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. 2013;8(2):e56561.
doi: 10.1371/journal.pone.0056561. Epub 2013 Feb 20.

Dishevelled Proteins Are Associated With Olfactory Sensory Neuron Presynaptic Terminals

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Free PMC article

Dishevelled Proteins Are Associated With Olfactory Sensory Neuron Presynaptic Terminals

Diego J Rodriguez-Gil et al. PLoS One. .
Free PMC article

Abstract

Olfactory sensory neurons (OSNs) project their axons from the olfactory epithelium toward the olfactory bulb (OB) in a heterogeneous and unsorted arrangement. However, as the axons approach the glomerular layer of the OB, axons from OSNs expressing the same odorant receptor (OR) sort and converge to form molecularly homogeneous glomeruli. Axon guidance cues, cell adhesion molecules, and OR induced activity have been implicated in the final targeting of OSN axons to specific glomeruli. Less understood, and often controversial, are the mechanisms used by OSN axons to initially navigate from the OE toward the OB. We previously demonstrated a role for Wnt and Frizzled (Fz) molecules in OSN axon extension and organization within the olfactory nerve. Building on that we now turned our attention to the downstream signaling cascades from Wnt-Fz interactions. Dishevelled (Dvl) is a key molecule downstream of Fz receptors. Three isoforms of Dvl with specific as well as overlapping functions are found in mammals. Here, we show that Dvl-1 expression is restricted to OSNs in the dorsal recess of the nasal cavity, and labels a unique subpopulation of glomeruli. Dvl-2 and Dvl-3 have a widespread distribution in both the OE and OB. Both Dvl-1 and Dvl-2 are associated with intra-glomerular pre-synaptic OSN terminals, suggesting a role in synapse formation/stabilization. Moreover, because Dvl proteins were observed in all OSN axons, we hypothesize that they are important determinants of OSN cell differentiation and axon extension.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Dvl-1 is expressed in zone 1 in the olfactory epithelium and restricted to the ONL and GL in the olfactory bulb.
Dvl-1 (green) is expressed as early as E13 (A) throughout the OE and it expression remains during embryonic and early postnatal development (B, C). By P7, mature olfactory sensory neurons and sustentacular cells show the higher levels of expression in the OE. D, E: Higher magnification images of E13 and P7 respectively. Dotted line marks the basal membrane of the OE; dashed line demarks the lower edge of Dvl-1 expressing OSNs. Dvl-1 in situ hybridization showed the same expression pattern at P4 in zone 1 (F) with no expression outside this zone (G). As expected, control sense probe did not show any staining in zone 1 (H) or outside (I). Dvl-1 was detected in the OB by E15 (J) in the ONL, and it extended to the developing GL by E17 (K). The expression remained in the ONL and GL after birth and was observed also in adult animals (L, M). Due to the lack of perfusion, at E15 and E17 an intense immunoflourescence could be detected in blood cells (J, K). Nuclei were counterstained with DRAQ5 (blue). EPL: external plexiform layer; GCL: granule cell layer; GL: glomerular layer; NC: Nasal cavity; NL: olfactory nerve layer; OB: Olfactory bulb. Scale bar = 100 µm (A–C), 50 µm (D–M).
Figure 2
Figure 2. Dvl-1 is expressed in a unique subpopulation of glomeruli.
A, B: Dvl-1 expression in the OB was analyzed in the context of two classical markers: NQO1 (zone I/dorsal OE) and OCAM (zones I-IV/ventral OE). All NQO1-expressing glomeruli showed Dvl-1 expression (A, B, D, F, G, H). A few OCAM-expressing glomeruli located in the dorsal part of the OB, also showed Dvl-1 expression (A, B, and open arrows in E, F, G). In the ventral part of the OB, only OCAM-expressing glomeruli were observed (A, B, C). In the transition NQO1/OCAM zone, those glomeruli expressing both markers, showed expression of Dvl-1 only in the NQO1 positive axons (A, B, and open arrowhead in H). Dvl-1 also revealed a subset of glomeruli negative for both classical markers (A, B, and arrows in G, I). In order to test whether these only Dvl-1 expressing glomeruli are functional, we stained for VGlut-2 (blue), Dvl-1 (green) and OCAM+NQO1 (both in red) (I). These glomeruli expressed VGlut-2 (arrows), consistent with functional activity. Scale bar  = 200 µm in A, B; 20 µm C–I. ONL: olfactory nerve layer; GL: glomerular layer; EPL: external plexiform layer; MCL: Mitral cell layer; GCL: granule cell layer.
