TY - JOUR
AU - Neuhaus, Eva M.
AB - Abstract The olfactory epithelium (OE) possesses unique lifelong neuroregenerative capacities and undergoes constitutive neurogenesis throughout mammalian lifespan. Two populations of stem cells, frequently dividing globose basal cells (GBCs) and quiescent horizontal basal cells (HBCs), readily replace olfactory neurons throughout lifetime. Although lineage commitment and neuronal differentiation of stem cells has already been described in terms of transcription factor expression, little is known about external factors balancing between differentiation and self-renewal. We show here that expression of the CXC-motif chemokine receptor 4 (CXCR4) distinguishes both types of stem cells. Extensive colocalization analysis revealed exclusive expression of CXCR4 in proliferating GBCs and their neuronal progenies. Moreover, only neuronal lineage cells were derived from CXCR4-CreER-tdTomato reporter mice in the OE. Furthermore, Cre-tdTomato mice specific for HBCs (Nestin+ and Cytokeratin14+) did not reduce CXCR4 expression when bred to mice bearing floxed CXCR4 alleles, and did not show labeling of the neuronal cells. CXCR4 and its ligand CXCL12 were markedly upregulated upon induction of GBC proliferation during injury-induced regeneration. in vivo overexpression of CXCL12 did downregulate CXCR4 levels, which results in reduced GBC maintenance and neuronal differentiation. We proved that these effects were caused by CXCR4 downregulation rather than over-activation by showing that the phenotypes of CXCL12-overexpressing mice were highly similar to the phenotypes of CXCR4 knockout mice. Our results demonstrate functional CXCR4 signaling in GBCs regulates cell cycle exit and neural differentiation. We propose that CXCR4/CXCL12 signaling is an essential regulator of olfactory neurogenesis and provide new insights into the dynamics of neurogenesis in the OE. CXCL12 is secreted by cells of the lamina propria and activates the chemokine receptor CXCR4 in globose basal cells of the olfactory epithelium. Receptor activation promotes division of these stem cells, and thereby regulates the size of the stem cell pool. Absence of CXCR4 signaling leads to exit from the cell cycle and increased neuronal differentiation. Open in new tabDownload slide Open in new tabDownload slide CXCL12, CXCR4, GBC, neurogenesis, olfactory epithelium, stem cell Significance statement Here the authors show that the chemokine CXCL12, secreted from the microenvironment of the olfactory stem cells, acts as paracrine factor to regulate homeostatic and injury-induced neurogenesis via CXCR4 in the olfactory system of postnatal mice. The authors thereby not only report one of the first extracellular signal influencing stem cell trajectories in the olfactory stem cell niche in vivo, but also the important role of chemokine signaling in regeneration of the olfactory epithelium. Existing well-characterized pharmacological tools to influence CXCR4 signaling may therefore help to improve autologous transplantation of olfactory stem cells, which are accessible by biopsy. INTRODUCTION The nervous system of mammals recovers poorly after injury, being unable to completely replace dying neurons and sufficiently reinnervate target structures. This tenet does not apply to olfactory neurons in the olfactory epithelium (OE), where lifelong neurogenesis and neuroregeneration occurs in all examined vertebrate species due to the persistence of self-renewing stem cells.1,2 Moreover, the adult mouse OE is capable of complete regeneration after the loss of mature cells.3 Similar to other tissues with continuously cycling stem cells for homeostasis,4 the OE contains two populations of stem cells. Continuous olfactory neurogenesis takes place through actively proliferating neurogenic progenitor cells called globose basal cells (GBCs).5,6 GBCs encompass sex determining region Y-box 2 (SOX2)+/Paired box 6 (Pax6) + stem-like cells that self-renew, Achaete-scute Family BHLH Transcription factor 1 (Ascl1) + transit-amplifying progenitors and Neurogin1+/Neurogenic differentiation 1 (NeuroD1) + immediate neuronal precursor cells. Another type of stem cell, the horizontal basal cells (HBCs), are quiescent reserve stem cells. These are nonproliferating under homeostatic conditions7 due to continued expression of the transcription factor p63.8 HBCs serve as a reservoir to replenish the actively cycling cells under conditions of extreme tissue damage, when notch signaling downregulates p63 expression and activates HBCs to multipotency.9 Extensive single cell RNA-sequencing and in vivo lineage-tracing helped to identify and characterize the transition states during tissue homeostasis10 and regeneration after injury.11 According to these studies, injury-induced HBCs multipotency likely arises from successive rounds of self-renewal leaving daughter cells that are competent to adopt different cell fates whereas the default state of HBCs is to form sustentacular cells, glia-like cells enwrapping the olfactory neurons. Despite the increasing knowledge concerning the fates of the different precursor cells and their characteristic transcription factor expression profiles, the nature of cues that direct activated stem and progenitor cells is still limited. Continuing interest in theses cues arises due to use of the OE as a model for adult neurogenesis, and the therapeutic potential of accessible autologous neural stem cells. Transplantation of cultured HBCs into the nasal cavity leads to rebuilding of the epithelium after injury, but the fraction of neuronal progeny is dependent on the in vitro culture conditions of the transplanted cells.12 Wnt signaling seems to bias activated HBCs toward a neuronal fate, while not having an influence on activation per se.10 Wnt signaling also stimulates olfactory ensheathing cells to enhance the proliferation and differentiation of neural stem cells in a secretory manner,13 and neuronal differentiation of GBCs was enhanced in medium conditioned by a cell line derived from the lamina propria.14 Moreover, application of fibroblast growth factor 2 (FGF2) and transforming growth factor α (TGFα) on an olfactory cell line influenced neurogenesis.15 Together these results show that neuronal differentiation depends on external cues, partially secreted by cells in the lamina propria. In the adult central nervous system, homeostatic neurogenic processes are limited to the subgranular zone of the hippocampus and the subventricular zone in the lateral wall of the ventricle.16 Neurogenesis is regulated by the chemokine CXCL12 and its cognate receptor CXCR4.17 CXCR4 is expressed in neuronal progenitor cells,18 and activation of CXCR4 supports neurogenesis in the adult dentate gyrus.19 Induced conditional deletion of Cxcr4 in neuronal stem cells leads to reduced neurogenesis and ectopic placement of newly formed granule neurons.20 Activation of CXCR4 maintains neuronal stem cells in a quiescent state and promotes the survival, but not the proliferation of cultured neuronal stem cells,21 while blockage of CXCR4 signal transduction downregulates neuronal stem cell self-renewal ability in culture.22 The CXCL12/CXCR4 system also plays a role in neuronal differentiation, since loss of signaling causes precocious differentiation of neuronal precursor cells.22 Not least, CXCL12 plays a role as a neurotransmitter regulating the strength of GABAergic inputs to neural progenitors in the dentate gyrus.23 Association of CXCL12 with CXCR4 activates different signaling pathways.24,25 In the present study, we demonstrate that CXCR4 is highly expressed in neuronal committed stem cells from adult mouse OE. CXCR4 signaling impairs the self-renewal ability of GBCs, which results in exhaustion of their stemness characteristic(s), induces cell cycle exit and promotes neural differentiation. METHODS Animal breeding and treatment Animal experiments were conducted in accordance to the EC directive 86/609/European Economic Community guidelines for animal experiments and permitted by the local government (Thüringer Landesamt für Lebensmittelsicherheit und Verbraucherschutz). Mice were kept under 12 hours light/dark cycles with ad libitum access to food and water. C57BL6/6J wild type mice were originally purchased from Charles River Laboratories (Sulzfeld, GER) and sacrificed at P8, P28, P56 (2 