April 2016
Volume 57, Issue 4
Open Access
Retinal Cell Biology  |   April 2016
Neuropilin-1–Expressing Microglia Are Associated With Nascent Retinal Vasculature Yet Dispensable for Developmental Angiogenesis
Author Affiliations & Notes
  • Agnieszka Dejda
    Department of Biochemistry and Molecular Medicine, Maisonneuve-Rosemont Hospital Research Centre, University of Montreal, Montreal, Quebec, Canada
    Department of Ophthalmology, Maisonneuve-Rosemont Hospital Research Centre, University of Montreal, Montreal, Quebec, Canada
  • Gaelle Mawambo
    Department of Biochemistry and Molecular Medicine, Maisonneuve-Rosemont Hospital Research Centre, University of Montreal, Montreal, Quebec, Canada
  • Jean-Francois Daudelin
    Department of Medicine Microbiology, Infectiology and Immunology, Maisonneuve-Rosemont Hospital Research Centre, University of Montreal, Montreal, Quebec, Canada
  • Khalil Miloudi
    Department of Neurology-Neurosurgery, McGill University, Montreal, Quebec, Canada
  • Naoufal Akla
    Department of Ophthalmology, Maisonneuve-Rosemont Hospital Research Centre, University of Montreal, Montreal, Quebec, Canada
  • Chintan Patel
    Department of Ophthalmology, Maisonneuve-Rosemont Hospital Research Centre, University of Montreal, Montreal, Quebec, Canada
  • Elisabeth M. M. A. Andriessen
    Department of Ophthalmology, Maisonneuve-Rosemont Hospital Research Centre, University of Montreal, Montreal, Quebec, Canada
  • Nathalie Labrecque
    Department of Medicine Microbiology, Infectiology and Immunology, Maisonneuve-Rosemont Hospital Research Centre, University of Montreal, Montreal, Quebec, Canada
  • Florian Sennlaub
    Institut National de la Santé et de la Recherche Médicale, Paris, France
    Institut de la Vision, Paris, France
  • Przemyslaw Sapieha
    Department of Biochemistry and Molecular Medicine, Maisonneuve-Rosemont Hospital Research Centre, University of Montreal, Montreal, Quebec, Canada
    Department of Ophthalmology, Maisonneuve-Rosemont Hospital Research Centre, University of Montreal, Montreal, Quebec, Canada
    Department of Neurology-Neurosurgery, McGill University, Montreal, Quebec, Canada
  • Correspondence: Przemyslaw (Mike) Sapieha, Maisonneuve-Rosemont Hospital Research Centre, 5415 Assomption Boulevard, Montreal, Quebec, H1T 2M4, Canada; mike.sapieha@umontreal.ca
Investigative Ophthalmology & Visual Science April 2016, Vol.57, 1530-1536. doi:10.1167/iovs.15-18598
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      Agnieszka Dejda, Gaelle Mawambo, Jean-Francois Daudelin, Khalil Miloudi, Naoufal Akla, Chintan Patel, Elisabeth M. M. A. Andriessen, Nathalie Labrecque, Florian Sennlaub, Przemyslaw Sapieha; Neuropilin-1–Expressing Microglia Are Associated With Nascent Retinal Vasculature Yet Dispensable for Developmental Angiogenesis. Invest. Ophthalmol. Vis. Sci. 2016;57(4):1530-1536. doi: 10.1167/iovs.15-18598.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: Neuropilin-1 (NRP-1) is a transmembrane receptor that is critical for vascular development within the central nervous system (CNS). It binds and influences signaling of several key angiogenic factors, such as VEGF-165, semaphorin 3A, platelet derived growth factor, and more. Neuropilin-1 is expressed by neurons and endothelial cells as well as a subpopulation of proangiogenic macrophages/microglia that are thought to interact with endothelial tip cells to promote vascular anastomosis during brain vascularization. We previously demonstrated a significant role for NRP-1 in macrophage chemotaxis and showed that NRP-1–expressing microglia are major contributors to pathologic retinal angiogenesis. Given this influence on CNS angiogenesis, we now investigated the involvement of microglia-resident NRP-1 in developmental retinal vascularization.

Methods: We followed NRP-1 expressing microglia during retinal development. We used LysM-cre myeloid lineage-driver cre mice to reduce expression of NRP-1 in retinal myeloid-derived cells and performed a comprehensive morphometric analysis of retinal vasculature during development.

Results: We provide evidence that NRP-1+ microglia are present throughout the retina during vascular development with a preference for the non-vascularized retina. Using LysM-Cre/Nrp1fl/fl mice, we reduced NRP-1 expression by ~65% in retinal microglia and demonstrate that deficiency in NRP-1 in these microglia does not impair retinal angiogenesis.

Conclusions: Our data draw a dichotomous role for NRP-1 in cells of myeloid lineage where it is dispensable for adequate retinal developmental vascularization yet obligate for pathologic retinal angiogenesis.

