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Ryoichiro Kageyama - One of the best experts on this subject based on the ideXlab platform.
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oscillatory expression of HES1 regulates cell proliferation and neuronal differentiation in the embryonic brain
Development, 2020Co-Authors: Shohei Ochi, Yui Imaizumi, Hiromi Shimojo, Hitoshi Miyachi, Ryoichiro KageyamaAbstract:ABSTRACT The expression of the transcriptional repressor HES1 oscillates in many cell types, including neural progenitor cells (NPCs), but the significance of HES1 oscillations in development is not fully understood. To examine the effect of altered oscillatory dynamics of HES1, we generated two types of HES1 knock-in mice, a shortened (type-1) and an elongated (type-2) HES1 gene, and examined their phenotypes focusing on neural development. Although both mutations affected HES1 oscillations, the type-1 mutation dampened HES1 oscillations more severely, resulting in much lower amplitudes. The average levels of HES1 expression in type-1 mutant NPCs were also lower than in wild-type NPCs but similar to or slightly higher than those in HES1 heterozygous mutant mice, which exhibit no apparent defects. Whereas type-2 mutant mice were apparently normal, type-1 mutant mice displayed smaller brains than wild-type mice and upregulated proneural gene expression. Furthermore, proliferation of NPCs decreased and cell death increased in type-1 mutant embryos. When Hes3 and Hes5 were additionally deleted, neuronal differentiation was also accelerated, leading to microcephaly. Thus, robust HES1 oscillations are required for maintenance and proliferation of NPCs and the normal timing of neurogenesis, thereby regulating brain morphogenesis.
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simultaneous requirements for HES1 in retinal neurogenesis and optic cup stalk boundary maintenance
The Journal of Neuroscience, 2020Co-Authors: Bernadett Bosze, Ryoichiro Kageyama, Myungsoon Moon, Nadean L. BrownAbstract:The bHLH transcription factor HES1 is a key downstream effector for the Notch signaling pathway. During embryogenesis neural progenitors express low levels of HES1 in an oscillating pattern, whereas glial brain boundary regions (e.g., isthmus) have high, sustained HES1 levels that suppress neuronal fates. Here, we show that in the embryonic mouse retina, the optic nerve head and stalk express high HES1, with the ONH constituting a boundary between the neural retina and glial cells that ultimately line the optic stalk. Using two Cre drivers with distinct spatiotemporal expression we conditionally inactivated HES1, to delineate the requirements for this transcriptional repressor during retinal neurogenesis versus patterning of the optic cup and stalk. Throughout retinal neurogenesis, HES1 maintains proliferation and blocks retinal ganglion cell formation, but surprisingly we found it also promotes cone photoreceptor genesis. In the postnatal eye, HES1 inactivation with Rax-Cre resulted in increased bipolar neurons and a mispositioning of Muller glia. Our results indicate that Notch pathway regulation of cone genesis is more complex than previously assumed, and reveal a novel role for HES1 in maintaining the optic cup–stalk boundary. SIGNIFICANCE STATEMENT The bHLH repressor HES1 regulates the timing of neurogenesis, rate of progenitor cell division, gliogenesis, and maintains tissue compartment boundaries. This study expands current eye development models by showing Notch-independent roles for HES1 in the developing optic nerve head (ONH). Defects in ONH formation result in optic nerve coloboma; our work now inserts HES1 into the genetic hierarchy regulating optic fissure closure. Given that HES1 acts analogously in the ONH as the brain isthmus, it prompts future investigation of the ONH as a signaling factor center, or local organizer. Embryonic development of the ONH region has been poorly studied, which is surprising given it is where the pan-ocular disease glaucoma is widely believed to inflict damage on RGC axons.
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HES1 expression in mature neurons in the adult mouse brain is required for normal behaviors
Scientific Reports, 2019Co-Authors: Tadanobu Matsuzaki, Toshiyuki Ohtsuka, Toru Yoshihara, Ryoichiro KageyamaAbstract:HES1 regulates the maintenance and proliferation of neural stem/progenitor cells as an essential effector of the Notch signaling pathway. Although Notch signaling is also involved in the functions of mature neurons in learning and memory and in the risk factors for mental disorders such as schizophrenia and bipolar disorder, the in-vivo role of HES1 in mature neurons remains unknown. Here, we found that HES1 is expressed by subsets of both excitatory and inhibitory neurons in the adult mouse brain, and that HES1 expression is induced by neuronal stimulation. Furthermore, inactivation of HES1 in excitatory neurons resulted in abnormal fear and anxiety behaviors concomitantly with higher neuronal excitability in the amygdala, while inactivation of HES1 in inhibitory neurons resulted in increased sociability and perseverative tendencies. These results indicated that HES1 is functionally important for normal behaviors not only in excitatory neurons but also in inhibitory neurons in the adult brain.
