Differential timing of neurogenesis underlies dorsal-ventral topographic projection of olfactory sensory neurons
- Naoki Ihara†2,
- Bao Ligao2,
- Yuji Ikegaya2, 3 and
- Haruki Takeuchi2, 4Email authorView ORCID ID profile
© The Author(s). 2017
Received: 13 December 2016
Accepted: 6 February 2017
Published: 13 February 2017
The mammalian primary olfactory system has a spatially-ordered projection in which olfactory sensory neurons (OSNs) located in the dorsomedial (DM) and ventrolateral (VL) region of the olfactory epithelium (OE) send their axons to the dorsal and ventral region of the olfactory bulb (OB), respectively. We previously found that OSN axonal projections occur sequentially, from the DM to the VL region of the OE. The differential timing of axonal projections is important for olfactory map formation because early-arriving OSN axons secrete guidance cues at the OB to help navigate late-arriving OSN axons. We hypothesized that the differential timing of axonal projections is regulated by the timing of OSN neurogenesis. To test this idea, we investigated spatiotemporal patterns of OSN neurogenesis during olfactory development.
Methods and results
To determine the time of OSN origin, we used two thymidine analogs, BrdU and EdU, which can be incorporated into cells in the S-phase of the cell-cycle. We injected these two analogs at different developmental time points and analyzed distribution patterns of labeled OSNs. We found that OSNs with different dates of origin were differentially distributed in the OE. The majority of OSNs generated at the early stage of development were located in the DM region of the OE, whereas OSNs generated at the later stage of development were preferentially located in the VL region of the OE.
These results indicate that the number of OSNs is sequentially increased from the DM to the VL axis of the OE. Moreover, the temporal sequence of OSN proliferation correlates with that of axonal extension and emergence of glomerular structures in the OB. Thus, we propose that the timing of OSN neurogenesis regulates that of OSN axonal projection and thereby helps preserve the topographic order of the olfactory glomerular map along the dorsal–ventral axis of the OB.
KeywordsOlfactory receptor Olfactory sensory neuron Neural circuit formation Topographic map Zonal organization Neurogenesis
In the mouse olfactory system, olfactory receptor (OR) genes form a multigene family comprising >1000 genes . Of this rich repertoire of genes, an individual olfactory sensory neuron (OSN) expresses only one functional OR . OSN axons that express a given type of OR converge to a few spatially invariant glomeruli in the olfactory bulb (OB) [3–5], generating an olfactory topographic map. The development of the olfactory topographic map comprises initial global targeting and subsequent activity-dependent refinement [6, 7]. The global targeting process is genetically determined and regulated by two independent mechanisms that control different axes of axonal extension in the OB. Axonal projections along the anterior–posterior axis are dependent on the expressed OR species . Several axon-guidance molecules, such as Neuropilin-1 and Plexin-A1, whose expression levels are regulated by expressed OR molecules have been proposed to participate in anterior–posterior targeting [9–11]. In contrast, as for the dorsal–ventral (D–V) targeting of OSN axons, there is a close correlation between OSN positions in the olfactory epithelium (OE) and glomerular locations in the OB . The OE can be roughly divided into two domains on the basis of genetic markers: dorsal (D)-zone and ventral (V)-zone. D-zone OSNs, which are located in the dorsomedial region of the OE, send their axons to the dorsal region of the OB, whereas V-zone OSNs, which are located in the ventrolateral region of the OE, project their axons to the ventral region of the OB. The expression patterns of OR genes are different between the D- and V-zones. In the D-zone, the expression patterns of OR genes are randomly distributed throughout the OE . However, V-zone specific OR genes show spatially limited expression. Each OR gene possesses a unique expression domain and these domains are arranged in a continuous and overlapping manner within the V-zone [14–16]. Several studies have shown that the topographic order of glomerular locations along the D–V axis of the ventral OB is determined by anatomical locations of OSNs in the V-zone [12, 14].
The topographic projection along the D–V axis of the OB is maintained by axon-axon and axon-target interactions. Several axon guidance molecules have been proposed to be involved in OSN axonal projections along the D–V axis [17–20]. We have previously demonstrated that Neuropilin-2 (Nrp2)/Sema3F repulsive interactions between OSN axons play important roles in preserving the topographic order along the D–V axis of the OB . Nrp2 and Sema3F show complementary, gradient expressions in the OE. More specifically, the expression of Sema3F is high in the D-zone and low in the V-zone, while Nrp2 shows the opposite gradient. We also showed through gain and loss of function experiments that these molecules are necessary for the dorsal–ventral topographic projection in the OB. Further, we found a temporal difference in axonal extensions , whereby OSN axons project to the OB from in sequential dorsal to ventral order. These sequential projections are quite important to maintain the topographic order of the olfactory map because early-arriving D-zone OSN axons guide late-arriving V-zone OSN axons by secreting Sema3F. However, it is not clear how the differential timing of these axonal projections is regulated. We hypothesized that the timing of OSN production would be different depending on the location of OSNs in the OE. To test this, we examined spatiotemporal patterns of OSN neurogenesis during olfactory development.
