mdka expression and Mdka immunostaining during retinal development and in adults.

In situ hybridization showing mdka expression at stages between 30hpf and adults (A-F). Mdka immunostaining at stages corresponding to the in situ hybridizations are also illustrated (G-H and images merged DAPI counterstain below). At 30 hpf, Mdka immunofluorescence in strongest at the basal surface of the retina (panel G, arrows). At 48hpf, Mdka antibodies transiently label the nascent inner plexiform layer (panel H, arrows). At 120hpf, when differentiated photoreceptors are present, Mdka immunostaining is present in horizontal cells (arrows) and the outer nuclear layer (brackets). Though tested numerous times, the level of Mdka immunofluorescence dramatically decreases between 7–10 dpf (panel J, J′). Note that at this time point horizontal cells express mdka (panel D). In the juvenile retina, Mdka immunostaining returns and both horizontal cells (arrows, panel K, K′) and the overlying rod photoreceptors in the ONL are intensely stained. This juvenile pattern of immunostaining is present in adults, but the overall intensity of the immunostaining is reduced, and the immunostaining of horizontal cells is consolidated to a small immunofluorescent plaque lying adjacent to each DAPI-stained nucleus (arrows, panel L′). hpf, hours post fertilizations; dpf, days post fertilization; mo, months; ONL, outer nuclear layer; INL, inner nuclear layer; IPL, inner plexiform layer; CMZ, circumferential marginal zone. Scale bar in panel J′ equals 20 µm and corresponds to images in columns A-D and G-J. Scale bar in panel L′ equals 10 µm and corresponds to images in panels E, F, K, K′, L and L′.

Mdka immunostaining of horizontal cells is intracellular and colocalizes with markers of the Golgi complex.

In fish injected with the Cx55.5:EGFP construct, immunolabels for both Mdka and EGFP colocalize to the cytoplasm of horizontal cells (arrowhead, panel A). In the adult retina, Mdka immunostaining colocalizes with the antibody marker of the Golgi complex (column B; and insets). ONL: outer nuclear layer; INL: inner nuclear layer. Scale bars equal 10 µm.

Circadian regulation of Mdka immunostaining in horizontal cells.

At the onset of the subjective day, Mdka immunostaining of horizontal cells is maximum (column A), decreases throughout the subjective day (column B) and reaches a minimum during the subjective night (column C). In contrast, there is no corresponding substantial change in the intensity of immunostaining within the endfeet of the Müller glia (D-F). ONL: outer nuclear layer; INL: inner nuclear layer; GCL: ganglion cell layer; NFL: nerve fiber layer. Scale bar = 10 µm.

Mdka protein localization following photoreceptor ablation.

In unlesioned retinas, Mdka immunostaining is localized to the horizontal cells and endfeet of glutamine synthetase (GS)-positive Müller glia (row A). At 4 dpl, Mdka antibodies label the radial processes of Müller glia (row B, arrows). Note the increased Mdka immunostaining in the endfeet of the Müller glia in lesioned retinas (cf. rows A and B). Also at 4 dpl, Mdka immunostaining is localized to each of the EdU-positive nuclei in both the INL and ONL (row C, arrowheads). ONL: outer nuclear layer; INL: inner nuclear layer; dpl: days post lesion. Scale bars = 25 µm.

Mdka knockdown reduces the number of injury-induced progenitors.

Progenitors were labeled with BrdU in the retinas of control (panel A) and experimental fish (panel B) at 3 dpl. Note the radial clusters of progenitors in the control retinas (arrows in A) and their relative absence in experimental retinas (panel B). BrdU-positive cells were counted, and this is graphically represented in panel C. INL: inner nuclear layer; GCL: ganglion cell layer; BrdU: Bromodeoxyuridine. Scale bar = 25 µm. ** p<0.01.

Knockdown of Mdka reduces the number of regenerated rod photoreceptors.

Graphs in panels A and B represent the number of regenerated rods and cones, respectively, counted within the photolytic lesions of experimental and control fish. Bromodeoxyuridine; rho: rhodopsin; pde6c: phosphodiesterase6c. *p-value < 0.05.