Alteration of Nrp1 signaling at different stages of olfactory neuron maturation promotes glomerular shifts along distinct axes in the olfactory bulb

Abstract

Building the topographic map in the mammalian olfactory bulb is explained by a model based on two axes along which sensory neurons are guided: one dorsoventral and one anteroposterior. This latter axis relies on specific expression levels of Nrp1. To evaluate the role of this receptor in this process, we used an in vivo genetic approach to decrease or suppress Nrp1 in specific neuronal populations and at different time points during axonal targeting. We observed, in neurons that express the M71 or M72 odorant receptors, that Nrp1 inactivation leads to two distinct wiring alterations, depending on the time at which Nrp1 expression is altered: first, a surprising dorsal shift of the M71 and M72 glomeruli, which often fuse with their contralateral counterparts, and second the formation of anteriorized glomeruli. The two phenotypes are partly recapitulated in mice lacking the Nrp1 ligand Sema3A and in mice whose sensory neurons express an Nrp1 mutant unable to bind Sema3A. Using a mosaic conditional approach, we show that M71 axonal fibers can bypass the Nrp1 signals that define their target area, since they are hijacked and coalesce with Nrp1-deficient M71-expressing axons that target elsewhere. Together, these findings show drastically different axonal targeting outcomes dependent on the timing at which Nrp1/Sema3A signaling is altered.

Keywords: Axon guidance; Mouse; Neuropilin 1; Olfaction; Olfr151; Olfr160; Semaphorin 3A.

Fig. 1.

Nrp1 expression patterns in the olfactory bulb. (A) Schematic of a mouse head showing the horizontal and coronal section planes used in B and C-K, respectively. A, anterior; L, lateral; P, posterior; M, medial; D, dorsal; V, ventral; nl, nerve layer; gl, glomerular layer; OB, olfactory bulb. (B) Immunostaining for Nrp1 (red) on a horizontal section of the medial part of a mouse OB (P0). High expression of Nrp1 is observed in the posterior part of the OB glomerular layer. Dashed line demarcates the boundary of the glomerular and nerve layers; individual glomeruli are circled. Blue, DAPI. (C-K) Immunostaining on coronal sections of mouse OBs showing single glomeruli innervated by M71, M72 or MOR23 sensory neurons (P11). Green in the left column shows expression in axonal fibers of β-galactosidase (M71^lacZ/lacZ or M72^lacZ/lacZ mice) or GFP (MOR23^GFP/GFP mice); red in the middle column indicates expression of Nrp1. The combined image is shown in the right column. Scale bars: 50 µm.

Fig. 2.

Axonal projections of M71-, M72- and MOR23-expressing OSNs in a late Nrp1 conditional deletion context. (A-D) Dorsal and medial whole-mount views of X-Gal-stained Nrp1^+/+;Omp^+/cre;M71^+/lacZ (P34) or Nrp1^+/+;Omp^+/cre;M72^lacZ/lacZ (P40) OBs. A, anterior; L, lateral; P, posterior; M, medial; D, dorsal; V, ventral. Scale bar in A is 1 mm (scale is identical for all dorsal OB views). Note that the glomeruli are independent. Scale bar in B is 1 mm (scale is identical for all medial OB views). (E) Medial whole-mount view of an Nrp1^+/+;Omp^+/cre;MOR23^GFP/GFP (P28) OB under epifluorescence. (F-I) Dorsal and medial whole-mount views of X-Gal-stained Nrp1^flox/flox;Omp^+/cre;M71^+/lacZ (P36) or Nrp1^flox/flox;Omp^+/cre;M72^lacZ/lacZ (P32) OBs showing a dorsalization of the glomeruli. (J) Medial whole-mount view of an Nrp1^flox/flox;Omp^+/cre;MOR23^GFP/GFP (P28) OB under epifluorescence, showing no obvious targeting alterations. (K,M) Comparison of the frequency of the specific phenotypes affecting M71 or M72 glomeruli assigned to each of the following categories: independent (indep.), linked or fused glomeruli. The frequencies are compared between controls and late-deleted Nrp1 (*P<0.001, χ² test). In addition, the presence or absence of an anterior glomerulus in each OB (control and late-deleted Nrp1) is indicated. (L,N,O) Mapping of all M71, M72 and MOR23 glomeruli (control and Nrp1 late-deleted populations) on a medial representation of the OB, showing the dorsalization of the M71 and M72 glomeruli. Glomeruli (from single OBs) linked to each other are connected by colored lines. A dot circled in black represents the mean position of glomeruli from each population. Ellipses represent the glomerular dispersion of each glomerular population. The dorsal part of the snout was considered as the horizontal line for all medial OB images, which corresponds to a 113° angle with the cribriform plate (L). Data corresponding to control animals (n=20 for M71, n=28 for M72 and n=12 for MOR23) are depicted in orange, and data from Nrp1 late-deleted animals (Nrp1^flox/flox;Omp^+/cre; n=10 for M71, n=22 for M72 and n=20 for MOR23) are in magenta. Insets show close-ups from the glomerular targeting sites.

