Article
Reference
Alteration of Nrp1 signaling at different stages of olfactory neuron maturation promotes glomerular shifts along distinct axes in the
olfactory bulb
ASSENS, Alexis, et al.
Abstract
The building of the topographic map in the mammalian olfactory bulb is explained by a model based on two axes along which sensory neurons are guided: one dorso-ventral and the other antero-posterior. This latter axis relies on specific expression levels of Neuropilin 1 (Nrp1). To evaluate the role played by this receptor in this process, we used an in vivo genetic approach to decrease or suppress it in specific neuronal populations and at different time points during axonal targeting. We observed, in neurons that express either the M71 or the M72 odorant receptors, that the inactivation of Nrp1 leads to two distinct wiring alterations, whose incidence depends on the time at which Nrp1 expression is altered: first, a surprising dorsal shift of the M71 and M72 glomeruli that 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 Semaphorin 3A (Sema3A), and in mice whose sensory neurons express a Nrp1 mutant unable to bind Sema3A. Finally, by using a mosaic conditional approach, we show that M71 axonal [...]
ASSENS, Alexis, et al. Alteration of Nrp1 signaling at different stages of olfactory neuron maturation promotes glomerular shifts along distinct axes in the olfactory bulb. Development, 2016, vol. 143, p. 3817-3825
DOI : 10.1242/dev.138941
Available at:
http://archive-ouverte.unige.ch/unige:88120
Disclaimer: layout of this document may differ from the published version.
Alteration of Nrp1 signaling at different stages of olfactory neuron maturation promotes glomerular shifts along distinct axes in the olfactory bulb
Alexis Assens1,2, Julien Dal Col1,2, Anthony Njoku1,2, Quentin Dietschi1,2, Chenda Kan1,2, Paul Feinstein3, Alan Carleton2,4,*, and Ivan Rodriguez1,2,*
1Department of Genetics and Evolution, University of Geneva, Geneva 1205, Switzerland
2Geneva Neuroscience Center, University of Geneva, Geneva 1205, Switzerland
3Department of Biological Sciences, Hunter College and The Graduate Center Biochemistry, Biology and Biopsychology and Behavioral Neuroscience Programs, CUNY, New York, New York, United States of America
4Department of Basic Neurosciences, School of Medicine, University of Geneva, Geneva 1205, Switzerland
Keywords: olfaction, axon guidance, mouse
* authors for correspondence (ivan.rodriguez@unige.ch, alan.carleton@unige.ch)
http://dev.biologists.org/lookup/doi/10.1242/dev.138941 Access the most recent version at
Development Advance Online Articles. First posted online on 30 August 2016 as 10.1242/dev.138941
Summary
The building of the topographic map in the mammalian olfactory bulb is explained by a model based on two axes along which sensory neurons are guided: one dorso-ventral and the other antero-posterior. This latter axis relies on specific expression levels of Neuropilin 1 (Nrp1). To evaluate the role played by this receptor in this process, we used an in vivo genetic approach to decrease or suppress it in specific neuronal populations and at different time points during axonal targeting. We observed, in neurons that express either the M71 or the M72 odorant receptors, that the inactivation of Nrp1 leads to two distinct wiring alterations, whose incidence depends on the time at which Nrp1 expression is altered: first, a surprising dorsal shift of the M71 and M72 glomeruli that 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 Semaphorin 3A (Sema3A), and in mice whose sensory neurons express a Nrp1 mutant unable to bind Sema3A.
Finally, by 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 somewhere else. Together, these findings show drastically different axonal targeting outcomes dependent on the timing at which Nrp1/Sema3A signaling is altered.
Introduction
In vertebrates, topographically organized maps in the brain process various sensory modalities (Accolla et al., 2007; Carleton et al., 2010; Luo and Flanagan, 2007; Vincis et al., 2012). These maps usually reflect an organization that is already present at the level of the sensory structures, but not always (O'Leary et al., 1999). The olfactory system is one of these exceptions, since functionally identical sensors, that is neurons expressing the same odorant receptor gene, are scattered in the sensory epithelium of the nose (Buck and Axel, 1991). It is only at the level of the sensory neuron axonal projections in the olfactory bulb (OB) that an organized topographical map emerges: to each sensory population expressing a given odorant receptor correspond generally two glomeruli in each OB (Mombaerts et al., 1996; Ressler et al., 1994; Vassar et al., 1994). These two glomeruli are located in stereotyped locations, which reflect a mirror image defined by a symmetry axis cutting the bulb approximately sagittally. Given the size of the odorant receptor gene repertoires in mammalian genomes (over 1000 odorant receptor genes in the mouse) and the thousands of sensory neurons expressing each of these receptor genes, the system faces a remarkable wiring and pathfinding problem. This complex task is to be completed in utero and perinatally, but also during the life of the animal, since olfactory sensory neurons (OSNs) are constantly renewed through adulthood.
Significant progress has been made in our understanding of how olfactory sensory neurons target to the adequate glomeruli in the bulb. Different mechanisms are at work to establish the bulbar map, and involve both activity- dependent sorting of axonal projections and genetically determined cues (Chen and Flanagan, 2006; Imai and Sakano, 2008; Sakano, 2010). Two levels of guidance have been identified. The first directs axons to their target zones in the bulb, and the second guides local axon sorting which ends in glomerular segregation (Feinstein et al., 2004; Feinstein and Mombaerts, 2004; Imai et al., 2006; Imai et al., 2009; Nakashima et al., 2013; Nishizumi and Sakano, 2015;
Rodriguez-Gil et al., 2015; Serizawa et al., 2006; Takeuchi et al., 2010; Wang et al., 1998). The current model explaining global targeting (at least on the medial side
of the bulb) proposes the existence of guidance cues that determine the position of the target along two axes in the bulb: one dorso-ventral and the other antero- posterior. The dorso-ventral axis roughly correlates with the anatomical distribution of the sensory neurons in the neuroepithelium, and with the graded and complementary expression of Neuropilin 2 and Semaphorin 3F along this axis (Takeuchi et al., 2010). Neurons located in the dorsal zone of the nasal cavity target to the dorsal bulb, while those located more ventrally project to more ventral parts. A second axis directs sensory fibers along the antero-posterior length of the bulb. Elegant studies have suggested that this anteroposterior targeting is dependent on the level of cAMP that is produced by the spontaneous activity (that is agonist-independent activity) of the odorant receptor expressed by each sensory neuron (Imai et al., 2006; Imai et al., 2009; Nakashima et al., 2013). These cAMP concentrations are then translated into specific levels of Nrp1 (among others guidance molecules), that are responsible for guiding olfactory axons along the antero-posterior axis of the bulb (Imai, 2012). The timing during the maturation of the olfactory sensory neuron at which Nrp1 plays this role has however not been defined.
