A Subset of Olfactory Sensory Neurons Express Forkhead Box J1-Driven eGFP

Abstract

Forkhead box protein J1 (FOXJ1), a member of the forkhead family transcription factors, is a transcriptional regulator of motile ciliogenesis. The nasal respiratory epithelium, but not olfactory epithelium, is lined with FOXJ1-expressing multiciliated epithelial cells with motile cilia. In a transgenic mouse where an enhanced green fluorescent protein (eGFP) transgene is driven by the human FOXJ1 promoter, robust eGFP expression is observed not only in the multiciliated cells of the respiratory epithelium but in a distinctive small subset of olfactory sensory neurons in the olfactory epithelium. These eGFP-positive cells lie at the extreme apical part of the neuronal layer and are most numerous in dorsal-medial regions of olfactory epithelium. Interestingly, we observed a corresponding small number of glomeruli in the olfactory bulb wherein eGFP-labeled axons terminate, suggesting that the population of eGFP+ receptor cells expresses a limited number of olfactory receptors. Similarly, a subset of vomeronasal sensory neurons expresses eGFP and is distributed throughout the full height of the vomeronasal sensory epithelium. In keeping with this broad distribution of labeled vomeronasal receptor cells, eGFP-labeled axons terminate in many glomeruli in both anterior and posterior portions of the accessory olfactory bulb. These findings suggest that Foxj1-driven eGFP marks a specific population of olfactory and vomeronasal sensory neurons, although neither receptor cell population possess motile cilia.

Keywords: FOXJ1; cilia; forkhead transcription factors; olfactory bulb; olfactory receptor neurons.

Figure 1.

RE and OE expression of Foxj1-driven eGFP. Foxj1-driven eGFP (green) robustly labels multiciliated epithelial cells. (A,B) Multiciliated epithelial cells of the nasal RE express Foxj1-driven eGFP throughout the cell body and cilia marked by AT (magenta). (C) Solitary chemosensory cells, identified by staining for GNAT3, do not express eGFP. (D) Goblet cells, visualized by MUC5B immunoreactivity, are distinct from eGFP-expressing multiciliated cells. (E) FOXJ1 antibody (magenta) reveals protein is present within the nuclei of eGFP-labeled multiciliated cells. Images are maximum z-projections of 5–10 µm substacks. (F) eGFP-expressing neuronal cell bodies are located apically within the OE (arrows) and are immunoreactive for OMP. (F’) Identical image to panel A but showing only the OMP label (magenta). (G, H) eGFP expression in the dendrite, dendritic knob, and cilia of the OSN as viewed in longitudinal section (G) and when the surface of the epithelium is viewed en face (H). (I) The nuclei and cell bodies of the eGFP-expressing OSNs are not immunoreactive for FOXJ1 (magenta). (I’) Identical image to panel D but showing only the FOXJ1 label. Images are maximum z-projections of 5–10 μm substacks.

Figure 2.

The majority of eGFP-expressing OSNs are restricted zonally (dorso-medial) within the MOE. This zonal organization is conserved throughout the MOE as depicted in transverse sections from different regions ((A) more anterior; (B) more posterior). Two eGFP-targeted glomeruli are visible in (B, arrows). Insets are magnifications of region denoted by the white box. Inset scale bars, 100 µm. Images are maximum z-projections of a ×20 tile scans. (C, D) 3D reconstructions of whole-mount ethmoturbinates showing eGFP (C) and eGFP plus OMP (D) immunoreactivity. The respiratory epithelium is clearly defined as dense eGFP immunoreactivity, whereas the olfactory epithelium is defined by sparse, punctate eGFP immunoreactivity indicative of OSNs. OSNs are most dense in dorsal regions of the turbinates.

Figure 3.

Foxj1-driven eGFP (green) positive neurons converge onto individual glomeruli within the MOB. (A) Sagittal ×20 tile scan of a section of the MOB shows an individual glomerulus targeted by eGFP (white box). Dorsal is toward the top of the page. (B) Higher magnification image of the region depicted in the white box in A shows eGFP neurons converging onto a single glomerulus within the OB. Whereas eGFP-expressing astrocytes are present in all glomeruli, the targeted glomerulus has a higher co-incidence of eGFP and OMP. (C–F) Quantification of glomerular fluorescence intensity reveals eGFP-targeted glomeruli. (C) Glomerulus in MOB showing neuropil labeled by OMP only. (D) Glomerulus in MOB showing neuropil double labeled for OMP and eGFP. (E) Colocalization analysis of 25 glomeruli, including the 5 that were qualitatively determined as targeted by eGFP-expressing axons. (F) eGFP to OMP fluorescence ratio in all imaged glomeruli (1631). (G–I) Numerous branched cells in the olfactory bulb expressed eGFP. Staining with GFAP antibody (magenta, main image; white inserts at right) confirms their identity as a type of astrocyte as reported previously (Jacquet et al. 2009). (G) Double-label image of the olfactory bulb showing double-label branched cells. Box indicates the cell enlarged in panels (H) and (I). (H) eGFP labeling shows a large multipolar cell. (I) Superimposed GFAP label (white) shows label surrounding the nucleus and extending into the proximal extensions of the soma as is typical of GFAP label of astrocytes.

Figure 4.

Foxj1-driven eGFP (green) expression in the accessory olfactory system. (A) Cross section of the VNO reveals the SE with scattered eGFP expression and the lumen of the VNO. eGFP expression is seen in both the superficial and basal regions of the SE. eGFP expression also occurs in scattered cells in the nonsensory epithelium of the VNO but at much lower levels relative to the SE. OMP-immunoreactivity; magenta. Nuclear counterstain (DRAQ5); blue. (B) High magnification of the apical portion of the SE showing robust eGFP expression throughout the VNO sensory neurons. (C) VNO showing FOXJ1 immunoreactivity and endogenous eGFP expression. VNO sensory neurons are not immunoreactive for FOXJ1, whereas the cells of the nonsensory epithelium show nuclear immunoreactivity to FOXJ1 (arrow). (C’) Same image as (C), showing only FOXJ1 immunoreactivity. (D) Region of respiratory epithelium captured in the same frame as (C), showing strong FOXJ1 immunoreactivity within eGFP-expressing multiciliated cells. (D’) Same image as D but only showing FOXJ1 immunoreactivity. (E, F) Lateral view of 2 bisected skulls showing eGFP-expressing fibers of the accessory olfactory nerves running from the VNO toward the AOB. In (F), the eGFP fluorescence image is overlaid on a color photograph. Septal organ (arrow) is devoid of eGFP expression. Note that the VNO was removed in panel (F) prior to imaging. (G) Longitudinal section through the AOB. Glomeruli in both the anterior and posterior AOB show differential expression of Foxj1-driven eGFP and OMP (magenta) within individual glomeruli.