Fig. 3.

Late and early expression of olfactory Cre drivers. (A) Strategy to generate the Gng8^cre allele. The position of the probe used for Southern blot is indicated. HA, homology arm; i, internal ribosome entry site (IRES). (B) Dorsal parts of the septal olfactory neuroepithelium of Gng8^+/cre;R26^+/flRFP, M71^+/cre; R26^+/flRFP or Omp^+/cre;R26^+/flRFP animals immunostained for RFP (red) and counterstained with DAPI (blue). A schematic coronal view of the turbinates is shown in the middle image, with a black square indicating the area where neurons were observed. sust., sustentacular; OSNs, olfactory sensory neurons. The most basally located RFP-positive neurons are indicated by cyan dots and correspond to the positions along the apicobasal axis indicated in C. Scale bar: 50 µm. (C) Positions of the most basal RFP-positive cells observed along the apicobasal axis (cyan dots, as illustrated in B) of the neuroepithelium of Gng8^+/cre;R26^+/flRFP mice (n=232 cells from two mice), M71^+/cre;R26^+/flRFP (n=264 cells from two mice) and Omp^+/cre;R26^+/flRFP (n=160 cells from two mice). Means and s.d. are indicated by thick and thin horizontal black lines, respectively. The gray shaded area represents the fifth percentile.

Fig. 4.

Axonal projections of M71-, M72- and MOR23-expressing OSNs in an early Nrp1 conditional deletion context. (A-D) Dorsal and medial whole-mount views of X-Gal-stained Nrp1^flox/flox;Gng8^+/+;M71^+/lacZ (P25) and Nrp1^flox/flox;Gng8^+/+;M72^lacZ/lacZ (P23) OBs. A, anterior; L, lateral; P, posterior; M, medial; D, dorsal; V, ventral. (E) Medial whole-mount view of an Nrp1^flox/flox;Gng8^+/+;MOR23^+/GFP (P30) OB under epifluorescence. Scale bars: 1 mm in A for all dorsal OB views; 1 mm in B for all medial OB views. (F) Close-up of the glomerular projection site shown in E. Scale bar: 400 µm. (G-J) Dorsal and medial whole-mount views of X-Gal-stained Nrp1^flox/flox;Gng8^+/cre;M71^+/lacZ (P25) or Nrp1^flox/flox;Gng8^+/cre;M72^+/lacZ (P25) OBs showing a dorsalization of the glomeruli and an additional anterior glomerulus. (K,L) Medial whole-mount views of Nrp1^flox/flox;Gng8^+/cre;MOR23^GFP/GFP (P30, K; P54, L) OBs under epifluorescence showing an additional anterior glomerulus. (M,O) Comparison of the frequency of the specific phenotypes affecting M71 or M72 glomeruli assigned to the independent, linked or fused categories. The frequencies are compared between controls and early-deleted Nrp1 (*P<0.001, χ² test). In addition, the presence or absence of an anterior glomerulus in each OB (control and early-deleted Nrp1) is indicated and their respective frequencies are compared (*P<0.001, χ² test). (Q) Comparison of the frequency of the specific phenotypes affecting MOR23 glomeruli, assigned to each of the following categories: wild-type (wt), wild-type and anterior, or anterior-only glomeruli. The frequencies are compared between controls and early-deleted Nrp1 (*P<0.001, χ² test). (N,P,R) Mapping of all M71, M72 and MOR23 glomeruli (control and Nrp1 early-deleted populations) on a medial representation of the OB. Glomeruli (from single OBs) linked to each other are connected by colored lines. A dot circled in black represents the mean position of glomeruli from each population. Ellipses represent the glomerular dispersion of each glomerular population. M71- and M72-expressing OSN populations in Nrp1 early-deleted animals have two projection sites: one dorsally located relative to the control population (often fusing with contralateral projecting fibers) and one anterior. In most OBs of early-deleted Nrp1 mice the MOR23-expressing fiber projection pattern is divided into one anteroventralized glomerulus and one located at the same position as in controls. Data corresponding to control animals (n=8 for M71, n=5 for M72 and n=14 for MOR23) are depicted in orange, and data from early-deleted Nrp1 animals (Nrp1^flox/flox;Gng8^+/cre, Nrp1^flox/flox;Gng8^cre/cre; n=12 for M71, n=22 for M72 and n=18 for MOR23) are in magenta. Insets show close-ups from the glomerular targeting sites.