To precisely evaluate the role played by Nrp1 in the establishment of the bulbar topographical map, we followed the path of genetically defined olfactory sensory populations deleted for Nrp1 at different stages during the building of the bulbar map, and competing with functionally identical sensory populations expressing Nrp1. Our data show a critical time- dependent function played by Nrp1 in the establishment of the olfactory axonal map topography.
Materials and Methods
Mice
The Gng8cre allele was generated by gene targeting in E14 embryonic stem (ES) cells. The targeting vector (TV) was built by modifying via recombineering techniques a bacterial artificial chromosome (BAC, bMQ314G20) containing the Gng8 coding sequence. The final TV contained a 5’ homology arm (HA) corresponding to 8.9Kb of genomic sequence ending with the Gng8 stop codon.
This 5’HA was followed by a Pac1-flanked reporter cassette comprising an internal ribosome entry site (IRES) sequence followed by the Cre recombinase coding sequence and an Frt-NeoR-Frt (FNF) selection cassette. The 3’ HA corresponding to 5.3Kb of genomic sequence downstream the Gng8 stop codon followed the reporter cassette. The linearized TV was electroporated into ES cells and G418-resistant colonies were screened by Southern blot using a 469nt- long RFLP probe starting with the start codon of the Ptgir gene located 11Kb downstream of Gng8 (Fig. 3). Positive clones showed a 14Kb EcoRV fragment while the WT allele produced a 46Kb band on gel. The FNF selection cassette was further excised by crossing Gng8cre mice with a transgenic mouse line expressing the FLP recombinase in the germ line. All animals presented in this study lacked the FNF cassette. All mice were bred on a mixed C57BL/6-CBA background, except for Sema3A null mice (generated by crossing mice bearing Sema3aflox and CMVcre alleles) that were bred on a CD-1 background to increase survival of mutant pups (Sibbe et al., 2007; Vieira et al., 2007). The MOR23(Olfr16)GFP, M72(Olfr160)lacZ, M71(Olfr151)lacZ, M71(Olfr151)GFP, M71(Olfr151)cre, OMPcre, CMVcre, Nrp1flox, Nrp1sema, Sema3aflox, Adcy3del, R26flRFP alleles were previously described (Dupe et al., 1997; Feinstein et al., 2004; Feinstein and Mombaerts, 2004; Gu et al., 2003; Li et al., 2004; Luche et al., 2007; Taniguchi et al., 1997;
Vassalli et al., 2002; Wong et al., 2000; Zheng et al., 2000). Age- and sex-matched littermates were used when comparing mutant and wild-type animals.
All animal procedures were done in accordance with the guidelines and regulations of the institution and of the state of Geneva.
Whole-mount analyses
Animals were dissected and either directly imaged for fluorescence or fixed with 4% paraformaldehyde and stained with X-Gal as previously described (Rodriguez et al., 1999). No association was observed between projection patterns of the right and the left bulbs of a given animal. Therefore, position data were pooled between right and left bulbs. Whole-mount images from X-Gal staining were taken on a Leica MZFLIII stereo microscope. For fluorescent reporters, images were taken either with a Zeiss SteREO Lumar V12 stereo microscope or a Zeiss LSM700 confocal microscope. All medial-view images were cropped and tilted to align the dorsal part of the nasal cavity with the image top frame.
Immunohistochemistry
After dissection, head samples were degased and fixed overnight in 4%
paraformaldehyde, then transferred to 15% sucrose for 12 hours, followed by 30% sucrose for 12 hours. Samples were subsequently embedded in O.C.T.
(VWR), frozen and 14-16µm cryosections were prepared using a cryostat. Slides were conserved at -80°C until use. For immunostaining, slides were pre- incubated for 30 minutes in blocking solution at room temperature (normal goat serum 2%, BSA 3%, triton X100 0.1% in PBS) followed by an incubation overnight at 4°C with the primary antibodies diluted in the same blocking solution. Primary antibodies used were: goat anti-Nrp1 (1:100, R&D AF566), chicken anti-GFP (1:400 Abcam ab13970), rabbit anti-RFP (1:400; Abcam ab62341) and chicken anti-betagalactosidase (1:500, Abcam ab9361). Slides were then washed three times for 15 minutes with Triton X100 0.1% in PBS. A secondary incubation of 90 minutes at room temperature with one of the following antibodies was performed: donkey anti-goat A555 (1:400, Invitrogen A21432), goat anti-chicken A488 (1:400, Invitrogen A11039) and goat anti- rabbit A555 (1:400, Invitrogen A21428). Finally, slides were washed three times for 15 minutes with Triton X100 0.1% in PBS, treated 5 minutes with DAPI (1:5000 in PBS) and mounted with DABCO in glycerol. Images were acquired using either a Zeiss Axioplan 2 or a Leica DM5500 microscopes.
Standardized OB maps
To generate standardized OB images of mapped glomerular positions, we first generated a two dimensional prototypic OB medial outline out of 80 representative images of medial OBs taken from 7 weeks-old C57BL6 male mice.
The OB outline of these 80 images was traced in Adobe Illustrator, compiled in an ImageJ stack (Schneider et al., 2012), and processed using the “stackreg”
alignment plugin (Thevenaz et al., 1998) to generate a reference outline. The OB outline and the glomerular positions of all experimental medial OB images were then aligned to this reference using the same procedure. M71 axonal projections in OBs from mice bearing M71lacZ/LacZ or M71+/LacZ alleles were considered equally since we did not observe any differences between them (data not shown). The same applies to mice bearing the M72lacZ/LacZ or M72+/LacZ alleles.
Centrographic measures
(x;y) coordinates of each glomerulus were extracted by exporting Adobe Illustrator image files (.ai) into scalable vector graphics files (.svg). Datasets containing the coordinates of each glomerular population were then built for each genotype and for each glomerular population. These datasets were then imported into RStudio, and the "aspace" module (Randy Bui, 2012) was used to compute the mean coordinates (X,Y) of a selected glomerular population as well as a standard deviational ellipse (X, Y, width, height and rotation angle), that represents the glomerular dispersion in two dimensions. One standard deviational ellipse covers approximately 68 percent of the glomeruli when a spatial normal distribution is observed (i.e. most glomeruli condensed in the center, fewer in the periphery).
Results
Nrp1 expression in olfactory sensory neurons.
Nrp1 is differentially transcribed by OSN populations. As previously reported, robust expression is observed in neurons that target the medial and the lateral parts of the bulb, with those located antero-laterally and postero-medially expressing Nrp1 the most strongly (Fig. 1A, B)(Miller et al., 2010; Pasterkamp et al., 1998; Schwarting et al., 2000). Glomeruli located on the medial side of the bulb exhibit a gradient of Nrp1 expression along the antero-posterior axis (Fig.
