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

Wholemount brightfield and fluorescent images showing <i>nrd:egfp</i> trangene expression in adult Tg(<i>nrd:egfp</i>)<i>/albino</i> zebrafish.

<p>(A) A multiple brightfield image overlay showing the entire adult fish. Arrowhead indicates the location of the pineal gland and cerebelum (shown in Panels E and F). The boxes and corresponding panel letter indicate the location of the higher magnification images shown in Panels C–K. (B) Corresponding fluorescent image to Panel A, showing EGFP expression in the eye (white arrow) and the pineal (white arrowhead). (C–D) Brightfield and corresponding fluorescent image showing EGFP expression in the eye. (E) Brightfield image of the dorsal side of the head showing the pineal gland (arrow), telecephalon (Te), and cerebellum (Ce, arrowhead). (F) Corresponding fluorescent image showing EGFP expression in the pineal gland (arrow) and cerebellum (arrowhead). (G–I) Brightfield, overlay, and fluorescent images of the anus and its expression of the transgene. (J–K) Brightfield and corresponding fluorescent image showing EGFP expression in nerves located near the girdle of the tail fin. The box indicates the location of image shown in Panel L. (L) A high magnification fluorescent image of a branch of the nerve shown in Panel K, most likely of the posterior lateral line, showing EGFP expression in each of the terminating synaptic buttons. Scale bar: 2 mm (A).</p

Retinal sections from embryonic Tg(<i>nrd:egfp</i>)<i>/albino</i> zebrafish immunolabeled with PCNA (red) and EGFP (green).

<p>(A) At 42 hpf, EGFP is detected throughout the retinal neuroepithelium in the central retina. PCNA immunolocalization, showing proliferating cells, is primarily restricted to the CMZ (arrows). In the overlapping region of PCNA and EGFP co-labeling can be visualized (arrowhead). (B) At 72 hpf, EGFP is detected in a subset of ganglion, amacrine and bipolar cells, and is not present in the outer nuclear layer. Proliferating cells are restricted to the CMZ (arrows), and there is no evidence of PCNA and EGFP co-immunolabeling. (C) At 96 hpf, there is persistent expression of EGFP detected in a subset of ganglion, amacrine and bipolar cells. Proliferating cells are restricted to the CMZ (arrows) and do not co-label with the transgene. Scale bars: 25 microns (A) and 50 microns (B, C).</p

Wholemount brightfield and flourescent images showing <i>nrd:egfp</i> transgene expression in the adult caudal tail fin.

<p>(A) Brightfield image of the adult caudal fin. (B) Corresponding fluorescent image to panel A. EGFP expression is visualized in the nerve coursing through each bony hemiray of the caudal fin, however at this level of magnification, it is difficult to visualize. (C–C′) High magnification brightfield and corresponding fluorescent overlay showing multiple bony lepidotrichia. The arrows point to the nerve running within each bony hemiray and arrowheads point to EGFP-positive ganglia associated with the nerve. (D and D′) A section of a single bony ray immunolabeled with EGFP to show the transgene and co-labeled with TO-PRO-3 to show all nuclei (magenta). The white line in Panel D shows the location of the nerve. Ep = Epithelium, Bn = bony ray, CT = connective tissue.</p

Retinal sections from adult Tg(<i>nrd:egfp</i>)/<i>albino</i> zebrafish over a time course of light treatment and immunolabeled with EGFP (green) to visualize the <i>nrd:egfp</i> transgene and co-labeled with either PCNA (A, C, D, I, J, J′, K, L, L′, M, N) or Glutamine Synthetase (B, G, H).

