Figure 3.

Images of retina from Tg(gfap:GFP) (A, B, C) zebrafish showing localization of anti-cytokeratin and GFP (A, 60× oil immersion, NA 1.4; B, C, 20× water immersion, NA 0.95), and (D) a diagram showing the ribbon structure of zebrafish optic nerve. 3A. A portion of retina labeled with anti-cytokeratin (magenta), DAPI (blue) and GFP. The inner limiting membrane (ilm) appears brightly decorated with anti-cytokeratin, which also less intensely labels cytoplasm of cells in the inner plexiform layer (example at arrow). The foot processes (fp) and inner segments of the Müller glia cells are illuminated by GFP and the nuclei of the RGCs (rgcn) by DAPI. The calibration bar represents 45 μm. 3B. Image of the optic nerve from the same section as shown in Figures 3A and 3C. Anti-cytokeratin labeling (magenta, arrows) can be seen in the cytoplasm of the optic nerve astrocytes that form the neurolemma of the ribbon-like optic nerve (see Figure 3D). Note the absence of GFP expression. The calibration bar represents 175 μm. 3C shows a section of retina that includes the optic nerve head, with the dotted line highlighting the portion of the section where the optic nerve appears exiting the retina. Cells resembling optic nerve astrocytes showing anti-cytokeratin labeled cytoplasm (magenta; example at arrow) appear to stream from the nerve into the retina. Note how the GFP-expressing Müller glial cells appear to form or strongly interact with the physiological cup of the optic nerve head, but then are excluded from the optic nerve itself. The calibration line represents 50 μm. Figure D is a diagram (adapted with permission from Figure 2D, Macdonald et al. [12]) illustrating the ribbon nature of the nerve, and the reticular astrocytes (green with red nuclei) forming the neurolemma and extending processes to the nodes (arrow, blue) in the myelin sheath formed by oligodendrocytes (O) on the RGC axons (n). The pattern of anti-cytokeratin staining seen in Figure 3B is most consistent with the Macdonald et al. [12] description of optic nerve astrocytes and cytokeratin distribution.