1B)(Imai et al., 2006). However, this is true at a global anteroposterior level, not always at the glomerular level, since one finds glomeruli that are intensely marked for Nrp1 surrounded by glomeruli expressing barely detectable levels of Nrp1 (Fig 1B, D, G, J). To precisely evaluate the role played by Nrp1 in the building of the olfactory map, we analyzed the axonal projections of three different Nrp1-expressing sensory populations. We chose M71, M72 and MOR23- expressing neurons because a) they express Nrp1 (Fig. 1C-K)(Dal Col et al., 2007), b) their axonal projections are robust, stereotyped, and have already been extensively studied, in particular by using the knock-in alleles M71lacZ , M72lacZ and MOR23GFP (Feinstein et al., 2004; Feinstein and Mombaerts, 2004; Vassalli et al., 2002), and c) their projections are located in areas easily visualized by whole mount analyses.
Depletion of Nrp1
To disrupt Nrp1 expression, we opted for an approach to specifically remove Nrp1 from OSNs. We made use of a mouse line bearing Nrp1flox/flox alleles (Gu et al., 2003) crossed to a mouse expressing a Cre recombinase specifically in maturing OSNs (via an OMPcre allele)(Li et al., 2004). To validate the OMP-driven deletion approach, bulbs from Nrp1flox/flox;OMP+/cre mice were stained with a Nrp1 antibody; a lack of Nrp1 staining was observed in the glomerular layer (Fig. S1A- G). M71lacZ;Nrp1flox/flox;OMP+/cre, M72lacZ;Nrp1flox/flox;OMP+/cre and
MOR23GFP;Nrp1flox/flox;OMP+/cre mice were generated (n=10, 22 and 20 respectively). The M71, M72 and MOR23 axonal projections were analyzed by whole mount analyses, and were compared to their respective controls (n=20, 28 and 12 respectively, Fig. 2). Homogenous glomeruli were formed by M71, M72 and MOR23 neurons. While no major targeting alteration was observed in MOR23GFP;Nrp1flox/flox;OMP+/cre mice (Fig. 2J,O), a drastic phenotype was observed in M71lacZ;Nrp1flox/flox;OMP+/cre and M72lacZ;Nrp1flox/flox;OMP+/cre mice, in which glomeruli were shifted dorsally (Fig. 2F-I,L,N). M72 axonal fibers often crossed the dorsal midline to innervate the contralateral M72 glomerulus (Fig. 2H,I,M).
M71-expressing neurons exhibited an even more drastic dorsalization of their glomeruli since the lateral and the medial glomeruli were fused on the dorsal part of the bulb (Fig. 2F,G,K). Considering a position of the mouse head in which the dorsal part of the snout is horizontal (which corresponds to an angle of +113 degrees for the cribriform plate, Fig. 2L), the M71 and M72 mean glomerular shift corresponds to a dorsalization of 490±210 and 468±127 m (±SD) with a +7 and +15 degree relative to the dorso-ventral axis respectively (Fig. S4A,B).
This shift was very different from the one we were expecting, since previously observed I7 glomeruli lacking Nrp1, on which the antero-posterior model was built, were anteriorized (Imai et al., 2009). This published and our own approach differed in the timing at which Nrp1 was excised since the Cre recombinase was under the control of a promoter active in mid-mature neurons (the I7 OR) or in late-mature neurons (OMP) respectively. We thus hypothesized that the timing at which Nrp1 was deleted could be critical in the mistargeting. We therefore chose to expand our Cre-mediated Nrp1 allele inactivation to target the different phases of OSN maturation.
Nrp1 deletion at different time points during axonal targeting
For an early deletion of Nrp1 during OSN maturation, we chose the Gng8 (G8) gene that is expressed in precursor and immature olfactory neurons (Hanchate et al., 2015; Ryba and Tirindelli, 1995). To this aim, we generated a knock-in mouse line bearing a Gng8cre allele, in which a polycistronic cassette containing
an IRES followed by the Cre recombinase CDS was inserted by homologous recombination immediately after the Gng8 stop codon (Fig. 3A). To evaluate this novel transgenic line, it was crossed with a mouse line bearing a R26flRFP reporter allele to generate Gng8cre;R26flRFP mice. Red fluorescence in the olfactory epithelium was evaluated. In Gng8cre;R26flRFP mice, over 90% of the olfactory epithelium was labeled, including the whole apical and medial layers and the upper part of the basal layer (Fig. 3B, Fig. S2A-B). This was expected since neurogenesis takes place at the very basal part of the epithelium, with neurons moving apically while differentiating. To further validate the Gng8-mediated early Nrp1 deletion approach, bulbs from Nrp1flox/flox; Gng8cre/cre mice were stained with a Nrp1 antibody; a lack of Nrp1 staining was observed in both the glomerular and the nerve layers (Fig. S1H-J). For expression of the Cre recombinase at middle stage of the axonal projection process, we opted for a knock-in mouse line bearing an M71cre allele that left the M71 CDS intact (Fig. 3B).
OR transcription can be observed prior to OMP expression (Hanchate et al., 2015; Rodriguez-Gil et al., 2015). To control for the stage at which the specific knock-in lines express the Cre recombinase, Gng8cre;R26flRFP, M71cre;R26flRFP and OMPcre;R26flRFP mice were analyzed, and the positions along the apico-basal axis of the septal sensory epithelium of the youngest red-fluorescent neurons was located (that is those closest to the basal lamina with a one neuron wide apico- basal sliding window, Fig. 3C). As expected, a coverage of the different OSN developmental phases was observed, with Gng8cre being apparently transcribed before M71cre, and M71cre before OMPcre (Fig. 3C).
Early deletion of Nrp1
In order to evaluate the effects of an excision of the floxed Nrp1 exon as early as possible, M71lacZ;Nrp1flox/flox;Gng8+/cre, M72lacZ;Nrp1flox/flox;Gng8+/cre and MOR23GFP;Nrp1flox/flox;Gng8+/cre mice were generated (n=12, 22 and 18 respectively) and compared to their respective controls (n=8, 5 and 14 respectively, Fig. 4). We initially assessed a potential general disruption of the glomerular arrangement by analyzing sections of P0 olfactory bulbs from
Nrp1flox/flox;Gng8cre/cre mice. We did not observe obvious alterations (data not shown). Given the early inactivation of the Nrp1 gene with Gng8cre, we expected a similar, although more pronounced dorsalization phenotype than the one observed using the OMPcre driver. In Nrp1flox/flox;Gng8+/cre mice, we indeed observed a fusion of the lateral and medial M71 glomeruli (Fig. 4G,H,M,N), and a higher frequency of contacts between the contralateral M72 glomeruli (Fig.