months) and 6 months. CXCR4loxP; R26CAG-LSL-tdT mice26 were crossbred with Tg(KRT14-cre)1Amc mice, purchased from Charles River Laboratories (Sulzfeld, GER, #00478227). In these mice, the transgene is composed of a Cre recombinase gene under the control of a human Cytokeratin 14 (CK14, Krt14) promoter. The allele is representative of founders exhibiting high levels of transgene expression. We generated TgKRT14(−cre); CXCR4loxP; R26CAG-LSL-tdT (KRT14(−cre) CXCR4loxP tdT) mice in order to delete Cxcr4 in KRT14-expressing HBCs. Expression of tdT was used to control for the expected expression of Krt14 in HBCs. CXCR4loxP; R26CAG-LSL-tdT mice were also crossbred with Tg(Nes-cre/ERT2) mice (Jackson stock number 016261, kindly provided by S. Keiner and O. W. Witte, Jena). The Nestin-cre/ERT2 transgene was designed with the rat nestin (Nes) promoter driving expression of a CreERT2 fusion gene. The transgene directs cre expression in the nestin-expressing cells in the brain, which are generally restricted to the neurogenic regions (subventricular zone and subgranular zone). TgNes-creERT2; CXCR4loxP; R26CAG-LSL-tdT animals were generated in order to delete Cxcr4 in Nestin-expressing basal cells after tamoxifen induction, expression of tdT was used to control the expression of Nestin in basal cells of the OE. Twenty-eight-days-old mice were fed with tamoxifen food (SNIFF, 400 mg/kg Tamoxifen-Citrat) for 3 weeks to induce CreER expression. For lineage tracing of CXCR4-expressing cells, we used a Cxcr4CreER(T2)-IRES-eGFP (Cxcr4CreER) knockin allele26 to breed Cxcr4CreER/Wt;R26CAG-LSL-tdT mice. In these mice, CreER was inserted into exon1 of Cxcr4, abolishing expression of functional CXCR4. Cxcr4CreER/CreER mice are fully deficient in CXCR4.26 The IRES-eGFP sequence was added to CreER for visualization of cells expressing Cxcr4. To generate an inducible Cxcr4 knockout, we bred Cxcr4CreER/Wt;R26CAG-LSL-tdT with CXCR4loxP mice to generate Cxcr4CreER/loxP;R26CAG-LSL-tdT mice. Adult mice were treated with tamoxifen (Sigma-Aldrich, Missouri; 10 mg/mL in corn oil) intraperitoneally for five consecutive days and sacrificed 28 days after the last tamoxifen administration. To generate a constitutive Cxcr4 knockout, we bred Cxcr4CreER/CreER mice and sacrificed the pregnant animals at E18. Transgenic CXCL12-RFP mice, expressing CXCL12-RFP under to CXCL12 promotor were generated by Prof. R. J. Miller23 and bred on a C57BL6/J background. Monomeric red fluorescence protein 1 (mRFP1) was inserted at the end of the CXCL12 coding sequence to generate a CXCL12-mRFP1 fusion construct in a CXCL12-containing BAC clone (RP23-203H21).23 CXCL12-RFP-transgenic mice contain two to five additional copies of functional Cxcl12 and have been used before as a model for Cxcl12 overexpression.28 In situ hybridization confirmed that Cxcl12 was orthotopically overexpressed in the central nervous system. For regeneration experiments, mice at an age of 2 and 6 months were treated once with 50 mg/kg methimazole (Sigma-Aldrich, St. Louis, Missouri) or 0.9% sodium chloride as a control intraperitoneally and euthanized with an overdose of isoflurane at 3, 14, or 28 days post injury (dpi). Tissue preparation For all experiments, mice were decapitated after euthanasia. For young mice (P8) whole heads were fixed in 4% paraformaldehyde (PFA; Carl Roth, Karlsruhe, GER) for 24 hours at 4°C for immunofluorescence or directly frozen in 2-methylbutane for in situ hybridization. In order to extract the whole OE of older mice, vomeronasal bones, zygomatic bones and plate, nasal bones, the lower jaw, inscisors and the anterior maxillae were removed according to a modified protocol from Dunston et al. 29 The OE was fixed for immunofluorescence in 4% PFA for 24 hours at 4°C, stored subsequently for cryopreservation in 30% sucrose (in PBS) for at least 24 hours at 4°C and frozen in 2-methylbutane (Carl Roth, Karlsruhe, GER). For in situ hybridization fresh tissues were directly frozen in 2-methylbutane without any fixation. All tissues were fixed in tissue freezing medium (Leica Microsystems, Wetzlar, GER) on a specimen disk and sectioned coronary at 18 μm thickness. Immunofluorescence In brief, slides were washed 3 × 10 minutes Tris buffered saline (TBS) and incubated in 0.1 mol/L citrate buffer (0.1 mol/L tri-sodium-citrate-dehydrate, 0.5% Tween-20. pH 6) at 97-99°C for 15 minutes to unmask epitopes. Unspecific binding sites were blocked in TBS Plus (2% Bovine Serum Albumin (BSA; Thermo Fisher Scientific Germany Ltd & Co KG, Bonn, GER), 3% Normal Donkey Serum (Merck, Darmstadt, GER), 0.1% Triton in TBS) for at least 1 hour. The slides were incubated with the primary antibody over night at 4°C (Table S1). After washing, the slides were incubated in the secondary antibody solution for 2 hours (1:500 in TBS Plus, Table S2). For quantification, 3-5 animals were analyzed per group and same sized Z-stacks were taken using a confocal laser scanning microscope with TCS SPE system (Leica DM2500. Leica Microsystems, Wetzlar, GER). Three comparable regions of septum, ectoturbinate and endoturbinate were analyzed in four sections at intervals of 160 μm. Cell counts were normalized to 1 mm epithelium. The distances of the most basally located OMP-positive cell bodies to the basal lamina were measured in the images of WT and CXCL12-RFP mice, and percentages were calculated based on the thickness of epithelium. Images were further processed using LAS AF (Leica Microsystems), Image J and Photoshop CS6 (Adobe Systems, California). In situ hybridization The radioactive sense and antisense riboprobes for mouse CXCR4 (mCXCR4, GI 10956330) were constructed by using 35S-UTP (Perkin Elmer, Massachusetts; NET380001MC). For transcription 100 mM dethiothreitol, 20 mM NTP-U-C, 10× transcription buffer and RNA polymerase (Roche Deutschland Holding GmbH, Grenzach-Wyhlen, GER; T7: 1088176001, SP6: 10810274001) as well as the linearized plasmid were mixed on ice and added to the lyophilized 35S-UTP. The transcription mix was incubated for 2 hours at 37°C followed by hydrolysis in 0.2 M sodium carbonate for 28 minutes at 60°C. The reaction was stopped by adding acetic acid on ice. The probes were purified using P-30 Mikro-Bio-Spin columns (Biorad, California). One microliter of the probe was diluted in water and radioactivity was measured in a Szinticounter Packard 16001R (Perkin Elmer, Massachusetts). The radioactive riboprobes were diluted in hybridization buffer (containing 0.6 M sodium chloride, 1 mM ethylenediaminetetraacetitc acid (EDTA), 10 mM Tris-HCl (pH 7.5), 20 mg/mL tRNA (Roche Deutschland Holding GmbH, Grenzach-Wyhlen, GER; 109 541), 1× Denhardt-Solution (Sigma Aldrich, Missouri) and 10% dextran sulfate mixed in formamide) to get a concentration of 0.05 Mio counts/μL. For prehybridization, sections were completely dried after thawing, fixed in 4% PFA for 1 hour and washed in phosphate buffered saline (PBS). Afterwards, the sections were permeabilized in 0.4% Triton X (in PBS), followed by trithanolamine buffer (pH 8) and acetic anhydride in Trithanolamine buffer. The sections were washed in PBS again, dehydrated in an isopropanol series and dried. For hybridization, sections were incubated with 1 μg/mL radioactive CXCR4 riboprobe in hybridization buffer for 20 hours at 60°C in a humid chamber containing 50% formamide. Afterwards, sections were washed in 2× Tri-sodium-citrate-dihydrate (SSC) and 1× SSC followed by an incubation in RNAse solution for 30 minutes at 42°C. The slides were washed consecutively in 1× SSC, 0.2× SSC and water followed by dehydration in isopropanol. In the dark room, dried slides were dipped into prewarmed NTB-Emulsion (IBS-Integra Biosciences AG, Zizers, CH; 10 542 844), dried for 1 day and stored 3-4 weeks protected from light at 4°C. Finally, coated slides were developed in preheated Kodak Professional D-19 Developer (45°C; Kodak, New York; 1 464 593) and fixed in Carestream Kodak Processing Chemicals Kodak Fixer (Sigma Aldrich, Missouri; P8307). After washing, sections were counterstained with cresyl violet for better visualization. Bright field images were performed on a light microscope (Zeiss Axio Imager A1, Carl Zeiss, Oberkochen, GER). Quantitative real-time polymerase chain reaction Mice were decapitated and skin and bones were removed. The olfactory mucosa was immediately frozen in liquid nitrogen. The RNA was isolated from the tissue with Purelink RNA Mini Kit (Thermo Fisher Scientific Germany Ltd. & Co. KG, Bonn, GER; 12183018A). Afterwards, cDNA was synthesized from RNA samples using the High Capacity cDNA Kit (Thermo Fisher Scientific Germany Ltd. & Co. KG, Bonn, GER; 4 368 814). Quantitative polymerase chain reaction (PCR) was performed in a Quant Studio3 Real Time PCR Cycler (Thermo Fisher Scientific Germany Ltd. & Co. KG, Bonn, GER) using Power Up SYBR Green Master Mix (Thermo Fisher Scientific Germany Ltd. & Co. KG, Bonn, GER; A25742). Per experimental group, three animals were analyzed in triplicates and three independent runs. Primers purchased from Qiagen (Qiagen N.V., Hilden, GER; GAPDH (QT01658692), CXCR4 (QT00249305), CXCL12 (QT00161112)). PCR conditions were performed as follows: 2 minutes 50°C, 2 minutes 95°C, 15 seconds 95°C, 1 minute 55°C, 15 seconds 95°C, 60°C 1 minute, 15 seconds 95°C for 44 cycles. Data were stored in the Thermo Fisher Cloud and calculated using the delta delta threshold cycle (ddCt) method, followed by representation of the ratio (2−ddCt) in Microsoft Excel.31 Western blot OEs were collected from 4 weeks old mice, homogenized using ceramic beads and sonicated in lysis buffer. Protein samples were inverted on a wheel for 1,5 hours at 4°C and centrifuged for 30 minutes at 14 800g at 4°C. In order to enrich receptor amounts, supernatants were incubated with wheat germ agglutinin agarose beads (Merck, Darmstadt, GER; 61768) for 1,5 hours at 4°C, washed and eluted at 43°C at 800 rpm. After transfer to nitrocellulose, immunoblots were blocked with 5% milk powder in TBS-T and incubated with primary anti-CXCR4 antibody or anti-UMB-2 (nonactivated CXCR4) antibody, followed by secondary horse radish peroxidase-coupled antibodies (Table S2). ECL Select (Sigma Aldrich, Missouri; RPN2235) was used for detection. Stripped immunoblots were incubated with antitransferrin receptor antibody. The intensity ratio of CXCR4/transferrin receptor or UMB-2 (nonactivated CXCR4)/transferrin receptor of the WT controls were set as 100% for comparison. Statistical analysis Statistical analysis was performed in GraphPad Prism 5.01, data were represented as mean ± SEM. Data were tested for normal distribution and homogeneity. Statistical significance was set at *P < .05 and analyzed using one-way ANOVA or two-way ANOVA, and Bonferroni post hoc test or Student's t-test. RESULTS Expression of CXCR4 in the OE Given that CXCR4 regulates stem and progenitor cells in diverse neuronal and non-neuronal tissues, we studied its expression in the OE, a major neurogenic niche. In situ hybridization and immunohistochemistry consistently showed strong Cxcr4 expression in the lower one-third of the OE at postnatal day (P)8 (99 ± 15 cells/mm; Figure 1A-C). In older animals (P28; P56), Cxcr4 mRNA and CXCR4 protein were still high, but were concentrated near the basal lamina (44 and 18 cells/mm at P28 and P56, respectively, Figure 1A-C). Since CXCR4 is involved in cell proliferation in different tissues, we investigated its colocalization with minichromosome maintenance complex component 2 (MCM2), a marker for proliferating cells.32 Distribution and number of MCM2+ cells were very similar to the number of CXCR4-expressing cells (Figure 1B,D). Almost all CXCR4-expressing cells (>99%) were proliferating, and 86% of MCM2-positive cells expressed CXCR4 at P8. Comparison of CXCR4 expression in olfactory progenitor cells and neuronal stem cells in the subgranular zone of the hippocampus revealed that CXCR4 is expressed at a strikingly high level in the OE (Figure 1E,F). FIGURE 1 Open in new tabDownload slide Expression of CXCR4 in the OE. A, Bright field micrographs of the OE of P8, P28, and P56 old mice after in situ hybridization with 35S-labeled probes for CXCR4. CXCR4 mRNA decreases with age, small amounts of mRNA in the lamina propria cluster in axon bundles. CXCR4 sense-probe (not shown) does not show a signal. Scale bar = 50 μm. B, Confocal images of coronal OE sections showing colocalization of CXCR4 (green) and MCM2 (red, proliferation marker). Scale bar = 20 μm. C and D, Quantification of CXCR4-positive cells (C) and MCM2-positive cells (D). Cell counts were analyzed using One-way ANOVA and Bonferroni post hoc test (error bars represent SEM, *P < .05). E, Immunostaining of CXCR4 in the OE (P56) and F, in the hippocampus (P56) shows markedly stronger Cxcr4 expression in the OE, left scale bar = 100 μm; right scale bar = 20 μm. Staining, exposure and laser intensities were the same in (E) and (F). Dotted lines represents basal lamina, expect for (F) where the line represents the border to the hilus FIGURE 1 Open in new tabDownload slide Expression of CXCR4 in the OE. A, Bright field micrographs of the OE of P8, P28, and P56 old mice after in situ hybridization with 35S-labeled probes for CXCR4. CXCR4 mRNA decreases with age, small amounts of mRNA in the lamina propria cluster in axon bundles. CXCR4 sense-probe (not shown) does not show a signal. Scale bar = 50 μm. B, Confocal images of coronal OE sections showing colocalization of CXCR4 (green) and MCM2 (red, proliferation marker). Scale bar = 20 μm. C and D, Quantification of CXCR4-positive cells (C) and MCM2-positive cells (D). Cell counts were analyzed using One-way ANOVA and Bonferroni post hoc test (error bars represent SEM, *P < .05). E, Immunostaining of CXCR4 in the OE (P56) and F, in the hippocampus (P56) shows markedly stronger Cxcr4 expression in the OE, left scale bar = 100 μm; right scale bar = 20 μm. Staining, exposure and laser intensities were the same in (E) and (F). Dotted lines represents basal lamina, expect for (F) where the line represents the border to the hilus CXCR4 expression in the neuronal lineage of progenitor cells To identify the cell type(s) expressing Cxcr4 in the OE, we assessed its colocalization with different markers. Proteins which are expressed in HBCs such as cytokeratin 5 (CK5; KRT5), cytokeratin 14 (KRT14) or intercellular adhesion molecule 1 (ICAM1)33,34 did not colocalize with CXCR4 (CK5 in Figure 2A,C,D, other markers not shown). In contrast, exocyst complex component 8 (SEC8; EXOC4) which identifies GBCs and immediate neuronal progenitor cells,35 overlapped extensively with CXCR4 (Figure 2A,C). The most mature subtype of progenitor cells and particularly immature neurons expressing growth associated protein 43 (GAP43) showed only little colocalization with CXCR4 (Figure 2B,C,D). CXCR4 was almost undetectable in olfactory marker protein (OMP)-positive mature neurons (Figure 2B,C). Under homeostatic conditions, we never detected CXCR4 in cytokeratin 18-positive sustentacular cells. Further colocalization analyses revealed expression of CXCR4 in the diverse neuronal lineage-restricted precursors, that is, in SOX2, c-KIT (CD117) and ASCL1-positive GBCs and in NEUROD1-positive immediate neuronal progenitor cells 1/2 (INP1/2) cells (Figure 2D). Taken together, these findings indicate that CXCR4 is present in precursors of the neuronal lineage, but not in mature neurons or olfactory glia cells. FIGURE 2 Open in new tabDownload slide CXCR4 expression in the neuronal lineage of precursor cells. Colabeling of (A) CXCR4, GBCs (SEC8), and HBCs (CK5) and (B) CXCR4, immature neurons (GAP43) and mature neurons (OMP), P8. C, Quantification of CXCR4 expression in different cells. (n = 3 animals, error bars represent SEM). D, Colocalization of CXCR4 with SOX2, c-KIT, ASCL1, NEUROD1 and GAP43, marking different stages of maturation. CXCR4 was almost not colocalized with CK5 in HBCs. E, OE of Krt14-Cre;CXCR4loxP;R26CAG-LSL-tdT mice (P8); tdTomato (red) expression under the control of the Krt14 promoter (CK14) did result in labeling of HBCs and sustentacular cells; Tomato expressing red cells were not labeled with CXCR4 or SEC8. Cxcr4 expression level was unaltered compared with the wild type (WT) epithelium. F, OE of Cxcr4CreER-IRES-GFP/Wt;R26CAG-LSL-tdT mice (P28) pulsed with Tamoxifen at P5. In these mice, GFP is expressed in GBCs, indicating Cxcr4 promoter activity, but not in neurons or neuronal progenitors. TdTomato, which is present progenies of the GFP expressing cells, did label GBCs (SEC8) and neurons (OMP), but not HBCs (CK5). Shown are confocal images; dotted lines represent basal lamina; scale bar = 20 μm except (D): 5 μm FIGURE 2 Open in new tabDownload