Fig. 4. Retinal flat-mounts showing RGCs and optic fiber layers from E16.5 retinas. (A) Low magnification of a wild-type retina labeled with anti-NFL (axons, green) and propidium iodide (nuclei, red). Axons were well fasciculated and were growing counter-radially into the optic disk (OD). (B) An enlarged view of the retina from the area highlighted in A. (C) Same image as B with the green channel (axons) removed. Note numerous nuclei in the ganglion cell layer. (D) Low magnification of Brn3b^–/– retina labeled with the same antibodies as in A. (E) An enlarged view of the retina from the area highlighted in D. The axon number was greatly reduced when compared with that of a wild-type retina. Axons were not fasciculated and many did not enter the OD. (F) Same image as E with the green channel (axons) removed. The density of nuclei was not reduced when compared with that of the wild type. (G) Low magnification of a Brn3b/Brn3c double mutant retina labeled with the same antibodies as in A. Axons were barely detectable (arrows). (H) An enlarged view from the highlighted area shown in G. The axon number was further reduced when compared with E, but misrouted axons were not observed. The thick green-labeled tubule structures were capillaries that label non-specifically. (I) Same image as that in H with green channel removed. A slightly reduced nuclei number was detected in the vacant areas, but the reduction was not significant when compared with Brn3b^–/– retinas.