Here, we have shown that both humans and mice with CHD7 deficiency have impaired olfaction. We observed Chd7 expression during development in restricted cell types of the olfactory epithelium and olfactory bulb, and Chd7 expression in the adult mouse olfactory epithelium in proliferating basal cells and in pro-neuronal basal cells. We also found that Chd7 deficient mice have a significant reduction in basal cell proliferation, which translates into a reduction in both basal cells and olfactory sensory neurons. Figure depicts a diagram of the olfactory epithelium in both wild-type and Chd7 mutant mice. The reduction in olfactory sensory neurons caused by Chd7 deficiency leads to regional variability in cilia density and disorganization of the Chd7 mutant olfactory epithelium, which may influence tight junctions required for signal transduction. Additionally, Chd7 deficient mice have a reduced capacity for regeneration of olfactory sensory neurons following chemical ablation of the olfactory epithelium. These data together suggest a critical role for CHD7 in olfactory tissues not only during development but also into adulthood.
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 (more ...)
Our data provide the first evidence that CHD7 functions in cellular proliferation and neuronal differentiation in the olfactory epithelium. The mechanism by which CHD7 regulates cell cycle progression and stem cell differentiation is not yet understood. DNA chromatin structure has a vital role in gene regulation, cellular proliferation and maintenance of the differentiated state. However, little is known about the cell and tissue specific functions of chromodomain proteins. Protein–protein interactions involving cell cycle progression and gene expression have been reported for some CHD family members (50
). Proteins such as histone deacetylases and nuclear receptor corepressor 1 are involved in protein interactions with CHD proteins (50
) and are also known to be critical for neuronal differentiation (57
). In a recent study using ChIP–chip analysis, CHD7 was shown to bind to methylated histone H3 K4 enhancer regions of numerous genes in the mammalian genome (59
). These data suggest a role for CHD7 in regulating transcription, potentially affecting multiple developmental processes during cell fate specification.
CHD7 and other chromodomain proteins are also thought to regulate access to chromatin by binding and unwinding it (25
). CHD7 could work in conjunction with other transcription factors involved in cell cycle progression and neuronal differentiation. Otx2, a paired-like homeodomain transcription factor, is critical for normal cellular proliferation and neuronal differentiation (60
mutant mice lack the pro-neuronal transcription factor Mash1
). Mash1 induces the expression of later bHLH transcription factors like Ngn1
, driving cell cycle exit and neuronal differentiation (33
). We found that CHD7 colocalizes with BrdU-positive proliferating cells and with Mash1 and NeuroD in basal cells. Since Otx2 is involved in cellular proliferation and induces the expression of Mash1 and NeuroD, CHD7 may be necessary for access to Otx2 target genes in the olfactory epithelium. Our data indicate that Chd7
deficiency may impact the expression and/or function of transcription factors dependent upon CHD7 for access to target genes through chromatin modifications.
Neural progenitor cells in the adult olfactory epithelium possess an extensive capacity for ongoing cellular proliferation and differentiation. These neural progenitors must be tightly regulated developmentally, temporally and spatially to maintain the integrity of the epithelium over time. Chd7
is expressed in both the embryonic and adult olfactory epithelium, and targets of CHD7 are likely to include factors involved in the regulation of neurogenesis, neuronal regeneration and cell cycle progression. CHD7 could be involved in the expression of morphogens such as the bone morphogenetic proteins (BMPs) or fibroblast growth factors (FGFs). Previous studies have shown that BMP4 has dosage dependent opposing effects on neurogenesis in the olfactory epithelium (61
). BMP2, BMP4 and BMP7 at high concentrations have anti-neurogenic effects on progenitors (61
). However, low concentrations of BMP4 increase neurogenesis in the olfactory epithelium, whereas BMP2 and BMP7 retain their anti-neurogenic effects at low concentrations (61
). Although low concentrations of BMP4 increase neurogenesis in the olfactory epithelium, BMP4 does not cause an increase in cellular proliferation (61
), in contrast to the decreased cellular proliferation we observed with Chd7
deficiency. FGF2 induces neurogenesis and increases cellular proliferation of progenitors in the olfactory epithelium (61
). Together these data suggest that BMP and FGF signaling may be sensitive to changes in CHD7
dosage in the olfactory epithelium.