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deubiquitinating enzymes regulate HES1 stability and neuronal differentiation
FEBS Journal, 2015Co-Authors: Taeko Kobayashi, Yumiko Iwamoto, Kazuhiro Takashima, Akihiro Isomura, Yoichi Kosodo, Koichi Kawakami, Tomoki Nishioka, Kozo Kaibuchi, Ryoichiro KageyamaAbstract:Hairy and enhancer of split 1 (HES1), a basic helix-loop-helix transcriptional repressor protein, regulates the maintenance of neural stem/progenitor cells by repressing proneural gene expression via Notch signaling. Previous studies showed that HES1 expression oscillates in both mouse embryonic stem cells and neural stem cells, and that the oscillation contributes to their potency and differentiation fates. This oscillatory expression depends on the stability of HES1, which is rapidly degraded by the ubiquitin/proteasome pathway. However, the detailed molecular mechanisms governing HES1 stability remain unknown. We analyzed HES1-interacting deubiquitinases purified from mouse embryonic stem cells using an HES1-specific antibody, and identified the ubiquitin-specific protease 27x (Usp27x) as a new regulator of HES1. We found that HES1 was deubiquitinated and stabilized by Usp27x and its homologs ubiquitin-specific protease 22 (Usp22) and ubiquitin-specific protease 51 (Usp51). Knockdown of Usp22 shortened the half-life of HES1, delayed its oscillation, and enhanced neuronal differentiation in mouse developing brain, whereas mis-expression of Usp27x reduced neuronal differentiation. These results suggest that these deubiquitinases modulate HES1 protein dynamics by removing ubiquitin molecules, and thereby regulate neuronal differentiation of stem cells.
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HES1 and hes5 regulate vascular remodeling and arterial specification of endothelial cells in brain vascular development
Mechanisms of Development, 2013Co-Authors: Masashi Kitagawa, Toshiyuki Ohtsuka, Ryoichiro Kageyama, Masato Hojo, Itaru Imayoshi, Masanori Goto, Mitsushige Ando, Susumu MiyamotoAbstract:Abstract The vascular system is the first organ to form in the developing mammalian embryo. The Notch signaling pathway is an evolutionarily conserved signaling mechanism essential for proper embryonic development in almost all vertebrate organs. The analysis of targeted mouse mutants has demonstrated essential roles of the Notch signaling pathway in embryonic vascular development. However, Notch signaling-deficient mice have so far not been examined in detail in the head region. The bHLH genes HES1 and Hes5 are essential effectors for Notch signaling, which regulate the maintenance of progenitor cells and the timing of their differentiation in various tissues and organs. Here, we report that endothelial-specific HES1 and Hes5 mutant embryos exhibited defective vascular remodeling in the brain. In addition, arterial identity of endothelial cells was partially lost in the brain of these mutant mice. These data suggest that HES1 and Hes5 regulate vascular remodeling and arterial fate specification of endothelial cells in the development of the brain. HES1 and Hes5 represent critical transducers of Notch signals in brain vascular development.
Toshiyuki Ohtsuka - One of the best experts on this subject based on the ideXlab platform.
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HES1 expression in mature neurons in the adult mouse brain is required for normal behaviors
Scientific Reports, 2019Co-Authors: Tadanobu Matsuzaki, Toshiyuki Ohtsuka, Toru Yoshihara, Ryoichiro KageyamaAbstract:HES1 regulates the maintenance and proliferation of neural stem/progenitor cells as an essential effector of the Notch signaling pathway. Although Notch signaling is also involved in the functions of mature neurons in learning and memory and in the risk factors for mental disorders such as schizophrenia and bipolar disorder, the in-vivo role of HES1 in mature neurons remains unknown. Here, we found that HES1 is expressed by subsets of both excitatory and inhibitory neurons in the adult mouse brain, and that HES1 expression is induced by neuronal stimulation. Furthermore, inactivation of HES1 in excitatory neurons resulted in abnormal fear and anxiety behaviors concomitantly with higher neuronal excitability in the amygdala, while inactivation of HES1 in inhibitory neurons resulted in increased sociability and perseverative tendencies. These results indicated that HES1 is functionally important for normal behaviors not only in excitatory neurons but also in inhibitory neurons in the adult brain.
