Defects in neural stem cell proliferation and olfaction in Chd7 deficient mice indicate a mechanism for hyposmia in human CHARGE syndrome - PubMed (original) (raw)

. 2009 Jun 1;18(11):1909-23.

doi: 10.1093/hmg/ddp112. Epub 2009 Mar 11.

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Defects in neural stem cell proliferation and olfaction in Chd7 deficient mice indicate a mechanism for hyposmia in human CHARGE syndrome

W S Layman et al. Hum Mol Genet. 2009.

Abstract

Mutations in CHD7, a chromodomain gene, are present in a majority of individuals with CHARGE syndrome, a multiple anomaly disorder characterized by ocular Coloboma, Heart defects, Atresia of the choanae, Retarded growth and development, Genital hypoplasia and Ear anomalies. The clinical features of CHARGE syndrome are highly variable and incompletely penetrant. Olfactory dysfunction is a common feature in CHARGE syndrome and has been potentially linked to primary olfactory bulb defects, but no data confirming this mechanistic link have been reported. On the basis of these observations, we hypothesized that loss of Chd7 disrupts mammalian olfactory tissue development and function. We found severe defects in olfaction in individuals with CHD7 mutations and CHARGE, and loss of odor evoked electro-olfactogram responses in Chd7 deficient mice, suggesting reduced olfaction is due to a dysfunctional olfactory epithelium. Chd7 expression was high in basal olfactory epithelial neural stem cells and down-regulated in mature olfactory sensory neurons. We observed smaller olfactory bulbs, reduced olfactory sensory neurons, and disorganized epithelial ultrastructure in Chd7 mutant mice, despite apparently normal functional cilia and sustentacular cells. Significant reductions in the proliferation of neural stem cells and regeneration of olfactory sensory neurons in the mature Chd7(Gt/+) olfactory epithelium indicate critical roles for Chd7 in regulating neurogenesis. These studies provide evidence that mammalian olfactory dysfunction due to Chd7 haploinsufficiency is linked to primary defects in olfactory neural stem cell proliferation and may influence olfactory bulb development.

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Figures

Figure 1.

Figure 1.

Chd7 Gt/+ mice have severely impaired olfaction. (A) Electro-olfactogram tracings from adult wild-type and Chd7 Gt/+ mice. The olfactory epithelium from each mouse was exposed to four different concentrations of amyl acetate. (B) Histogram of electro-olfactogram responses from wild-type (open bars; n = 8) and Chd7 Gt/+ (solid bars; n = 7) mice shows four different concentrations of amyl acetate and five additional odorants tested at 10−3

m

. Each response was normalized and expressed as a percentage of a pure amyl acetate pulse given during the same trace. AA, amyl acetate; 8-AL, octanal; 7-AL, haptaldehyde; 6-AL, hexanal; Eug, eugenol; Car, carvone. *P < 0.05, **P < 0.005 and ***P < 0.001 as determined by unpaired Student’s _t_-test.

Figure 2.

Figure 2.

Chd7 is expressed in developing and mature olfactory tissues. X-gal staining shows Chd7 expression in the embryonic olfactory epithelium (A) and bulb (B). Immunofluorescence with anti-CHD7 shows Chd7 expression in embryonic E12.5 olfactory epithelium (C and D) and bulb (E and F) and in the adult olfactory epithelium (G–L). Chd7 expression in the adult olfactory epithelium is regionally variable (G and H), with most CHD7-positive cells present in the basal and apical regions of the olfactory epithelium (I and J). A few regions of the olfactory epithelium contain CHD7-postive cells which span the olfactory epithelium (basal to apical), typically in crypt regions where the epithelium undergoes acute turns in orientation (K and L). White dotted lines in (I–L) indicate apical and basal surfaces of the epithelium.

Figure 3.

Figure 3.

