Figure 3. Sema3G is expressed exclusively in ECs in the mouse retina.

(A) Schematic illustration of retinal preparations for RT-qPCR and RNA in situ hybridization. (B) Sema3G mRNA expression in the retina at different time points after birth (n = 3–5 mice). (C) Representative images of RNA in situ hybridization for Sema3G mRNA on retinal sections at P6, P10, P14, and P20 of WT mice. Sema3G is expressed by blood vessels in the superficial, intermediate, and deep layers (white arrowheads). (D) Representative images of double fluorescence RNA in situ hybridization for Sema3G mRNA (red) and CD31 mRNA (green) in combination with immunofluorescence for lectin in P20 WT retinas. (E) Schematic illustration of the structure of retinal layers and the distribution of vessels in retinal sections. (F) Immunoblot analysis of Sema3G protein levels in lysates of primary human retinal microvascular ECs (HRMECs), mouse brain microvascular ECs (bEnd.3 cells), immortalized vascular ECs (EA.hy926), and primary human umbilical vein ECs (HUVECs). (G) Schematic illustration of the vascular network in flat-mounted retinas. (H) Representative images of RNA in situ hybridization for Sema3G mRNA and immunofluorescence for isolectin B4 (IB4) in whole-mounted retinas of WT mice at P20. Sema3G colocalizes with IB4 in microvessels (left panel) and large blood vessels (right panel). (I) Representative images of RNA in situ hybridization for Sema3G mRNA, in combination with immunofluorescence for Iba-1 (a microglial marker) and GFAP (an astroglial marker) in whole-mount retinas of WT mice at P20. Error bars represent mean ± SEM. **P < 0.01; ***P < 0.001; 2-tailed Student’s t tests. Scale bars: 100 μm (C and D) and 50 μm (H and I); magnified images: 50 μm (D). GCL, ganglion cell layer; NBL, neuroblast layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer.