Mol. Cells 2018; 41(7): 695-702
Published online July 11, 2018
https://doi.org/10.14348/molcells.2018.0230
© The Korean Society for Molecular and Cellular Biology
Correspondence to : *Correspondence: kimhp@yuhs.ac (HPK); bokj@yuhs.ac (JB)
The inner ear is a complex sensory organ responsible for hearing and balance. Formation of the inner ear is dependent on tight regulation of spatial and temporal expression of genes that direct a series of developmental processes. Recently, epigenetic regulation has emerged as a crucial regulator of the development of various organs. However, what roles higher-order chromatin organization and its regulator molecules play in inner ear development are unclear. CCCTC-binding factor (CTCF) is a highly conserved 11-zinc finger protein that regulates the three-dimensional architecture of chromatin, and is involved in various gene regulation processes. To delineate the role of CTCF in inner ear development, the present study investigated inner ear-specific
Keywords CTCF, inner ear development, neurogenesis,
The inner ear is a complex sensory organ composed of five sensory patches specialized for hearing and balance. The organ of Corti in the cochlea is responsible for hearing. In the vestibule, three cristae (anterior, lateral, and posterior) detect angular movements and two maculae (utricle and saccule) detect linear acceleration. These five sensory patches house mechanosensory hair cells that recognize and convert the mechanical stimuli originating from sound or angular/linear movements into electrical signals. These signals are then conveyed to neurons of the cochlear and vestibular ganglia, which transmit the signals to the brain. Formation of a functional inner ear requires tight regulation of genes important to developmental processes, including fate determination, differentiation, and morphogenesis (Wu and Kelley, 2012).
Recently, epigenetic regulations have been shown to play important roles in development and disease (Ong and Corces, 2014). Epigenetic mechanisms influence gene expression patterns by modulating DNA methylation status and histone modifications. An essential epigenetic component, CCCTC-binding factor (CTCF) plays a primary role in the global organization of chromatic architecture. CTCF has been implicated in various cellular processes, including transcriptional regulation, insulation, X chromosome inactivation, and RNA splicing (Ong and Corces, 2014; Phillips and Corces, 2009). Furthermore, CTCF has been found to play crucial roles in limb and brain development: Specific deletion of
In this study, we applied a conditional knockout (cKO) mouse system to determine the role of CTCF in inner ear development. Our results highlight an essential association between CTCF and otic neurogenesis and posit CTCF as a crucial homeostatic regulator of inner ear by regulating a master neurogenic regulator,
The mice utilized in this study carried a conditional Ctcf allele (
Paint-fill analysis,
Total RNA was extracted from control and
P19 cells were maintained in alpha minimum essential medium (Welgene, Korea) supplemented with 10% heat-inactivated fetal bovine serum (Welgene) and 100 U/ml of penicillin/streptomycin (Welgene).
ChIP assays were performed as described previously (Park et al., 2016). The sequences of the primers for the
To investigate the role of CTCF in inner ear development, we conditionally deleted
As stated above, inactivation of
We then examined whether development of the sensory patches or spiral ganglion neurons residing within the inner ear are compromised in the malformed inner ears of
Next, we sought to determine whether the development of spiral ganglion neurons is affected by the loss of CTCF.
The complete lack of spiral ganglion neurons in the inner ears of
Additionally, we analyzed the expression domains of the genes significantly altered by loss of
In light of the above, we wondered whether the gene expression changes arising in the absence of CTCF are specific to neurogenic genes. When examined by whole-mount
Our analyses of the inner ear from
Genome-wide CTCF occupancy patterns, which have been mapped across more than 100 mammalian cell types, revealed prominent CTCF binding to the −14.4 kb and −7.6 kb transcription start sites of
As histone modification contributes to distinct chromatin states and gene expression, we attempted to determine whether CTCF is involved in histone modifications of the
In this study, by analyzing inner ear-specific
Neurosensory specficiation in the anterior aspect of the inner ear primordium is one of the earliest and most important processes in inner ear development (Wu and Kelley, 2012). This specification establishes the anteroposterior axis of the developing inner ear, which guides the subsequent development of sensory patches of the inner ear. The anterior crista and utricular macula are specified within the neurosensory domain, whereas the posterior crista is specified outside the domain. This neurosensory specification is directed by anteriorly restricted expression of genes important for sensory specification, such as
Epigenetic regulations in inner ear development have recently been reported. In the inner ear of chickens, epigenetic modifiers, such as the DNA methyltransferase DNMT3A and the histone demethylase KDM4B, were shown to be important for proper formation and invagination of the otic placode, respectively (Roellig and Bronner, 2016; Uribe et al., 2015). In the present study, we demonstrated that the chromatin architecture protein CTCF also plays a critical role in otic neurogenesis by regulating
In our
Depletion of
In summary, we identified a role for CTCF during inner ear development by analyzing developing inner ears of
Mol. Cells 2018; 41(7): 695-702
Published online July 31, 2018 https://doi.org/10.14348/molcells.2018.0230
Copyright © The Korean Society for Molecular and Cellular Biology.
