Semaphorin 3F expression pattern in outer retina and RPE

Abstract

Purpose : Semaphorins are known modulators of angiogenesis and axonal pathfinding. Recent studies have identified a novel role for soluble class 3 semaphorins in the inner and outer retina. Two class 3 semaphorins (3A and 3F) with a very distinct retinal distribution pattern have recently been described. Sema3A is found exclusively in the inner retina whereas Sema3F is predominantly expressed in the outer retina. Based on the potent anti-angiogenic effect and the localization of Sema3F, a contribution to physiologic avascularity of the outer retina has been proposed. The exact localization of Sema3F expression on mRNA and protein levels as well as the distribution of Sema3F expression in the RPE could give new insights into the role of Sema3F in the outer retina.

Methods : In situ hybridization and immunohistochemistry for Sema3F were performed on eyes from C57/BIJ6 mice at postnatal day (P) 21. In addition, a modified Ussing chamber with a porcine RPE-choroid tissue culture was used to separate the apical (retina facing) and basal (choroid facing) sides of the RPE monolayer for secretion analysis. The supernatant of the apical and basal chamber were collected after 1 and 3 days for western blot analysis.

Results : In situ hybridization showed a strong expression of Sema3F in both the outer nuclear layer as well as the RPE. Immunohistochemistry (IHC) revealed no protein signal in the outer nuclear layer but at the basolateral side of the retinal pigment epithelium. This was confirmed in the Ussing chamber experiments showing a stronger signal on the basal side of the RPE compared to the apical side.

Conclusions : The in situ hybridization results confirm a distinct expression pattern for Sema3F in the outer retina as previously suggested by laser capture microdissection data. Immunohistochemistry detected a signal on the basolateral side of the RPE. A possible explanation for the missing detection of Sema3F protein in the outer nuclear layer could be that only the neuropilin receptor-bound fraction of soluble Sema3F is amenable to IHC detection. The Ussing chamber experiments revealed a stronger secretion of Sema3F to the basal side of the RPE, in line with a basolateral localization of Sema3F found on IHC. The localization in the outer retina and the basolateral RPE secretion supports the hypothesis that Sema3F forms an anti-angiogenic barrier in the outer retina which might be relevant in the pathophysiology of neovascular AMD.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.