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HES1 and hes5 regulate vascular remodeling and arterial specification of endothelial cells in brain vascular development
Mechanisms of Development, 2013Co-Authors: Masashi Kitagawa, Toshiyuki Ohtsuka, Ryoichiro Kageyama, Masato Hojo, Itaru Imayoshi, Masanori Goto, Mitsushige Ando, Susumu MiyamotoAbstract:Abstract The vascular system is the first organ to form in the developing mammalian embryo. The Notch signaling pathway is an evolutionarily conserved signaling mechanism essential for proper embryonic development in almost all vertebrate organs. The analysis of targeted mouse mutants has demonstrated essential roles of the Notch signaling pathway in embryonic vascular development. However, Notch signaling-deficient mice have so far not been examined in detail in the head region. The bHLH genes HES1 and Hes5 are essential effectors for Notch signaling, which regulate the maintenance of progenitor cells and the timing of their differentiation in various tissues and organs. Here, we report that endothelial-specific HES1 and Hes5 mutant embryos exhibited defective vascular remodeling in the brain. In addition, arterial identity of endothelial cells was partially lost in the brain of these mutant mice. These data suggest that HES1 and Hes5 regulate vascular remodeling and arterial fate specification of endothelial cells in the development of the brain. HES1 and Hes5 represent critical transducers of Notch signals in brain vascular development.
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microrna9 regulates neural stem cell differentiation by controlling HES1 expression dynamics in the developing brain
Genes to Cells, 2012Co-Authors: Toshiyuki Ohtsuka, Aitor Gonzalez, Ryoichiro KageyamaAbstract:Earlier studies show that HES1 expression is oscillatory in neural stem cells but sustained and high in the roof plate and the floor plate, and that such different dynamics of HES1 expression (oscillatory versus sustained) regulate different proliferation and differentiation characteristics of these cells (active in neural stem cells but rather dormant in roof/floor plate cells). The mechanism of how different dynamics of HES1 expression is controlled remains to be determined. Here, we found that the seed sequence of microRNA-9 (miR-9) is complementary to the 3′-UTR sequence of HES1 mRNA. MiR-9 is highly expressed in the ventricular zone of the developing brain, which contains neural stem cells, but it is not expressed in the roof plate or the floor plate. Over-expression of miR-9 negatively regulates the HES1 protein expression by interacting with the 3′-UTR of HES1 mRNA, thereby inducing cell cycle exit and neuronal differentiation. Conversely, knockdown of miR-9 inhibits neuronal differentiation. Furthermore, knockdown of miR-9 inhibits the oscillatory expression of HES1 mRNA in neural stem cells. These results indicate that miR-9 regulates the proliferation and differentiation of neural stem cells by controlling the dynamics of HES1 expression in the developing brain.
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notch HES1 pathway contributes to the cochlear prosensory formation potentially through the transcriptional down regulation of p27kip1
Journal of Neuroscience Research, 2009Co-Authors: Junko Murata, Toshiyuki Ohtsuka, Akinori Tokunaga, Suetaka Nishiike, Hidenori Inohara, Hideyuki Okano, Ryoichiro KageyamaAbstract:The Notch signaling pathway has a crucial role in the differentiation of hair cells and supporting cells by mediating “lateral inhibition” via the ligands Delta-like1 (Dll1) and Jagged2 (Jag2) and the effectors HES1 and Hes5 during mammalian inner ear development. Recently, another Notch ligand, Jagged1 (Jag1)-dependent Notch activation, has been revealed to be important for the determination of the prosensory region in the earlier stage before cell differentiation. However, little is known about the effectors of the Notch pathway in this context. P27Kip1, a cyclin-dependent kinase inhibitor, is also known to demarcate the prosensory region in the cochlear primordium, which consists of the sensory progenitors that have completed their terminal mitoses. HES1 reportedly promotes precursor cell proliferation through the transcriptional down-regulation of p27Kip1 in the thymus, liver, and brain. In this study, we observed HES1 as a mediator between the Notch signaling pathway and the regulation of proliferation of sensory precursor cells by p27Kip1 in the developing cochlea. We showed that HES1, but not Hes5, was weakly expressed at the time of onset of p27Kip1. The expression pattern of HES1 prior to cell differentiation was similar to that of activated Notch1. P27Kip1 was up-regulated and BrdU-positive S-phase cells were reduced in the developing cochlear epithelium of HES1 null mice. These results suggest that the Notch-HES1 pathway may contribute to the adequate proliferation of sensory precursor cells via the potential transcriptional down-regulation of p27Kip1 expression and play a pivotal role in the correct prosensory determination. © 2009 Wiley-Liss, Inc.