Chd7 is expressed in basal stem cells in the mature olfactory epithelium. The adult olfactory epithelium from wild-type and Chd7 Gt/+ mice is stained with anti-CHD7 and antibodies against OMP (A and B), Mash1 (C and D), NeuroD (E and F), Reep6 (G and H) and Sus1 (I and J). CHD7-positive cells in the apical and basal domains do not colocalize with anti-OMP (A and B). White dotted lines in (C–F) indicate the apical and basal surfaces of the epithelium. CHD7-positive cells express the pro-neuronal basal stem cell marker Mash1 and the immature olfactory sensory neuron marker NeuroD. CHD7-positive cells colocalize with nuclei of the sustentacular cell markers Reep6 and Sus1 (I and J). Arrow (I) indicates a CHD7-positive/Sus1-positive cell with a microvillar-like cell morphology.

Figure 4.

Figure 4.

Chd7 mutant mice have olfactory bulb hypoplasia. Brains were dissected and imaged from 6-week-old Chd7 +/+ (n = 5) and Chd7 Gt/+ mice (n = 5) (A and B). Measurements were taken of the brain (b), telencephalon (t), olfactory bulb length (l) and olfactory bulb width (w). Chd7 Gt/+ mice have significantly smaller brains, telencephalons and olfactory bulb lengths compared with wild-type littermates (I). Significant differences in olfactory bulb length taken as a percent of the brain and telencephalon indicate that Chd7 mutant mice (closed bars) have olfactory bulb hypoplasia compared with wild-type littermates (open bars) (J). Cross-sections of the olfactory bulbs stained with hematoxylin and eosin (C and D) show no difference in the layer composition of Chd7 Gt/+ olfactory bulbs. Anti-OMP in the olfactory bulb stains the glomeruli in both wild-type and Chd7 Gt/+ mice (E and F). Dopaminergic interneurons surrounding the glomeruli express tyrosine hydroxylase (TH) in response to electrical activity from olfactory sensory neurons in wild-type mice, whereas TH label is decreased in Chd7 Gt/+ mice (G and H). *P < 0.05 and **P < 0.01 as determined by unpaired Student’s _t_-test. Abbreviations: GL, glomerular layer; ePL, external plexiform layer; MCL, mitral cell layer and GCL, granule cell layer.

Figure 5.

Figure 5.

Olfactory sensory neurons and sustentacular cells are physiologically intact in early postnatal Chd7 Gt/+ mice. Odor-evoked calcium responses are similar in postnatal day 3 wild-type (n = 4) and Chd7 Gt/+ (n = 4) mouse olfactory epithelial slices. Representative confocal images (A–C) from a single fluo-4 AM-loaded Chd7 Gt/+ mouse olfactory epithelial slice (A) before, (B) during or (C) after odorant application. Representative responding neurons are outlined in (A–C). Average time course data (D) were generated from recordings of 22 wild-type and 23 Chd7 Gt/+ neurons imaged over six slices. No significant difference was observed between wild-type and Chd7 Gt/+ mice as determined by an unpaired Student’s _t_-test. Histogram representation of the average peak odor-evoked response (E and F) of the neurons measured in (D). Glutathione _S_-transferase activity (G) was examined in 6-week-old wild-type (closed circles, n = 3) and Chd7 Gt/+ (closed squares, n = 3) mice. A representative pair of 6-week-old wild-type and Chd7 Gt/+ littermates showed no significant difference in sustentacular cell activity as determined by an unpaired Student’s _t_-test. Adenylyl cyclase activity was examined in the presence or absence of 10 µ

m

forskolin (H) in 6-week-old wild-type (n = 8) and Chd7 Gt/+ (n = 8) littermate mice. No significant difference was observed in either the basal or forskolin-stimulated cyclase activity as determined by an unpaired Student’s _t-_test. Data are presented as the mean ± SEM from three separate experiments, where each condition was tested in duplicate. ***P < 0.001 as measured by one-way ANOVA.

Figure 6.

Figure 6.

Components of the olfactory cilia are intact in Chd7 Gt/+ mice. Olfactory cilia proteins were labeled in wild-type and Chd7 Gt/+ mice by immunofluorescence with anti-acetylated α-tubulin (A–D), anti-Gγ13 (E and F) and anti-adenylyl cyclase III (G and H). Regional decreases in immunofluorescence label were consistently observed in Chd7 Gt/+ mice for all cilia markers, as represented by acetylated α-tubulin label (A and B). White dotted lines in (C–F) indicate the basal surface of the epithelium. Immunofluorescence using antibodies against cilia proteins indicated that although regional decreases in label existed, all cilia components analyzed were present in Chd7 Gt/+ olfactory cilia compared with wild-type littermates.