<p>(A) At 48 hours after light onset, almost all rod photoreceptors have been ablated and proliferating cells can be seen in the in the INL. Nuclei are labeled in blue with TO-PRO-3 (TP3). (B) At this time point, Müller glial cells express Glutamine Synthetase (G.S., red, arrow), and do not co-label with EGFP (arrowhead). (C) At 72 hours after light onset, clusters of proliferating progenitors begin to migrate towards the ONL (arrowheads). (D) At 96 hours post light onset, PCNA-positive progenitors (arrowheads) are present in both in INL and ONL, with occasional aberrant migration to the GCL. (E–F) At 72 and 96 hours after light onset, respectively, clusters of progenitors weakly express EGFP (arrowheads). (F) At 96 hours after light onset, EGFP is observed in a newly-formed and disorganized ONL. (G–H) At 72 and 96 hours after light onset, respectively, weakly-EGFP-positive clusters in the INL (arrowheads) down-regulated Glutamine Synthetase. Müller glial cells that did not re-enter the cell cycle strongly express G.S. (arrows), but are EGFP-negative. (I–J′) At 72 hours after light onset, weakly-EGFP-positive cells in both the INL and ONL co-label with PCNA. The box in I represents the PCNA and EGFP labeling shown in J and J′, respectively. (K–L′) At 96 hours after light onset, weakly-EGFP-positive cells in both the INL (arrowheads) and ONL continue to co-label with PCNA. The box in K represents the PCNA and EGFP labeling shown in L and L′, respectively. (M) At 7 days after light onset, a subset of PCNA-positive progenitors in the ONL co-label with EGFP (N) At 11 days after light onset, only a few PCNA-positive progenitors remain in the ONL. EGFP can be visualized in rod photoreceptors and newly-formed rod inner segments (arrowhead).</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

Retinal sections from adult Tg(<i>nrd:egfp</i>)/<i>albino</i> zebrafish at 96 hours after light onset showing transgene expression (green) and EdU labeling (red).

<p>(A). Schematic representation of EdU injections during the light time course with corresponding immunolocalization shown in Panels B–D′. (B–B′) EGFP and EGFP/EdU co-labeling, respectively, showing weakly-EGFP-positive cells in the INL (arrowheads) and ONL co-label with EdU. The boxes in B′ represent the panels shown in C–D′. (C) Higher magnification image of the box shown in the top right of Panel B′. Note that the weakly-EGFP-positive progenitors co-label with EdU (arrowheads), but strongly-EGFP-positive rod nuclei (arrow) are EdU-negative. (D–D′) Higher magnification image of the box shown in the left of Panel B′, showing EGFP and EdU immunolocalization, respectively, in a cluster of INL progenitors. (E) Schematic representation of a single EdU injection prior starting the light treatment in order to label a subset of the progenitors. (F–F′). High magnification confocal microscopy showing EGFP and EGFP/EdU co-labeling in the ONL at 96 hours after light onset. An individual EdU-positive cell in the ONL (arrowhead) co-labels with weak EGFP expression. The strongly-EGFP-positive cell, in contrast, is EdU-negative (arrow).</p

High magnification images of retinal sections from adult Tg(<i>nrd:egfp</i>)<i>/albino</i> zebrafish over a time course of light treatment.

<p>Sections were immunolabeled with EGFP (green) to visualize the <i>nrd:egfp</i> transgene and Zpr-3 (red) to visualize rod photoreceptors. (A) Prior to light treatment (0 hr), EGFP co-labels with Zpr-3 and is observed in rod photoreceptor soma, rod inner segments (RIS) and rod outer segments (ROS). (B) At 48 hours after light onset, the ROS and RIS are almost completely destroyed and only a few EGFP-positive cells remain in the ONL. (C) At 72 hours after light onset, newly-formed rod progenitor cells are present in the ONL. These could be readily discerned from existing rod photoreceptors due to their comparably weak expression of the transgene (inset shows new rod progenitor on the left). (D) At 96 hours after light onset, a greater number of new regenerated cells are present in the ONL, although it still somewhat disorganized. (E) At 7 days after light onset, newly differentiated rod photoreceptors appear more organized and greater in abundance. (F) At 11 days after light onset, EGFP is expressed in the newly formed rod photoreceptors and co-labels with Zpr-3-positive and newly-formed RIS and R0S. Scale bar: 50 microns (A–F).</p

Section from Tg(<i>nrd:egfp</i>)<i>/albino</i> zebrafish showing <i>nrd:egfp</i> trangene expression in the endocrine pancreas (A–A′″) and gut (B–C). (A–A′″).

<p>Immunolocalization of EGFP (A, green) co-labels with Insulin (A′, red) in the endocrine pancreas (A′″, En). Co-labeling with TO-PRO-3 shows all nuclei (A′″, blue) and indicates the surrounding exocrine pancreas (A′″, Ex) and adjacent lumen of the gut (A′″, Lu). (B) EGFP expression can be visualized in enteroendocrine cells within each villus and in the surrounding smooth muscle. The adjacent pancreas is also visible (arrow). (C) High magnification image of the box in panel B.</p