Fig. 5.

Reciprocal hijacking of Nrp1-expressing and Nrp1-deficient fibers. (A) Dorsal whole-mount view of Nrp1^+/flox;M71^cre/GFP;R26^+/flRFP (P45) OBs under epifluorescence. A, anterior; L, lateral; P, posterior; M, medial. (B-D) Close-ups of the lateral and the medial glomeruli from A. (E,I) Dorsal whole-mount views of Nrp1^flox/flox;M71^cre/GFP;R26^+/flRFP (E, P45; I, P51) OBs under epifluorescence. (F-H) Close-ups of the lateral, medial and fused glomeruli from E. Lateral and medial glomeruli are co-innervated by green (Nrp1-expressing) and red (Nrp1-deficient) fibers. (J-L) Close-ups of the anterior and fused glomeruli from I. Anterior glomeruli are innervated mainly by Nrp1-deficient (red) fibers. (M-O) High magnification of Nrp1^flox/flox;M71^cre/GFP;R26^flRFP/flRFP fused glomeruli showing heterogeneous innervation of the fused glomeruli. (P) Comparison of the frequency of the specific phenotypes affecting M71 glomeruli assigned to the independent or linked/fused categories for the indicated genotypes. The frequencies are compared between Nrp1^flox/flox;M71^cre/GFP;R26^flRFP and their controls (*P<0.001, χ² test). In addition, the presence or absence of an anterior glomerulus in each OB is indicated and the frequencies between Nrp1^flox/flox;M71^cre/GFP;R26^flRFP and their controls are compared (*P<0.001, χ² test). Data corresponding to control animals (Nrp1^+/+;M71^cre/GFP;R26^+/flRFP, Nrp1^+/flox;M71^cre/GFP; R26^{+ or flRFP/flRFP}, n=15) are depicted in orange, and data from Nrp1^flox/flox;M71^cre/cre;R26^{+ or flRFP/flRFP} (n=22) and Nrp1^flox/flox;M71^cre/GFP;R26^{+ or flRFP/flRFP} (n=62) are in magenta. Scale bars: 1 mm in A,E,I; 100 μm in B-D,F-H,J-O.

Fig. 6.

Expression of an Nrp1 variant unable to bind Sema3A, or constitutive deletion of Sema3a, partially recapitulates the early Nrp1 deletion phenotype of M72-expressing fibers. (A-D) Dorsal and medial whole-mount views of X-Gal-stained Nrp1^+/sema;M72^+/lacZ (P33, A,B) and Sema3a^+/del;M72^lacZ/lacZ (P31, C,D) OBs. A, anterior; L, lateral; P, posterior; M, medial; D, dorsal; V, ventral. Scale bars: 1 mm in A for all dorsal OB views; 1 mm in B for all medial OB views. (E-H) Dorsal and medial whole-mount views of X-Gal-stained Nrp1^sema/sema;M72^lacZ/lacZ (P30, E,F) or Sema3a^del/del;M72^+/lacZ (P38, G,H) OBs showing a dorsalization of the glomeruli and an additional anterior glomerulus. (I,K) Comparison of the frequency of the specific phenotypes affecting M72 glomeruli assigned to the independent or linked/fused categories. The frequencies are compared between Nrp1^sema/sema, Sema3a^del/del and their respective controls (*P<0.001 for both, χ² test). In addition, the presence or absence of an anterior glomerulus in each OB (Nrp1^sema/sema, Sema3a^del/del and their respective controls) is indicated and their respective frequencies are compared (*P<0.001 for both, χ² test). (J,L) Mapping of M72 glomeruli (Nrp1^sema/sema, Sema3a^del/del and their respective control populations) on a medial representation of the OB. Glomeruli (from single OBs) linked to each other are connected by colored lines. A dot circled in black represents the mean position of glomeruli from each population. Ellipses represent the dispersion of each glomerular population. Data corresponding to control animals (Nrp1^+/sema, n=14 and Sema3a^+/del, n=14) are depicted in orange, and data from Nrp1^sema/sema (n=18) and Sema3a^del/del (n=8) mice are in magenta. Insets show close-ups from the glomerular targeting sites.