Adult c-Kit(+) progenitor cells are necessary for maintenance and regeneration of olfactory neurons

Abstract

The olfactory epithelium houses chemosensory neurons, which transmit odor information from the nose to the brain. In adult mammals, the olfactory epithelium is a uniquely robust neuroproliferative zone, with the ability to replenish its neuronal and non-neuronal populations due to the presence of germinal basal cells. The stem and progenitor cells of these germinal layers, and their regulatory mechanisms, remain incompletely defined. Here we show that progenitor cells expressing c-Kit, a receptor tyrosine kinase marking stem cells in a variety of embryonic tissues, are required for maintenance of the adult neuroepithelium. Mouse genetic fate-mapping analyses show that embryonically, a c-Kit(+) population contributes to olfactory neurogenesis. In adults under conditions of normal turnover, there is relatively sparse c-Kit(+) progenitor cell (ckPC) activity. However, after experimentally induced neuroepithelial injury, ckPCs are activated such that they reconstitute the neuronal population. There are also occasional non-neuronal cells found to arise from ckPCs. Moreover, the selective depletion of the ckPC population, utilizing temporally controlled targeted diphtheria toxin A expression, results in failure of neurogenesis after experimental injury. Analysis of this model indicates that most ckPCs reside among the globose basal cell populations and act downstream of horizontal basal cells, which can serve as stem cells. Identification of the requirement for olfactory c-Kit-expressing progenitors in olfactory maintenance provides new insight into the mechanisms involved in adult olfactory neurogenesis. Additionally, we define an important and previously unrecognized site of adult c-Kit activity.

Keywords: growth factor; neurogenesis; olfactory epithelium; receptor tyrosine kinase; stem cells.

Figure 1

ckPC fate mapping during embryonic and postnatal peripheral olfactory development. (A, B) Genetic strategies for the inducible GFP or bgal labeling of progeny from c-Kit-expressing cells. (C, D) Representative section through olfactory region of c-Kit^CreERT2/+ ;GFP reporter embryo induced with tamoxifen from E9.5–10.5 and killed at E12.5, revealing scattered GFP labeled cells in olfactory epithelium with bipolar neuronal morphology (arrow). Arrowhead marks dendrite ending in a dendritic knob at the nasal airspace (asterisk). (E) In olfactory epithelium from c-Kit^CreERT2/+;GFP reporter mouse, tamoxifen-induced postnatal day 1–3, killed day 7, c-Kit expressing progenitors produce neurons. Arrow marks layer of GFP (+) olfactory neurons; note the dendrites extending apically (double arrow), ending in dendritic knobs. There are several GFP (+) basal cells and immature neurons deep in the olfactory epithelium. Sustentacular cell layer is not labeled. Asterisk marks top of epithelium at the nasal airspace. (F, G) Embryos tamoxifen-induced from E10.5–12.5 killed at E18.5, examined as whole mount view of the anterior head bisected sagittally; anterior is towards right, dorsal is towards top of field. (F) In c-Kit^CreERT2/+ ;R26R^LacZ tissue, note extensive x-gal labeling in olfactory epithelium (OE) region along the turbinates. (G) Negative control littermate c-Kit^{CreERT2−/−} ;R26R^LacZ; x-gal reaction results in no background staining. (H) Control tissues from adult c-Kit^CreERT2/+ ;R26R^LacZ mouse that never received tamoxifen, processed for x-gal reaction, reveal no labeling in olfactory epithelium, confirming absence of non-specific Cre activity. Bar=50 μm; dashed line marks basal lamina in all images.

Figure 2

C-Kit expressing progenitors exhibit prominent activity in olfactory tissue during the postnatal period, producing neurons and glands. C-Kit^CreERT2/+;R26R^LacZ mice treated with tamoxifen on postnatal day (PD) 2 to induce bgal expression and fixed at 1 month have a mean of 96.8 ± 23 (s.d.) x-gal (+) cells per 0.5 mm of OE (n=3 mice). (A) On low magnification overview of x-gal stained coronal sections, bgal (blue) expression is evident in the OE across the nasal cavity, with reporter-labeled Bowman’s glands (arrows) scattered along the septal and turbinate mucosa. (B) Examination of boxed region in A at high magnification shows detail of a typical x-gal (+) ckPC-derived Bowman’s gland acinar unit and duct (arrows), along with labeled ckPC-derived OE cells among the neuronal layers. (C–F) Sections were processed for dual visualization of bgal (magenta) and cell type-specific markers (green) to confirm ckPC-derived cell phenotypes. (C) Most of the bgal (+) OE cells reside within the OMP (+) mature olfactory neuron layers (bracket) at 1 month following tamoxifen induction. (D) The Gap43 (+) immature neuronal layers are deeper in the OE; only a single Gap43 (+)/bgal (+) cell is evident (arrow) in this field. (E) CK5 marks the horizontal basal cell layer, which does not contain bgal-labeled cells; nuclei are labeled with DAPI (blue, right panel). (F) A bgal (+) gland and duct unit expresses CK18 (arrows); in olfactory mucosa, CK18 is selectively expressed by sustentacular cell bodies at the apical layer and their cytoplasmic process which extend basally, and by the duct and acinar cells of Bowman’s glands. Dashed line indicates basal lamina, bar=25 μm.

