Neuregulin1 and ErbB expression in the uninjured and regenerating olfactory mucosa

Abstract

Neuregulin1, a protein involved in signaling through the ErbB receptors, is required for the proper development of multiple organ systems. A complete understanding of the expression profile of Neuregulin1 is complicated by the presence of multiple isoform variants that result from extensive alternative splicing. Remarkably, these numerous protein products display a wide range of divergent functional roles, making the characterization of tissue-specific isoforms critical to understanding signaling. Recent evidence suggests an important role for Neuregulin1 signaling during olfactory epithelium development and regeneration. In order to understand the physiological consequences of this signaling, we sought to identify the isoform-specific and cell type-specific expression pattern of Neuregulin1 in the adult olfactory mucosa using a combination of RT-qPCR, FACS, and immunohistochemistry. To complement this information, we also analyzed the cell-type specific expression patterns of the ErbB receptors using immunohistochemistry. We found that multiple Neuregulin1 isoforms, containing predominantly the Type I and Type III N-termini, are expressed in the uninjured olfactory mucosa. Specifically, we found that Type III Neuregulin1 is highly expressed in mature olfactory sensory neurons and Type I Neuregulin1 is highly expressed in duct gland cells. Surprisingly, the divergent localization of these Neuregulin isoforms and their corresponding ErbB receptors does not support a role for active signaling during normal turnover and maintenance of the olfactory mucosa. Conversely, we found that injury to the olfactory epithelium specifically upregulates the Neuregulin1 Type I isoform bringing the expression pattern adjacent to cells expressing both ErbB2 and ErbB3 which is compatible with active signaling, supporting a functional role for Neuregulin1 specifically during regeneration.

Keywords: ErbB; Isoform analysis; Neuregulin1; Olfactory epithelium.

Fig. 1. Olfactory epithelium and lamina propria have differing expression preferences of Nrg1 isoforms, and react to injury differently

(A) Schematic outlining the known exons that are differentially spliced in the Nrg1 gene. For clarity, a consistent color scheme was used for the exons which are part of the three main variable regions (N terminal, EGF, and C terminal). (B,C) Each variable exon is clustered in their respective domain and the expression levels in the olfactory epithelium (B) or lamina propria (C) are depicted as a percentage of total Nrg1 (n = 3 for each tissue type). Black bars represent the levels expressed during uninjured normal tissue, while white bars represent levels in tissue two days post injury from inhaled methyl bromide gas. (SEM error bars, T-test, *p < 0.05, **p < 0.001) (D,E) The full-length isoform composition and percentage of total Nrg1 in olfactory epithelium (D) or lamina propria (E) is depicted as a nested pie chart. The size of each slice is representative of the relative amount of the labeled exon. Contiguous fragments moving from the center to outside represents a full length isoform. For example, in the olfactory epithelium, a small (6.8%) of total Nrg1 is Type I-α-a. For clarity in the figure, the EGF variable domain is placed on the larger, outside ring.

Fig. 2. Expression of variable exons of Nrg1 in FACS-purified cell type-specific populations

qRT-PCR results of RNA extracted from (A) Duct/Gland cells, (B) Mature Neurons, (C) HBCs, (D) OECs, (E) Immature Neurons, (F) GBCs, (G), LP Fibroblasts. CT values each exon were normalized to β-actin, averaged, and plotted as relative to whole mucosa (WM). (SEM error bars, n = 3 (all except immature neurons, n = 7).

Fig. 3. Spatial Expression Pattern of Nrg1 Type I and Type III in the normal olfactory mucosa

(A) An antibody against Nrg1 Type 1 shows expression in Sox9(+) duct/gland cells in the lamina propria. (B) An antibody against Nrg1 Type III shows expression in OMP(+) mature OSNs, and (C) absence from Tuj(+) immature OSNs. Arrows indicate Type I (+) Duct/Gland cells, while arrowheads indicate location of the basal lamina. Insets highlight regions of co-localization. Nuclei are visualized with DAPI. Scale bar = 50 μm.

Fig. 4. Spatial expression pattern of Nrg1 Receptor ErbB3 in the normal olfactory mucosa

(A) An antibody against ErbB3 shows expression in Sox9(+) duct/gland cells and (B) absence from PGP9.5(+) OSNs and (C) CK14(+) HBCs. (D–F) ErbB3 also labels TrpM5(+) microvillar cells on the apical layer of the epithelium. Arrows indicate co-localization. Insets highlight regions of co-localization. Nuclei are visualized with DAPI. Scale bar = 20 μm.

Fig. 5. Spatial expression pattern of Nrg1 Receptor ErbB2 in the normal olfactory mucosa

(A–C) Co-staining of ErbB2 receptor with a Neurog1-eGFP reporter, a marker for downstream neural progenitors. (D–G) Co-staining of ErbB2 with the more upstream progenitor markers Ascl1 (Mash1), NeuroD1, Pax6, and CD54, respectively. (H,I) ErbB2 co-stains with the proliferative marker Ki67 in addition to overlapping with the immature neuronal marker NCAM. (J,K) ErbB2 does not colocalize with the mature neuronal marker OMP or the duct/gland marker ECAD. Nuclei are visualized with DAPI. Scale bar = 20 μm.

Fig. 6. Nrg1 Type I expression in the olfactory mucosa following MeBr lesion

(A) Nrg1 Type 1 expression at 1–4 days post lesion (dpl), showing restriction from CD54(+) HBCs and (B–C) increased expression in the olfactory epithelium. (E) Areas of the mucosa that are subjected to a harsher lesion have increased levels of Nrg1 Type I present in the epithelium at 7 dpl compared to (F) epithelium that has had a less harsh lesion. Arrows indicate Nrg1 Type I expression in the epithelium. Nuclei are visualized with DAPI. Scale bar = 20 μm.

Fig. 7. Dynamics of ErbB3 expression in the olfactory mucosa after MeBr lesion

(A) ErbB3 staining shows expression in Sox9(+) duct/gland cells in the lamina propria 1 dpl. (B,C) ErbB3 expression is seen in duct/gland cells at 2–3 dpl, and is enhanced in non-duct/gland cells by 4 dpl. (E) ErbB3 is again expressed primarily in duct/gland cells by 7 dpl. (F,G) ErbB3 is not expressed in CK14(+) HBCs at 1–2 dpl, but (H,I) appears co-localized with a small, apically-situated CK14(+) population at 3–4 dpl. (J) by 7 dpl, ErbB3 is again restricted from all CK14(+) HBCs. (K–M) ErbB2 is not detected at 1–3 dpl, (N) but at 4 dpl, ErbB2 and ErbB3 are co-expressed. (O) By 7 dpl, ErbB2 and ErbB3 are again mutually exclusive. (P) Nrg1 Type I and ErbB3 are expressed in different populations of cells in the lamina propria at 1 dpl, but (Q–S) from 2–4 dpl, they are co-localized in both lamina propria and epithelial populations of duct/gland cells. (T) At 7 dpl, Nrg1 Type I expression is once again restricted from the OE and no longer co-localizes with ErbB3. Insets highlight regions of co-localization. Nuclei are visualized with DAPI. Scale bar = 20 μm.