Materials and methods
All experimental procedures were performed with the approval of the Animal Experiment Ethics Committee at the University of Tokyo and according to the University of Tokyo guidelines for the care and use of laboratory animals.
EdU and BrdU injections
5-Ethynyl-2-deooxyuridine (EdU; Thermo Fisher Scientific) or 5-Bromo-2′-deoxyuridine (BrdU; Sigma-Aldrich) was intraperitoneally injected into mice at embryonic days 11.5, 12.5, 13.5, 15.5, and 17.5, and postnatal days 0 and 2 (50 mg/kg).
In situ hybridization and immunostaining
Immunostaining was performed according to previously described methods . Primary antibodies used are as follows: mouse anti-BrdU antibodies (1:500, Sigma-Aldrich); rabbit anti-OMACS antibodies (1:500); goat anti-OCAM antibodies (1:500, R&D systems). Anti-OMACS antibodies were generated by immunizing rabbits with KLH-conjugated synthetic peptides corresponding to 7–29 amino acid residue of the OMACS gene (operon biotechnologies). EdU signals were detected with the Click-iT EdU imaging kit (Thermostat Fisher Scientifics). To detect BrdU signal, sections were pre-treated for 1 h in 1.2 N HCl at 37 °C and then rinsed with 0.1 M borate buffer (pH 8.5). After washout with phosphate-buffered saline (PBS), sections were incubated in a detection solution for 30 min. Slides were then washed three times in PBS for 5 min and immunostained with anti-BrdU antibodies.
Image acquisition and statistical analyses
Optical and fluorescent images were photographed with a BZ-X700 microscope (Keyence). Images of three coronal OE slices were acquired and the number of Br(E)dU-positive OSNs located within the NCAM-positive OE layer was manually counted. To define the OR-zones, serial OE sections at a thickness of 10 μm were used. Each OE section was subjected to in situ hybridization using one of four OR probes (M72, P2, I7 and MOR28). After taking images, expression patterns of the OR genes were compared to determine the boundaries between the OR-zones. Percentages of labeled OSNs within the OR-zones were calculated as the number of labeled OSNs within each OR-zone divided by that in the entire OE region. All statistical analyses were performed with Origin Software (OriginLab).
What is the significance of the differential timing of OSN neurogenesis? The timing of neurogenesis is a determinant for cell-fate specification and precise neural circuitry [26, 27]. For example, it has been reported that the timing of neurogenesis determines the location of secondary olfactory mitral cells as well as their axonal projections . However, in the primary olfactory system, region-specific OR gene expression is already observed at E13.5 [29, 30]. Furthermore, other regional markers, such as Nrp2, OMACS, and OCAM, are expressed as early as E12.5 [21, 24]. These observations indicate that the fate of OSNs is already specified at the early embryonic stage. Thus, it is unlikely that the timing of neurogenesis determines the expression of ORs nor does it determine the projection site of OSN axons.
In contrast to the V-zone of the OE, we were unable to find a positional difference in the rate of OSN production within the D-zone of the OE (Fig. 2b). It could be that different mechanisms are responsible for mediating OSN neurogenesis in the D-zone of the OE. In this study, we observed a burst of proliferation twice in the D-zone: the first one was at E12.5 and the second one was at E17.5 (Figs. 1d and 2c). D-zone ORs are classified into two phylogenetically different groups; Class I and Class II . These two groups constitute distinct glomerular domains in the dorsal OB; the anterior region comprises Class I-ORs while the posterior region comprises Class II-ORs [13, 37]. We assumed that the first burst of proliferation reflects the proliferation for Class I OR-expressing OSNs, and the second one reflects that for Class II OR-expressing OSNs. Comparing the difference in the timing of neurogenesis between Class I OR- and Class II OR-expressing OSNs may give us some insight into the mechanisms underlying the formation of these domains in the OB.
Olfactory-specific cell adhesion molecule
Olfactory-specific medium acyl-CoA synthetase (OMACS)
Olfactory sensory neuron
We thank A. Nakashima for her help in preparing the manuscript.
This work was supported by the Takeda Science Foundation, JST-PREST and JSPS KAKENHI Grant Number, 16H06144.
Availability of data and materials
The datasets generated and analysed during the current study are available in the figshare repository (https://doi.org/10.6084/m9.figshare.4580509.v1).
E, NI and HT conceived the experiments. E, NI, YI and HT wrote the manuscript. E, NI and BL performed the experiments and analyzed the data. All authors contributed to the writing and provided helpful comments. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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