6

Determine retinal cell type of the GFP-expressing cells using immunohistochemistry method. GFP-expressing retinal tissues at three developmental stages (E10, E14, and E18) were sectioned and stained with retinal cell type specific antibodies, e.g., Xap-1 (A-C) and Visinin (D-F) for photoreceptor cells, Lim1+2 for horizontal cells (G-I), Pkcα for bipolar cells (J-L), Brn3a for ganglion cells (M-O), and Vimentin for Müller glial cells (P-R). For each set of images (A-R) the entire retina cross section is shown to allow for the laminar location to be easily visualized. The image on the right shows a merged high magnification image and a pair of separate images showing the antibody staining and GFP fluorescence. The white-boxed region is shown in higher magnification on the right. Double labeled cells are indicated by arrowheads at higher power. Staining with Xap-1 (A-C) and Visinin (D-F) confirmed the identity of GFP-expressing cells in the photoreceptor layer as cone photoreceptors. Lim1+2 labeling consistently labeled cells on the outermost region of the INL. Antibody staining with Pkcα failed to label any GFP-expressing cells at E10 (J). The Pkcα positive GFP-expressing cells were first seen at E14. Pkcα staining increased in both frequency and intensity in GFP-expressing cells at E18. Double labeled cells showed round cell bodies at both E14 and E18 and two distinct processes extending in opposite directions, characteristic of bipolar cells, were regularly observed at E18 (L, GFP). Antibody staining with Brn3a confirms that the GFP-expressing cells (arrowheads in M-O) are ganglion cells. As previously reported Brn3a does not label all ganglion cells. This was seen in E14 tissue (N) where GFP-expressing cells that show ganglion cell morphology and location but no Brn3a labeling (asterisk). Vimentin showed strong labeling at E10 (P) however failed to show double labeling with GFP. Double labeling at E14 (Q) was very rare and slightly more frequent at E18 (R). In both cases double labeling was only seen in the processes of the cells (Q-R, arrowheads).