DEV01929 Supplementary Material

Files in this Data Supplement:

• Movie 1 - Movie 1. 3D rotating view of the zebrafish brain at 30 hpf. 3D projection of a laser scanning confocal Z-series of a 30 hpf forebrain labeled for axons (anti-AT, green) and astroglia (anti-Gfap, red). The AC and POC directly overlap regions lined with astroglia in the telencephalon (up) and diencephalon, respectively. RGC axons have just left the eyes (either side of the image) and are growing towards the midline adjacent to the POC. Refer to Fig. 1A,B for orientation of commissures.

• Movie 2 - Movie 2. 3D rotating view of the zebrafish forebrain at 36 hpf. 3D projection of a laser scanning confocal Z-series of a 36 hpf forebrain labeled for axons (anti-AT, green) and astroglia (anti-Gfap, red). Astroglia are positioned superficial to the AC, POC and optic chiasm. At this age, RGC axons have just crossed the midline. The optic chiasm forms at the midline touching an astroglial cluster just anterior to the POC and associated glial bridge. The AC and POC now resemble ribbons that are oriented perpendicular to the pial surface. Refer to Fig. 2F for orientation.

• Movie 3 - Movie 3. Close-up 3D movie of the POC/chiasm region at 40 hpf. Axons were labeled with anti-AT (green) and astroglia with anti-Gfap (red). A Z-series was collected using an epifluorescent compound microscope and the movie was generated using Open Lab and Volocity software (Improvision). The starting image is a face-on view of the POC/chiasm region, with the optic chiasm positioned above the POC. The movie first zooms into the image and then rotates back and forth. Note the overlapping position of axons and astroglia. The image then completes a 180° turn, such that the view is from ‘inside’ the brain. Note the astroglial cells extend long processes into the brain, and both the POC and RGC axons course directly through these radial-like fibers. Refer to Fig. 1A for orientation.