slide CXCR4 expression in the neuronal lineage of precursor cells. Colabeling of (A) CXCR4, GBCs (SEC8), and HBCs (CK5) and (B) CXCR4, immature neurons (GAP43) and mature neurons (OMP), P8. C, Quantification of CXCR4 expression in different cells. (n = 3 animals, error bars represent SEM). D, Colocalization of CXCR4 with SOX2, c-KIT, ASCL1, NEUROD1 and GAP43, marking different stages of maturation. CXCR4 was almost not colocalized with CK5 in HBCs. E, OE of Krt14-Cre;CXCR4loxP;R26CAG-LSL-tdT mice (P8); tdTomato (red) expression under the control of the Krt14 promoter (CK14) did result in labeling of HBCs and sustentacular cells; Tomato expressing red cells were not labeled with CXCR4 or SEC8. Cxcr4 expression level was unaltered compared with the wild type (WT) epithelium. F, OE of Cxcr4CreER-IRES-GFP/Wt;R26CAG-LSL-tdT mice (P28) pulsed with Tamoxifen at P5. In these mice, GFP is expressed in GBCs, indicating Cxcr4 promoter activity, but not in neurons or neuronal progenitors. TdTomato, which is present progenies of the GFP expressing cells, did label GBCs (SEC8) and neurons (OMP), but not HBCs (CK5). Shown are confocal images; dotted lines represent basal lamina; scale bar = 20 μm except (D): 5 μm To confirm the notion that Cxcr4 expression is restricted to the neuronal lineage in the OE, we employed Cre-recombinase-mediated lineage tracing. First, we examined transgenic mice expressing Cre-recombinase under the control of the KRT14 promoter (Tg(KRT14-cre)1Amc). In Tg(KRT14-cre);R26CAG-LSL-tdT mice, tdTomato (tdT) labeled HBCs and sustentacular cells, but not GBCs or neuronal populations (Figure 2E). This observation is consistent with KRT14 being expressed in HBCs, and the default state of Krt14-positive resting HBCs to form sustentacular cells under homeostatic conditions.10 Although Wnt-dependent HBC activation could specify GBC neural progenitors under steady-state conditions, tdTomato signal did not overlap with CXCR4 (Figure S1). Moreover, KRT14Cre;Cxcr4loxP;R26CAG-LSL-tdT mice generated to abolish expression of the CXCR4 gene in HBCs revealed completely unaltered expression levels of CXCR4 in the OE (Figures 2E and S1). In addition, the number of GBCs, identified by SEC8-labeling, was unchanged. Next, we used a recently generated Cxcr4CreER(T2)-IRES-eGFP(Cxcr4CreER) knock-in allele26 to trace the Cxcr4 lineage in the OE. P28 Cxcr4CreER/Wt;R26CAG-LSL-tdT mice that received tamoxifen at P5 exhibited tdT-labeling in several neuronal precursor cells and in mature neurons (Figure 2F). While few microvillar cells, which originate from the same GBCs as the neurons,10 could also be detected, sustentacular cells did not express the marker protein. The IRES-eGFP construct, which was included in the Cxcr4CreER allele for visualization of cells presently expressing Cxcr4,26 recapitulated Cxcr4 expression in GBCs (Figure 2F). As expected, expression of dtT at detectable levels lagged behind the expression of green fluorescent protein (GFP), since GFP-positive GBCs were scarcely labeled with dtT. Since the CreER sequence disrupts the Cxcr4 coding region in the Cxcr4CreER allele, Cxcr4CreER/Wt mice represent a heterozygous Cxcr4 knockout model. Although heterozygous Cxcr4 deletion decreased CXCR4 levels in migrating interneurons,36 the level of CXCR4 protein in the OE was not altered in Cxcr4CreER/WT animals (Figures 2F and S1). Moreover, no morphologic abnormalities were obvious, and the number of SEC8-positive GBCs did not change compared with the WT OE (Figure 2F). As judged by expression of cell proliferation markers and lineage tracing, CXCR4-cells comprise actively proliferating progenitor cell types in the neuronal lineage. We moreover assume that Cxcr4CreER is a valid tool for fate-mapping in the OE. Lesion induces CXCR4 expression in the OE We next studied Cxcr4 expression in an injury-repair model of the OE. We experimentally destroyed the OE of adult (2 and 6 months old) mice by intraperitoneal methimazole injection,37,38 and analyzed the regeneration 1, 3, 14, and 28 dpi (Figure 3A). Quantitative PCR revealed that Cxcr4 mRNA increased markedly in the olfactory mucosa (composed of the OE and the underlying lamina propria) during regeneration (14 dpi), and returned to slightly elevated baseline levels at 28 dpi (Figure 3B). Histological assessment by in situ hybridization and immunostaining revealed a massive expansion of CXCR4-positive cells at 14 dpi (Figure 3C-E). Early in the regeneration phase (3 dpi), when HBCs start proliferating, few CXCR4-positive cells were present and expression of the mRNA was sparse. There was no difference between control animals without injection and animals injected with sodium chloride (data not shown). After the epithelium had almost regained its normal thickness at 28 dpi, the number of CXCR4-positive cells was only slightly elevated compared with the control conditions (Figure 3C,E). FIGURE 3 Open in new tabDownload slide CXCR4 during injury-induced regeneration. A, Experimental scheme. B, qPCR of CXCR4 from OE of 2 and 6 months old mice, three animals per group, three replicates each. C, Quantification of immunolabeled CXCR4-positive cells. D, In situ hybridization with 35S-labeled probes for CXCR4. CXCR4 mRNA was markedly increased at 14 dpi. CXCR4 sense-probe (not shown) did not show any signal. Scale bar = 50 μm. E-G, Confocal images of coronal OE sections (2 months). E, Upregulation of CXCR4 (green) during regeneration. F, Nonactivated (nonphosphorylated) CXCR4, only a small fraction of the receptor is present in intracellular compartments. G, CXCL12. Dotted lines represent basal lamina. Scale bar = 20 μm. Two-way ANOVA and Bonferroni post hoc test, error bars represent SEM, *P < .05 compared with controls FIGURE 3 Open in new tabDownload slide CXCR4 during injury-induced regeneration. A, Experimental scheme. B, qPCR of CXCR4 from OE of 2 and 6 months old mice, three animals per group, three replicates each. C, Quantification of immunolabeled CXCR4-positive cells. D, In situ hybridization with 35S-labeled probes for CXCR4. CXCR4 mRNA was markedly increased at 14 dpi. CXCR4 sense-probe (not shown) did not show any signal. Scale bar = 50 μm. E-G, Confocal images of coronal OE sections (2 months). E, Upregulation of CXCR4 (green) during regeneration. F, Nonactivated (nonphosphorylated) CXCR4, only a small fraction of the receptor is present in intracellular compartments. G, CXCL12. Dotted lines represent basal lamina. Scale bar = 20 μm. Two-way ANOVA and Bonferroni post hoc test, error bars represent SEM, *P < .05 compared with controls To figure out whether CXCR4 was not only upregulated, but also activated during regeneration we made use of the anti-CXCR4 antibody UMB-2 (CXCR4 [nonactivated]), which recognizes the C-terminal CXCR4 epitope only when the 346SSS348 cluster is not phosphorylated.39 Since phosphorylation of 346SSS348 occurs upon receptor stimulation, the UMB-2 signal is lost in activated receptors. Weak labeling with the UMB-2 antibody during regeneration indicated massive activation of the receptor (Figure 3F). Moreover, expression of the CXCR4 ligand CXCL12 was increased in the OE and in the lamina propria (Figure 3G). Major CXCR4 upregulation coincides with GBC expansion after injury During the first days after injury, HBCs initiate a transcriptional program to re-establish the barrier function, which includes upregulation of keratins. Accordantly, nearly all remaining cells expressed the HBC marker Cytokeratin 5 (CK5) (Figure 4A). Absence of MCM2 at 1 dpi indicates that the cell did not proliferate yet (at this early stage) (Figure 4B). At the same time, specification of committed progenitors starts with the upregulation of SOX2, a stem cell transcription factor of activated HBCs.11,40 At 1 dpi, we detected SOX2 and the GBC marker SEC8 in very few cells, but these few cells already coexpressed CXCR4 (Figure 4C). At 3 dpi, a major proportion of the cells was proliferating (ie, they were MCM2+), and more cells transitioned into SOX2-positive neuronal precursors (Figure 4B,C). Concomitantly, expression of the GBC marker SEC8 and CXCR4 increased (Figure 4D), showing that activated HBCs already transitioned into GBCs. Some CXCR4 positive early GBCs seem to have weak Krt5 labeling remaining, possibly because these cells emerged from the activated HBCs recently and still have small