Fig. 6

p53 stabilization upon loss of Gnl2 and NS causes apoptosis and differentially affects retinal differentiation. (A) Western blot analysis of p53 (top panel) shows stabilization of p53 protein in gnl2 and ns mutants at 48 hpf (left and middle panel) and ns mutants at 96 hpf (right panel) in comparison to low levels of p53 protein in WT embryos. As a control, stabilization of p53 upon exposure to 25 Gy of γ-irradiation is shown. Irradiation of gnl2 and ns mutants does not visibly increase p53 levels. The loading control for actin is shown in the bottom panel. (B–G) Tunel assay showing presence of apoptotic cells in gnl2 (C; n = 6) and ns mutants (D; n = 6) at 48 hpf with the highest incidence in the optic tectum. In ns mutants, apoptosis was less pronounced as compared to gnl2 mutants. (E–G) Upon injection of 1 ng of p53 MO, apoptosis is dramatically reduced in gnl2 (F; n = 13) and ns mutants (G; n = 16). Injection of p53 MO has no effect on cell survival in WT embryos (E; n = 57). (H–S) Confocal views of immunolabeling with zn8 antibody (green) that marks mature RGCs, with DAPI nuclear counterstaining (blue). (K–M) and (Q–S) show single zn8 fluorescence. Dashed lines indicate the outline of the lens. At 72 hpf, the GCL in WT (H and K) and single p53 mutants (n = 4; N and Q) has extended around the lens in a cup-like shape (arrows in K and Q). In contrast, gnl2 (n = 5; I and L) and ns (n = 5; J and M) mutants lack a fully formed GCL (arrows in L and M) and RGCs are present only in the central retina. In gnl2/p53 compound mutants (n = 3; O and R), differentiated RGCs were present only in the central retina and failed to extend around the lens (arrows in R) similar to single gnl2 mutants (arrows in L). The optic nerve size was equivalent between single gnl2 and compound gnl2/p53 mutants (arrowheads in L and R). Similarly, the GCL in ns/p53 compound mutants (n = 5; P and S) fails to extend around the lens properly (arrows in S) and the diameter of the optic nerve is similar to that of single ns mutants (arrowheads in M and S). (B–J and N–P) Lateral view, anterior to the left, dorsal up. (K–M and Q–S) Dorsal view, anterior to the left. di, diencephalon; h, hindbrain; ot, optic tectum; t, telencephalon; uic, uninjected control. Scalebar (B–G) 125 μm, (H–P) 25 μm.