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Axon Fasciculation in the Developing Olfactory Nerve

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Axon Fasciculation in the Developing Olfactory Nerve

Alexandra M Miller et al. Neural Dev.

Abstract

Olfactory sensory neuron (OSN) axons exit the olfactory epithelium (OE) and extend toward the olfactory bulb (OB) where they coalesce into glomeruli. Each OSN expresses only 1 of approximately 1,200 odor receptors (ORs). OSNs expressing the same OR are distributed in restricted zones of the OE. However, within a zone, the OSNs expressing a specific OR are not contiguous - distribution appears stochastic. Upon reaching the OB the OSN axons expressing the same OR reproducibly coalesce into two to three glomeruli. While ORs appear necessary for appropriate convergence of axons, a variety of adhesion associated molecules and activity-dependent mechanisms are also implicated. Recent data suggest pre-target OSN axon sorting may influence glomerular convergence. Here, using regional and OR-specific markers, we addressed the spatio-temporal properties associated with the onset of homotypic fasciculation in embryonic mice and assessed the degree to which subpopulations of axons remain segregated as they extend toward the nascent OB. We show that immediately upon crossing the basal lamina, axons uniformly turn sharply, usually at an approximately 90° angle toward the OB. Molecularly defined subpopulations of axons show evidence of spatial segregation within the nascent nerve by embryonic day 12, within 48 hours of the first OSN axons crossing the basal lamina, but at least 72 hours before synapse formation in the developing OB. Homotypic fasciculation of OSN axons expressing the same OR appears to be a hierarchical process. While regional segregation occurs in the mesenchyme, the final convergence of OR-specific subpopulations does not occur until the axons reach the inner nerve layer of the OB.