amounts of the HBC-specific protein. Few scattered CXCR4 expressing cells were SOX2-negative at 3 dpi, indicating that these cells already completed the transition into SOX2-negative GBCs. The colocalization of CXCR4 with SEC8 and SOX2 early during regeneration is consistent with the proposed role of CXCR4 in the neuronal lineage. FIGURE 4 Open in new tabDownload slide CXCR4 expression during regeneration coincides with the appearance of GBCs. Confocal images of coronal OE sections from 2 months old mice, 1 and 3 days after methimazole treatment. A, All cells expressed CK5 at this times points, few cells expressed CXCR4 at 3 dpi. B, Few cells started MCM2 expression at 1 dpi, at 3 dpi nearly all cells were MCM2-positive. C, Some cells started expressing SOX2 at 1 dpi, at 3 dpi nearly all cells were SOX2-positive. D, At 1 dpi, few cells started expressing Sec8; the number of SEC8-positive cells increased at 3 dpi. E, CXCR4 and MCM2 at 14 and 28 dpi. F, Quantification of CXCR4-positive cells coexpressing MCM2, most (~90%) CXCR4-positive cells were proliferating. G, CXCR4, SEC8 and CK5. H, Quantification of CXCR4-immunolabeled cells coexpressing SEC8. Nearly all (>90%) SEC8-positive GBCs coexpressed CXCR4. Colabeling with CK5 was absent. I, CXCR4 and GAP43. J, Quantification of CXCR4-immunolabeled cells coexpressing GAP43. Only few of the GAP43-positive immature neurons coexpressed CXCR4. K, Colabeling of CXCR4 with OMP was absent. Cell counts were analyzed using Two-way ANOVA and Bonferroni post hoc test; error bars represent SEM, *P < .05 compared with controls; n = 3 animals. Upper dotted lines represent the apical border of the epithelium; structures above this line are remnants and debries that are not yet washed off the surface of the epithelium. Lower dotted line represents the basal lamina. Scale bar = 5 μm (A-D), 20 μm (E, G, I) FIGURE 4 Open in new tabDownload slide CXCR4 expression during regeneration coincides with the appearance of GBCs. Confocal images of coronal OE sections from 2 months old mice, 1 and 3 days after methimazole treatment. A, All cells expressed CK5 at this times points, few cells expressed CXCR4 at 3 dpi. B, Few cells started MCM2 expression at 1 dpi, at 3 dpi nearly all cells were MCM2-positive. C, Some cells started expressing SOX2 at 1 dpi, at 3 dpi nearly all cells were SOX2-positive. D, At 1 dpi, few cells started expressing Sec8; the number of SEC8-positive cells increased at 3 dpi. E, CXCR4 and MCM2 at 14 and 28 dpi. F, Quantification of CXCR4-positive cells coexpressing MCM2, most (~90%) CXCR4-positive cells were proliferating. G, CXCR4, SEC8 and CK5. H, Quantification of CXCR4-immunolabeled cells coexpressing SEC8. Nearly all (>90%) SEC8-positive GBCs coexpressed CXCR4. Colabeling with CK5 was absent. I, CXCR4 and GAP43. J, Quantification of CXCR4-immunolabeled cells coexpressing GAP43. Only few of the GAP43-positive immature neurons coexpressed CXCR4. K, Colabeling of CXCR4 with OMP was absent. Cell counts were analyzed using Two-way ANOVA and Bonferroni post hoc test; error bars represent SEM, *P < .05 compared with controls; n = 3 animals. Upper dotted lines represent the apical border of the epithelium; structures above this line are remnants and debries that are not yet washed off the surface of the epithelium. Lower dotted line represents the basal lamina. Scale bar = 5 μm (A-D), 20 μm (E, G, I) At 14 dpi, we observed a massive increase in proliferation, re-establishment, and expansion of GBCs (14 dpi, Figures 4E-J, S2, and S3), together with a maximal induction of CXCR4 levels. The increased proliferation rate persisted until 4 weeks after injury, and the majority of the proliferative cells in the regeneration phase expressed CXCR4 (28 dpi, Figure 4E,F). Nearly all CXCR4-positive cells were GBCs, identified by SEC8 (Figure 4G,H). In contrast to the situation immediately after injury, CK5-expression was restricted to a single row of CXCR4-negative HBCs (Figure 4G). The immature neurons (GAP43) coexpressed CXCR4, but CXCR4 expression vanished once the cells had matured and started to express OMP (Figures 4I,J, S2, and S3). Together, expansion of GBCs for regeneration of the injured epithelium coincides with massive CXCR4 upregulation. Alteration of CXCR4 signaling by overexpression of CXCL12 The CXCL12-CXCR4 signaling pathway has key roles during development, and Cxcr4 knockout embryos die before birth due to defects in vascularization.41-43 Cxcr4 knockout animals are therefore not available for investigations on adult neurogenesis, and heterozygous knockout of Cxcr4 did not alter expression levels of the receptor (Figure 2F). We therefore aimed at generating inducible conditional Cxcr4 knockout mice by combining Cxcr4loxP and Cxcr4CreER (Cxcr4CreER/loxP;R26CAG-LSL-tdT). Tamoxifen application should induce Cxcr4 deletion and tdTomato signal in cells with an active Cxcr4 promoter, and thus generate traceable Cxcr4-deficient cells. Unexpectedly, we did not observe differences in Cxcr4 expression in the OE 4 weeks after TAM treatment (Figure S1), despite abolished CXCR4 expression in monocytes from these mice.26 This may be due to differences in the transcription and/or translation rates of Cxcr4 and Cre-recombinase and dynamic maturation of Cxcr4-expressing progenitors into CXCR4-negative neurons. In addition, KRT14Cre;Cxcr4loxP/loxP;R26CAG-LSL-tdT, as well as NesCreER;Cxcr4loxP/loxP;R26CAG-LSL-tdT animals, which express Cre under the control of the KRT14 and Nestin promoter, respectively, showed completely unaltered expression levels of Cxcr4 in the OE (Figure S1). Since we were not able to abolish Cxcr4 expression in olfactory progenitors, we sought to perturb CXCR4 signaling. To this end, we employed bacterial artificial chromosome (BAC) transgenic mice with additional Cxcl12 copies under the Cxcl12 promoter generating a CXCL12-red fluorescent protein (RFP) fusion protein.23 CXCL12-RFP was distributed in the basal part of the epithelium (Figure 5A), where CXCR4-expressing cells are located. PCR analysis revealed that the amount of CXCL12 mRNA in the OE was approximately doubled compared with WT animals (Figure 5B). Overexpressed CXCL12 slightly spread throughout the basal parts of the OE, whereas CXCL12 in WT mice was predominantly restricted to HBCs. A possible explanation is that the increased amount of the ligand overwhelms the capacity of CXCR4 in GBCs to internalize CXCL12, resulting in otherwise not detectable amounts of CXCL12 in the surroundings of the cells. FIGURE 5 Open in new tabDownload slide CXCL12 overexpression downregulates CXCR4. A, Confocal images of coronal OE sections of WT and CXCL12-RFP mice (P8) expressing RFP under the control of the Cxcl12 promoter. RFP immunolabeling showed distribution in the basal and central parts of the epithelium. B, qPCR for CXCL12 mRNA from OE (P8), n = 3 animals per group, Student's t-test, error bars represent SEM, *P < .05. C, CXCR4 antibody labeling showing lower amounts of CXCR4 in CXCL12-overexpressing mice (P8, P56). D, Quantification of CXCR4-positive cells in cryosections. (n = 3 animals per group, Student's t-test, error bars represent SEM, *P < .05). E and F, Western blots of OE showing CXCR4 (E) and nonactivated CXCR4 (UMB-2) protein levels. Intensities of the CXCR4 bands relative to Transferrin-receptor as control are given. (n = 3 animals per group, Student's t-test, error bars present SEM, *P < .05). G, Immunostaining of nonactivated CXCR4 (UMB-2) showing that the vast majority of the remaining receptors were activated. H, High magnification, CXCR4 (red), nonactivated CXCR4 (green), nonphosphorylated receptors were not present on the cell surface in CXCL12-RFP mice. I, In situ hybridization with digoxigenin-labeled probes for CXCR4 in CXCL12-RFP transgenic and WT mice. CXCR4 mRNA was markedly decreased in CXCL12-RFP transgenic mice compared with WT. Dotted lines represent basal lamina. Scale bar = 20 μm except (H) 5 μm FIGURE 5 Open in new tabDownload slide CXCL12 overexpression downregulates CXCR4. A, Confocal images of coronal OE sections of WT and CXCL12-RFP mice (P8) expressing RFP under the control of the Cxcl12 promoter. RFP immunolabeling showed distribution in the basal and central parts of the epithelium. B, qPCR for CXCL12 mRNA from OE (P8), n = 3 animals per group, Student's t-test, error bars represent SEM, *P < .05. C, CXCR4 