Vascularization of central nervous system (CNS) tissue, such as the retina, requires timed and coordinated, multicellular crosstalk between sprouting blood vessels, glia, neurons, and immune cells.15 Proper integration of signals driving developmental CNS angiogenesis ensures formation of healthy vascular networks in time to provide metabolic support to neural progenitors and maturing neural tissue.6 
An obligate receptor for CNS angiogenesis that is expressed on several cell populations involved in developmental vascularization is neuropilin-1 (NRP-1). Neuropilin-1 is a transmembrane glycoprotein that binds, integrates, and modulates signaling of key angiogenic factors, such as VEGF, semaphorin 3A, TGF-β1, platelet derived growth factor (PDGF), and more.7,8 Moreover, independently of the growth factors, NRP-1 forms a complex with ABL1, which promotes ECM-induced phosphorylation of integrin targets associated with endothelial actin remodeling.5 Although originally identified as an adhesion molecule in the nervous system,9 it subsequently has been reported on endothelial cells,10 including tip and stalk cells and proangiogenic macrophages.11,12 The essential role of NRP-1 in CNS angiogenesis was established in NRP-1–null mice, which show high embryonic mortality due to serious defects in blood vessel formation.13,14 Similarly, specific knockout of Nrp1 in endothelial cells results in severe vascular disruption and defective cardiovascular development15 and mutant mice with impaired NRP-VEGF binding and reduced NRP-1 expression show higher postnatal mortality and deficiencies in myocardial vascularization, retinal angiogenesis, and arteriogenesis.16 
Interestingly, yolk sac–derived macrophages expressing NRP-1 have been localized in close proximity to endothelial tip cells promoting vascular anastomosis during brain vascularization,11 yet selective targeting of NRP-1 on these macrophages does not affect normal blood vessel growth in the brain.12 These macrophages/microglia, are part of a larger family of tissue-resident macrophages that develop in the embryo before the appearance of hematopoietic stem cells (HSCs)1719 and colonize CNS tissues (including the retina11,20) before blood vessels are established.21,22 
Given the contribution of myeloid cells to retinal vascular development4 and the essential role for NRP-1+ mononuclear phagocytes (MNPs)/microglia during pathologic retinal angiogenesis,23 we investigated the contribution of NRP-1+ microglia to developmental retinal angiogenesis. Using myeloid lineage affiliated lysozyme gene cre-driver mice (LysM-cre)24 we reduced NRP-1 expression on microglia by over 65%. Comprehensive morphometric analysis revealed that deletion of NRP-1 in these cells does not impair development of retinal vasculature. 
Materials and Methods
Animals
All studies were performed according to the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the Animal Care Committee of the University of Montreal in agreement with the guidelines established by the Canadian Council on Animal Care. C57BL/6 wild-type (WT), LysM-Cre (Lyz2tm1(cre)Ifo/J; no. 004781), Neuropilin 1-floxed (Nrp1tm2Ddg/J; no. 005247), and ROSA26EYFP-floxed (Gt(ROSA)26Sortm1(EYFP)Cos/J; no. 006148) mice were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). We generated two lines of myeloid-specific transgenic mice by breeding LysM-Cre mice (Cre-recombinase expressed in myeloid cell lineage) with ROSA26EYFP-floxed and Neuropilin 1-floxed mice. This resulted in a mouse with EYFP expressing myeloid cells and a mouse with attenuated Nrp1 in myeloid cells. 
FACS of Digested Retinas
