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Lavan Khandan, Harald J Junge; The Role of Localized Norrin Deposition in Retinal Vascular Development. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):3391.
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© ARVO (1962-2015); The Authors (2016-present)
Norrin/Frizzled4 signaling is essential for the vascularization of the retina and blood-retina barrier function. Norrin is a non-Wnt ligand for Frizzled4 and induces canonical beta-catenin signaling in vascular endothelial cells. Little is known about the tissue distribution of the matricellular ligand Norrin, or the function of its distribution. Here, we test the hypothesis that Norrin is deposited to matrix-rich layers in the retina, and that this enrichment plays a role in guiding nascent blood vessels into appropriate retinal layers.
We treated murine retinal tissue sections with alkaline phosphatase (AP) tagged Norrin and incubated sections with a chromogenic AP substrate to visualize Norrin deposition. We used standard molecular cloning techniques to systematically modify the Norrin coding sequence and conduct a structure-function analysis with respect to tissue binding, Frizzled4 binding (immuno-precipitations) and signaling (TOPflash reporter assays). We quantified binding of wild type and mutant Norrin-AP to retinal sections using UV-VIS spectroscopic measurements of analytes recovered from treated retinal sections.
We found that Norrin binding to retinal tissue occurs predominantly in the retinal layers that attract vasculature. Of four tested vertebrate Norrin proteins, only Xenopus laevis Norrin showed strongly reduced binding to murine retinal tissues. By analyzing a series of chimeras of human and frog Norrin we identified a region harboring a determinant of Norrin tissue binding. Xenopus Norrin-AP bound poorly to murine retinal sections. However, introducing the determinant of retinal tissue binding from human Norrin into frog Norrin conferred strong tissue binding to the frog protein. These two proteins performed similarly in binding Frizzed4 and eliciting signaling.
Our results suggest a model where Norrin is deposited into matrix rich layers of the retina and recruits developing blood vessels. Our structure-function analysis reveals a determinant in the Norrin protein sequence that mediates tissue binding independent of Frizzled4 binding. Our findings should allow us to determine the functional significance of tissue-binding by intravitreally administering purified wild type or mutant Norrin to Norrin knockout mice and assessing effects on intraretinal capillary formation and blood-retina barrier formation.
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