Fig 2: Mononuclear phagocyte-resident NRP1 expression in LysM-Cre/Nrp1fl/fl AGating scheme explaining the identification of the Ly6G-, F4/80+, CD11b+, and NRP1+ mononuclear phagocytes in retinas and sclera-choroid-RPE cell complexes. 1. Gating of live cells, 2. Removal of doublets, 3. Selection of viable cells, 4. Exclusion of neutrophils, 5. Gating of mononuclear phagocytes, 6. Gating of NRP1+ mononuclear macrophages.BFACS histogram of APC-conjugated rat IgG2A isotype control (red) versus anti-mNRP1 APC-conjugated rat IgG2A (R&D systems) (blue).CFACS histogram of FMO (fluorescence minus one) versus anti-mNRP1 APC-conjugated rat IgG2A.D, EQuantification of NRP1-positive microglia (Ly6G-, F4/80+, CD11b+, CX3CR1hi, CD45lo, NRP1+) in retinas and sclera-choroid-RPE cell complexes in Naïve (non-burned) mice (D); n = 4 and at D3 (E); n = 4.F, GQuantification of mononuclear phagocytes (Ly6G-, F4/80+, CD11b+) in retinas and sclera-choroid-RPE cell complexes in Naïve (non-burned) mice (F); n = 5 (LysM-Cre/Nrp1+/+), 4 (LysM-Cre/Nrp1fl/fl) and at D3 (G); n = 4.HCompilation of representative compressed Z-stack confocal images of FITC–dextran-labeled CNV and IB4-stained laser impact area from LysM-Cre/Nrp1+/+ and LysM-Cre/Nrp1fl/fl mice at D7. Scale bar: 20 µm.I–KQuantification of area of FITC–dextran-labeled CNV (I), isolectin B4 (IB4)-stained laser impact area (J) and the ratio of FITC/IB4 per laser burn (K) relative to LysM-Cre/Nrp1+/+ at D7; n = 14 burns (LysM-Cre/Nrp1+/+), n = 20 burns (LysM-Cre/Nrp1fl/fl). Data information: All comparisons between groups were analyzed using a Student’s unpaired t-test; **P < 0.01, ***P < 0.001; error bars represent mean ± SEM; exact P-values listed in Table 2.

Fig 3: Epifluorescence whole-mount images of bulbs of transgenic mice expressing rat OR I7 from a mouse MOR23 promoter and gap-YFP with and without Nrp1. Images of medial and lateral views of bulbs were taken with a Nikon SMZ25 stereomicroscope. Signal represents the intrinsic fluorescence of YFP. Dorsal is up, ventral is down. A, Views on the medial and lateral aspects of a left bulb (left two images) and a right bulb (right two images) of two I7-Cre-YFP Tg mice at PD17. Axons coalesce into a single glomerulus. B, Views on the medial and lateral aspects of the right bulbs of six I7-Cre-YFP Tg × n5247-flox littermates at PD14. Images are pairwise for an individual mouse. Glomeruli are indicated with yellow arrows. Axons coalesce into multiple glomeruli, in particular in the lateral aspect.

Fig 4: Three-dimensional reconstructions of bulbs of transgenic mice expressing rat OR I7 from a mouse MOR23 promoter and gap-YFP with and without Nrp1. For PD14, three bulbs of three I7-Cre-YFP Tg littermates (indicated in shades of gray) and eight bulbs of seven I7-Cre-YFP Tg × n5247-flox littermates (indicated in distinct colors) were reconstructed. For one mouse, both the left and right bulbs (green and turquoise) are included in the merged bulb. For PD21, three bulbs of three I7-Cre-YFP Tg × n5247-flox littermates were reconstructed. Views are medial, lateral, and ventral. A, Merged bulb of three I7-Cre-YFP Tg bulbs at PD14. B, Merged bulb of three I7-Cre-YFP Tg bulbs at PD14 together with eight I7-Cre-YFP Tg × n5247-flox bulbs at PD14. Because of the large number of glomeruli depicted, the gray-transparent outer layer of the bulb shown in Figures 3 and 9 is here omitted. Medially, I7-Cre-YFP Tg × n5247-flox glomeruli are shifted anteriorly and ventrally and scattered over a sector that is anchored on the tightly clustered I7-Cre-YFP Tg glomeruli. Laterally, I7-Cre-YFP Tg × n5247-flox glomeruli are shifted dorsally and ventrally and scattered over a belt that is centered on the tightly clustered I7-Cre-YFP Tg glomeruli. This belt surrounds the anterior bulb as a U-shape but is not continuous ventrally, as can be seen in the ventral view. C, Merged bulb of three I7-Cre-YFP Tg × n5247-flox bulbs at PD21. These mice also contain the reporter ROSA-STOP-lacZ, but the taulacZ marker is not visualized. The number and degree of scattering of glomeruli is more pronounced than at PD14.

Fig 5: Proposed mechanism of action of microglial Nrp1 on OPC proliferation.Resting ramified microglia (homeostatic microglia) that do not express Nrp1 (left, light pink) become activated and upregulate Nrp1. Nrp1 on activated microglia stimulates phosphorylation of PDGFRa on adjacent OPCs (gray) and promotes PDGF AA-dependent OPC proliferation. Question mark above the gray box indicates that the mechanism that causes Nrp1 upregulation on activated microglia is unknown.