Retinas from WT, ROSA26EYFPfl/fl LysM-Cre, LysM-Cre/ROSA26EYFPfl/fl, or LysM-Cre/Nrp1fl/fl mice were homogenized in a solution of 750 U/mL DNaseI (Sigma-Aldrich Corp., St. Louis, MO, USA) and 0.5 mg/mL of collagenase D (Roche, Basel, Switzerland) for 15 minutes at 37°C with gentle shaking. Homogenates then were filtered through a 70-μm cell strainer and washed in PBS + 3% fetal bovine serum. Cells were incubated with LEAF purified anti-mouse CD16/32 (101310; BioLegend, San Diego, CA, USA) for 15 minutes at room temperature to block Fc receptors. Cells then were incubated for 30 minutes at room temperature with the following antibodies: Alexa Fluor 700 anti-mouse CD45.2 (109822; BioLegend), FITC anti-mouse/human CD11b (101206; BioLegend), PE/CY7 anti-mouse Ly-6G/Ly-6C (Gr-1; 108416; BioLegend), Pacific blue anti-mouse F4/80 (123124; BioLegend), 7AAD (51-68981E; BD Biosciences, San Jose, CA, USA), anti-mouse CX3CR1 Phycoerythrin conjugated Goat IgG (FAB5825P; R&D Systems, Inc., Minneapolis, MN, USA), or Control Goat IgG Isotype Control Phycoerythrin conjugated (IC108P; R&D Systems), and anti-mNeuropilin-1 Allophycocyanin conjugated Rat IgG2A (FAB5994A; R&D Systems), or Rat IgG2A Isotype Control Allophycocyanin conjugated (IC006A; R&D Systems). Fluorescence-activated cell sorting (FACS) was performed on a LSRII (BD Biosciences) device and data were analyzed using FlowJo software (version 7.6.5; FlowJo, Ashland, OR, USA). 
Immunohistochemistry
Flatmounted retinas or retinal cryosections were stained with Rhodamine labeled Griffonia (Bandeiraea) Simplicifolia Lectin I (RL-1102; Vector Laboratories, Inc., Burlingame, CA, USA) in 1 mM CaCl2 in PBS for retinal vasculature and in some cases with anti-IBA1 (rabbit polyclonal; Wako Chemicals USA, Inc., Cape Charles, VA, USA) and anti-rat Neuropilin-1 (goat IgG; R&D Systems) antibodies. 
Quantifications
Cells positive for IBA-1 (microglia) and EYFP- or NRP-1 expressing microglia were quantified in total of 24 images taken per flat mounted retina (two images in avascular area, two images of vascular front and two images in vascular plexus in each of the four patels). Quantification of filopodia along the angiogenic front and branch points of the vascular plexus was performed on eight random images (2 per patel) of the area of interest taken per retina. 
Statistical Analyses
Data are presented as mean ± SEM. We used Student's t-test to compare different groups; a P < 0.05 was considered statistically significant. 
Results
NRP-1+ Microglia Are Present Throughout the Retina During Vascular Development
During development, retinal microglia are in close proximity to vascular tip cells, supporting growth4,20,25 and anastomosis.11 Given the obligate role of NRP-1+ MNPs/microglia in pathologic retinal angiogenesis associated with ischemic retinopathies,23 we sought to determine their presence and distribution during physiological retinal vascularization in the first postnatal week when the superficial vascular layer develops. 
Immunofluorescence of retinas from WT mice from P2 to P6 revealed that NRP-1+ microglia (colabeled with IBA1) are present throughout the retina during vascular development in nonvascularized and vascularized areas as well as at the vascular front (Fig. 1A). A representative P6 retina is shown in Figure 1B and 3-dimensional (3D) reconstructions of confocal z-stacks in Figure 1C confirming robust NRP-1 expression on microglia throughout regions of the retina and on developing vasculature (stained with lectin). Quantification of NRP-1+ microglia throughout the retina (Fig. 1A) revealed the highest concentration of NRP-1+ microglia in zones that had yet to be vascularized and lower densities in vascularized areas (Fig. 1D). The total number of microglia (IBA1+ cells) from P2 to P6 either does not significantly vary between avascular and vascularized areas or is higher in vascularized zones compared to avascular areas (Fig. 1E). This specific distribution of NRP-1+ microglia suggests specialized roles during retinal development. 
Figure 1
 