4I,J,O,P). The M71 and M72 dorsal glomerular shift corresponds to a distance of 698±84 and 537±229 m with an angle of -5 and +8 respectively (Fig. S4C,D), relative to the dorso-ventral axis. Surprisingly, we also observed the presence of an anteriorized glomerulus in M71, M72, and MOR23-labeled mice with an early deletion of Nrp1 (Fig. 4G-R). Relative to the endogenous glomeruli and following the antero-posterior axis (i.e., relative to the dorsal part of the snout), the mean position of the M71, M72 and MOR23 supplementary anterior glomeruli were at a distance of 1827±97, 1688±227 and 442±98m with an angle of +30, +33 and -41 degrees. Since the number of OSNs expressing a given receptor may affect their targeting or at least their propensity to form a stable glomerulus, we evaluated the possible alteration of OSN numbers in a Nrp1 null background. All X-Gal-stained OSNs located on the rostral part of the dorsal turbinate II up to the groove linking it to the basal turbinate II were counted on whole mounts of Gng8cre;Nrp1+/+ and Gng8cre;Nrp1flox/flox MOEs. We found no significant difference between the two genotypes (189±32 (Nrp1 wt) vs 178±28 (Nrp1 null), n=4 and n=3 P50 mice respectively).
As a parallel approach to the removal of Nrp1 at different stages of OSN development, we forced a reduction of Nrp1 expression in OSNs. We assumed that in mice bearing a single functional Nrp1 allele, the number of Nrp1 transcripts would be decreased. We analyzed the axonal projections of M72 fibers in OMP+/cre;Nrp1+/+;M72lacZ and OMP+/cre;Nrp1+/flox;M72lacZ mice. We found no significant differences in glomerular position between mice bearing one or two Nrp1 alleles (Fig. S3A-D), which either suggests that the readout of Nrp1 levels is not fine tuned, or that there is compensatory expression of Nrp1 from the remaining allele when one is ineffective.
OR-driven deletion of Nrp1
Although precisely timed during OSN development and restricted to OSNs, the late (OMP-driven) and early (Gng8-driven) Nrp1 deletion did affect all OSNs. To investigate the possible contribution of the non-labeled and possibly misrouted Nrp1-deficient OSNs in the observed phenotypes, we took advantage of the monogenic expression of OR genes to exclusively inactivate Nrp1 in OSNs expressing the M71 OR gene, a highly transcribed gene which starts to be expressed early during OSN development (Fig. 3C). R26flRFP;Nrp1flox/flox ;M71cre/cre mice were generated. 100% of the M71 lateral and medial glomeruli were fused dorsally, and the presence of a very rostral glomerulus was observed (n=22, Fig.
5P). This phenotype was very similar to the one observed in M71lacZ;Nrp1flox/flox;Gng8+/cre mice.
In addition, we also took advantage of the monoallelic expression of OR genes, and generated compound heterozygotes by adding an M71 wild-type allele driving the expression of a GFP fluorophore. R26flRFP;Nrp1flox/flox ;M71cre/GFP mice were thus generated, allowing in the same animal to visualize the projections of Nrp1-deficient M71 axons (in red) and of M71 wild-type axons (in green)(Fig. 5).
We observed the formation by Nrp1-deficient fibers of dorsally-located M71 glomeruli (either linked or fused) in more than 90% of the OB analyzed (n=62, Fig. 5E-L,P, Fig. S5A,B), similarly to what was observed in M71lacZ;Nrp1flox/flox;Gng8+/cre and M71lacZ;Nrp1flox/flox;OMP+/cre mice. Wild-type M71 fibers of R26flRFP;Nrp1flox/flox ;M71cre/GFP mice did often project to two glomeruli, whose position was relatively similar to the one observed in wild-type mice.
However, a significant proportion of the Nrp1-deficient fibers innervated the wild-type M71 glomeruli (Fig. 5F-H, Fig. S5A-C). Even more surprising, numerous Nrp1-expressing M71 axons were rerouted towards the dorsal Nrp1- deficient glomerulus (Fig. 5F,H,K-O, Fig. S5B). Finally, a small anterior glomerulus formed by Nrp1-deficient fibers was observed (Fig. 5I,J,P), reminiscent of what was seen in early Nrp1-deleted mice. In this context, the formation of an anterior glomerulus and the dorsalization must come about
through cell autonomous mechanisms and not depend on any other populations of axons with Nrp1 having been deleted.
Sema3A versus Nrp1 deletion
Sema3A and VEGF165 represent the two main ligands for Nrp1 (Schwarz and Ruhrberg, 2010). Given the widespread role played by Sema3A in the development of the nervous system, and in particular of the olfactory system (Imai et al., 2009; Pasterkamp et al., 1998; Schwarting et al., 2000; Schwarting et al., 2004; Taniguchi et al., 2003), we evaluated its role in the projection of M71 and M72-expressing fibers. We took two parallel and complementary approaches. On one side, we used a Nrp1 hypomorphic mutant allele (Nrp1sema) whose product is unable to recognize Sema3A but still able to respond to VEGF (Renzi et al., 2000), and on the other, a constitutive Sema3A null allele (Sema3Adel)(Fig. 6, Fig. S6). M71GFP;Nrp1sema/sema mice (n=18, FigS6B) and M72lacZ;Nrp1sema/sema mice (n=12, Fig6E,F) were analyzed. Similarly to what was observed in Nrp1flox/flox;Gng8+/cre animals, lateral and medial axonal projections of M71 and M72 did form an anteriorized glomerulus and projected to a dorsalized glomerulus relative to the wild-type glomerular position (Fig. 6E,F,I,J; Fig. S6B,D).
A similar pattern was observed in M71GFP;Sema3Adel/del and M72lacZ;Sema3Adel/del mice (n=9, Fig. S6C,D and n=8, Fig. 6G,H,K,L, respectively). However, M71 and M72 projection patterns in Nrp1sema/sema and Sema3Adel/del animals were more variable and disorganized (dorsalized glomeruli were often observed as pairs or triplets and not frequently linked or fused) than the ones observed in late or early Nrp1-deleted mice (Fig. 6E-L, Fig. S6B-D). Interestingly, the projection pattern of M72-expressing fibers in constitutive (Sema3Adel/del or Nrp1sema/sema) or in the early Nrp1-deletion context is reminiscent of what is observed in Adcy3del/del animals (Fig. S7)(Dal Col et al., 2007). This is consistent with what we know about this olfactory cyclase, which is not only critical for olfactory transduction, but which we previously showed to be necessary for Nrp1 expression in olfactory sensory neurons (Dal Col et al., 2007).
Discussion
Here, we dissected the role played by Nrp1 in the establishment of the topographic map built by axonal projections of olfactory sensory neurons. By using parallel genetic approaches, in vivo, we singled out and followed the axonal fibers of three functionally different sensory populations that were each defined by the expression of a specific odorant receptor gene. We altered the function of Nrp1 in these populations at different steps during axonal targeting, and in different competitive environments. We observed that, as predicted by the current model explaining the antero-posterior positioning of glomeruli in the bulb, M71-, M72- and MOR23-expressing sensory neurons formed anteriorized glomeruli relative to their wild-type position when the interaction between Nrp1 and Sema3A was impaired constitutively or early during OSN maturation.