deficiency does not appear to affect the structure, function or localization of olfactory cilia components. However, we observed regional cilia reductions in Chd7Gt/+
mice that could contribute to defects in odorant detection. These data indicate that unlike other mouse models with defects in olfaction, olfactory dysfunction in Chd7Gt/+
mice is not caused by defects in cilia structure, function (41
) or protein transport (40
). Instead, reduced numbers of olfactory sensory neurons and cilia could contribute to impaired olfaction in mature Chd7Gt/+
mice, perhaps through altered electrical signal transmission. The apparently normal calcium responses in neurons of the neonatal Chd7
mutant mouse olfactory epithelium suggest that the developing mutant epithelium is relatively normal. Therefore, later defects in electro-olfactogram, olfactory bulb tyrosine hydroxylase label, reduced olfactory bulb size and reduced olfactory performance in CHARGE patients could be a result of ongoing abnormalities in neural stem cell proliferation and reduced/disorganized olfactory sensory neurons. Future studies of developing Chd7
mutant olfactory tissues and olfactory behaviors in Chd7
mutant mice should help to clarify these pleiotropic effects.
Olfactory dysfunction in CHARGE has typically been associated with defects in the olfactory bulb which ranged from hypoplasia to complete absence of one or both lobes of the olfactory bulb (15
mutant mice have olfactory bulb hypoplasia, consistent with the observed human phenotype. However, the olfactory bulb defects observed in CHARGE individuals often consist of more severe hypoplasia or complete absence of one or both olfactory bulb lobes. Since we detected olfactory hypoplasia in young adult mice (6 weeks), it is also possible that the olfactory bulb hypoplasia could become progressively more severe as the animals age. Defects in the olfactory epithelium have not previously been analyzed in humans or mice with CHD7
). We found that Chd7Gt/+
mice have severely impaired olfaction by electro-olfactogram, which measures odorant detection directly from the surface of the epithelium independent of the olfactory bulb. Our data indicate that olfactory dysfunction in CHARGE individuals may be attributed to primary defects in the olfactory epithelium, and raise the possibility that reduced sensory input from olfactory sensory neurons could contribute to later and more severe olfactory bulb defects.
Prior studies of olfaction in CHARGE have also been limited because the mutation status of the individuals was not reported (15
) in all but one study of a female with CHARGE and Kallmann (19
), and in a recent report of three individuals ascertained on the basis of a Kallmann syndrome phenotype (63
). The eight patients in our study have CHD7
mutations that span the gene and one functional domain (SNF domain) of the CHD7 protein (Table ). Our study is the first report of measured reduction in olfaction in CHARGE patients with known CHD7
mutations. We chose the B-SIT to analyze olfactory dysfunction in CHARGE individuals, because it is a rapid test that is readily available and inexpensive, especially compared with physiological measures of olfactory function in humans (64
). The B-SIT is easy to perform, even for children. However, the B-SIT is not reliable for distinguishing degrees of hyposmia and anosmia (64
). Also, our results could be potentially influenced by cognitive impairment, as is true for the University of Pennsylvania Smell Identification Test, from which the B-SIT is based. Our B-SIT data were obtained from individuals who took the test at home, which could also have influenced test results. Despite these limitations, the B-SIT showed reduced olfaction in a majority of CHD7
mutation-positive CHARGE individuals. A better understanding of the mechanisms involved in the pathogenesis of CHARGE will help facilitate both diagnosis and therapies for CHARGE syndrome. Our data suggest that the B-SIT could be used in a clinical setting as an additional diagnostic tool for evaluating children and adults with suspected CHARGE phenotypes.
We have identified a novel role for CHD7 in neural stem cells of the olfactory epithelium, which could provide insight into a similar role for Chd7
in regulating cell cycle and cell fate specification in other sensory and non-sensory tissues. Our data also demonstrate a novel mechanism for olfactory dysfunction in mammals caused by reduced olfactory sensory neurons. How does a reduction in olfactory sensory neurons lead to impaired olfaction? We hypothesize that fewer olfactory sensory neurons are insufficient to generate an electrical potential, leading to reduced neuronal electrical transmission to the olfactory bulb. Reduced tyrosine hydroxylase in the Chd7Gt/+
olfactory bulb is consistent with this notion. A reduction in olfactory sensory neurons in Chd7Gt/+
mice may also alter the integrity of the olfactory epithelium such that critical cell–cell contacts are disrupted, impairing the ability of the olfactory sensory neurons to process and maintain electrical signals. It will be important to identify whether there are critical genes dysregulated by loss of Chd7
function. We expect some genes regulated by CHD7 to influence proliferation, whereas others may control aspects of cellular differentiation. Generation and characterization of conditional Chd7
null mutants could also enable further analysis of the roles of CHD7 during olfactory development. Since Chd7
null mice are embryonic lethal by E11 (21
), tissue specific and inducible knockouts would enable research on homozygous phenotypes that are currently not amenable for study in heterozygous mouse models. Our work also opens new questions for future research on the function of CHD7 in stem cells in the olfactory system and elsewhere.