Neuropilin-1–expressing microglia are present throughout the retina during vascular development. (A) Schematic depiction of retinal flatmount during vascularization illustrating areas of analysis, such as vascularized and nonvascularized zones, as well as the vascular front. (B) Confocal images of lectin– (vessel and microglia stain), IBA-1–(microglia), and NRP-1–stained flatmounted retinas from WT mice at P6. The Figures show nonvascular area (top), vascular front (middle), and superficial vascular plexus (bottom). Representative images of three independent experiments are shown. (C) Shown is a 3D reconstruction of the retina from a WT mouse at P6. Bar graphs showing a percentage of NRP-1+ microglia (D) and a total number of microglia (E) in different regions of the retinas collected between P2 and P6 ± SEM. n = 3 to 6; *P < 0.05, **P < 0.001, ***P < 0.0001. Scale bar: 50 μm.
Figure 1
 
Neuropilin-1–expressing microglia are present throughout the retina during vascular development. (A) Schematic depiction of retinal flatmount during vascularization illustrating areas of analysis, such as vascularized and nonvascularized zones, as well as the vascular front. (B) Confocal images of lectin– (vessel and microglia stain), IBA-1–(microglia), and NRP-1–stained flatmounted retinas from WT mice at P6. The Figures show nonvascular area (top), vascular front (middle), and superficial vascular plexus (bottom). Representative images of three independent experiments are shown. (C) Shown is a 3D reconstruction of the retina from a WT mouse at P6. Bar graphs showing a percentage of NRP-1+ microglia (D) and a total number of microglia (E) in different regions of the retinas collected between P2 and P6 ± SEM. n = 3 to 6; *P < 0.05, **P < 0.001, ***P < 0.0001. Scale bar: 50 μm.
LysM Is Expressed in a Subset of Retinal Microglia That Are Evenly Spread Throughout the Retina During Vascular Development
We have demonstrated previously that infiltrating NRP-1+ MNPs, which express microglial markers when in the retina, are essential for pathologic angiogenesis.23 These MNPs expressed the myeloid-lineage lysozyme 2 gene and are targeted by LysM-Cre driver mice.24 We hence investigated the presence of LysM expressing cells during retinal development by intercrossing ROSA26EYFP-floxed mice with LysM-Cre mice. LysM-Cre/ROSA26EYFPfl/fl mice express EYFP in cells of myeloid lineage. To validate this transgenic mouse, we first performed FACS analysis on whole mouse retinas collected from LysM-Cre/ROSA26EYFPfl/fl, control ROSA26EYFPfl/fl or C57BL/6 (WT) mice. Analysis by FACS revealed that approximately 30% of retinal microglia detected at P2 when vascularization is already initiated with EYFP+ and, hence, recombined with LysM-Cre (Figs. 2D, 2E). Noteworthy, 66% of NRP-1+ microglia are EYFP+ indicating that the LysM-Cre driver mouse will at least ablate expression in these retinal microglia (Supplemental Fig. S1). We defined microglia as Gr-1, F4/80+, CD11b+, CX3CR1hi, and CD45lo expressing cells (Supplemental Fig. S2, Figs. 2A–C).23 
Figure 2
 
LysM-Cre is expressed in a subset of retinal microglia that are evenly spread throughout the retina during vascular development. Representative FACS plots from P2 retinas depicting that (A) 96.5% (in WT), (B) 96.6% (in ROSA26EYFPfl/fl), and (C) 94.6% (in LysM-Cre/ROSA26EYFPfl/fl) of Gr-1−/F4/80+/CD11b+cells express high levels of CX3CR1 and intermediate/low levels of CD45 consistent with a microglial phenotype. (D) In retinas from LysM-Cre/ROSA26EYFPfl/fl mice, these microglia cells express EYFP, not present in WT and ROSA26EYFPfl/fl retinas. (E) Bar graph showing percentage quantification of EYFP-positive compared to EYFP-negative microglia in LysM-Cre/ROSA26EYFPfl/fl ± SEM. n = 5 (each “n” comprises four retinas); ***P < 0.0001. (F) Confocal images of lectin– (vessel and microglia stain) and IBA-1 (microglia marker)–stained flatmounted retinas from LysM-Cre/ROSA26EYFPfl/fl mice at P5. Figures show nonvascular area (top), vascular front (middle), and superficial vascular plexus (bottom). Representative images of three independent experiments are shown. (G) Figure shows 3D reconstructions of the representative images of the retina. (H) Bar graphs showing a percentage of EYFP positive microglia in different regions of the LysM-Cre/ROSA26EYFPfl/fl retinas collected between P2 and P6 ± SEM; n = 3 to 4. Scale bar: 50 μm.
Figure 2
 