However, in addition to these anterior glomeruli, we observed that both M71 and M72-expressing neurons also formed medial glomeruli located at their wild- type antero-posterior position. Surprisingly, these glomeruli exhibited a dorsal shift to such an extent that the medial and the lateral glomeruli were often fused.
In a previous study, Nrp1 was shown to play a role in sorting fibers inside axon bundles projecting toward the OB (Imai et al., 2009). In this published report, in a pan-OSN Nrp1 knock-out background, axonal projections of I7-expressing OSNs exhibited split projections, one glomerulus located in its wild type position, and the other anteriorized relative to this latter. When the Nrp1 deletion was restricted to a specific OSN population (I7-expressing OSNs), a single anteriorized glomerulus was observed (Imai et al., 2009). The hypothesis provided to explain the difference between the two phenotypes was that the relative Nrp1 levels among axons determine OSN projection sites. However, our observation of an anterior in addition to a posterior glomerulus in Nrp1flox/flox;M71cre/GFP;R26flRFP bulbs is difficult to reconcile with this view. We propose hereafter other explanations, not necessarily exclusive, for the presence of the two well defined and stereotyped M71, M72 or MOR23 glomeruli on the medial or dorsal sides of the Nrp1-deficient bulb. First, two different populations of neurons expressing the same odorant receptor but differing in their
expression of other proteins may coexist in the olfactory sensory epithelium and may be revealed in a Nrp1-deficient context. Second, the anterior or dorsal projections could reflect axons that targeted the bulb at different times during development. The two target positions would be maintained during the life of the animal, the early projections guiding later incoming fibers. Finally, depending on their location in the neuroepithelium, sensory neurons, which are likely to encounter different signals at different times during fiber migration, could simply be prone to innervate different domains in the absence of Nrp1.
The absence of an anterior glomerulus in a Nrp1 late-deletion context suggest that young sensory neurons are differentially sensitive to Nrp1 expression relative to older ones, or at least that the lack of Nrp1-derived signals leads to different outcomes depending on when the signals fade. Sensory neurons would thus target anteriorly when Nrp1 is deleted early during their differentiation, and dorsally when deleted late. The presence of two glomeruli in M71lacZ;Nrp1flox/flox;Gng8+/cre mice would thus reflect neurons that lost Nrp1 as soon as Gng8 got expressed, and other neurons whose Nrp1flox alleles took longer to be recombined.
We previously described an altered M72 and MOR23 projection pattern in Adcy3del/del mice. Among various transcriptional differences in guidance-related genes between Adcy3+/+ and Adcy3del/del olfactory sensory neurons, we found (among other guidance molecules) Nrp1 to be significantly downregulated.
Interestingly, the Nrp1-deficient phenotype reported here is very similar to the one observed in Adcy3del/del mice, suggesting that the downregulation of Nrp1 plays a critical role in the Adcy3del/del axonal guidance alterations.
We reported here the presence of a dorsal fused glomerulus in Nrp1flox/flox;M71cre/GFP;R26flRFP mice. Given that we did not observe M71 glomeruli in the dorsal OB of wild-type mice, the OB dorsal domain may be repulsive to Nrp1-positive fibers (e.g. M71-expressing neurons). However, the presence of Nrp1-positive fibers in the dorsal fused glomerulus of these Nrp1flox/flox;M71cre/GFP;R26flRFP mice indicates that this potential repulsion may be
overcome by other signals, such as potential homotypic interactions between like-fibers (i.e. fibers expressing the same OR).
As previously discussed, the current model of "global" axonal targeting of olfactory sensory neurons involves two axes on the OB: a Nrp2-dependent dorso-ventral axis and a Nrp1-dependent antero-posterior axis. The rostralization of the glomeruli we report here is compatible with this model, but the dorsalization of M71 and M72 glomeruli suggests an additional role played by Nrp1 in the targeting along the dorso-ventral axis. However, considering two fixed and orthogonal axes to explain OSN targeting may tackle the problem with an inadequate tool, that although pleasurable to our topographical view of the world, and although useful as a descriptor or even as a working hypothesis, may not really reflect cues provided by the biological signals that direct the targeting of olfactory sensory axons in the bulb.
Acknowledgements
This work was supported by grants from the Swiss National Science Foundation 31003A_149753 and 310030E-135910 to IR, CR33I13_143723 to IR and AC, and 31003A_153410 to AC, and from the Research Centers in Minority Institutions Program grant from the National Institute on Minority Health and Health Disparities (MD007599) and NIH SC1 GM088114 to P.F. A.A., J.DC., A.N., Q.D., and C.K., P.F. carried out experimental studies. A.A., J.DC., A.N., Q.D., C.K., P.F., A.C. and I.R. performed data analyses. I.R. and A.C. conceived the study, and I.R. wrote the bulk of the manuscript. All authors discussed the results and commented on the manuscript.
Figures
Figure 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. (B) Immunostaining for Nrp1 (red) on a horizontal section of the medial part of a mouse OB (PND 0). High expression of Nrp1 is observed in the posterior part of the OB glomerular layer. Scale bar is 50 µm. (C-K) Immunostainings on coronal sections of mouse OBs showing single glomeruli innervated by M71, M72 or MOR23 sensory neurons (PND 11). The green color in the left column shows the expression in axonal fibers of betagalactosidase (M71lacZ/lacZ or M72lacZ/lacZ mice), or GFP (MOR23GFP/GFP mice); the red color in the middle column indicates the
expression of Nrp1. The combined image is shown on the right column. Scale bar is 50µm in C-K.
Figure 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 (PND 34) or Nrp1+/+;OMP+/cre;M72lacZ/lacZ (PND 40) OBs.
A, anterior; L, lateral; P, posterior; M, medial; D, dorsal; V, ventral. Scale bar in A is 1mm (scale is identical for all dorsal OB views). Note that the glomeruli are independent. Scale bar in B is 1mm (scale is identical for all medial OB views).
(E) Medial whole-mount view of a Nrp1+/+;OMP+/cre;MOR23GFP/GFP (PND 28) OB under epifluorescence. (F-I) Dorsal and medial whole-mount views of X-gal- stained Nrp1flox/flox;OMP+/cre;M71+/lacZ (PND 36) or Nrp1flox/flox;OMP+/cre;M72lacZ/lacZ (PND 32) OBs showing a dorsalization of the glomeruli. (J) Medial whole-mount view of a Nrp1flox/flox;OMP+/cre;MOR23GFP/GFP (PND 28) OB under epifluorescence, showing no obvious targeting alteration. (K,M) Comparison of the frequency of the specific phenotypes affecting M71 or M72 glomeruli, assigned to each of the following categories: indep. (independent), linked or fused glomeruli. The
frequencies are compared between controls and Nrp1 late-deleted (*P<0.001, χ2- test). In addition, the presence or absence of an anterior glomerulus in each OB (control and Nrp1 late-deleted) 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, while data from Nrp1 late-deleted animals (Nrp1flox/flox;OMP+/cre, n=10 for M71, n=22 for M72 and n=20 for MOR23) are in magenta. Close-ups from the glomerular targeting sites are shown.