Figure 3

Adult c-Kit expressing progenitor cells are activated during injury-induced epithelial reconstitution. (A, B) Un-lesioned adult c-Kit^CreERT2/+ ;R26R^LacZ mice were treated with tamoxifen for two days to induce bgal expression and then recovered 1 week; (A) nasal tissue sections were reacted with x-gal to permit reporter enzyme visualization (blue label). Arrow indicates a cluster of c-Kit derived cells in basal region, with the position and morphology of globose basal cells and immature neurons, reflecting the sporadic activity of c-Kit (+) progenitors during normal olfactory neuronal turnover. (B) Probing un-lesioned adult olfactory tissue for c-Kit protein expression also reveals sporadic basal cell labeling (arrow). (C, D) Mice were given a single dose of methimazole to induce olfactory lesion, and maintained on tamoxifen during epithelial reconstitution prior to sacrifice 10 days later. X-gal labeled cells are evident throughout the new neuroepithelium (C, blue), and antibody to c-Kit (D) robustly labels cells in the basal germinal zone of the regenerating epithelium, consistent with expression by progenitor GBCs; also a single microvillar or sustentacular cell in this field (arrow) is c-Kit (+). (E) Quantification of c-Kit-derived OE cells from un-lesioned versus 10 day post-methimazole lesioned tissue, *** indicated p<0.001, t-test, n=3 mice. (F, G) Additional examples of adult methimazole lesioned sections; (F) antibody to bgal confirms the specificity of x-gal reactivity. (G) Occasional fields have bgal (+) cells present in the lamina propria (arrow), with the location and morphology of Bowman’s glands. Nuclei are labeled by DAPI (blue). Dashed line indicates basal lamina, bar=100 μm.

Figure 4

C-Kit and SCF protein expression in regenerating adult OE, at 10 days following methimazole lesion. (A) Low magnification view indicates the general pattern of c-Kit immunoreactivity, scattered along the basal regions of the OE (arrow). (B–D) PCNA and c-Kit protein co-localize, indicating c-Kit expression in proliferative basal cells. (E, F) Kit ligand, SCF, is detectable locally. SCF expression in reconstituting olfactory tissue localizes to the sustentacular/microvillar cell layer, and to bone marrow; bracket in (E) marks the layer of sustentacular cell nuclei at the apical position of the epithelium. Arrow indicates a bone marrow cell with intense SCF label. Cell layers are marked: Sus, sustentacular cells; Neu, neurons; BCs, basal cells. (F) By Western blot probed for SCF, a predominate band of about 43 kD is detected in neonatal (lane a) and adult (lane b) olfactory extracts. Nuclei are labeled by DAPI (blue). Dashed line indicates basal lamina; bar=50 μm in A and E, 25 μm D.

Figure 5

Neurons arising from proliferative c-Kit (+) progenitors co-express the Cre reporter and neurotubulin. The experimental model for c-Kit fate mapping following olfactory lesion is outlined, c-Kit^CreERT2/+;R26R^LacZ mice. (A–C) Cells in reconstituting OE that derived from c-Kit (+) progenitors are labeled with antibody to bgal (magenta); many bgal (+) cells are situated in the neuronal layers of the OE and are co-labeled by Tuj1 (green, arrows), indicating that they are immature neurons. (D–F) Mice were given a single injection of BrdU 2 hours prior to sacrifice, to visualize actively mitotic cells. Co-immunolabeling with antibodies to BrdU (green) and c-Kit protein (magenta) indicates that 42 ± 9.4% (n=8 sections) of c-Kit (+) cells are also BrdU (+) (arrows), consistent with many c-Kit (+) cells functioning as proliferative GBCs following lesion. (G–I) Reconstituting olfactory tissue 10 days following methimazole lesion was also probed for c-Kit protein (red or magenta) and CK5 (green), a marker for HBCs, indicating that most c-Kit-expressing cells in the OE are GBCs. In this confocal z-stack image, the solid arrow marks a basal cell adjacent to the basal lamina that co-expresses c-Kit and CK5. Dashed arrows mark examples of more typical c-Kit (+) cells, which are CK5(−) and are situated in the GBC zone, superficial to the CK5-labeled horizontal cell layer. Occasional co-expression of c-Kit with HBC markers may be a reflection that the receptor is not strictly confined to “downstream” lineage-committed neuronal progenitors. HBCs often have a reactive pyramidal shape during OE reconstitution following methimazole, rather than their thin, flat morphology when quiescent. Dashed line indicates basal lamina; bar=25 μm in A–F, 50 μm in G–I; DAPI nuclear stain is blue in H, I.

