TNFα is detected in cones at 5 wpf and in Müller glia at 7 wpf in gosh mutants. (A) Quantitative PCR histogram of 3, 5, and 7 wpf of control and gosh samples. 3 wpf of control and gosh samples displayed similar levels of TNFα mRNA. Upregulation of TNFα mRNA at 5 and 7 wpf in gosh samples relative to control samples. (B–M) Paraffin sections of wild-type sibling and gosh mutant retinas were labeled with antibodies against TNFα and GS at 3, 5, and 7 wpf. Müller glia are visualized as cells with nuclei located in the INL and radial processes that span the apico-basal axis of the neural retina. Wild-type sibling retinas exhibit undetectable levels of TNFα (B,D). gosh mutant retinas show no TNFα immunoreactivity at 3 wpf, as in wild-type retinas (C); however, degenerating cones display labeling against TNFα at 5 wpf (E, arrows). Circles in (E) denote autofluorescence. Nuclei of Müller glia are localized in the INL (white arrowhead) and their basal and apical processes reach the basal region of the retinal ganglion cell layer and the outer limiting membrane (black arrowhead) in the ONL in 7-wpf wild-type retinas (F,G). TNFα immunoreactivity is absent, although background signals are observed in the cone outer segment. However, in gosh mutant retina, strong signals are detected in dying cones (H, asterisks) and weaker signal in Müller glia (white arrowhead) and their cell process (H,I). (J,K) indicate high magnification images of the ONL shown in (H, asteriks). (L,M) indicate high magnification images of basal foot shown in (H, dotted box). The outer limiting membrane (J,K, black arrowhead) and the basal feet of Müller glia (L,M) are strongly stained. Amacrine and ganglion cells show faint TNFα-positive signal (H,I). n: nuclei of cone photoreceptors (ns, p > 0.05; ∗p < 0.05).
|
