Figure 1
Figure 1. NRP1 expression during hindbrain vascularization in the mouse. (A-F) Whole-mount immunofluorescence labeling of the mouse embryo hindbrain; scale bar (A-D and F) 50 μm. (A) Maximal projection (xy) of a confocal z stack through the E10.5 subventricular zone shows NRP1 on IB4-positive endothelial stalk and tip cells (wavy arrow and arrowhead, respectively; yellow indicates double labeling). NRP1 is also prominent on tip-cell filopodia (IB4 low) and neural progenitors (IB4 negative). (A′) Virtual transverse (xz) section through the z stack in (A) at the level indicated with a stippled line. (B,B′) Single NRP1 channel of (A,A′). Some IB4-positive tissue macrophages (arrow in A) are poorly ramified at E10.5 and do not obviously express NRP1 (compare A with B). (C) Single xy scan of the confocal z stack projection in (B) at the level indicated with a stippled line in (B'); clusters of tip cell filopodia protrude into the neural progenitor layer (arrowheads). (D-E) Confocal z stack through the E11.5 subventricular zone shows that IB4-positive endothelial cells and IBA1/IB4-positive ramified tissue macrophages express NRP1; purple indicates co-labeling with NRP1 and IB4. The boxed area in (D) is indicated at higher magnification in (D′) and as the single NRP1 channel in (E). NRP1 is high on filopodia-studded tip cells and on IBA1-enriched macrophage processes (arrowheads and clear arrows, respectively). NRP1 appears lower in neural progenitors at E11.5 (D) compared with E10.5 (A). (F) Z stack through the subventricular zone at E11.5 shows VEGFR2 expression on IB4-positive endothelial stalk and tip cells (wavy arrow and arrowhead, respectively; yellow indicates double labeling). VEGFR2 is high on filopodia but not obviously expressed by tissue macrophages or neural progenitors. (G) Schematic representation of NRP1 and VEGFR2 distribution and hypothetical interactions in hindbrain cell types during angiogenesis. NRP1 (light green) is co-expressed with VEGFR2 (dark green) on endothelial stalk and tip cells (red), which is a prerequisite for homotypic interactions. NRP1 is also expressed by neural progenitors (orange) and tissue macrophages (Mφ, blue), and therefore heterotypically and in trans relative to VEGFR2 in endothelial cells.

NRP1 expression during hindbrain vascularization in the mouse. (A-F) Whole-mount immunofluorescence labeling of the mouse embryo hindbrain; scale bar (A-D and F) 50 μm. (A) Maximal projection (xy) of a confocal z stack through the E10.5 subventricular zone shows NRP1 on IB4-positive endothelial stalk and tip cells (wavy arrow and arrowhead, respectively; yellow indicates double labeling). NRP1 is also prominent on tip-cell filopodia (IB4 low) and neural progenitors (IB4 negative). (A′) Virtual transverse (xz) section through the z stack in (A) at the level indicated with a stippled line. (B,B′) Single NRP1 channel of (A,A′). Some IB4-positive tissue macrophages (arrow in A) are poorly ramified at E10.5 and do not obviously express NRP1 (compare A with B). (C) Single xy scan of the confocal z stack projection in (B) at the level indicated with a stippled line in (B'); clusters of tip cell filopodia protrude into the neural progenitor layer (arrowheads). (D-E) Confocal z stack through the E11.5 subventricular zone shows that IB4-positive endothelial cells and IBA1/IB4-positive ramified tissue macrophages express NRP1; purple indicates co-labeling with NRP1 and IB4. The boxed area in (D) is indicated at higher magnification in (D′) and as the single NRP1 channel in (E). NRP1 is high on filopodia-studded tip cells and on IBA1-enriched macrophage processes (arrowheads and clear arrows, respectively). NRP1 appears lower in neural progenitors at E11.5 (D) compared with E10.5 (A). (F) Z stack through the subventricular zone at E11.5 shows VEGFR2 expression on IB4-positive endothelial stalk and tip cells (wavy arrow and arrowhead, respectively; yellow indicates double labeling). VEGFR2 is high on filopodia but not obviously expressed by tissue macrophages or neural progenitors. (G) Schematic representation of NRP1 and VEGFR2 distribution and hypothetical interactions in hindbrain cell types during angiogenesis. NRP1 (light green) is co-expressed with VEGFR2 (dark green) on endothelial stalk and tip cells (red), which is a prerequisite for homotypic interactions. NRP1 is also expressed by neural progenitors (orange) and tissue macrophages (Mφ, blue), and therefore heterotypically and in trans relative to VEGFR2 in endothelial cells.

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