LysM-Cre is expressed in a subset of retinal microglia that are evenly spread throughout the retina during vascular development. Representative FACS plots from P2 retinas depicting that (A) 96.5% (in WT), (B) 96.6% (in ROSA26EYFPfl/fl), and (C) 94.6% (in LysM-Cre/ROSA26EYFPfl/fl) of Gr-1−/F4/80+/CD11b+cells express high levels of CX3CR1 and intermediate/low levels of CD45 consistent with a microglial phenotype. (D) In retinas from LysM-Cre/ROSA26EYFPfl/fl mice, these microglia cells express EYFP, not present in WT and ROSA26EYFPfl/fl retinas. (E) Bar graph showing percentage quantification of EYFP-positive compared to EYFP-negative microglia in LysM-Cre/ROSA26EYFPfl/fl ± SEM. n = 5 (each “n” comprises four retinas); ***P < 0.0001. (F) Confocal images of lectin– (vessel and microglia stain) and IBA-1 (microglia marker)–stained flatmounted retinas from LysM-Cre/ROSA26EYFPfl/fl mice at P5. Figures show nonvascular area (top), vascular front (middle), and superficial vascular plexus (bottom). Representative images of three independent experiments are shown. (G) Figure shows 3D reconstructions of the representative images of the retina. (H) Bar graphs showing a percentage of EYFP positive microglia in different regions of the LysM-Cre/ROSA26EYFPfl/fl retinas collected between P2 and P6 ± SEM; n = 3 to 4. Scale bar: 50 μm.
To gain insight on the distribution pattern of these microglia, we performed immunofluorescence analysis on LysM-Cre/ROSA26EYFPfl/fl retinas collected during retinal vascularization between P2 and P6. Retinas were stained with a microglial marker (IBA-1; magenta) and vessel/microglial marker (lectin; red). Confocal microscopy revealed EYFP+ microglia evenly distributed in the inner retina over nonvascularized and vascularized areas as well as at the vascular front where they were associated with nascent vessels (Figs. 2F–G). Quantitative analysis confirmed an equal distribution of EYFP+ microglia throughout the retina with no significant bias towards a given region of the retina during vascular development (Fig. 2H). The LysM-Cre driver mouse does not efficiently induce recombination in microglia of yolk sac origin as LyzM expression commences on embryonic day 11.5.26 However, we noted that approximately 35% of retinal microglia were EYFP+ suggesting a potential non-yolk sac origin. 
LysM-Cre/Nrp1fl/fl Mice Show Significantly Reduced Levels of NRP-1 on Retinal Microglia
Analysis by FACS of whole retinas at P2 (Gating scheme in Supplemental Fig. S2) revealed equal numbers of total retinal microglia (Gr-1, F4/80+, CD11b+, CX3CR1hi, and CD45lo) between WT and LysM-Cre/Nrp1fl/fl mice (Figs. 3A–C) suggesting that during physiological development NRP-1 is not required for mobilization of myeloid cells to the retina and does not influence microglial mitosis. This is in contrast to states of heightened inflammation associated with pathologic retinal angiogenesis, such as in oxygen-induced retinopathy where NRP-1 has an obligate role for myeloid cells chemotaxis.23 Importantly, LysM-Cre/Nrp1fl/fl mice showed a 65% reduction in number of retinal microglia expressing NRP-1 (Fig. 3D). The observed drop in NRP-1+ microglia in LysM-Cre/Nrp1fl/fl mice occurs throughout the retina given the distribution of NRP-1+ microglia (Supplemental Fig. S3). In addition, mean fluorescence intensity (MFI) obtained by FACS indicates significant attenuation of NRP-1 expression per cell (Supplemental Fig. S4). Experimental approaches (no permeabilization and targeting of extracellular epitopes of NRP-1) allowed us to track extracellular domain of NRP-1 receptor. These data confirmed that LysM-Cre/Nrp1fl/fl mice are a valid model to assess the role of microglial NRP-1+ during vascular development of the retina. 
Figure 3
 
LysM-Cre/Nrp1fl/fl show significantly reduced levels of NRP-1+ on retinal microglia. Representative FACS plots from P2 retinas depicting that (A) 95% (in WT) and (B) 94.7% (in LysM-Cre/Nrp1fl/fl) of Gr-1−/F4/80+/CD11b+ cells express high levels of CX3CR1 and intermediate/low levels of CD45 consistent with a microglial phenotype. (C) Retinas from WT and LysM-Cre/Nrp1fl/fl mice show equal numbers of total retinal microglia (Gr-1, F4/80+, CD11b+, CX3CR1hi, and CD45lo). The data are expressed in percentage ± SEM; n = 4 to 9. (D) The microglia express NRP-1 in WT retinas and have significantly reduced NRP-1 expression in retinas from LysM-Cre/Nrp1fl/fl mice. The data are expressed in percentage of NRP-1+ microglia ± SEM; n = 4 to 9. ***P < 0.0001.
Figure 3
 
LysM-Cre/Nrp1fl/fl show significantly reduced levels of NRP-1+ on retinal microglia. Representative FACS plots from P2 retinas depicting that (A) 95% (in WT) and (B) 94.7% (in LysM-Cre/Nrp1fl/fl) of Gr-1−/F4/80+/CD11b+ cells express high levels of CX3CR1 and intermediate/low levels of CD45 consistent with a microglial phenotype. (C) Retinas from WT and LysM-Cre/Nrp1fl/fl mice show equal numbers of total retinal microglia (Gr-1, F4/80+, CD11b+, CX3CR1hi, and CD45lo). The data are expressed in percentage ± SEM; n = 4 to 9. (D) The microglia express NRP-1 in WT retinas and have significantly reduced NRP-1 expression in retinas from LysM-Cre/Nrp1fl/fl mice. The data are expressed in percentage of NRP-1+ microglia ± SEM; n = 4 to 9. ***P < 0.0001.
Cell-Specific Attenuation of Nrp1 in Myeloid Lineage Does Not Impair Development of Mouse Retinal Vasculature
Given the abundance of NRP-1+ microglia in the inner retina during retinal vascularization, we sought to determine their role in developmental angiogenesis. The superficial retinal vasculature forms during the first week of postnatal life, growing outward from the optic nerve head and following structural network and growth factors provided by astrocytes and Muller glia.2,5,22,27 We first assessed rates of retinal vascular growth from P2 to P7 in control WT or LysM-Cre/ NRP-1+/+ mice versus LysM-Cre/Nrp1fl/fl mice (Fig. 4A). Analysis of lectin-stained retinas did not reveal any significant differences in the pace of formation of inner retinal vasculature between the tested groups at any given time point (Fig. 4B). Upon completion of the superficial retinal vasculature at P7, vessels sprout into the retina towards the inner nuclear layer and form the deep and subsequently the intermediate vascular plexus by the end of the third postnatal week.27,28 Evaluation of retinal cross-sections at P21 revealed that all three vascular layers developed in LysM-Cre/Nrp1fl/fl similarly to control mice (Fig. 4C). In addition, neuroretinal lamination remains unaffected as determined at P7 (Supplemental Fig. S5) and P21 (Fig. 4C). 
Figure 4
 
