Figure 4

Figure 4

Barhl1a:GFP labels a population of new-born RGCs and their axons. (A, A’) Single optical slices (Z-stacks) extracted from a movie frame of a retina of a Tg(barhl1a:GFP;atoh7:gap43-RFP) embryo at ~35 and ~45 hpf, respectively, showing the distribution of Barhl1a:GFP (green) and Atoh7:gap43-RFP (magenta) cells across the basal-apical surface of the retinal neuroepithelium. For both images anterior is to the left, dorsal is to the top. The outline of the retina is shown in the gray channel. Asterisks highlight the GFP-negative (gap43-RFP -positive) cells whereas the arrowheads point at the GFP positive cells. (B) Quantification of the percentage of GFP-positive (GFP + ve) and GFP-negative (GFP-ve) cells performed at 50 hpf revealed that 54% of RGCs are expressing Barhl1a:GFP (1391 total counted cells from n = 7 retinae). (C-C”) A single optical slice (Z-stack) in frontal view (dorsal is top) through the retina of a 50 hpf, Tg(barhl1a:GFP;atoh7:gap43-RFP) embryo counterstained with DAPI (C”), anti-GFP (C, green) and the RGC marker anti-Zn5 (C’, magenta) to brightly label both Barhl1a:GFP and the GCL. Asterisks highlight Barhl1a:GFP-negative (gap43-RFP/Zn5 -positive) RGCs. (D-D”) Frontal view of a 3D reconstruction of the head of an Tg(barhl1a:GFP;atoh7:gap43-RFP) embryo at 50 hpf (dorsal is to the top). Atoh7:gap43-RFP labelled retina and optic nerve are shown in magenta. At this stage, Barhl1a-GFP cells (green) have fully differentiated and extended their axons out of the retina forming the optic nerve. The optic nerves cross contra-laterally to form the optic chiasm (white arrowhead, D’) and reach their targets in the brain. The embryo has been counterstained with DAPI as shown in gray in (D). ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. Scale bars: (A-A’) 20 µm, (C-C”) 20 µm, (D-D”) 50 µm.