Figure 7.

Figure 7.

Olfactory sensory neurons in Chd7 Gt/+ mutant mice are disorganized and reduced in number. Scanning electron micrographs of the olfactory epithelium show loss of the orderly arrangement of olfactory sensory neurons in (B) Chd7 Gt/+ mice (n = 4) compared with (A) wild-type littermates (_n_=4). Olfactory cilia are present in wild-type and Chd7 Gt/+ mice; however, Chd7 Gt/+ mice have variable distribution of cilia on the apical surface (C and D). Transmission electron micrographs from Chd7 Gt/+ mice (n = 3) compared with wild-type littermates (n = 3) show that olfactory sensory neurons from Chd7 Gt/+ mice properly extend dendrites to the apical surface and have cilia which project along the nasal mucosa (E and F). Light microscopy of olfactory epithelial tissues processed for TEM (G and H) shows a reduction in olfactory sensory neurons. Cell counts of TEM tissue sections (I) show a significant reduction in Chd7 Gt/+ (closed bars) olfactory sensory neurons and basal cells compared with wild-type (open bars) littermates *P < 0.05 and ***P < 0.001 as determined by unpaired Student’s _t_-test. Abbreviation: OSNs, olfactory sensory neurons.

Figure 8.

Figure 8.

Olfactory epithelial basal cell proliferation is reduced in Chd7 Gt/+ mice. Adult wild-type and Chd7 Gt/+ mice were exposed to BrdU 30 min prior to tissue collection, then processed for double immunofluorescence with anti-BrdU and anti-CHD7. Many CHD7-positive basal cells are actively dividing, based on co-localization between anti-CHD7 and anti-BrdU (A–D). Chd7 is highly expressed in crypt regions (marked by arrows) which also appear to be regions of high proliferation in the olfactory epithelium (A and B). White dotted lines in (C and D) indicate the apical surface of the epithelium. Cell counts (wild-type=open bars, Chd7 _Gt/+_=closed bars) show a 50% reduction in the number of BrdU-positive cells in the Chd7 Gt/+ olfactory epithelium and a 50% reduction in the number of all CHD7-positive cells (basal and apical). *P < 0.05 and ***P < 0.001 as determined by unpaired Student’s _t-_test.

Figure 9.

Figure 9.

Chd7 Gt/+ mice exhibit altered regeneration of olfactory sensory neurons in the olfactory epithelium after chemical ablation. A 6-week-old wild-type (n = 9) and Chd7 Gt/+ mice (n = 8) were given an intranasal infusion of 1% Triton X-100 in saline then sacrificed 2, 4 or 8 weeks later. The olfactory epithelium at 4 weeks post-ablation (A and B) shows olfactory sensory neurons in both wild-type (n = 4) and Chd7 Gt/+ (n = 4) littermates, with some disorganization and reduced OMP label. At 8 weeks post-ablation (C–F), wild-type mice (n = 4) exhibit a normal appearing olfactory epithelium, whereas Chd7 Gt/+ mice (n = 4) have regionally restricted OMP staining and OSN regeneration.

Figure 10.

Figure 10.

Diagram of the Chd7 mutant mouse olfactory epithelium. Wild-type (A) and Chd7 mutant (B) mouse olfactory epithelium is depicted with all of the main cell types including olfactory sensory neurons (blue), sustentacular cells (dark gray), globose basal cells (GBCs; purple), horizontal basal cells (HBCs; pink) and microvillar cells (white). Chd7 mutant mice have ∼30% fewer olfactory sensory neurons and 50% fewer GBCs compared with wild-type. Red bars indicate regions of normal cilia density (solid) and sparse cilia density (dashed). Chd7 mutants have fewer olfactory sensory neurons, which causes gaps between the olfactory sensory neurons and a loss of support leading to a disorganized appearance. Abbreviation: OSNs, olfactory sensory neurons.

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