Fig. 3

Photoreceptor progenitor proliferation is regulated by six7.

(A) EdU (red) and six7-in situ labeling (blue) in a retinal cryosection from 48-hpf WT embryos. Note the expression of six7 is coincident with proliferating cells in the ONL. (B) EdU labeling in retinal cryosections of WT, six7-MO control and six7 morphants at 52 hpf. Note that EdU labeled cells persist in the central retina in six7 morphants. (C) Retinal cryosections of WT and six7 morphants labeled with EdU at 48 hpf and immunolabeled for rods (4C12, green) at 4 dpf. Proliferative cells are restricted to the ONL and differentiate as photoreceptors in WT and six7-knockdown retinas. Proliferative cells colabeled for rod markers in the central retina, (arrowheads, inset). (D) Retinal cryosections of WT and six7-morphants immunolabeled for phospho-histone 3 (PH3) at 48 hpf and (E) graph showing the number of PH3-positive cells in the INL and ONL per sections (WT, n = 5; and six7-MO1, n = 4; 3–4 sections/retina). Mitosis levels increased significantly in the ONL in six7-morphants. No significant changes were observed in the INL. (***p = 0.0007, Student’s t test). (F) Retinal cryosections of WT and ljr^p23ahub at 3 dpf co-labeled for TUNEL (red) and rods (4C12, green), nuclei counterstained with SYTO61. (G) Quantification of TUNEL positive cells in the ONL, INL and GCL of WT (n = 7) and ljr^p23ahub (n = 9); (*p<0.05, *****p<0.0001, Student’s t test calculated on log transformed data). (H) Transverse retinal sections from WT and six7-MO1 injected animals at 3-dpf co-labeled for TUNEL (red) and rods (4C12, green), nuclei counterstained with SYTO61. (I) Quantification of TUNEL positive cells in the ONL, INL and GCL of control (n = 7) and six7-MO1 injected embryos (n = 6); (*p<0.05, Student’s t test calculated on log transformed data). Error bars represent SD. Dorsal is up. ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer; CMZ, ciliary marginal zone.