Figure 6

Depletion of c-Kit-expressing cells in adult olfactory mucosa results in failed olfactory neurogenesis. (A) Genetic strategy for the selective ablation of the c-Kit (+) cell population under temporal control of tamoxifen administration. In c-Kit^CreERT2/+ ;ROSA26^eGFP-DTA mice following tamoxifen administration (TAM), c-Kit (+) cells will express diphtheria toxin (DTA), causing selective cell death. The methimazole lesion paradigm was combined with DTA-mediated ckPC-specific ablation to test directly the requirement for ckPCs in olfactory regeneration. (B) Histologic analysis reveals failed reconstitution in the absence of ckPCs. Epithelium is thinner and poorly reconstituted in tamoxifen-treated tissue (B₁); Tuj-1 staining (red) shows marked reduction in olfactory neurons, versus typical abundant labeling of new neurons during epithelial reconstitution in littermate of identical genotype treated with methimazole-only (B). (B₂, B₃) Closer views of boxed regions show detail of Tuj-1 labeled soma, dendrites and dendritic knobs present in normally-regenerating controls, and absent in ckPC-depleted tissue. Bracket marks epithelial height, which is markedly thinner in ckPC-depleted tissue. (C, D) Differentiated olfactory neurons, marked by OMP expression (red), are prominent by 10 days post-lesion in normal epithelial reconstitution (C, arrows) after methimazole, but nearly absent in the ckPC-depleted epithelium (D); cellular debris (asterisks) is present in nasal airspace due to recent lesion. (E) Quantification of TuJ-1 labeled and OMP labeled neurons reflects the significant neurogenic failure after lesion in animals lacking ckPCs; *** indicates p<0.001, **** indicates p<0.0001, t-test, n=3 mice. Error bars indicate SEM. MZ=methimazole. Bar=50 μm.

Figure 7

Non-neuronal cells types are present in ckPC-depleted regenerating olfactory epithelium. (A) Non-neuronal sustentacular cells (Sus) and HBCs (BC) express Pax6 (brown), stratified in apical and basal regions in normally-reconstituted epithelium after lesion (A), in control mice treated with methimazole only. Unlabeled Pax6 (−) neuronal layers (Neu) are present in the middle regions of the epithelium. (B) During failed neurogenesis following methimazole treatment in ckPC-depleted mice, the abnormal OE is predominantly comprised by Pax6 (+) cells, with the Pax6 (−) neuronal layers absent. (C) HBCs, also identified by expression of cytokeratin 5 (CK5, red) are identifiable in the typical layer adjacent to the basal lamina (dashed line) in c-Kit^CreERT2/+;ROSA26^eGFP-DTA mice treated with the lesion and ckPC depletion paradigm, indicating that ckPCs act downstream of HBCs. Bar= 50 μm. (D) OE thickness was measured from tissue sections obtained from c-Kit^CreERT2/+;ROSA26^eGFP-DTA mice in different conditions: (1) no treatment (control), (2) daily tamoxifen for 10 days, (3) 10 days following methimazole lesion, or (4) 10 days following methimazole lesion with daily tamoxifen. Importantly, TAM treatment alone in this model results in only minimal decrease in OE thickness. Quantification indicates significant differences in mean epithelial thickness. Error bars indicate SEM; *** indicates p<0.001, t-test, n=3 mice. Tamoxifen (TAM); Methimazole (MZ); No treatment (No Tx).

Figure 8

Schematic diagram of adult olfactory mucosa and proposed role of ckPCs in neuroepithelial maintenance. The basal germinal zone of the neuroepithelium includes horizontal basal cells (HBCs) and globose basal cells (GBCs), a subset of which express the c-Kit receptor and function as ckPCs. Complex lineage relationships among stem and progenitor cells support adult epithelial maintenance, producing immature olfactory sensory neurons (OSNi), which differentiate into mature bipolar receptor neurons (OSNm). Sustentacular and microvillar cells (Sus/mv) are situated apically, and Bowman’s Gland acinar and duct cells are indicated. Sus/mv cells produce Stem Cell Factor (SCF), the ligand for the c-Kit receptor. Curved and straight arrows reflect previously described relationships among OE cell types. BL=basal lamina.