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the first HES1 dimer inhibitors from natural products
Bioorganic & Medicinal Chemistry Letters, 2009Co-Authors: Midori A Arai, Toshiyuki Ohtsuka, Ryoichiro Kageyama, Ayako Masada, Masami IshibashiAbstract:In the present study, we developed a high-throughput screening system for small molecule-inhibitors of the basic helix-loop-helix (bHLH) transcriptional repressor factor HES1. Successful dimerization of HES1 immobilized on a microplate and fluorophore (Cy3)-labelled HES1 was confirmed. Using this system, several natural products were identified as the first HES1 dimer inhibitors. Of these, two compounds which were isolated from myxomycetes (true slime molds) inhibited HES1 from N box-dependent suppression of the gene expression in C3H10T1/2 cells.
Takahiro Nakamura - One of the best experts on this subject based on the ideXlab platform.
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HES1 regulates corneal development and the function of corneal epithelial stem progenitor cells
Stem Cells, 2008Co-Authors: Toshiyuki Ohtsuka, Hiroshi Kokubu, Takahiro Nakamura, Eiichi Sekiyama, Leanne J Cooper, Nigel J Fullwood, Yann Barrandon, Ryoichiro KageyamaAbstract:HES1, a major target gene in Notch signaling, regulates the fate and differentiation of various cell types in many developmental systems. To gain a novel insight into the role of HES1 in corneal tissue, we performed gain-of-function and loss-of-function studies. We show that corneal development was severely disturbed in HES1-null mice. HES1-null corneas manifested abnormal junctional specialization, cell differentiation, and less cell proliferation ability. Worthy of note, HES1 is expressed mainly in the corneal epithelial stem/progenitor cells and is not detected in the differentiated corneal epithelial cells. Expression of HES1 is closely linked with corneal epithelial stem/progenitor cell proliferation activity in vivo. Moreover, forced HES1 expression inhibits the differentiation of corneal epithelial stem/progenitor cells and maintains these cells' undifferentiated state. Our data provide the first evidence that HES1 regulates corneal development and the homeostatic function of corneal epithelial stem/progenitor cells.
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HES1 Regulates Corneal Development and the Function of Corneal Epithelial Stem/Progenitor Cells
Stem Cells, 2008Co-Authors: Takahiro Nakamura, Toshiyuki Ohtsuka, Ryoichiro Kageyama, Hiroshi Kokubu, Eiichi Sekiyama, Leanne J Cooper, Nigel J Fullwood, Yann Barrandon, Shigeru KinoshitaAbstract:HES1, a major target gene in Notch signaling, regulates the fate and differentiation of various cell types in many developmental systems. To gain a novel insight into the role of HES1 in corneal tissue, we performed gain-of-function and loss-of-function studies. We show that corneal development was severely disturbed in HES1-null mice. HES1-null corneas manifested abnormal junctional specialization, cell differentiation, and less cell proliferation ability. Worthy of note, HES1 is expressed mainly in the corneal epithelial stem/progenitor cells and is not detected in the differentiated corneal epithelial cells. Expression of HES1 is closely linked with corneal epithelial stem/progenitor cell proliferation activity in vivo. Moreover, forced HES1 expression inhibits the differentiation of corneal epithelial stem/progenitor cells and maintains these cells' undifferentiated state. Our data provide the first evidence that HES1 regulates corneal development and the homeostatic function of corneal epithelial stem/progenitor cells.
Hiroshi Kokubu - One of the best experts on this subject based on the ideXlab platform.
