Neuropilin-1 and the Positions of Glomeruli in the Mouse Olfactory Bulb

Configurations of M71 glomeruli in bulbs of conditional Nrp1 knockout mice. A, Triple-mutant mice n5247MCZ. B, Quadruple-mutant mice NMRCZ. The occurrence of configurations I–VI is listed per age. Red numbers indicate the most frequent configuration for a given age. Positions of labeled glomeruli in the various configurations are illustrated schematically with green dots, and in the case of NMRCZ, with orange dots for glomeruli that consist clearly of both RFP⁺ and GFP⁺ axons, and green dots for glomeruli formed predominantly by GFP⁺ axons. No glomeruli formed by only RFP⁺ axons were observed. The data are pooled from whole-mount images and coronal sections. Pairwise χ² tests with Bonferroni correction for multiple comparisons were performed on successive age pairs for both strains: n5247MCZ, PD0 vs. PD14 (*), PD14 vs. PD21 [not significant (n.s.)], PD21 vs. PD105 (***); NMCZ, PD7 vs. PD14 (n.s.), PD14 vs. PD21 (n.s.), PD21 vs. PD35 (n.s.), PD35 vs. PD45 (n.s.), PD45 vs. PD 70 (*). The pre–multiple-comparison sigma values are designated as *p < 0.05, **p < 0.01, and ***p < 0.001.

Three-dimensional reconstructions of bulbs of PD21 gene-targeted mice expressing M71 with and without Nrp1. Serial block-face two-photon tomography was carried out to image the intrinsic fluorescence in four bulbs of four homozygous M71-IRES-tauYFP mice at PD21, and in six bulbs of six triple-mutant n5247MCZ mice at PD21. Each individual mouse is indicated with a distinct color. The gray outer area represents the surface of the glomerular layer, and the black inner area the regions of the bulb below the glomerular layer. A, Dorsal view, comparable to the view in Figure 1. The medial M71 glomeruli are poorly visible in this dorsal view, because they reside in a flat, medial domain of the bulb. B, Dorsomedial view. The ectopic anterior glomeruli reside in the rostral tip of the bulb. Both the medial and lateral glomeruli are visible here by making the glomerular layer transparent. The bulb is tilted slightly laterally to expose the medial glomeruli better. C, Close-ups of views oriented in such a way that the individual glomeruli are separated clearly: dorsal, anterior, and medial domains of the bulb.

Confocal and whole-mount imaging of M71 glomeruli in triple-mutant NMCZ and quadruple-mutant NMRCZ mice. A, B, Coronal 12-µm sections of bulbs of NMCZ mice at PD21 imaged with a Zeiss LSM 710 confocal microscope, using the intrinsic fluorescence of GFP and counterstaining of nuclei with DAPI. The box indicated with a white stippled line in the left images is magnified in the right images. A, Ectopic dorsal glomerulus, large. B, Ectopic anterior glomerulus, small and located deeper in the glomerular layer. C–H, Whole-mount bulbs of an NMRCZ mouse at PD59 imaged in wide-field on a Nikon SMZ25 stereofluorescence microscope. C, Dorsal view of the left and right bulbs, with the left bulb exhibiting configuration VI, and the right bulb, configuration V. Not all glomeruli are visible in this view. D, The right bulb from C (white box) is tilted to the right to provide a better view of the medial aspect and demonstrate the configuration consisting of an ectopic anterior glomerulus (blue box), a medial glomerulus (red box), and an ectopic dorsal glomerulus (orange box). E, Magnified view of the ectopic anterior glomerulus and consisting of GFP⁺ axons, as shown in the blue box in D. F, Magnified view of the two dorsal glomeruli of the left bulb, in the yellow box in C. The left glomerulus exhibits a mixed, comparable contribution of GFP⁺ and RFP⁺ axons. The right glomerulus consists mostly of GFP⁺ axons, with a compartment of RFP+ axons. G, Magnified view of the mixed GFP⁺ RFP⁺ medial glomerulus of the right bulb, as shown in the red box in D. H, Magnified view of the ectopic dorsal glomerulus consisting predominantly of GFP⁺ axons, as shown in the orange box in D. A small compartment of this glomerulus is innervated by an axon bundle containing also RFP⁺ axons (pink arrow). I, J, 12-µm sections of bulbs of NMRCZ mice at PD56 and PD70, respectively, imaged with a Zeiss LSM 710 confocal microscope, using the intrinsic fluorescence of GFP and RFP and counterstaining of nuclei with DAPI. I, Ectopic dorsal glomerulus consisting of RFP⁺ and GFP⁺ axons. J, Magnified view of an ectopic-anterior glomerulus consisting of GFP⁺ axons. Scale bars, A left, 100 µm; A right, 50 µm; B left, 100 µm; B right, 50 µm; C and D, 300 µm; E, 20 µm; F, 50 µm; G, 25 µm; H, 50 µm; and I and J, 25 µm.