Figure 3. Late and early expression of olfactory Cre drivers.
(A) Strategy to generate the Gng8cre allele. The solid bar represents the probe used for southern blot. 5’HA, 5’ homology arm; 3’HA, 3’ homology arm; i, IRES (internal ribosome entry site). (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. 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. cells, sustentacular cells; OSNs, olfactory sensory neurons. The most basally-located RFP-positive neurons are indicated by cyan dots, and correspond to the positions along the baso-apical axis indicated in (C).
Scale bar is 50µm. (C) Positions of the most basal RFP-positive cells observed along the baso-apical axis (cyan dots, as illustrated in (B)) of the neuroepithelium of Gng8+/cre;R26+/flRFP mice (n=232 cells from 2 mice), M71+/cre;R26+/flRFP (n=264 cells from 2 mice) and OMP+/cre;R26+/flRFP (n=160 cells from 2 mice). Means and standard deviations are indicated by thick and thin horizontal black lines, respectively. The grey shaded area represents the 5th percentile.
Figure 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 Nrp1flox/flox;Gng8+/+;M71+/lacZ (PND 25) or Nrp1flox/flox;Gng8+/+;M72lacZ/lacZ (PND 23) OBs. A, anterior; L, lateral; P, posterior; M, medial; D, dorsal; V, ventral. (E) Medial whole-mount view Nrp1flox/flox;Gng8+/+;MOR23+/GFP (PND 30) OB under epifluorescence. Scale bar in A is 1mm (scale is identical for all dorsal OB views).
Scale bar in B is 1mm (scale is identical for all medial OB views). (F) Close-up of the glomerular projection site shown in E. Scale bar is 400µm (G-J) Dorsal and medial whole-mount views of X-gal-stained Nrp1flox/flox;Gng8+/cre;M71+/lacZ (PND 25) or Nrp1flox/flox;Gng8+/cre;M72+/lacZ (PND 25) OBs showing a dorsalization of the glomeruli and an additional anterior glomerulus. (K,L) Medial whole-mount views of Nrp1flox/flox;Gng8+/cre;MOR23GFP/GFP (PND 30 left, PND 54 right) 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 each of the following categories: indep. (independent), linked or fused glomeruli. The frequencies are compared between controls and Nrp1
early-deleted (*P<0.001, χ2-test). In addition, the presence or absence of an anterior glomerulus in each OB (control and Nrp1 early-deleted) 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: wt (wildtype), wt+anterior (wildtype and anterio) or anterior only glomeruli. The frequencies are compared between controls and Nrp1 early-deleted (*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 projections sites: one dorsally located relative to the control population (often fusing with contralateral projecting fibers) and another one anterior. The MOR23-expressing fibers projection pattern is in most OBs of early-deleted Nrp1 mice divided into one antero-ventralized glomerulus and one located at the same position relative to controls. Data corresponding to control animals (n=8 for M71, n=5 for M72 and n=14 for MOR23) are depicted in orange, while data from early Nrp1-deleted animals (Nrp1flox/flox;Gng8+/cre, Nrp1flox/flox;Gng8cre/cre, n=12 for M71, n=22 for M72 and n=18 for MOR23) are in magenta. Close-ups from the glomerular targeting sites are shown.
Figure 5. Reciprocal hijacking of Nrp1-expressing and Nrp1-deficient fibers.
(A) Dorsal whole-mount view of Nrp1+/flox;M71cre/GFP; R26+/flRFP (PND 45) 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,F) Dorsal whole-mount views of Nrp1flox/flox;M71cre/GFP;R26+/flRFP (PND 45 left, PND 51 right) OBs under epifluorescence. (G-I) Close-ups of the lateral, medial and fused glomeruli from E.
Lateral and medial glomeruli are coinnervated by green and red fibers (Nrp1- expressing and -deficient fibers respectively). (J-L) Close-ups of the anterior and fused glomeruli from F. Anterior glomeruli are innervated mainly by Nrp1- deficient fibers (i.e. red fibers). (M-O) High resolution close-ups of Nrp1flox/flox;M71cre/GFP;R26flRFP/flRFP fused glomeruli showing an heterogeneous innervation of the fused glomeruli. (P) Comparison of the frequency of the specific phenotypes affecting M71 glomeruli, assigned to each of the following categories: indep. (independent) and linked or fused glomeruli for Nrp1flox/flox;M71cre/cre;R26flRFP;R26flRFP mice and Nrp1flox/flox;M71cre/GFP;R26flRFP and their controls). The frequencies are compared between Nrp1flox/flox;M71cre/GFP;R26flRFP 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 Nrp1flox/flox;M71cre/GFP; R26flRFP and their controls are compared (*P<0.001, χ2-test). Data corresponding to control animals (Nrp1+/+;M71cre/GFP;R26+/flRFP, Nrp1+/flox;M71cre/GFP;R26+ or flRFP/flRFP, n=15) are depicted in orange, while data from Nrp1flox/flox;M71cre/cre;R26+ or flRFP/flRFP (n=22) and Nrp1flox/flox;M71cre/GFP;R26+ or flRFP/flRFP (n=62) are in magenta. Scale bar is 1mm in A, E, F. Scale bars is 100μm in B-D, G-I, J-L, M-O.
Figure 6. Expression of a Nrp1 variant unable to bind Sema3A, or constitutive deletion of Sema3A, partially recapitulate 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 (PND 33, left) or Sema3a+/del;M72lacZ/lacZ (PND 31, right) OBs. A, anterior; L, lateral; P, posterior; M, medial; D, dorsal; V, ventral. Scale bar in A is 1mm (scale is identical for all dorsal OB views). Scale bar in B is 1mm (scale is identical for all medial OB views). (E-H) Dorsal and medial whole-mount views of X-gal stained Nrp1sema/sema;M72lacZ/lacZ (PND 30, left) or Sema3adel/del;M72+/lacZ (PND 38 right) 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 each of the following categories:
independent and linked or fused glomeruli. The frequencies are compared between Nrp1sema/sema, Sema3Adel/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 (Nrp1sema/sema, Sema3Adel/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 (Nrp1sema/sema, Sema3Adel/del and their respective controls 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. Data corresponding to control animals (Nrp1+/sema, n=14 and Sema3A+/del, n=14) are depicted in orange, while data from Nrp1sema/sema mice (n=18) and Sema3Adel/del mice (n=8) are in magenta. Close-ups from the glomerular targeting sites are shown.