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HES1 regulates corneal development and the function of corneal epithelial stem progenitor cells
Stem Cells, 2008Co-Authors: Toshiyuki Ohtsuka, Hiroshi Kokubu, Takahiro Nakamura, Eiichi Sekiyama, Leanne J Cooper, Nigel J Fullwood, Yann Barrandon, Ryoichiro KageyamaAbstract:HES1, a major target gene in Notch signaling, regulates the fate and differentiation of various cell types in many developmental systems. To gain a novel insight into the role of HES1 in corneal tissue, we performed gain-of-function and loss-of-function studies. We show that corneal development was severely disturbed in HES1-null mice. HES1-null corneas manifested abnormal junctional specialization, cell differentiation, and less cell proliferation ability. Worthy of note, HES1 is expressed mainly in the corneal epithelial stem/progenitor cells and is not detected in the differentiated corneal epithelial cells. Expression of HES1 is closely linked with corneal epithelial stem/progenitor cell proliferation activity in vivo. Moreover, forced HES1 expression inhibits the differentiation of corneal epithelial stem/progenitor cells and maintains these cells' undifferentiated state. Our data provide the first evidence that HES1 regulates corneal development and the homeostatic function of corneal epithelial stem/progenitor cells.
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HES1 Regulates Corneal Development and the Function of Corneal Epithelial Stem/Progenitor Cells
Stem Cells, 2008Co-Authors: Takahiro Nakamura, Toshiyuki Ohtsuka, Ryoichiro Kageyama, Hiroshi Kokubu, Eiichi Sekiyama, Leanne J Cooper, Nigel J Fullwood, Yann Barrandon, Shigeru KinoshitaAbstract:HES1, a major target gene in Notch signaling, regulates the fate and differentiation of various cell types in many developmental systems. To gain a novel insight into the role of HES1 in corneal tissue, we performed gain-of-function and loss-of-function studies. We show that corneal development was severely disturbed in HES1-null mice. HES1-null corneas manifested abnormal junctional specialization, cell differentiation, and less cell proliferation ability. Worthy of note, HES1 is expressed mainly in the corneal epithelial stem/progenitor cells and is not detected in the differentiated corneal epithelial cells. Expression of HES1 is closely linked with corneal epithelial stem/progenitor cell proliferation activity in vivo. Moreover, forced HES1 expression inhibits the differentiation of corneal epithelial stem/progenitor cells and maintains these cells' undifferentiated state. Our data provide the first evidence that HES1 regulates corneal development and the homeostatic function of corneal epithelial stem/progenitor cells.
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HES1 and hes5 control the progenitor pool intermediate lobe specification and posterior lobe formation in the pituitary development
Molecular Endocrinology, 2007Co-Authors: Aya Kita, Toshiyuki Ohtsuka, Ryoichiro Kageyama, Masato Hojo, Masashi Kitagawa, Itaru Imayoshi, Hiroshi Kokubu, Ryosuke Ohsawa, Nobuo HashimotoAbstract:The pituitary gland is composed of two distinct entities: the adenohypophysis, including the anterior and intermediate lobes, and the neurohypophysis, known as the posterior lobe. This critical endocrine organ is essential for homeostasis, metabolism, reproduction, and growth. The pituitary development requires the control of proliferation and differentiation of progenitor cells. Although multiple signaling molecules and transcription factors are required for the proper pituitary development, the mechanisms that regulate the fate of progenitor cells remain to be elucidated. Hes genes, known as Notch effectors, play a crucial role in specifying cellular fates during the development of various tissues and organs. Here, we report that mice deficient for HES1 and Hes5 display severe pituitary hypoplasia caused by accelerated differentiation of progenitor cells. In addition, this hypoplastic pituitary gland (adenohypophysis) lacks the intermediate lobe and exhibits the features of the anterior lobe only. HES1 ...
Francois Guillemot - One of the best experts on this subject based on the ideXlab platform.
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id sustains HES1 expression to inhibit precocious neurogenesis by releasing negative autoregulation of HES1
Developmental Cell, 2007Co-Authors: Nengyin Sheng, Francois Guillemot, Ryoichiro Kageyama, Wei Bian, Yoshifumi Yokota, Robert Benezra, Naihe JingAbstract:Negative bHLH transcription factor HES1 can inhibit neural stem cells (NSCs) from precocious neurogenesis through repressing proneural gene expression; therefore, sustenance of HES1 expression is crucial for NSC pool maintenance. Here we find that Ids, the dominant-negative regulators of proneural proteins, are expressed prior to proneural genes and share an overlapping expression pattern with HES1 in the early neural tube of chick embryos. Overexpression of Id2 in the chick hindbrain upregulates HES1 expression and inhibits proneural gene expression and neuronal differentiation. By contrast, HES1 expression decreases, proneural gene expression expands, and neurogenesis occurs precociously in Id1;Id3 double knockout mice and in Id1-3 RNAi-electroporated chick embryos. Mechanistic studies show that Id proteins interact directly with HES1 and release the negative feedback autoregulation of HES1 without interfering with its ability to affect other target genes. These results indicate that Id proteins participate in NSC maintenance through sustaining HES1 expression in early embryos.
