Maturation of the Olfactory Sensory Neuron and Its Cilia

Abstract

Olfactory sensory neurons (OSNs) are bipolar neurons, unusual because they turn over continuously and have a multiciliated dendrite. The extensive changes in gene expression accompanying OSN differentiation in mice are largely known, especially the transcriptional regulators responsible for altering gene expression, revealing much about how differentiation proceeds. Basal progenitor cells of the olfactory epithelium transition into nascent OSNs marked by Cxcr4 expression and the initial extension of basal and apical neurites. Nascent OSNs become immature OSNs within 24-48 h. Immature OSN differentiation requires about a week and at least 2 stages. Early-stage immature OSNs initiate expression of genes encoding key transcriptional regulators and structural proteins necessary for further neuritogenesis. Late-stage immature OSNs begin expressing genes encoding proteins important for energy production and neuronal homeostasis that carry over into mature OSNs. The transition to maturity depends on massive expression of one allele of one odorant receptor gene, and this results in expression of the last 8% of genes expressed by mature OSNs. Many of these genes encode proteins necessary for mature function of axons and synapses or for completing the elaboration of non-motile cilia, which began extending from the newly formed dendritic knobs of immature OSNs. The cilia from adjoining OSNs form a meshwork in the olfactory mucus and are the site of olfactory transduction. Immature OSNs also have a primary cilium, but its role is unknown, unlike the critical role in proliferation and differentiation played by the primary cilium of the olfactory epithelium's horizontal basal cell.

Keywords: differentiation; neural development; olfactory receptor; smell; transcriptional regulation.

Figure 1.

The cell types of the olfactory epithelium. The olfactory epithelium is separated from the underlying lamina propria by a basal lamina. Horizontal basal cells (HBCs, dark green) lie flat on the basal lamina. HBCs are multipotent progenitor cells capable of giving rise to globose basal cells (GBCs) that sit apical to the HBCs and are capable of differentiating directly into sustentacular cells (gold). Immature sustentacular cells (yellow) are depicted as residing (transiently) in the GBC cell layer. At least 3 subtypes of GBCs exist in the OSN lineage: a multipotent GBC (light green), which gives rise to a transit-amplifying GBC expressing the neural fate transcription factor Ascl1 (lightest blue), and then an immediate neuronal precursor GBC expressing the transcription factor Neurog1 (light blue). Immediate neuronal precursor GBCs differentiate into nascent OSNs (light blue) whose cell bodies lie apical to the GBCs. Nascent OSNs rapidly transition into immature OSNs (medium blue), whose cell bodies are apical to the nascent OSNs. Immature OSNs differentiate into mature OSNs (dark blue), the most abundant cell type in this tissue. At the apical surface of the olfactory epithelium are the cell bodies of the sustentacular cells (gold). Sustentacular cells completely surround the cell body and dendrite of each mature OSN. Each sustentacular cell extends a process terminating in an end foot at the basal lamina. Microvillar cells, Bowman’s glands, and resident macrophages are not depicted.

Figure 2.

In situ hybridization for Cxcr4, Gap43, and Omp mRNAs locate the cell body layers of nascent OSNs (A), immature OSNs (B), and mature OSNs (C), respectively, in the olfactory epithelium of mice (3–4 weeks of age). Scale bar = 20 µm. Images produced by J. McIntyre, S. Bose, and W. Titlow.

Figure 3.

Cxcr4⁺ nascent OSNs are the first step in the differentiation of the immediate neuronal precursor (INP) type of GBC into a neuronal phenotype (McIntyre et al. 2010). (A) Cxcr4⁺ nascent OSNs (red) do not yet express Ncam1 (green), but they already have neurites. Examples of Cxcr4⁺ basal axons joining fascicles of the olfactory nerve (ON) are marked with blue arrows. Examples of Cxcr4⁺ apical dendrites are marked with white arrows. BV, blood vessel; NC, nasal cavity air space; dashed line, position of the basal lamina. (B) Developmental distance estimates for the OSN lineage produced from single-cell RNA-seq data (Fletcher et al. 2017). Cells enriched for Cxcr4 mRNA are in the clusters labeled as INP2 and INP3. (C) Heatmaps display the average scaled expression profile for gene clusters (numbered at left) whose expression defines clusters of cells (organized as columns) according to their developmental positions in the OSN lineage shown in (B) (Fletcher et al. 2017). The row labeled as CC depicts the patterns of abundance of 40 cell cycle-associated mRNAs and marks the transition to postmitotic phenotypes.