Figures

Figure 1
Figure 1
Development of the olfactory nerve pathway. (A-C) Sagittal sections of CD1 embryos stained for GAP-43 (green)/NCAM (red)/DRAQ5 (blue) at E12 (A), E13 (B), and E15 (C). An orientation compass is shown in (A). Scale bars = 200 μm. OE, olfactory epithelium; ON, olfactory nerve; pOB, presumptive olfactory bulb.
Figure 2
Figure 2
Homotypic axon segregation occurs by E12. Sagittal sections of CD1 embryos. (A, B) NQO1 (green)/OCAM (red). NQO1+ cells are clustered caudally, mostly within the OCAM+ region of the nerve. (C-E) NRP-1 (green)/NCAM (red): (C) lateral section - open arrowhead indicates NRP-1+ region; (D) medial section; (E) three-dimensional reconstruction from the lateral side of the nerve. The asterisk indicates the central region of the nerve devoid of NRP-1 staining. An orientation compass is shown in (B), for (A-D) and in (E). Scale bar shown in (D) = 50 μm in (A, B) and 100 μm in (C, D).
Figure 3
Figure 3
At E15, axons expressing NRP-1 continue to distinctly compartmentalize in the olfactory nerve. Sagittal sections of CD1 embryos. (A-C') NRP-1 (green)/NCAM (red)/DRAQ5 (blue). (A) Lateral section. The open arrow indicates lack of segregation in fascicles targeting the caudal nerve. (B) Lateral-medial section. (C) Medial section. The open arrowhead shows the lack of segregation in rostral fascicles coming up to join the nerve. (C') High magnification of boxed region in (C). An orientation compass is shown in (A). Scale bars in (A-C) = 50 μm, C' = 10 μm.
Figure 4
Figure 4
At E15, established regional markers show that pre-target axon sorting in the olfactory nerve occurs early in development. Sagittal sections of CD1 embryos. All panels are stained with the nuclear marker DRAQ5 (blue). (A) NQO1 (red)/OCAM (green). (B) Dolichos biflorus agglutinin (DBA; green)/NCAM (red). Open arrow indicates focal DBA positive axons. (C) Wisteria floribunda agglutinin (WFA; green)/NCAM (red). Open arrow indicates focal WFA positive axons. (D) Robo2 (green)/NCAM (red). Open arrow indicates Robo2/NCAM positive fascicle. Asterisk indicates Robo2 staining in the mitral cell and external plexiform layers of the olfactory bulb. An orientation compass is shown in (A). Scale bars shown in D = 100 μm for (A-D).
Figure 5
Figure 5
Levels of NRP-1 and DBA expression are changed in the absence of adenylyl cyclase 3. (A-D) Coronal sections from AC3 heterozygous versus homozygous knock-out mice at E15. (A, B) NRP-1 (green)/NCAM (red)/DRAQ5 (blue); (C, D) DBA(green). (A, A') Coronal cross-section of the OB from a heterozygous AC3 mouse stained for NRP-1. (B-B') Coronal section from a homozygous AC3 knockout mouse stained for NRP-1. (A') High-magnification of boxed area in (A). (B') High-magnification of boxed area in (B). (C) Coronal section of the OB from a heterozygous AC3 knockout mouse stained for DBA. (D) Coronal section from a homozygous AC3 knockout mouse stained for DBA. An orientation compass is shown in (A'). Scale bars = 100 μm in (A-D) and 25 μm in (A'-D').
Figure 6
Figure 6
Trajectory of the OSN axons crossing the basal lamina. Sagittal sections of P2-lacZ/M72-GFP embryos. (A-E) Z-series projections taken from sagittal sections of E16 heterozygous P2-lacZ (red) and M72-GFP (green) mice. (A, D, E) P2 OSNs within the OE. The axons turn sharply towards the bulb as they exit the basal lamina (open arrow in (A)). (B) Three OSNs (two P2, one M72) within the OE. (C) Two M72 axons within the OE. Sometimes the axons initially turn the wrong way and have to correct their trajectory as they travel towards the bulb. Scale bar in (A-E) = 50 μm.
Figure 7
Figure 7
OR-specific subpopulations of OSN axons show evidence of regional segregation within the olfactory nerve. Sagittal sections of P2-lacZ/M72-GFP embryos. (A-C, A'-C') Z-series projections taken from sagittal sections of E16 P2-lacZ or M72-GFP mice. (A) Low-powered montage of the olfactory nerve pathway of a heterozygous P2-lacZ mouse; (A') high-magnification image of the left-hand boxed area in (A); (A'') high magnification image of the right-hand boxed area in (A), showing the P2+ axons approaching the inner nerve layer of the OB. (B) Low-powered montage of the olfactory nerve pathway of a homozygous M72-GFP mouse; (B') high-magnification image of the boxed area in (B). Open arrowheads indicate incomplete homotypic fasciculation. (C) Low-powered montage of the olfactory nerve pathway of a heterozygous P2-lacZ mouse; (C') high-magnification image of the boxed area in (C). Orientation compass shown in (A). Scale bars in (A, B, B', C, C') = 100 μm and in (A', A'') = 5 μm.
Figure 8
Figure 8
OR-specific subpopulations are regionally restricted within the olfactory nerve pathway. Sagittal sections of P2-lacZ/M72-GFP embryos. (A-C) Z-series projections taken from sagittal sections of E16 heterozygous P2-lacZ (red) and M72-GFP (green) mice. (A) Sagittal low-powered montage allowing visualization of two subpopulations of OSNs, P2 (red) and M72 (green) axons in the olfactory nerve; (A') high-magnification image of the boxed area in (A). (B) Sagittal low-powered montage of P2/M72 axons within the nerve. (C) Coronal low-powered image demonstrating the clustering of P2 axons and M72 axons; (C') high-magnification image of the boxed area in (C). An orientation compass is shown in (A) for (A, B) and in (C) for (C, C'). Scale bars = 25 μm.
Figure 9
Figure 9
OSN axon fasciculation in the olfactory nerve pathway. Sagittal sections of P2-lacZ/M72-GFP embryos. (A-D) Z-series projections taken from sagittal sections of E16 P2-lacZ or M72-GFP mice. Low-powered montages of the olfactory nerve pathway of (A) a heterozygous P2-lacZ mouse and (B, C) taken from a homozygous M72-GFP mouse. (B', C') High-magnification view of boxed areas in (B, C). (D) Low-powered montages of the olfactory nerve pathway taken from a P2-lacZ heterozygous mouse; (D') high-magnification view of the boxed area in (D). An orientation compass is shown in (A). Scale bars in (A, B, C, D) = 50 μm and in (B', C', D') = 10 μm.
Figure 10
Figure 10
Hierarchical model of OSN axon sorting. OR-specific OSNs (each color represents an individual OR) are randomly distributed throughout one of approximately four zones along the dorsal-ventral axis in the OE, with no known organization along the anterior-posterior axis. OSNs each express some currently unknown constellation of guidance molecules (each pattern represents a set of guidance molecules). The OSN axons sort regionally in the mesenchyme according to the guidance molecules they express independent of their OR specificity. After the OSN axons cross into the inner nerve layer of the OB, OR-specific sorting occurs resulting in final convergence into a glomerulus.

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References

    1. Bozza T, Vassalli A, Fuss S, Zhang JJ, Weiland B, Pacifico R, Feinstein P, Mombaerts P. Mapping of class I and class II odorant receptors to glomerular domains by two distinct types of olfactory sensory neurons in the mouse. Neuron. 2009;61:220–233. doi: 10.1016/j.neuron.2008.11.010. - DOI - PMC - PubMed
    1. Imai T, Yamazaki T, Kobayakawa R, Kobayakawa K, Abe T, Suzuki M, Sakano H. Pre-target axon sorting establishes the neural map topography. Science. 2009;325:585–590. doi: 10.1126/science.1173596. - DOI - PubMed
    1. Yoshihara Y, Kawasaki M, Tamada A, Fujita H, Hayashi H, Kagamiyama H, Mori K. OCAM: A new member of the neural cell adhesion molecule family related to zone-to-zone projection of olfactory and vomeronasal axons. J Neurosci. 1997;17:5830–5842. - PMC - PubMed
    1. Walz A, Mombaerts P, Greer CA, Treloar HB. Disrupted compartmental organization of axons and dendrites within olfactory glomeruli of mice deficient in the olfactory cell adhesion molecule, OCAM. Mol Cell Neurosci. 2006;32:1–14. doi: 10.1016/j.mcn.2006.01.013. - DOI - PubMed
    1. Cuschieri A, Bannister LH. The development of the olfactory mucosa in the mouse: light microscopy. J Anat. 1975;119:277–286. - PMC - PubMed

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