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roles of the basic helix loop helix genes HES1 and hes5 in expansion of neural stem cells of the developing brain
Journal of Biological Chemistry, 2001Co-Authors: Toshiyuki Ohtsuka, Francois Guillemot, Masami Sakamoto, Ryoichiro KageyamaAbstract:Abstract Neural stem cells, which differentiate into neurons and glia, are present in the ventricular zone of the embryonal brain. The precise mechanism by which neural stem cells are maintained during embryogenesis remains to be determined. Here, we found that transient misexpression of the basic helix-loop-helix genes HES1 and Hes5 keeps embryonal telencephalic cells undifferentiated although they have been shown to induce gliogenesis in the retina. These telencephalic cells later differentiate into neurons and astroglia when Hes expression is down-regulated, suggesting that HES1- andHes5- expressing cells are maintained as neural stem cells during embryogenesis. Conversely, in the absence of HES1and Hes5, neural stem cells are not properly maintained, generating fewer and smaller neurospheres than the wild type. These results indicate that HES1 and Hes5 play an important role in the maintenance of neural stem cells but not in gliogenesis in the embryonal telencephalon.
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HES1 and hes5 activities are required for the normal development of the hair cells in the mammalian inner ear
The Journal of Neuroscience, 2001Co-Authors: Azel Zine, Francois Guillemot, Ryoichiro Kageyama, Alexandre Aubert, Stavros Therianos, Francois De RibaupierreAbstract:The mammalian inner ear contains two sensory organs, the cochlea and vestibule. Their sensory neuroepithelia are characterized by a mosaic of hair cells and supporting cells. Cochlear hair cells differentiate in four rows: a single row of inner hair cells (IHCs) and three rows of outer hair cells (OHCs). Recent studies have shown that Math1, a mammalian homolog of Drosophila atonal is a positive regulator of hair cell differentiation. The basic helix–loop–helix (bHLH) genes HES1 and Hes5 (mammalian hairy and Enhancer-of-split homologs) can influence cell fate determination by acting as negative regulators to inhibit the action of bHLH-positive regulators. We show by using reverse transcription-PCR analysis that HES1 , Hes5 , and Math1 are expressed in the developing mouse cochleae. In situ hybridization revealed a widespread expression of HES1 in the greater epithelial ridge (GER) and in lesser epithelial ridge (LER) regions. Hes5 is predominantly expressed in the LER, in supporting cells, and in a narrow band of cells within the GER. Examination of cochleae from HES1 −/− mice showed a significant increase in the number of IHCs, whereas cochleae from Hes5 −/− mice showed a significant increase in the number of OHCs. In the vestibular system, targeted deletion of HES1 and to a lesser extent Hes5 lead to formation of supernumerary hair cells in the saccule and utricle. The supernumerary hair cells in the mutant mice showed an upregulation of Math1. These data indicate that HES1 and Hes5 participate together for the control of inner ear hair cell production, likely through the negative regulation of Math1.
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HES1 and hes5 as notch effectors in mammalian neuronal differentiation
The EMBO Journal, 1999Co-Authors: Toshiyuki Ohtsuka, Makoto Ishibashi, Gerald Gradwohl, Shigetada Nakanishi, Francois Guillemot, Ryoichiro KageyamaAbstract:While the transmembrane protein Notch plays an important role in various aspects of development, and diseases including tumors and neurological disorders, the intracellular pathway of mammalian Notch remains very elusive. To understand the intracellular pathway of mammalian Notch, the role of the bHLH genes HES1 and Hes5 (mammalian hairy and Enhancer-of-split homologues) was examined by retrovirally misexpressing the constitutively active form of Notch (caNotch) in neural precursor cells prepared from wild-type, HES1-null, Hes5-null and HES1-Hes5 double-null mouse embryos. We found that caNotch, which induced the endogenous HES1 and Hes5 expression, inhibited neuronal differentiation in the wild-type, HES1-null and Hes5-null background, but not in the HES1-Hes5 double-null background. These results demonstrate that HES1 and Hes5 are essential Notch effectors in regulation of mammalian neuronal differentiation.