References
Accolla, R., Bathellier, B., Petersen, C.C., and Carleton, A. (2007). Differential spatial representation of taste modalities in the rat gustatory cortex. J Neurosci 27, 1396-1404.
Buck, L., and Axel, R. (1991). A novel multigene family may encode odorant receptors: a molecular basis for odor recognition. Cell 65, 175-187.
Carleton, A., Accolla, R., and Simon, S.A. (2010). Coding in the mammalian gustatory system. Trends in neurosciences 33, 326-334.
Chen, Y., and Flanagan, J.G. (2006). Follow your nose: axon pathfinding in olfactory map formation. Cell 127, 881-884.
Dal Col, J.A., Matsuo, T., Storm, D.R., and Rodriguez, I. (2007). Adenylyl cyclase- dependent axonal targeting in the olfactory system. Development 134, 2481- 2489.
Dupe, V., Davenne, M., Brocard, J., Dolle, P., Mark, M., Dierich, A., Chambon, P., and Rijli, F.M. (1997). In vivo functional analysis of the Hoxa-1 3' retinoic acid response element (3'RARE). Development 124, 399-410.
Feinstein, P., Bozza, T., Rodriguez, I., Vassalli, A., and Mombaerts, P. (2004). Axon guidance of mouse olfactory sensory neurons by odorant receptors and the beta2 adrenergic receptor. Cell 117, 833-846.
Feinstein, P., and Mombaerts, P. (2004). A contextual model for axonal sorting into glomeruli in the mouse olfactory system. Cell 117, 817-831.
Gu, C., Rodriguez, E.R., Reimert, D.V., Shu, T., Fritzsch, B., Richards, L.J., Kolodkin, A.L., and Ginty, D.D. (2003). Neuropilin-1 conveys semaphorin and VEGF signaling during neural and cardiovascular development. Dev Cell 5, 45-57.
Hanchate, N.K., Kondoh, K., Lu, Z., Kuang, D., Ye, X., Qiu, X., Pachter, L., Trapnell, C., and Buck, L.B. (2015). Single-cell transcriptomics reveals receptor transformations during olfactory neurogenesis. Science 350, 1251-1255.
Imai, T. (2012). Positional information in neural map development: lessons from the olfactory system. Dev Growth Differ 54, 358-365.
Imai, T., and Sakano, H. (2008). Odorant receptor-mediated signaling in the mouse. Curr Opin Neurobiol 18, 251-260.
Imai, T., Suzuki, M., and Sakano, H. (2006). Odorant Receptor-Derived cAMP Signals Direct Axonal Targeting. Science 314, 657-661.
Imai, T., Yamazaki, T., Kobayakawa, R., Kobayakawa, K., Abe, T., Suzuki, M., and Sakano, H. (2009). Pre-target axon sorting establishes the neural map topography. Science 325, 585-590.
Li, J., Ishii, T., Feinstein, P., and Mombaerts, P. (2004). Odorant receptor gene choice is reset by nuclear transfer from mouse olfactory sensory neurons. Nature 428, 393-399.
Luche, H., Weber, O., Nageswara Rao, T., Blum, C., and Fehling, H.J. (2007).
Faithful activation of an extra-bright red fluorescent protein in "knock-in" Cre- reporter mice ideally suited for lineage tracing studies. Eur J Immunol 37, 43-53.
Luo, L., and Flanagan, J.G. (2007). Development of continuous and discrete neural maps. Neuron 56, 284-300.
Miller, A.M., Maurer, L.R., Zou, D.J., Firestein, S., and Greer, C.A. (2010). Axon fasciculation in the developing olfactory nerve. Neural development 5, 20.
Mombaerts, P., Wang, F., Dulac, C., Chao, S.K., Nemes, A., Mendelsohn, M., Edmondson, J., and Axel, R. (1996). Visualizing an olfactory sensory map. Cell 87, 675-686.
Nakashima, A., Takeuchi, H., Imai, T., Saito, H., Kiyonari, H., Abe, T., Chen, M., Weinstein, L.S., Yu, C.R., Storm, D.R., et al. (2013). Agonist-independent GPCR activity regulates anterior-posterior targeting of olfactory sensory neurons. Cell 154, 1314-1325.
Nishizumi, H., and Sakano, H. (2015). Developmental regulation of neural map formation in the mouse olfactory system. Developmental neurobiology 75, 594- 607.
O'Leary, D.D., Yates, P.A., and McLaughlin, T. (1999). Molecular development of sensory maps: representing sights and smells in the brain. Cell 96, 255-269.
Pasterkamp, R.J., De Winter, F., Holtmaat, A.J., and Verhaagen, J. (1998). Evidence for a role of the chemorepellent semaphorin III and its receptor neuropilin-1 in the regeneration of primary olfactory axons. J Neurosci 18, 9962-9976.
Randy Bui, R.N.B., Tarmo K. Remmel (2012). aspace: A collection of functions for estimating centrographic statistics and computational geometries for spatial point patterns. R package version 3.2.
Renzi, M.J., Wexler, T.L., and Raper, J.A. (2000). Olfactory sensory axons expressing a dominant-negative semaphorin receptor enter the CNS early and overshoot their target. Neuron 28, 437-447.
Ressler, K.J., Sullivan, S.L., and Buck, L.B. (1994). Information coding in the olfactory system: evidence for a stereotyped and highly organized epitope map in the olfactory bulb. Cell 79, 1245-1255.
Rodriguez, I., Feinstein, P., and Mombaerts, P. (1999). Variable patterns of axonal projections of sensory neurons in the mouse vomeronasal system. Cell 97, 199- 208.
Rodriguez-Gil, D.J., Bartel, D.L., Jaspers, A.W., Mobley, A.S., Imamura, F., and Greer, C.A. (2015). Odorant receptors regulate the final glomerular coalescence of olfactory sensory neuron axons. Proc Natl Acad Sci U S A 112, 5821-5826.
Ryba, N.J., and Tirindelli, R. (1995). A novel GTP-binding protein gamma-subunit, G gamma 8, is expressed during neurogenesis in the olfactory and vomeronasal neuroepithelia. J Biol Chem 270, 6757-6767.
Sakano, H. (2010). Neural map formation in the mouse olfactory system. Neuron 67, 530-542.
Schneider, C.A., Rasband, W.S., and Eliceiri, K.W. (2012). NIH Image to ImageJ: 25 years of image analysis. Nat Methods 9, 671-675.
Schwarting, G.A., Kostek, C., Ahmad, N., Dibble, C., Pays, L., and Puschel, A.W.
(2000). Semaphorin 3A is required for guidance of olfactory axons in mice. J Neurosci 20, 7691-7697.
Schwarting, G.A., Raitcheva, D., Crandall, J.E., Burkhardt, C., and Puschel, A.W.