Cell-specific attenuation of Nrp1 in myeloid lineage does not impair development of mouse retinal vasculature. Retinas from WT, LysM-Cre/Nrp1+/+, and LysM-Cre/Nrp1fl/fl mice were collected between P2 and P7, and at P14 and P21. (A) Representative images of the whole flatmounted retinas stained with lectin. Red dotted lines indicate vascularized areas. (B) Bar graphs showing percentage of the retina covered with superficial vascular plexus ± SEM; n = 6 to 17. (C) Representative images of vertical sections of retinas at P21 showing formation of superficial, intermediate, and deep vascular plexuses. (D) Analysis of sprouting angiogenesis and vessel branching. Red squares show filopodia and red stars branch points. Representative images of (E) vascular front and (G) vascularized areas in flatmounted retinas stained with lectin. Bar graphs showing (F) number of filopodia per field ± SEM; n = 3 to 6 and (H) number of branch points per field ± SEM; n = 3 to 6. Each “n” means one retina and comprises four to eight photographed fields. Scale bars: 500 μm (A); 100 μm (C); 50 μm (E, G).
Figure 4
 
Cell-specific attenuation of Nrp1 in myeloid lineage does not impair development of mouse retinal vasculature. Retinas from WT, LysM-Cre/Nrp1+/+, and LysM-Cre/Nrp1fl/fl mice were collected between P2 and P7, and at P14 and P21. (A) Representative images of the whole flatmounted retinas stained with lectin. Red dotted lines indicate vascularized areas. (B) Bar graphs showing percentage of the retina covered with superficial vascular plexus ± SEM; n = 6 to 17. (C) Representative images of vertical sections of retinas at P21 showing formation of superficial, intermediate, and deep vascular plexuses. (D) Analysis of sprouting angiogenesis and vessel branching. Red squares show filopodia and red stars branch points. Representative images of (E) vascular front and (G) vascularized areas in flatmounted retinas stained with lectin. Bar graphs showing (F) number of filopodia per field ± SEM; n = 3 to 6 and (H) number of branch points per field ± SEM; n = 3 to 6. Each “n” means one retina and comprises four to eight photographed fields. Scale bars: 500 μm (A); 100 μm (C); 50 μm (E, G).
To evaluate minute discrepancies in sprouting angiogenesis and anastomosis, we next performed morphometric analysis of the superficial retinal vasculature during development (Fig. 4D).29 Quantitative analysis of confocal images of flatmounted retinas stained with lectin from P2 to P7 revealed comparable numbers of filopodia at the vascular front (Figs. 4E, 4F) and similar numbers of branch points in the superficial plexus (Figs. 4G, 4H) of LysM-Cre/Nrp1fl/fl and control mice. Collectively, our study demonstrates that while LysM-expressing NRP-1+ microglia populate the inner retina throughout vascular development, they are dispensable for retinal angiogenesis. 
Discussion
Our results demonstrated that cell-specific attenuation of NRP-1 in LysM-expressing microglia does not impair mouse retinal angiogenesis. We investigated the role of NRP-1+ microglia in retinal vascularization given that: (1) NRP-1+ microglia are present abundantly in the developing retina and associated with blood vessels, (2) NRP-1+ microglia are detected on sites of vascular anastomosis during retinal development,11 and (3) colony-stimulating factor 1 (CSF-1)–deficient mice with significantly reduced numbers of cells of monocytic lineage show lower numbers of branch points in their retinal vascular plexus.4 While only approximately 35% of retinal microglia during postnatal retinal development express LysM, they account for 65% of NRP-1-expressing microglia. Our findings that LysM-expressing NRP-1+ microglia are dispensable for proper retinal angiogenesis are complementary and in agreement with a recent study by Fantin et al.12 suggesting that yolk sac–derived NRP-1+ macrophages are also not essential for developmental angiogenesis in the hindbrain. Conversely, endothelial-resident NRP-1 is critical for proper CNS vascularization as deletion of endothelial NRP-1 reduces vessel branching in the hindbrain12,30 and limits tip formation and branching in the retina.31 Taken with previous work identifying the key contribution of NRP-1+ myeloid cells to pathologic retinal angiogenesis,23 data presented in this study drew a dichotomous role for NRP-1 in myeloid cells where it is dispensable for adequate retinal developmental vascularization yet obligate for pathologic retinal angiogenesis. 
Acknowledgments
Supported by a Canada Research Chair in Retinal Cell Biology, The Alcon Research Institute Young Investigator Award (PS), by operating grants from the Canadian Institutes of Health Research (221478; PS), the Canadian Diabetes Association (OG-3-11-3329-PS), and Natural Sciences and Engineering Research Council of Canada (418637), and by the Fondation HMR, Réseau en Recherche en Santé de la Vision du Quebec and the Fond en Recherche en Opthalmologie de l'UdM. 
Disclosure: A. Dejda, None; G. Mawambo, None; J.-F. Daudelin, None; K. Miloudi, None; N. Akla, None; C. Patel, None; E.M.M.A. Andriessen, None; N. Labrecque, None; F. Sennlaub, None; P. Sapieha, None 
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Figure 1
 