Figure 4.

The biological processes that distinguish major stages in the differentiation of mouse OSNs. Combining expression profiling data from purified populations of mature OSNs and immature OSNs with the changes in mRNA abundance in samples of olfactory mucosa 5 days after unilateral ablation of an olfactory bulb allow identification of biological processes associated with changes in the OSN transcriptome. The transition from a GBC through a nascent OSN into an early immature OSN consists of changes in gene expression that support increased capacity for processes that are necessary for neurite outgrowth. In late-stage immature OSNs further changes in gene expression provide increased capacity for several homeostatic mechanisms necessary to support high levels of neural activity and the production of nearly 10 000 distinct proteins. The expression of these genes carries over into mature OSNs. The final steps in the maturation of OSNs primarily involve expressing genes needed for the transmission of electrical signals down axons and across their synapses to targets in the glomeruli of the olfactory bulb.

Figure 5.

The abundance of 807 transcriptional regulator transcripts expressed by immature OSNs (Nickell et al. 2012) shows an inverse correlation between the effects of bulbectomy (Heron et al. 2013) and enrichment in samples of Omp⁺ mature OSNs (Saraiva et al. 2015). Expression in immature OSNs is defined as those transcripts identified by Nickell et al. (2012) as having a probability of expression in immature OSNs > 0.5 and not suffering contradictory evidence of enrichment in mature OSNs of >5-fold (Saraiva et al. 2015). The transcripts in set A tend to be expressed in both GBCs and immature OSNs. The transcripts in set B are relatively insensitive to bulbectomy because they are most abundant in immature OSNs, even though many are also expressed in mature OSNs. The transcripts in set C are found in both immature and mature OSNs.

Figure 6.

Locations of the cell bodies of cells undergoing apoptosis in the olfactory epithelium. (A) The apical (1.0) to basal (0.0) depth of 50 cell bodies immunoreactive for active caspase 3 from 2 phenotypically normal 123pCre:Lhx2^(fl/+) mice age postnatal day 26 (Zhang et al. 2016). (B) The peaks of caspase 3 immunoreactivity from (A) are plotted with the mean and ranges of the depths of the cell bodies of 5 cell types spanning the olfactory epithelium (Nickell et al. 2012).

Figure 7.

Anatomy and appearance of olfactory cilia. (A) Scanning electron microscopy images of the surface of the mouse olfactory epithelium. Scale bar = 10 µm. (B) En face imaging of olfactory cilia in the mouse olfactory epithelium. OSN cilia were labeled by MyrPalm–mCherry. The arrow highlights the dendritic knob, and the arrowhead highlights the OSN cilia. The red asterisk in the inset image indicates the proximal segment of the olfactory cilia. Scale bar = 10 µm.

Figure 8.

OSN ciliogenesis during embryonic development. As early as embryonic day 11 (E11), 1 single primary cilium of approximately 1 µm in length forms in the nascent OSN. The primary cilium continues to lengthen to 2 µm, and by E14, multiple cilia are present that are up to 3 µm in length and extend from immature OSNs that are marked by GAP43 expression. Over the next several days (E14–E19), OMP and the signal transduction proteins ACIII (Adcy3), Golf (Gnal), and the cyclic nucleotide-gated channel subunit Cnga2 (CNG) are expressed. Olfactory cilia continue to elongate to up to 100 µm and form the meshwork in the mucous layer where olfactory transduction occurs.