Amacrine cell-derived VEGF is required for development and maintenance of the retinal vasculature in mice

Abstract

Purpose: The retinal vasculature of many organisms including humans and mice consists of three distinct plexus layers. While it is clear that the inner retinal vascular layer develops over a pre-existing astrocytic network and that development of vascular and neuronal networks are co-dependent, it is unclear how the outer retinal vascular networks form. As retinal neurons populate the retina and mature, oxygen demands change and activation of the oxygen sensing VHL/HIF-α/VEGF pathway in maturing neurons may be a strong driving force for development and maintenance of the outer plexus layers. In this study, we examined the contribution of amacrine and horizontal cells due to their close proximity to the intermediate and outer retinal vascular layers.

Methods: Transgenic mice expressing Cre recombinase specifically in amacrine and horizontal cells (Ptf1a-Cre mice) were mated with floxed VHL, HIF-1α, HIF-2α and/or VEGF mice to generate conditional knockouts. Amacrine and horizontal cells were genetically ablated using Ptf1a-Cre and forced expression of diphtheria toxin (DT) receptors.

Results: We show that amacrine and horizontal cell processes tightly associate with intermediate and outer plexus retinal capillaries. Pseudo-hypoxia in Ptf1a-Cre; VHL mutants induces formation of a dense intermediate plexus compared to controls, while a dramatically attenuated intermediate plexus is observed in Ptf1a-Cre; VEGF and Ptf1a-Cre; HIF-1α mutants. Co-deletion of HIF-1α, but not HIF-2α, rescued the vascular phenotypes of Ptf1a-Cre; VHL KO mice. Amacrine and horizontal cell ablation by DT injection also suppressed the formation of the intermediate plexus and DT injection after the retinal vasculature had developed resulted in attenuation of the vasculature. In all of these genetic manipulations the deep plexus was less affected.

Conclusions: Dysregulated VEGF release from amacrine and horizontal cells results in formation of a very dense intermediate vascular plexus, while elimination of VEGF (or of amacrine and horizontal cells themselves) prevents its formation. These data demonstrate a novel function of amacrine cells, directing formation of the intermediate plexus layer. Horizontal cells, on the other hand, are likely strictly dependent on the vasculature, but do not determine its formation or maintenance.

Keywords: 698 retinal development • 416 amacrine cells • 748 vascular endothelial growth factor