Neuropilin-1–expressing microglia are present throughout the retina during vascular development. (A) Schematic depiction of retinal flatmount during vascularization illustrating areas of analysis, such as vascularized and nonvascularized zones, as well as the vascular front. (B) Confocal images of lectin– (vessel and microglia stain), IBA-1–(microglia), and NRP-1–stained flatmounted retinas from WT mice at P6. The Figures show nonvascular area (top), vascular front (middle), and superficial vascular plexus (bottom). Representative images of three independent experiments are shown. (C) Shown is a 3D reconstruction of the retina from a WT mouse at P6. Bar graphs showing a percentage of NRP-1+ microglia (D) and a total number of microglia (E) in different regions of the retinas collected between P2 and P6 ± SEM. n = 3 to 6; *P < 0.05, **P < 0.001, ***P < 0.0001. Scale bar: 50 μm.
Figure 1
 
Neuropilin-1–expressing microglia are present throughout the retina during vascular development. (A) Schematic depiction of retinal flatmount during vascularization illustrating areas of analysis, such as vascularized and nonvascularized zones, as well as the vascular front. (B) Confocal images of lectin– (vessel and microglia stain), IBA-1–(microglia), and NRP-1–stained flatmounted retinas from WT mice at P6. The Figures show nonvascular area (top), vascular front (middle), and superficial vascular plexus (bottom). Representative images of three independent experiments are shown. (C) Shown is a 3D reconstruction of the retina from a WT mouse at P6. Bar graphs showing a percentage of NRP-1+ microglia (D) and a total number of microglia (E) in different regions of the retinas collected between P2 and P6 ± SEM. n = 3 to 6; *P < 0.05, **P < 0.001, ***P < 0.0001. Scale bar: 50 μm.
Figure 2
 
LysM-Cre is expressed in a subset of retinal microglia that are evenly spread throughout the retina during vascular development. Representative FACS plots from P2 retinas depicting that (A) 96.5% (in WT), (B) 96.6% (in ROSA26EYFPfl/fl), and (C) 94.6% (in LysM-Cre/ROSA26EYFPfl/fl) of Gr-1−/F4/80+/CD11b+cells express high levels of CX3CR1 and intermediate/low levels of CD45 consistent with a microglial phenotype. (D) In retinas from LysM-Cre/ROSA26EYFPfl/fl mice, these microglia cells express EYFP, not present in WT and ROSA26EYFPfl/fl retinas. (E) Bar graph showing percentage quantification of EYFP-positive compared to EYFP-negative microglia in LysM-Cre/ROSA26EYFPfl/fl ± SEM. n = 5 (each “n” comprises four retinas); ***P < 0.0001. (F) Confocal images of lectin– (vessel and microglia stain) and IBA-1 (microglia marker)–stained flatmounted retinas from LysM-Cre/ROSA26EYFPfl/fl mice at P5. Figures show nonvascular area (top), vascular front (middle), and superficial vascular plexus (bottom). Representative images of three independent experiments are shown. (G) Figure shows 3D reconstructions of the representative images of the retina. (H) Bar graphs showing a percentage of EYFP positive microglia in different regions of the LysM-Cre/ROSA26EYFPfl/fl retinas collected between P2 and P6 ± SEM; n = 3 to 4. Scale bar: 50 μm.
Figure 2
 
LysM-Cre is expressed in a subset of retinal microglia that are evenly spread throughout the retina during vascular development. Representative FACS plots from P2 retinas depicting that (A) 96.5% (in WT), (B) 96.6% (in ROSA26EYFPfl/fl), and (C) 94.6% (in LysM-Cre/ROSA26EYFPfl/fl) of Gr-1−/F4/80+/CD11b+cells express high levels of CX3CR1 and intermediate/low levels of CD45 consistent with a microglial phenotype. (D) In retinas from LysM-Cre/ROSA26EYFPfl/fl mice, these microglia cells express EYFP, not present in WT and ROSA26EYFPfl/fl retinas. (E) Bar graph showing percentage quantification of EYFP-positive compared to EYFP-negative microglia in LysM-Cre/ROSA26EYFPfl/fl ± SEM. n = 5 (each “n” comprises four retinas); ***P < 0.0001. (F) Confocal images of lectin– (vessel and microglia stain) and IBA-1 (microglia marker)–stained flatmounted retinas from LysM-Cre/ROSA26EYFPfl/fl mice at P5. Figures show nonvascular area (top), vascular front (middle), and superficial vascular plexus (bottom). Representative images of three independent experiments are shown. (G) Figure shows 3D reconstructions of the representative images of the retina. (H) Bar graphs showing a percentage of EYFP positive microglia in different regions of the LysM-Cre/ROSA26EYFPfl/fl retinas collected between P2 and P6 ± SEM; n = 3 to 4. Scale bar: 50 μm.
Figure 3
 
