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Travis D'Cruz, Esther Gramage, Peter F Hitchcock; Midkine-a protein localization in the embryonic and adult retina of the zebrafish. Invest. Ophthalmol. Vis. Sci. 2014;55(13):705. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Midkine-a (Mdka) is a retinoic acid-induced, heparin-binding growth factor with multiple functions in neural development and repair. In the zebrafish retina, actively dividing retinal progenitor cells and Müller glia express mdka mRNA during development, while in the adult, mdka expression is restricted to the horizontal cells and is robustly regulated by the circadian rhythm. The purpose of this study was to determine the protein localization of Mdka in the developing and adult retinas of zebrafish.
Whole embryos and larvae and eye cups from adults were fixed with 4% paraformaldehyde and embedded in OCT. Sections were obtained from embryos at 30 and 48 hours post fertilization (hpf), larvae at 72, 96 and 120 hpf, and adult eyes. Fluorescence immunohistochemistry using polyclonal antibodies raised against Mdka, cell type-specific antibody markers and confocal microscopy were used to determine the localization of Mdka. Specificity of the antibody was determined by the selective loss of Mdka immunolabeling in sections and Western blots from embryos injected with morpholino oligonucleotides targeted to mdka.
At 30 hpf, Mdka is localized throughout the basal processes of neuroepithelial cells in the undifferentiated retina. At 48 and 72 hpf, it becomes restricted to the presumptive inner and outer plexiform layers. At 96 and 120 hpf, when the larval retina is fully differentiated and mdka expression is restricted to Müller glia and horizontal cells, Mdka protein is in the outer nuclear layer, but does not co-localize with cone-specific markers or the Müller glia marker glutamine synthetase. At the same time, Mdka forms a single small plaque of protein at the apical surface of each horizontal cell nucleus. In adults, the horizontal cell staining persists and is regulated by the circadian rhythm.
Matching the complex pattern of gene regulation, Mdka protein localization follows a complex temporal and spatial pattern in the zebrafish retina. Our results suggest that Mdka protein may be secreted by Müller glia into the extracellular space of the outer nuclear layer. However, for horizontal cells, the protein is accumulated in a specific location inside the cell and regulated by the circadian rhythm.
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