Numbers of labeled cells per mouse in mice with gene-targeted mutations in the M71 locus at PD21. Numbers are given ± SD and are Abercrombie corrected, as described in Bressel et al. (2016). A symbol represents an individual mouse. The numbers for M71-IRES-tauGFP (n = 5 mice) and M71-IRES-tauRFP2 (n = 3 mice) are taken from Bressel et al. (2016) for comparison. “M71-IRES-Cre heterozygous” and “M71-IRES-tauRFP2 heterozygous” are the numbers of labeled cells representing the two different M71 alleles in NMRCZ (n = 4 mice), and the sum of these numbers is given in “NMRCZ.” The symbols (asterisk, upward triangle, downward triangle, diamond) used for NMRCZ represent individual mice. Analysis of cell counts using one-way ANOVA found all homozygous vs. heterozygous pairs significant (p < 0.05) according to Tukey’s multiple comparison test, which is consistent with monoallelic expression of OR genes. All other comparisons (–/– vs. –/– pairs and +/– vs. +/– pairs) are not significant.

Posterior glomeruli in the vicinity of Gucy1b2⁺ glomeruli can be Nrp1⁺ or Nrp1^–. Fluorescence images of three horizontal sections of a bulb of a homozygous Gucy1b2-IRES-tauGFP mouse at 4 weeks were taken with a Zeiss LSM 710 confocal microscope. Each of the three Gucy1b2⁺ glomeruli is indicated with a yellow arrowhead. A, Section at a very ventral level, 0.13 mm from the bottom of the bulb, as shown schematically in D. Intrinsic GFP fluorescence (GFP*) is combined with immunofluorescence for Nrp1 (red) and Adcy3 (blue). Nuclear staining with DAPI is in white. Merged is all colors together. The top four panels show the entire section. The bottom four panels show high-magnification views of an area surrounding the GFP⁺ glomerulus. This glomerulus is Adcy3^– and Nrp1^–. The posterior part of the bulb at this very ventral level is devoid of Nrp1 immunofluorescence but contains numerous glomeruli that are Adcy3⁺. B, Section at a more dorsal level, 0.29 mm from the bottom of the bulb. The image on the left shows the entire section. The two panels on the right show high-magnification views of an area surrounding the GFP⁺ glomerulus. This extremely posterior GFP⁺ glomerulus is Adcy3^– and Nrp1^–. The glomerulus anterior to the GFP⁺ glomerulus is Adcy3⁺ and Nrp1⁺ (yellow arrow), but the glomerulus posterior to the GFP⁺ glomerulus is Adcy3⁺ and Nrp1^– (unfilled yellow arrowhead). C, Section at an intermediate dorsal-ventral level, 1.24 mm from the bottom of the bulb. The image on the left shows the entire section. The two panels on the right show high-magnification views of an area surrounding the GFP⁺ glomerulus. This very posterior GFP⁺ glomerulus is Adcy3^– and Nrp1^–. The glomeruli anterior to it are strongly Adcy3⁺ and Nrp1^–, and the most anterior glomerulus (indicated with an arrow) is Adcy3⁺ and weakly Nrp1⁺. D, Schematic diagram of a medial view on the bulb. The positions of the three glomeruli shown in A–C are indicated. Scale bars, A, top 200 μm, bottom 50 μm; B and C, left 200 μm, right 50 μm. a, anterior; p, posterior; m, medial; l, lateral; glom, glomerulus.

Three-dimensional reconstructions of bulbs with Nrp1 immunofluorescence levels in glomeruli of a bulb of a wild-type C57BL6/J mouse at PD14. After iDISCO treatment of the bulb, immunofluorescence with antibodies against Nrp1 and VGLUT2 was applied to the whole mount, followed by serial block-face two-photon tomography and 3D reconstruction. A, Overall Nrp1 immunofluorescence signal, medial view on the bulb. The lookup table from low (blue) to high (green) represents the intensity of the overall Nrp1 immunofluorescence signal, which is much stronger in axons than in glomeruli. The signal in glomeruli is mostly obscured by the signal in overlying axons. B, Segmented VGLUT2 immunofluorescence signal, shown in uniform green, binary signal, with no intensity information. VGLUT2 is a marker for OSN axon terminals within glomeruli. The layers of the bulb below the glomerular layer are rendered in black for contrast and orientation. C, Medial view of the reconstructed bulb. The Nrp1 signal is confined to glomeruli by generating a mask using the VGLUT2 signal. The lookup table from low (violet) to high (red) represents the intensity of the Nrp1 immunofluorescence signal in glomeruli. Generally, anterior glomeruli are Nrp1^low (from violet to blue), and generally, posterior glomeruli are Nrp1^high (from green over yellow to red). D, Lateral view of the reconstructed bulb. Generally, anterior glomeruli are Nrp1^low, and generally, posterior glomeruli are Nrp1^high.

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.

Example of a three-dimensional reconstruction of a bulb of a PD14 transgenic mouse expressing rat OR I7 from a mouse MOR23 promoter and gap-YFP. The right bulb of a mouse was reconstructed in 3D after epifluorescence whole-mount imaging with a Nikon SMZ25 stereomicroscope. The images of the medial and lateral aspects of this bulb are the same as in Figure 8, right bottom, and reproduced here to facilitate comparison with the 3D reconstruction. There are two labeled glomeruli in the medial aspect (compared to two in the epifluorescence whole-mount image, yellow arrows), and five labeled glomeruli in the lateral aspect (compared to four in the epifluorescence whole-mount image, yellow arrows).