Midkine-a Protein Localization in the Developing and Adult Retina of the Zebrafish and Its Function During Photoreceptor Regeneration

<div><p>Midkine is a heparin binding growth factor with important functions in neuronal development and survival, but little is known about its function in the retina. Previous studies show that in the developing zebrafish, Midkine-a (Mdka) regulates cell cycle kinetics in retinal progenitors, and following injury to the adult zebrafish retina, <i>mdka</i> is strongly upregulated in Müller glia and the injury-induced photoreceptor progenitors. Here we provide the first data describing Mdka protein localization during different stages of retinal development and during the regeneration of photoreceptors in adults. We also experimentally test the role of Mdka during photoreceptor regeneration. The immuno-localization of Mdka reflects the complex spatiotemporal pattern of gene expression and also reveals the apparent secretion and extracellular trafficking of this protein. During embryonic retinal development the Mdka antibodies label all mitotically active cells, but at the onset of neuronal differentiation, immunostaining is also localized to the nascent inner plexiform layer. Starting at five days post fertilization through the juvenile stage, Mdka immunostaining labels the cytoplasm of horizontal cells and the overlying somata of rod photoreceptors. Double immunolabeling shows that in adult horizontal cells, Mdka co-localizes with markers of the Golgi complex. Together, these data are interpreted to show that Mdka is synthesized in horizontal cells and secreted into the outer nuclear layer. In adults, Mdka is also present in the end feet of Müller glia. Similar to <i>mdka</i> gene expression, Mdka in horizontal cells is regulated by circadian rhythms. After the light-induced death of photoreceptors, Mdka immuonolabeling is localized to Müller glia, the intrinsic stem cells of the zebrafish retina, and proliferating photoreceptor progenitors. Knockdown of Mdka during photoreceptor regeneration results in less proliferation and diminished regeneration of rod photoreceptors. These data suggest that during photoreceptor regeneration Mdka regulates aspects of injury-induced cell proliferation.</p></div

Knockdown of Mdka reduces the number of regenerated rod photoreceptors.

<p>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; <i>rho</i>: <i>rhodopsin</i>; <i>pde6c</i>: <i>phosphodiesterase6c</i>. *p-value < 0.05.</p

Mdka knockdown reduces the number of injury-induced progenitors.

<p>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.</p

Mdka protein localization following photoreceptor ablation.

<p>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.</p

<i>mdka</i> expression and Mdka immunostaining during retinal development and in adults.

<p><i>In situ</i> hybridization showing <i>mdka</i> expression at stages between 30hpf and adults (A-F). Mdka immunostaining at stages corresponding to the <i>in situ</i> 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 <i>mdka</i> (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’.</p

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

<p>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.</p

Circadian regulation of Mdka immunostaining in horizontal cells.

<p>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.</p