antibody labeling showing lower amounts of CXCR4 in CXCL12-overexpressing mice (P8, P56). D, Quantification of CXCR4-positive cells in cryosections. (n = 3 animals per group, Student's t-test, error bars represent SEM, *P < .05). E and F, Western blots of OE showing CXCR4 (E) and nonactivated CXCR4 (UMB-2) protein levels. Intensities of the CXCR4 bands relative to Transferrin-receptor as control are given. (n = 3 animals per group, Student's t-test, error bars present SEM, *P < .05). G, Immunostaining of nonactivated CXCR4 (UMB-2) showing that the vast majority of the remaining receptors were activated. H, High magnification, CXCR4 (red), nonactivated CXCR4 (green), nonphosphorylated receptors were not present on the cell surface in CXCL12-RFP mice. I, In situ hybridization with digoxigenin-labeled probes for CXCR4 in CXCL12-RFP transgenic and WT mice. CXCR4 mRNA was markedly decreased in CXCL12-RFP transgenic mice compared with WT. Dotted lines represent basal lamina. Scale bar = 20 μm except (H) 5 μm Consistent with CXCL12 overexpression, the amount of nonactivated CXCR4 declined markedly (Figure 5G,F). Activation by CXCL12 induced CXCR4 internalization as indicated by a lower level of (nonstimulated) CXCR4 at the surface of GBCs (Figure 5G,H). Moreover, CXCL12 overexpression reduced the CXCR4 level in the OE (Figure 5F-I). This is based on Western blot showing a reduced protein level (Figure 5E) and immunohistology showing a reduced number of CXCR4-expressing cells (Figure 5C,D). Our findings in the OE are consistent with previous studies44,45 showing that increased levels of extracellular CXCL12 trigger CXCR4 activation, internalization, and degradation. In addition, we found that excessive CXCR4 stimulation also reduced Cxcr4 mRNA expression (Figure 5I). CXCR4 regulates proliferation and neuronal differentiation of GBCs Overexpression of CXCL12 caused reduced expression of Cyclin D1 (Figures 6A and S4), a key regulator of stem cell fate decision46 in GBCs. Consistent with the well-known function of Cyclin D1 in cell cycle progression,47 reduced expression of Cyclin D1 in olfactory progenitors lead to attenuated proliferation (MCM2, Figures 6B and S4). Accordingly, the total number of SEC8-positive GBCs was reduced in CXCL12-RFP-overexpressing mice (Figures 6C,D and S4). We did not observe an increase in the number apoptotic Caspase 3-positive cells, showing that the reduction in the number of GBCs did not result from premature cell death (Figure 6E). FIGURE 6 Open in new tabDownload slide Proliferation and differentiation of GBCs depends on CXCR4 signaling. Shown are confocal images of WT and CXCL12-RFP mice (P8). A, Cyclin D1, B, MCM2, C, SEC8 showing decreased numbers of GBCs and INPs due to CXCL12 overexpression. D, Quantification of the number of SEC8-positive cells (n = 3 animals, significance levels Student's t-test, error bars represent SEM, *P < .05). E, Labeling of apoptotic cells with Caspase 3 showing only few apoptotic cells in WT and CXCL12-RFP mice. F, Immature (GAP43), G, mature (OMP) olfactory neurons showing increased numbers of GBCs and INPs due to CXCL12 overexpression. H, Quantification of the number of OMP-positive neurons, n = 3 animals per group, Student's t-test, error bars represent SEM, *P < .05. I, Distance of basally located OMP-positive neurons to the basal lamina showing an expanded neuronal layer in CXCL12 overexpressing mice. (n = 3 animals per group, percentages based on OE thickness, Student's t-test, error bars represent SEM, *P < .05). J, Increased thickness of the ciliary layer (ACIII labeling) in CXCL12-overexpressing mice. K, Activated S6 (pS6) and L, activated CREB (pCREB). Dotted lines represent basal lamina. Scale bar = 20 μm; E, 5 μm FIGURE 6 Open in new tabDownload slide Proliferation and differentiation of GBCs depends on CXCR4 signaling. Shown are confocal images of WT and CXCL12-RFP mice (P8). A, Cyclin D1, B, MCM2, C, SEC8 showing decreased numbers of GBCs and INPs due to CXCL12 overexpression. D, Quantification of the number of SEC8-positive cells (n = 3 animals, significance levels Student's t-test, error bars represent SEM, *P < .05). E, Labeling of apoptotic cells with Caspase 3 showing only few apoptotic cells in WT and CXCL12-RFP mice. F, Immature (GAP43), G, mature (OMP) olfactory neurons showing increased numbers of GBCs and INPs due to CXCL12 overexpression. H, Quantification of the number of OMP-positive neurons, n = 3 animals per group, Student's t-test, error bars represent SEM, *P < .05. I, Distance of basally located OMP-positive neurons to the basal lamina showing an expanded neuronal layer in CXCL12 overexpressing mice. (n = 3 animals per group, percentages based on OE thickness, Student's t-test, error bars represent SEM, *P < .05). J, Increased thickness of the ciliary layer (ACIII labeling) in CXCL12-overexpressing mice. K, Activated S6 (pS6) and L, activated CREB (pCREB). Dotted lines represent basal lamina. Scale bar = 20 μm; E, 5 μm Reduced expression of Cyclin D1 causes lengthening of G1, and thereby triggers premature neurogenesis and decreases the generation and expansion of progenitor cells in the central nervous system.46 We therefore analyzed differentiation of GBCs into immature and mature olfactory neurons. GAP43 expression, as a marker for immature olfactory neurons, did not show major differences between WT and CXCL12-RFP-overexpressing mice (Figures 6F and S4). On the other hand, the number of OMP-expressing neurons was significantly increased in the mutants, and the neuronal cell bodies were shifted toward the basal lamina (Figures 6G-I and S4), possibly caused by the expansion of the population. Olfactory neurons express various ciliary signaling proteins, which are important for odorant sensing, among them adenylate cyclase type III (ACIII). We found the ACIII-positive ciliary layer was expanded in CXCL12-RFP mice, indicating an increased density of cilia covering the epithelial surface (Figures 6J and S4). Alternative markers for olfactory cilia such as Gαolf and cyclic nucleotide-gated channel alpha 2 channel were also increased in CXCL12-RFP mice (data not shown), showing that the underlying cause was the increase in the number of mature neurons rather than differences in ciliogenesis. CXCR4 signaling cascade Since CXCL12 overexpression markedly affected stem cell proliferation and neuronal differentiation, we next investigated the signaling cascade. CXCR4 signaling can involve activation of the mitogen activated protein kinases (MAPK) extracellular signal regulated kinases 1/2 (ERK1/2), but we did not observe any obvious differences in phosphorylated ERK1/2 (data not shown). CXCR4 may also signal via the phosphoinositide-3-kinase (PI3K)/serine threonine protein kinase B (Akt)/mechanistic target of Rapamycin (mTOR) pathway, a key transducer of signals that drive proliferation and inhibit differentiation of adult hippocampal neural progenitors. We therefore assayed phosphorylated S6 (pS6) as part of this signaling pathway: activated mTOR phosphorylates and activates 70S6K, which in turn phosphorylates S6, a component of the 40S ribosomal subunit.48 We found that CXCL12-RFP animals had markedly lower levels of pS6 at P8 and at P56 (Figures 6K and S4G), indicating reduced mTOR signaling. In addition, PI3K/Akt signaling stimulates cAMP response element-binding protein (CREB) in adult hippocampal neural progenitor cells,49 which affects cell proliferation, neurite outgrowth, and differentiation.50 Consistent with the reduced pS6 levels, we also observed decreased phosphorylation of CREB upon CXCL12 overexpression (Figures 6L and S4H). Both results together indicate reduced PI3K signaling, which points toward reduced CXCR4 signaling upstream of PI3K. The effects of the transgene is broader than the normal pattern of CXCR4 and extends through larger parts of the epithelium. The underlying cause has to await further experimentation, but could involve altered maturation of neuronal lineage cells due to loss of CXCR4-induced signaling. Other possible reasons may be that excess CXCL12, which is not complete internalized by GBCs, may influence progenitors and immature neurons due to very low expression levels of CXCR4, which escape here used nonamplified immunofluorescence detection. Overexpression of CXCL12 mimics knockout of Cxcr4 The reduced activation