Jeong-Oh Shin1,5, Jong-Joo Lee2,3,5, Mikyoung Kim2, Youn Wook Chung2, Hyehyun Min1, Jae-Yoon Kim1,3, Hyoung-Pyo Kim2,3,*, and Jinwoong Bok1,3,4,*
1Department of Anatomy, Yonsei University College of Medicine, Seoul 03722, Korea, 2Department of Environmental Medical Biology, Yonsei University College of Medicine, Seoul 03722, Korea, 3BK21 PLUS project for Medical Science, Yonsei University College of Medicine, Seoul 03722, Korea, 4Department of Otorhinolaryngology, Yonsei University College of Medicine, Seoul 03722, Korea
Correspondence to:*Correspondence: kimhp@yuhs.ac (HPK); bokj@yuhs.ac (JB)
The inner ear is a complex sensory organ responsible for hearing and balance. Formation of the inner ear is dependent on tight regulation of spatial and temporal expression of genes that direct a series of developmental processes. Recently, epigenetic regulation has emerged as a crucial regulator of the development of various organs. However, what roles higher-order chromatin organization and its regulator molecules play in inner ear development are unclear. CCCTC-binding factor (CTCF) is a highly conserved 11-zinc finger protein that regulates the three-dimensional architecture of chromatin, and is involved in various gene regulation processes. To delineate the role of CTCF in inner ear development, the present study investigated inner ear-specific
Keywords: CTCF, inner ear development, neurogenesis,
The inner ear is a complex sensory organ composed of five sensory patches specialized for hearing and balance. The organ of Corti in the cochlea is responsible for hearing. In the vestibule, three cristae (anterior, lateral, and posterior) detect angular movements and two maculae (utricle and saccule) detect linear acceleration. These five sensory patches house mechanosensory hair cells that recognize and convert the mechanical stimuli originating from sound or angular/linear movements into electrical signals. These signals are then conveyed to neurons of the cochlear and vestibular ganglia, which transmit the signals to the brain. Formation of a functional inner ear requires tight regulation of genes important to developmental processes, including fate determination, differentiation, and morphogenesis (Wu and Kelley, 2012).
Recently, epigenetic regulations have been shown to play important roles in development and disease (Ong and Corces, 2014). Epigenetic mechanisms influence gene expression patterns by modulating DNA methylation status and histone modifications. An essential epigenetic component, CCCTC-binding factor (CTCF) plays a primary role in the global organization of chromatic architecture. CTCF has been implicated in various cellular processes, including transcriptional regulation, insulation, X chromosome inactivation, and RNA splicing (Ong and Corces, 2014; Phillips and Corces, 2009). Furthermore, CTCF has been found to play crucial roles in limb and brain development: Specific deletion of
In this study, we applied a conditional knockout (cKO) mouse system to determine the role of CTCF in inner ear development. Our results highlight an essential association between CTCF and otic neurogenesis and posit CTCF as a crucial homeostatic regulator of inner ear by regulating a master neurogenic regulator,
The mice utilized in this study carried a conditional Ctcf allele (
Paint-fill analysis,
Total RNA was extracted from control and
P19 cells were maintained in alpha minimum essential medium (Welgene, Korea) supplemented with 10% heat-inactivated fetal bovine serum (Welgene) and 100 U/ml of penicillin/streptomycin (Welgene).
ChIP assays were performed as described previously (Park et al., 2016). The sequences of the primers for the
To investigate the role of CTCF in inner ear development, we conditionally deleted
As stated above, inactivation of
We then examined whether development of the sensory patches or spiral ganglion neurons residing within the inner ear are compromised in the malformed inner ears of
Next, we sought to determine whether the development of spiral ganglion neurons is affected by the loss of CTCF.
The complete lack of spiral ganglion neurons in the inner ears of
Additionally, we analyzed the expression domains of the genes significantly altered by loss of
In light of the above, we wondered whether the gene expression changes arising in the absence of CTCF are specific to neurogenic genes. When examined by whole-mount
Our analyses of the inner ear from
Genome-wide CTCF occupancy patterns, which have been mapped across more than 100 mammalian cell types, revealed prominent CTCF binding to the −14.4 kb and −7.6 kb transcription start sites of
As histone modification contributes to distinct chromatin states and gene expression, we attempted to determine whether CTCF is involved in histone modifications of the
In this study, by analyzing inner ear-specific
Neurosensory specficiation in the anterior aspect of the inner ear primordium is one of the earliest and most important processes in inner ear development (Wu and Kelley, 2012). This specification establishes the anteroposterior axis of the developing inner ear, which guides the subsequent development of sensory patches of the inner ear. The anterior crista and utricular macula are specified within the neurosensory domain, whereas the posterior crista is specified outside the domain. This neurosensory specification is directed by anteriorly restricted expression of genes important for sensory specification, such as
Epigenetic regulations in inner ear development have recently been reported. In the inner ear of chickens, epigenetic modifiers, such as the DNA methyltransferase DNMT3A and the histone demethylase KDM4B, were shown to be important for proper formation and invagination of the otic placode, respectively (Roellig and Bronner, 2016; Uribe et al., 2015). In the present study, we demonstrated that the chromatin architecture protein CTCF also plays a critical role in otic neurogenesis by regulating
In our
Depletion of
In summary, we identified a role for CTCF during inner ear development by analyzing developing inner ears of
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