(2004). Semaphorin 3A-mediated axon guidance regulates convergence and targeting of P2 odorant receptor axons. Eur J Neurosci 19, 1800-1810.
Schwarz, Q., and Ruhrberg, C. (2010). Neuropilin, you gotta let me know: should I stay or should I go? Cell Adh Migr 4, 61-66.
Serizawa, S., Miyamichi, K., Takeuchi, H., Yamagishi, Y., Suzuki, M., and Sakano, H.
(2006). A neuronal identity code for the odorant receptor-specific and activity- dependent axon sorting. Cell 127, 1057-1069.
Sibbe, M., Taniguchi, M., Schachner, M., and Bartsch, U. (2007). Development of the corticospinal tract in Semaphorin3A- and CD24-deficient mice. Neuroscience 150, 898-904.
Takeuchi, H., Inokuchi, K., Aoki, M., Suto, F., Tsuboi, A., Matsuda, I., Suzuki, M., Aiba, A., Serizawa, S., Yoshihara, Y., et al. (2010). Sequential arrival and graded secretion of Sema3F by olfactory neuron axons specify map topography at the bulb. Cell 141, 1056-1067.
Taniguchi, M., Nagao, H., Takahashi, Y.K., Yamaguchi, M., Mitsui, S., Yagi, T., Mori, K., and Shimizu, T. (2003). Distorted odor maps in the olfactory bulb of semaphorin 3A-deficient mice. J Neurosci 23, 1390-1397.
Taniguchi, M., Yuasa, S., Fujisawa, H., Naruse, I., Saga, S., Mishina, M., and Yagi, T.
(1997). Disruption of semaphorin III/D gene causes severe abnormality in peripheral nerve projection. Neuron 19, 519-530.
Thevenaz, P., Ruttimann, U.E., and Unser, M. (1998). A pyramid approach to subpixel registration based on intensity. IEEE Trans Image Process 7, 27-41.
Vassalli, A., Rothman, A., Feinstein, P., Zapotocky, M., and Mombaerts, P. (2002).
Minigenes impart odorant receptor-specific axon guidance in the olfactory bulb.
Neuron 35, 681-696.
Vassar, R., Chao, S.K., Sitcheran, R., Nunez, J.M., Vosshall, L.B., and Axel, R. (1994).
Topographic organization of sensory projections to the olfactory bulb. Cell 79, 981-991.
Vieira, J.M., Schwarz, Q., and Ruhrberg, C. (2007). Selective requirements for NRP1 ligands during neurovascular patterning. Development 134, 1833-1843.
Vincis, R., Gschwend, O., Bhaukaurally, K., Beroud, J., and Carleton, A. (2012).
Dense representation of natural odorants in the mouse olfactory bulb. Nat Neurosci 15, 537-539.
Wang, F., Nemes, A., Mendelsohn, M., and Axel, R. (1998). Odorant receptors govern the formation of a precise topographic map. Cell 93, 47-60.
Wong, S.T., Trinh, K., Hacker, B., Chan, G.C., Lowe, G., Gaggar, A., Xia, Z., Gold, G.H., and Storm, D.R. (2000). Disruption of the type III adenylyl cyclase gene leads to peripheral and behavioral anosmia in transgenic mice. Neuron 27, 487-497.
Zheng, C., Feinstein, P., Bozza, T., Rodriguez, I., and Mombaerts, P. (2000).
Peripheral olfactory projections are differentially affected in mice deficient in a cyclic nucleotide-gated channel subunit [In Process Citation]. Neuron 26, 81-91.
Figure S1. Nrp1 expression patterns in the olfactory bulb in a late and early Nrp1 conditional deletion context.
(A) Schematic of a mouse head showing the coronal section plane used in B-J. D, dorsal (B-D) Immunostaining for Nrp1 on a coronal section of the central part of a mouse OB (PND 0) of a wt mouse. Nrp1 expression is observed in both the nerve and glomerular layers of the OB. (E-G) Immunostaining for Nrp1 on a coronal section of the central part of a mouse OB (PND 0) of a OMP+/cre;Nrp1flox/flox mouse. Nrp1 expression is restricted to the nerve layer. (H-J) Immunostaining for Nrp1 on a coronal section of the central part of a mouse OB (PND 0) of a Gng8cre/cre;Nrp1flox/flox mouse. Nrp1 expression is absent from both the nerve layer and the glomeruli. Scale bar is 200 µm in B, E, H and 25µm in C, D, F, G, I, J.
Development 143: doi:10.1242/dev.138941: Supplementary information
Figure S2. Gng8cre expression pattern in the mouse olfactory system.
(A) RFP immunostaining on a coronal section of the main olfactory epithelium of a Gng8+/cre; R26+/flRFP animal (PND 15) showing widespread expression of Gng8cre in sensory neurons. D, dorsal; L, lateral; V, ventral; M, medial. Scale bar is 1mm. (B) Magnification of the inset shown in A. Scale bar is 200 μm.
Development 143: doi:10.1242/dev.138941: Supplementary information
Figure S3. Similar axonal projections of M72-expressing OSNs in a wt vs.
hemizygous Nrp1 conditional deletion context.
(A-B) Dorsal and medial whole-mount views of the OB of an X-gal-stained Nrp1+/flox;OMP+/cre;M72+/lacZ mouse (PND 55). (C) Frequencies of the specific phenotypes affecting M72 glomeruli assigned to each of various categories. (D) Mapping of M72 glomeruli (Nrp1+/+ and Nrp1+/flox) as in Fig. 6. Scale bar is 1mm.
Development 143: doi:10.1242/dev.138941: Supplementary information
Figure S4. Angular relationship between control and Nrp1-deleted M71 and M72 mean glomeruli.
(A,B) The mean position of M71 and M72 glomeruli (dots circled in black) from controls and Nrp1 late-deleted animals from Fig. 3 is reported. The bars indicate the length and the angle of the glomerular shifts between control and Nrp1-late deleted mice. (C,D) The mean position of M71 and M72 glomeruli from controls and Nrp1 early-deleted animals from Fig. 4 is shown. The bars indicate the length and the angle of the glomerular shifts between control and Nrp1-early deleted mice.
Development 143: doi:10.1242/dev.138941: Supplementary information
Figure S5. Early fasciculation of Nrp1-expressing and Nrp1-deficient fibers.
(A) Dorsal whole-mount view of a Nrp1flox/flox;M71cre/GFP;R26flRFP/flRFP (PND 40) OB. (B) High resolution close-up of the fused glomerulus from the left OB, with the green and red channels showed independently. (C) High resolution close-up of axonal fibers before glomerular convergence. Scale bar is 1mm in A, and 100μm in B, C.
Development 143: doi:10.1242/dev.138941: Supplementary information
Development 143: doi:10.1242/dev.138941: Supplementary information
