Axon growth and guidance genes identify nascent, immature, and mature olfactory sensory neurons

Abstract

Neurogenesis of projection neurons requires that axons be initiated, extended, and connected. Differences in the expression of axon growth and guidance genes must drive these events, but comprehensively characterizing these differences in a single neuronal type has not been accomplished. Guided by a catalog of gene expression in olfactory sensory neurons (OSNs), in situ hybridization and immunohistochemistry revealed that Cxcr4 and Dbn1, two axon initiation genes, marked the developmental transition from basal progenitor cells to immature OSNs in the olfactory epithelium. The CXCR4 immunoreactivity of these nascent OSNs overlapped partially with markers of proliferation of basal progenitor cells and partially with immunoreactivity for GAP43, the canonical marker of immature OSNs. Intracellular guidance cue signaling transcripts Ablim1, Crmp1, Dypsl2, Dpysl3, Dpysl5, Gap43, Marcskl1, and Stmn1-4 were specific to, or much more abundant in, the immature OSN layer. Receptors that mediate axonal inhibition or repulsion tended to be expressed in both immature and mature OSNs (Plxna1, Plxna4, Nrp2, Efna5) or specifically in mature OSNs (Plxna3, Unc5b, Efna3, Epha5, Epha7), although some were specific to immature OSNs (Plxnb1, Plxnb2, Plxdc2, Nrp1). Cell adhesion molecules were expressed either by both immature and mature OSNs (Dscam, Ncam1, Ncam2, Nrxn1) or solely by immature OSNs (Chl1, Nfasc1, Dscaml1). Given the loss of intracellular signaling protein expression, the continued expression of guidance cue receptors in mature OSNs is consistent with a change in the role of these receptors, perhaps to sending signals back to the cell body and nucleus.

Fig. 1

Messenger RNAs encoding proteins that regulate growth cone dynamics were expressed primarily in immature OSNs. A: Guide to the cell body layers of the olfactory epithelium. Ccnd1 labels a subset of basal cells; Gap43 labels immature OSNs; OMP labels mature OSNs. Sus, unlabeled sustentacular cell body layer; mOSN, mature OSN cell body layer; iOSN, immature OSN cell body layer; basal, basal cell layer. B–D: Ppp2cb, Marcksl1, and Ablim1 were detected in immature OSNs. E: Ablim2 was detected in immature and mature OSNs. F–H: Crmp1, Dpysl3, and Dpysl5 were detected in immature OSNs. I: Dpysl2 was detected in immature and mature OSNs. J,K: Stmn1 and Stmn2 were detected in immature OSNs. L,M: Stmn3 and Stmn4 were detected in immature and mature OSNs. N,O: Examples of the absence of labeling when sense probes were used. Lines, location of basal lamina. Scale bars = 20 μm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Fig. 2

A–G: Guidance cue receptor and cell adhesion molecule mRNAs expressed primarily by immature OSNs. A–G: Images of in situ hybridization for Plxnb1, Plxnb2, Plxdc2, Nrp1, Chl1, Nfasc, and Dscaml1. Lines, location of basal lamina. Scale bars = 20 μm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Fig. 3

Basally located cells express axon initiation mRNAs. A–D: Dbn1 (B) and Cxcr4 (D) mRNAs were expressed in a thin layer of cells that may partially overlaps with the basal end of the immature OSN layer marked by adjacent sections hybridized for Gap43 mRNA (A,C). E–G: Cells expressing Dbn1 (E) and Cxcr4 (G) formed a nearly continuous layer throughout the olfactory epithelium, compared with the clusters of cells positive for Neurog1 (F), the canonical marker of immediate neuronal precursors. Lines, location of basal lamina. Scale bars = 20 μm in A–D; 100 μm in E–G. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Fig. 4

CXCR4 immunoreactivity identifies cells located two to four cell diameters apical to the basal lamina. A–E: CXCR4-immunoreactive processes extended toward the apical surface of the olfactory epithelium (arrows), and some cells are immunoreactive for both CXCR4 and GAP43. C–E: A region where cells immunoreactive for both CXCR4 and GAP43 (asterisks) were abundant. The arrow indicates an apical process labeled by CXCR4 immunoreactivity. F,G: Basal processes immunoreactive for CXCR4 (arrowhead in F) crossed the basal lamina and entered olfactory nerve bundles, where they were associated with NCAM1-positive axons. H: Broader view of the integration of CXCR4-immunoreactive fibers (some identified by arrows) into the bundles of NCAM1-immunoreactive fascicles of the olfactory nerve. I: Example of CXCR4-immunoreactive fibers exiting the base of the olfactory epithelium. J,K: In P4 mice, in which the frequency of proliferating basal cells is higher than in older mice, some proliferating basal cells marked by phosphorylated histone H3 (pH-3) or by Mki67 were also marked by CXCR4 immunoreactivity (arrowheads), but others were not (arrows). Lines, location of basal lamina. Scale bars = 20 μm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Fig. 5

Cxcl12 was expressed beneath the olfactory epithelium in an age-dependent pattern. A: Cxcl12 was expressed in the lamina propria at age P0. B: At age P21, Cxcl12 was instead detected in cells within the bone underlying the lamina propria. Images from the nasal septum are shown. Lines, location of basal lamina. Scale bars = 20 μm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Fig. 6

Guidance cue receptor mRNAs expressed primarily in mature OSNs. A–E: Efna3, Epha5, Epha7, Plxna3, and Unc5b displayed this pattern of expression. Lines, location of basal lamina. Scale bars = 20 μm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Fig. 7

Guidance cue receptor and cell adhesion molecule mRNAs detected in both immature and mature OSNs. A–H: Plxna1, Plxna4, Efna5, Nrp2, Nrxn1, and Ncam1 displayed this pattern. Lines, location of basal lamina. Scale bars = 20 μm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]