LysM-Cre/Nrp1fl/fl show significantly reduced levels of NRP-1+ on retinal microglia. Representative FACS plots from P2 retinas depicting that (A) 95% (in WT) and (B) 94.7% (in LysM-Cre/Nrp1fl/fl) of Gr-1−/F4/80+/CD11b+ cells express high levels of CX3CR1 and intermediate/low levels of CD45 consistent with a microglial phenotype. (C) Retinas from WT and LysM-Cre/Nrp1fl/fl mice show equal numbers of total retinal microglia (Gr-1, F4/80+, CD11b+, CX3CR1hi, and CD45lo). The data are expressed in percentage ± SEM; n = 4 to 9. (D) The microglia express NRP-1 in WT retinas and have significantly reduced NRP-1 expression in retinas from LysM-Cre/Nrp1fl/fl mice. The data are expressed in percentage of NRP-1+ microglia ± SEM; n = 4 to 9. ***P < 0.0001.
Figure 3
 
LysM-Cre/Nrp1fl/fl show significantly reduced levels of NRP-1+ on retinal microglia. Representative FACS plots from P2 retinas depicting that (A) 95% (in WT) and (B) 94.7% (in LysM-Cre/Nrp1fl/fl) of Gr-1−/F4/80+/CD11b+ cells express high levels of CX3CR1 and intermediate/low levels of CD45 consistent with a microglial phenotype. (C) Retinas from WT and LysM-Cre/Nrp1fl/fl mice show equal numbers of total retinal microglia (Gr-1, F4/80+, CD11b+, CX3CR1hi, and CD45lo). The data are expressed in percentage ± SEM; n = 4 to 9. (D) The microglia express NRP-1 in WT retinas and have significantly reduced NRP-1 expression in retinas from LysM-Cre/Nrp1fl/fl mice. The data are expressed in percentage of NRP-1+ microglia ± SEM; n = 4 to 9. ***P < 0.0001.
Figure 4
 
Cell-specific attenuation of Nrp1 in myeloid lineage does not impair development of mouse retinal vasculature. Retinas from WT, LysM-Cre/Nrp1+/+, and LysM-Cre/Nrp1fl/fl mice were collected between P2 and P7, and at P14 and P21. (A) Representative images of the whole flatmounted retinas stained with lectin. Red dotted lines indicate vascularized areas. (B) Bar graphs showing percentage of the retina covered with superficial vascular plexus ± SEM; n = 6 to 17. (C) Representative images of vertical sections of retinas at P21 showing formation of superficial, intermediate, and deep vascular plexuses. (D) Analysis of sprouting angiogenesis and vessel branching. Red squares show filopodia and red stars branch points. Representative images of (E) vascular front and (G) vascularized areas in flatmounted retinas stained with lectin. Bar graphs showing (F) number of filopodia per field ± SEM; n = 3 to 6 and (H) number of branch points per field ± SEM; n = 3 to 6. Each “n” means one retina and comprises four to eight photographed fields. Scale bars: 500 μm (A); 100 μm (C); 50 μm (E, G).
Figure 4
 
Cell-specific attenuation of Nrp1 in myeloid lineage does not impair development of mouse retinal vasculature. Retinas from WT, LysM-Cre/Nrp1+/+, and LysM-Cre/Nrp1fl/fl mice were collected between P2 and P7, and at P14 and P21. (A) Representative images of the whole flatmounted retinas stained with lectin. Red dotted lines indicate vascularized areas. (B) Bar graphs showing percentage of the retina covered with superficial vascular plexus ± SEM; n = 6 to 17. (C) Representative images of vertical sections of retinas at P21 showing formation of superficial, intermediate, and deep vascular plexuses. (D) Analysis of sprouting angiogenesis and vessel branching. Red squares show filopodia and red stars branch points. Representative images of (E) vascular front and (G) vascularized areas in flatmounted retinas stained with lectin. Bar graphs showing (F) number of filopodia per field ± SEM; n = 3 to 6 and (H) number of branch points per field ± SEM; n = 3 to 6. Each “n” means one retina and comprises four to eight photographed fields. Scale bars: 500 μm (A); 100 μm (C); 50 μm (E, G).
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