of signaling proteins as well as the reduced Cyclin D1 expression point toward reduced CXCR4 signaling in the OE of CXCL12-overexpressing mice. This may be the consequence of excessive CXCR4 activation causing internalization, degradation, and desensitization of CXCR4.44,45 To test this assumption, we compared CXCL12-overexpressing to Cxcr4-deficient Cxcr4CreER/CreER mice. Since Cxcr4-deficiency is lethal before birth, we assessed the OE at embryonic day 18 (E18). CXCR4 signal was fully absent in Cxcr4CreER/CreER and markedly reduced in CXCL12-RFP mice compared with WT (Figure 7A). This indicates that Cxcr4CreER/CreER mice are a valid Cxcr4-deficiency model and that E18 CXCL12-RFP mice recapitulate the loss of CXCR4 present in postnatal CXCL12-RFP mice. Moreover, and very similar to postnatal mice, E18 CXCL12-RFP mice showed reduced expression of Cyclin D1, lower numbers of GBCs (SEC8) and increased numbers of mature neurons (OMP), which was recapitulated in E18 Cxcr4CreER/CreER mice (Figure 7B-F). In addition, the OE was thicker in both, E18 Cxcr4-deficient and E18 CXCL12-overexpressing mice compared with age-matched WT mice (Figure 7G). In conclusion, increased neuronal differentiation and decreased proliferation of stem cells in the OE of postnatal CXCL12-overexpressing mice most likely originates from reduced CXCR4 signaling. FIGURE 7 Open in new tabDownload slide CXCL12 overexpression mimics Cxcr4 knockout. Immunolabeling of coronal OE sections of late embryonic WT, Cxcr4 knockout (Cxcr4CreEr/CreER) and CXCL12-overexpressing mice (E18), confocal images. A, CXCR4, B, Cyclin D1, C, GBCs (SEC8), D, immature neurons (GAP43), and E, mature neurons (OMP). Staining patterns for E18 mice are similar to P8; Cxcr4 knockout and CXCL12-overexpression results in very similar phenotypes. Dotted lines represent basal lamina. Scale bar = 20 μm. F, Quantification of the number of mature neurons, increased numbers for Cxcr4 knockout and CXCL12-overexpression. G, Quantification of the thickness of the OE showing increased thickness due to Cxcr4 knockout and CXCL12-overexpression. n = 3 animals per group, Student's t-test, error bars represent SEM, *P < .05 FIGURE 7 Open in new tabDownload slide CXCL12 overexpression mimics Cxcr4 knockout. Immunolabeling of coronal OE sections of late embryonic WT, Cxcr4 knockout (Cxcr4CreEr/CreER) and CXCL12-overexpressing mice (E18), confocal images. A, CXCR4, B, Cyclin D1, C, GBCs (SEC8), D, immature neurons (GAP43), and E, mature neurons (OMP). Staining patterns for E18 mice are similar to P8; Cxcr4 knockout and CXCL12-overexpression results in very similar phenotypes. Dotted lines represent basal lamina. Scale bar = 20 μm. F, Quantification of the number of mature neurons, increased numbers for Cxcr4 knockout and CXCL12-overexpression. G, Quantification of the thickness of the OE showing increased thickness due to Cxcr4 knockout and CXCL12-overexpression. n = 3 animals per group, Student's t-test, error bars represent SEM, *P < .05 DISCUSSION The OE is a unique model to study adult neurogenesis, since replacement of neurons takes place constantly throughout life.1,2 Two neurocompetent stem cell populations exist. GBCs are heterogeneous progenitors, which repopulate cells lost to routine turnover,6 HBCs build a reserve population of mitotically quiescent cells adherent to the basal lamina.34 Following acute injury, HBCs release from the basal lamina, proliferate, and differentiate into GBCs, which then replace cells that were damaged due to lesion.7 Despite a wealth of knowledge on the different transcription factors controlling self-renewal and differentiation of stem cells, only little knowledge exists about the external factors regulating stem cell proliferation and differentiation. In the present study, we employed in vivo CXCR4 manipulation to show that chemokine signaling modulates maintenance and repair neurogenesis in the postnatal OE (summarized in Supporting Information Figure S5). CXCR4 is expressed in neuronal stem cells and immature granule neurons in neurogenic regions of the adult central nervous system.18,51,52 Stem cells in the subgranular zone of the dentate gyrus proliferate and differentiate into dentate granule cells, and CXCR4 signaling maintains this stem cell pool and regulates normal placement of immature granule neurons.20 Cells in the subventricular zone migrate down the rostral migratory stream into the olfactory bulb, where they switch to radial migration toward their final destination. CXCR4 activation increases this radial migration53 in a PI3K/Akt and MAPK dependent manner.54,55 We report here that Cxcr4 expression levels are notably stronger in GBCs, the proliferating neuronal precursor cell of the postnatal OE, compared with hippocampal stem cells. High expression levels of CXCR4 could be one reason for the well-known high rates of neurogenesis in the OE. In the dentate gyrus, environmental enrichment increased the proliferation, differentiation and survival of newly-formed neurons and enhanced the CXCR4 expression levels,56 and treatment with the CXCR4 antagonist AMD3100 revealed that this increase in neurogenesis depends on CXCR4.57 Colabeling and genetic lineage tracing revealed that Cxcr4 is exclusively expressed in all cells of the neuronal lineage. Cxcr4 seems to be expressed in the same type of neuronal stem cell as leucine-rich repeat-containing G-protein coupled receptor 5 (Lgr5),58 since progenies of Cxcr4 and Lgr5-expressing precursor cells are very similar. Interestingly, coexpression of both receptors also occurs in other types of stem cells, such as in gastric epithelial stem cells59 and stem-like cells of colorectal cancer.60 After methimazole-induced injury, otherwise quiescent HBCs are activated and generate all cellular subtypes of the OE. CXCR4 expression, under steady state conditions restricted to GBCs, starts in a subpopulation of injury-activated CK5-positive HBCs shortly after injury, at the time point of HBC decision to self-renew or to differentiate.11 OE lesion induced coexpression of marker proteins in both types of basal cells, HBCs and GBCs, has already been described.61 However, CXCR4 expression in HBCs is not long lasting, since we did not observe apparent CXCR4 colocalization with HBC markers at 14 or 28 days after injury, similar as in homeostatic conditions. Early CXCR4-expressing cells coexpress SEC8 and SOX2, a transcription factor that plays an essential role in the genesis of the neuronal lineage.11,40 It is tempting to speculate that the early CXCR4 activation directs the path for generating new GBCs. In addition, the ligand of CXCR4, CXCL12, was markedly upregulated during early phases of regeneration, resulting in massive activation of the receptor. This resembles the situation in the CNS after brain injury, where CXCL12 is upregulated and stimulates stem cell proliferation and progenitor migration to the ischemic area.62 Similar to what we observed after the OE injury, the amount of CXCL12 in the injured brain increased first, followed by an increase in the levels of CXCR4 mRNA and protein.63 Also under steady state conditions, the proliferating rate of stem cells in the subventricular zone depends on expression levels of CXCL12 in the vasculature.64 Ongoing regeneration and reconstruction of the epithelium after lesion coincides with a massive expansion of CXCR4-positive proliferating GBCs. Similar as in the homeostatic conditions, CXCR4 massively declined during neuronal differentiation and was absent in mature OMP-positive neurons. Notch1 signaling could be involved in driving Cxcr4 expression, since Notch1 has been shown to control GBC proliferation during injury-induced regeneration of the OE,9,65 and is known to increase the mRNA of Cxcr4 in tumor cells.66,67 Activation of Cxcr4 transcription could also be regulated by SOX11, a transcription factor enriched in GBCs and INPs,10 since SOX11 binds to regulatory regions of CXCR4 and directly upregulates Cxcr4 expression in lymphoma cells68 and in in mesenchymal stem cells.69 Investigation of CXCR4-dependent adult neurogenesis is not possible in animals with a global knockout of Cxcr4, since genetic deletion of the Cxcr4 gene results in embryonic lethality.41,42 Unexpectedly, several attempts to generate an OE-specific Cxcr4 knockout using various Cre Driver lines (Cxcr4CreER/loxP;R26CAG-LSL-tdT, KRT14Cre;Cxcr4loxP/loxP;R26CAG-LSL-tdT, NesCreER;Cxcr4loxP/loxP;R26CAG-LSL-tdT) were not successful. We therefore investigated mice overexpressing the ligand CXCL12.70 in vivo short term application of CXCL12 has recently been described to lead to increased CXCR4 signaling.71 On the other hand, continuous treatment with the ligand CXCL12 reduced the CXCR4 level in cortical interneurons,45 and CXCL12 accumulation triggers CXCR4 endocytosis and degradation, with defects in CXCR4-mediated functions.44 Moreover, CXCL12 overexpression in vivo caused loss of CXCR4 by lysosomal degradation and resulted in similar interneuron migration defects as Cxcl12 deficiency.28 In parallel to these observations, CXCL12-overexpressing mice showed markedly reduced CXCR4 protein and mRNA levels in the OE. The comparison of the OE of embryonic Cxcr4 deficient and CXCL12-overexpressing mice allowed us to conclude that the phenotypes caused by CXCL12 overexpression raised from reduced CXCR4 signaling. Similar as in other tissues, overexpression of CXCL12 results in a Cxcr4 knockout phenotype. CXCR4 expression also plays an important role for proliferation and differentiation of cultured adult neural progenitor cells from the subventricular zone and expression levels decreased concomitantly with lineage progression.72 This is consistent with the situation in the adult dentate gyrus, where loss of CXCR4 decreases the proliferation of stem cells and the production of progenitor cells.20,73 In the OE, CXCR4 downregulation also leads to a reduction of the number of proliferative stem cells and enhanced differentiation of mature neurons, suggestive of precocious maturation of GBCs. Maintenance of stemness of OE cells by CXCR4 signaling involves regulation of Cyclin D1 expression, since CXCR4 downregulation leads to reduced Cyclin D1 expression in basal cells. Cxcr4 mutant zebrafish were also found to exhibit poor expression of Cyclin D1, which functions to accelerate the G1/S transition to promote proliferation of dorsal forerunner cells.74 Moreover, increased Cyclin expression in cancer cells occurs upon CXCL12/CXCR4 signaling via activation of the PI3K signaling pathway, which then induces cell proliferation and invasion.75 Cyclin D1 is required for progression through the G1 phase of the cell cycle, which plays a decisive role in the regulation of cell proliferation and differentiation.76 The length of the G1 phase has been shown to determine the cellular fate of neuronal stem cells, and prolonging of the G1 phase can induce the cells into a neurogenic lineage.77 In the hippocampus, overexpression of CyclinD1 induced a constant expansion of neuronal progenitor cells, while suppressing neurogenesis.78 The CXCR4 signaling pathway clearly regulates the homeostasis between progenitor cell proliferation and differentiation in OE adult neurogenesis. Interestingly, a significant reduction in the expression of CXCR4 and its ligand CXCL12 occurs in the OE in patients with schizophrenia.79 Aberrant neurogenesis was proposed to contribute to the pathogenesis of schizophrenia, and olfactory dysfunctions are common in patients early in the course of the illness.80 The details of the CXCR4 mediated signaling cascade remain to be determined, but we observed a downregulation of CREB and S6 phosphorylation in the OE of CXCL12-RFP mice, consistent with reduced signaling of CXCR4. In the hippocampus of rats, CXCR4 expression also showed positive correlation with pCREB levels.56 CREB phosphorylation could be reduced due to lowered activation of AKT/protein kinase B downstream of phosphatidylinositol 3-kinase (PI3K).81 Reduced PI3K signaling would also explain the lowered levels of phosphorylated S6 protein, a component of the 40S ribosomal subunit48 and downstream target of 70S6K, a canonical effector mTOR signaling. Although S6 phosphorylation occurs in the mouse OE also following odor stimulation,82 differences in odorant signaling are unlikely since all animals were kept under identical conditions. Moreover, we did not observe differences in expression levels of Kirrel2, another activity marker of olfactory neurons. CONCLUSION With its relative simplicity and experimental accessibility, the postnatal OE provides an attractive system for studying the activation and specification events that occur during the differentiation of multiple cell lineages from an adult stem cell. This work describes the roles of CXCR4 signaling in maintaining normal cellular turnover as well as mediating restoration of the injured OE. We thereby provide new insight into the mechanisms underlying the maintenance or differentiation of progenitor/stem cells. Due to well-developed pharmacological tools to influence in situ CXCR4/CXCL12 signaling, these results may also be helpful to support expansion of cultured OE stem cells for transplantation therapies. ACKNOWLEDGMENTS We thank Christine Anders, Helga Bechmann, and Stefan Bechmann for excellent technical assistance. Silke Keiner and Otto Witte (Dept. Neurology, Jena University Hospital) provided Nestin-CreER mice; Praveen Ashok Kumar, Yves Werner, and Dagmar Schütz (Dept. Pharmacology, Jena University Hospital) provided Cxcr4CreER mice. This work was supported by the Deutsche Forschungsgemeinschaft (DFG). CONFLICT OF INTEREST The authors declared no potential conflicts of interest. AUTHOR CONTRIBUTIONS K.S.: conception and design, data collection, analysis and interpretation, manuscript writing, read and approved the final manuscript; J.K.: data collection and analysis, read and approved the final manuscript; R.S.: provision of study material, data interpretation, manuscript review, read and approved the final manuscript; E.M.N.: conception and design, data analysis and interpretation, manuscript writing, financial support, read and approved the final manuscript. DATA AVAILABILITY STATEMENT The data that support the findings of this study are available on request from the corresponding author. REFERENCES 1 Mackay-Sim A , Kittel P. Cell dynamics in the adult mouse olfactory epithelium: a quantitative autoradiographic study . J Neurosci . 1991 ; 11 : 979 - 984 . Google Scholar Crossref Search ADS PubMed WorldCat 2 Graziadei PP , Graziadei GA. Neurogenesis and neuron regeneration in the olfactory system of mammals. I. 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Google Scholar Crossref Search ADS PubMed WorldCat Abbreviations ACIII adenylate cyclase type III AKT serine threonine protein kinase B Ascl1 achaete-scute family BHLH transcription factor 1 BSA bovine serum albumin cAMP cyclic adenosine monophosphate CK5 cytokeratin 5 CREB cAMP response element-binding protein CXCL12 CXC-motif chemokine ligand 12 CXCR4 CXC-motif chemokine receptor 4 ddCt delta delta threshold cycle dpi days post injury EDTA ethylenediaminetetraacetitc acid ERK1/2 extracellular-signal regulated kinases 1/2 GAP43 growth associated protein 43 GAPDH glyceraldehyde 3-phosphate dehydrogenase GBC globose basal cell GFP green fluorescent protein Gαolf G-protein subunit alpha of olfactory sensory neurons HBC horizontal basal cell ICAM1 intercellular adhesion molecule 1 INP immediate neuronal progenitor cells KRT14 cytokeratin 14 LGR5 leucine-rich repeat-containing G-protein coupled receptor 5 MAPK mitogen-activated protein kinase MCM2 minichromosome maintenance complex component 2 mTOR mechanistic target of rapamycin NeuroD1 neurogenic differentiation 1 OE olfactory epithelium OMP olfactory marker protein P postnatal day p probability, significance value Pax6 paired box 6 PBS phosphate buffered saline PCR polymerase chain reaction PFA paraformaldehyde PI3K phosphoinositide 3-kinase pS6 phosphorylated S6 RFP red fluorescent protein SEC8 exocyst complex component 8 SOX2 sex determining region Y-box 2 SSC tri-sodium-citrate-dihydrate TBS tris buffered saline WT wild type Author notes Funding information Deutsche Forschungsgemeinschaft, Grant/Award Number: 391445343 ©2021 The Authors. 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TI - Chemokine signaling is required for homeostatic and injury-induced neurogenesis in the olfactory epithelium
JF - Stem Cells
DO - 10.1002/stem.3338
DA - 2021-05-01
UR - https://www.deepdyve.com/lp/oxford-university-press/chemokine-signaling-is-required-for-homeostatic-and-injury-induced-AWVb72GECm
SP - 617
EP - 635
VL - 39
IS - 5
DP - DeepDyve
ER -