Figure 3
Figure 3. Tracing analysis also showed Dvl-1 only expressing glomeruli, predominately in the dorso-lateral and dorso-medial domains.
The ventral domain was not included in order to increase clarity and emphasize the glomeruli expressing multiple markers. This analysis identified OCAM+ glomeruli (red arrow), OCAM+/Dvl-1+ glomeruli (yellow arrow), Dvl-1+ glomeruli (green arrow), NQO1+/Dvl-1+ glomeruli (turquoise arrow), OCAM+/NQO1+/Dvl-1+ glomeruli (white arrow). The X axis represents the line connecting all the glomeruli, stretched out linearly. We removed most of the ventral and lateral tracing (relative position 0–1500) because all these glomeruli were only OCAM positive.
Figure 4
Figure 4. Dvl-1 exhibits a punctate distribution in OSN axons, and is associated with synapses.
In order to test if the punctate distribution of Dvl-1 (A) is associated with synapses, we double stained for VGlut2 (B), synaptotagmin 1 (Syt 1, C), and synaptophysin (Syp, D) three classical presynaptic terminal markers. All three synaptic markers co-localized with Dvl-1 puncta (filled arrows in B–D). However, there were instances in which a Dvl-1 puncta did not colocalize suggesting that the expression of synaptic markers may vary among OSN terminals (open arrows in B–D). To rule out the expression of Dvl-1 in other populations of cells with glomerular localization we used GAD-67 GFP mice to assess periglomerular cells and Thy-1-YFP mice to assess mitral cells (E, F, respectively). Dvl-1 puncta were not present in any GAD-67 GFP or Thy-1 YFP processes, consistent with the notion that Dvl-1 is only in OSN axons in the glomeruli. Nuclei were counterstained with DRAQ5 (blue). Scale bar  = 10 µm in A, E, F; 5 µm in B–D.
Figure 5
Figure 5. Dvl-2 is expressed in OSN, in all layers through the olfactory bulb and is associated with synapses inside the glomeruli.
Dvl-2 is expressed as early as E13 (A) throughout the OE. During embryonic development some cells showed higher levels of expression than others (B, arrows) and after birth, Dvl-2 expression was restricted to the mature OSN layer of the OE (C, D). Dvl-2 expression was not restricted to the cytoplasm, but was also detected in the nucleus (D, inset). In the developing OB, Dvl-2 was observed as early as E13 (E), and it was detected up to adult (F–H). Even though Dvl-2 was detected in all layers, levels of expression differed between layers and in between animals of the same age (especially after birth). Glomeruli showed a punctate distribution of Dvl-2 (I) similar to that observed for Dvl-1. Dvl-2 expression was not restricted to the cytoplasm and processes, but it also showed nuclear expression, although this expression was not observed in all cells of any given cell type. Electron microscopy images of Dvl-2 stained sections showed that inside the glomerulus, Dvl-2 is in the presynaptic terminal (J). K: In agreement with the immunofluorescence, electron micrographs of periglomerular cells showed some nuclei with Dvl-2 expression (M), while others showed no evidence of Dvl-2 expression (L). Nuclei were counterstained with DRAQ5 (blue). Scale bar  = 20 µm in A–D; 50 µm in E–H; 10 µm in I; 200 nm in K; 1 µm in K. EPL: external plexiform layer; GCL: granule cell layer; GL: glomerular layer; MCL: Mitral cell layer; NC: Nasal cavity; OE, olfactory epithelium; OB: Olfactory bulb; ONL: olfactory nerve layer; V: ventricle.
Figure 6
Figure 6. Dvl-3 is expressed in OSN knobs and in all layers of the olfactory bulb.
Dvl-3 expression levels in OSN cell bodies remain low during embryonic development (A, B) and increase after birth (C). Dvl-3 was detected in OSN knobs (arrows in A – B), but only those knobs expressing levels higher that those of sustentacular cells were readily detectable. Z-stack projections (B, C) showed higher numbers of Dvl-3 expressing knobs. In the olfactory bulb Dvl-3 was observed through all the layers during embryonic development and postnatally (D – F). Nuclei were counterstained with DRAQ5 (blue). Scale bar = 20 µm in A, C; 10 µm in B; 50 µm in D–F. AOB: accessory olfactory bulb.

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