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, 518 (24), 4825-41

Composition of the Migratory Mass During Development of the Olfactory Nerve

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Composition of the Migratory Mass During Development of the Olfactory Nerve

Alexandra M Miller et al. J Comp Neurol.

Abstract

The embryonic development of the olfactory nerve includes the differentiation of cells within the olfactory placode, migration of cells into the mesenchyme from the placode, and extension of axons by the olfactory sensory neurons (OSNs). The coalition of both placode-derived migratory cells and OSN axons within the mesenchyme is collectively termed the "migratory mass." Here we address the sequence and coordination of the events that give rise to the migratory mass. Using neuronal and developmental markers, we show subpopulations of neurons emerging from the placode by embryonic day (E)10, a time at which the migratory mass is largely cellular and only a few isolated OSN axons are seen, prior to the first appearance of OSN axon fascicles at E11. These neurons also precede the emergence of the gonadotropin-releasing hormone neurons and ensheathing glia which are also resident in the mesenchyme as part of the migratory mass beginning at about E11. The data reported here begin to establish a spatiotemporal framework for the migration of molecularly heterogeneous placode-derived cells in the mesenchyme. The precocious emigration of the early arriving neurons in the mesenchyme suggests they may serve as "guidepost cells" that contribute to the establishment of a scaffold for the extension and coalescence of the OSN axons.

Figures

Figure 1
Figure 1
Development of the olfactory placode (OP) and olfactory bulb (OB). The distribution of cells is shown with the nuclear marker DRAQ5. A: E9.5/TS15; OP (arrow). B: E10/TS16; olfactory pit (arrow). C: E10.5/TS17; olfactory pit (arrow). D: E11/TS18; olfactory pit (arrow). E: E11.5/TS19. F: E12/TS20; presumptive olfactory bulb (arrow, asterisk). G: E12.5/TS20; presumptive olfactory bulb (arrow, asterisk). H: E13/TS21; presumptive olfactory bulb (arrow, asterisk). OPit, olfactory pit; pVNO, presumptive vomeronasal organ; M, mesenchyme; pOB, presumptive olfactory bulb; NC, nasal cavity; T, telencephalon. Scale bar = 200 μm in H (applies to all).
Figure 2
Figure 2
The MM cells are distinct from the GnRH+ cells, the OECs, and the OMP+ cells that emerge from the OE/pVNO at E12. Sagittal sections of DCX-GFP embryos. A: E12 DCX-GFP section stained for GnRH (purple). A′: GnRH. A″: DCX-GFP. B: E12 DCX-GFP section stained for BLBP (purple). B′: BLBP. B″: DCX-GFP. C: E12 DCX-GFP section stained for OMP (purple). C′: OMP. C″: DCX-GFP. Scale bars = 100 μm.
Figure 3
Figure 3
MM cells are present in the mesenchyme beginning at E10–10.5 and express diverse neuronal markers. DNER (green)/β-tubulin III (red)/DRAQ5 (blue). A: E10–10.5 sagittal section. Low-magnification image of the olfactory placode. A′: High-magnification image of the boxed area in A. MM cells (solid arrow) and OSNs (open arrow). Dotted line indicates basal lamina. B: E10–10.5 sagittal section. Low-magnification image showing a cluster of MM cells in the mesenchyme. B′: High-magnification image of the boxed area in B (filled arrow indicates cluster). Abbreviations as in Fig. 1. Scale bars = 50 μm in A, B; 10 μm in A′, B′.
Figure 4
Figure 4
MM cells emerge from the olfactory pit. GAP43 (green)/DRAQ5 (blue). E11.5 sagittal section. A: Low-magnification image of MM cells exiting the presumptive OE. B–F: Z-series of a high-magnification image of the boxed area from A, taken at 1-μm intervals. Dotted lines indicate position of the basal lamina. The MM cells within the olfactory pit (F; filled arrowhead) extend a leading process that contacts the cells already in the mesenchyme (F; open arrowhead). Abbreviations as in Fig. 1. Scale bars = 50μm in A; 20μm in F (applies to B–F).
Figure 5
Figure 5
Composition of the migratory mass. DCX-GFP sections stained with peripherin (red)/DNER (blue). E10 sagittal section. A: DCX-GFP/PRPH/DNER. Inset shows a single PRPH+ axon fascicle within the MM. A′: DCX-GFP/PRPH. A″: PRPH/DNER. B, B′, B″, C, C′, C″: Sagittal CD1 sections stained with DNER (green)/peripherin (red)/DRAQ5 (blue). (B) Low-magnification image of the MM at E11. (B′) High-magnification of the upper box shown in B. (B″) High-magnification of the lower box shown in B. (C) Low-magnification image of the MM at E12. (C′) High-magnification of the upper box shown in C. (C″) High-magnification of the lower box shown in C. The axons are seen directly apposed and wrapping the MM cells (filled arrows). Scale bars = 20 μm in A, A′, A″, B′, B″, C′, C″; 50 μm in B, C.
Figure 6
Figure 6
The mean ± SEM density of DCX+/Lhx2+ cells decreases between E12 and E13. DCX-GFP sections stained with Lhx2 (red)/DRAQ5 (blue). Sagittal sections. A–C: The expression level of both Lhx2 and DCX-GFP is variable. Cells with similar expression levels cluster (i.e., dorsal aspect of MM in A; circle). A few isolated MM cells express Lhx2 but not DCX-GFP. A′–C′: Lhx2 staining. A″–C″: DCX–GFP labeling. A‴-C‴: DCX-GFP sections stained with Lhx2 without the nuclear marker to demonstrate the colocalization. D: Cell counts of Lhx2+/DCX+ cells at E12 and E13. Scale bars = 25 μm in A–A‴, B–B‴; 10 μm in C–C‴.
Figure 7
Figure 7
MM cells, ensheathing cells, and OSN axons travel in clusters through the mesenchyme. Sagittal sections stained for DCX (red)/BLBP (green)/DRAQ5 (blue). A–C: Composition of the MM at E11, 12, and 12.5. Note the clustering of OSN axons, MM cells, and OECs in the mesenchyme at E12 and E12.5, respectively. D–G: Electron microscopy of the developing olfactory nerve pathway. D: Section through the mesenchyme of an E10 mouse. Two distinct cell morphologies are apparent (α and β). A few examples of likely axons (arrows) are seen, but no evidence of fasciculation. E, F: Examples of adherens junctions (E, arrows; F, circle) between adjacent cells within the mesenchyme at E10; see also circle in (D). G: Section through the mesenchyme of an E12 mouse. Fascicles of coalesced axons (yellow) are seen surrounded by the α (red) and β (blue) cells as well as an unidentified process (Δ, blue). A process (green) that appears to be an axon with an enlarged terminal at one end is seen interposed between the two clusters of axons. Scale bars = 10 μm in A–C; 5 μm in G (for D); 1.25 μm in G (for E); 2.5 μm in G (for F).
Figure 8
Figure 8
MM cells act as guidepost cells as the OSN axons transverse and organize within the mesenchyme. A: At 10.5, both OSNs (blue) and MM cells (red) differentiate within the developing OE and the MM cells begin to egress into the mesenchyme. B: By E11, the OSN axons (blue) begin crossing the basal lamina and contacting the mesenchymal MM cells. While OSN axon outgrowth is occurring, the MM cells (red) coalesce into an organized mass that parallels the nascent olfactory nerve pathway. C: By E12, the OSN axons (blue) have traversed the mesenchyme, in close apposition to the MM cells (red), until the axons contact and penetrate the telencephalic vesicle.

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