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Ryoichiro Kageyama

Publications and source records attributed to Ryoichiro Kageyama.

At least 19 recordsLinked to original sources
PubMed2006

Notch and HES5 are regulated during human cartilage differentiation.

The molecular mechanisms of cartilage differentiation are poorly understood. In a variety of tissues other than cartilage, members of the basic helix-loop-helix (bHLH) family of transcription factors have been demonstrated to play critical roles in differentiation. We have characterized the human bHLH gene HES5 and investigated its role during chondrogenesis. Blockage of the Notch signaling pathway with a gamma-secretase inhibitor has demonstrated that the human HES5 gene is a downstream marker of Notch signaling in articular chondrocytes. Markers for the Notch signaling pathway significantly decrease during cartilage differentiation in vitro. Cell proliferation assayed by using BrdU has revealed that blockage of Notch signaling is associated with significantly decreased proliferation. Northern blot and reverse transcription/polymerase chain reaction of a panel of various tissues have shown that HES5 is transcribed as a 5.4-kb mRNA that is ubiquitously expressed in diverse fetal and adult tissues. Articular cartilage from HES5(-/-) and wild-type mice has been analyzed by using various histological stains. No differences have been detected between the wild-type and HES5(-/-) mice. Our data thus indicate that the human HES5 gene is coupled to the Notch receptor family, that expression of Notch markers (including HES5) decreases during cartilage differentiation, and that the blockage of Notch signaling is associated with significantly decreased cell proliferation.

Adolescent↗
PubMed2006

Sustained Notch signaling in progenitors is required for sequential emergence of distinct cell lineages during organogenesis.

Mammalian organogenesis results from the concerted actions of signaling pathways in progenitor cells that induce a hierarchy of regulated transcription factors critical for organ and cell type determination. Here we demonstrate that sustained Notch activity is required for the temporal maintenance of specific cohorts of proliferating progenitors, which underlies the ability to specify late-arising cell lineages during pituitary organogenesis. Conditional deletion of Rbp-J, which encodes the major mediator of the Notch pathway, leads to premature differentiation of progenitor cells, a phenotype recapitulated by loss of the basic helix-loop-helix (bHLH) factor Hes1, as well as a conversion of the late (Pit1) lineage into the early (corticotrope) lineage. Notch signaling is required for maintaining expression of the tissue-specific paired-like homeodomain transcription factor, Prop1, which is required for generation of the Pit1 lineage. Attenuation of Notch signaling is necessary for terminal differentiation in post-mitotic Pit1+ cells, and the Notch-repressed Pit1 target gene, Math3, is specifically required for maturation and proliferation of the GH-producing somatotrope. Thus, sustained Notch signaling in progenitor cells is required to prevent conversion of the late-arising cell lineages to early-born cell lineages, permitting specification of diverse cell types, a strategy likely to be widely used in mammalian organogenesis.

Animals↗
PubMed2006

High levels of Cre expression in neuronal progenitors cause defects in brain development leading to microencephaly and hydrocephaly.

Hydrocephalus is a common and variegated pathology often emerging in newborn children after genotoxic insults during pregnancy (Hicks and D'Amato, 1980). Cre recombinase is known to have possible toxic effects that can compromise normal cell cycle and survival. Here we show, by using three independent nestin Cre transgenic lines, that high levels of Cre recombinase expression into the nucleus of neuronal progenitors can compromise normal brain development. The transgenics analyzed are the nestin Cre Balancer (Bal1) line, expressing the Cre recombinase with a nuclear localization signal, and two nestin CreER(T2) (Cre recombinase fused with a truncated estrogen receptor) mice lines with different levels of expression of a hybrid CreER(T2) recombinase that translocates into the nucleus after tamoxifen treatment. All homozygous Bal1 nestin Cre embryos displayed reduced neuronal proliferation, increased aneuploidy and cell death, as well as defects in ependymal lining and lamination of the cortex, leading to microencephaly and to a form of communicating hydrocephalus. An essentially overlapping phenotype was observed in the two nestin CreER(T2) transgenic lines after tamoxifen mediated-CreER(T2) translocation into the nucleus. Neither tamoxifen-treated wild-type nor nestin CreER(T2) oil-treated control mice displayed these defects. These results indicate that some forms of hydrocephalus may derive from a defect in neuronal precursors proliferation. Furthermore, they underscore the potential risks for developmental studies of high levels of nuclear Cre in neurogenic cells.

Aneuploidy↗
PubMed2006

Persistent and high levels of Hes1 expression regulate boundary formation in the developing central nervous system.

The developing central nervous system is partitioned into compartments by boundary cells, which have different properties than compartment cells, such as forming neuron-free zones, proliferating more slowly and acting as organizing centers. We now report that in mice the bHLH factor Hes1 is persistently expressed at high levels by boundary cells but at variable levels by non-boundary cells. Expression levels of Hes1 display an inverse correlation to those of the proneural bHLH factor Mash1, suggesting that downregulation of Hes1 leads to upregulation of Mash1 in non-boundary regions, whereas persistent and high Hes1 expression constitutively represses Mash1 in boundary regions. In agreement with this notion, in the absence of Hes1 and its related genes Hes3 and Hes5, proneural bHLH genes are ectopically expressed in boundaries, resulting in ectopic neurogenesis and disruption of the organizing centers. Conversely, persistent Hes1 expression in neural progenitors prepared from compartment regions blocks neurogenesis and reduces cell proliferation rates. These results indicate that the mode of Hes1 expression is different between boundary and non-boundary cells, and that persistent and high levels of Hes1 expression constitutively repress proneural bHLH gene expression and reduce cell proliferation rates, thereby forming boundaries that act as the organizing centers.

Animals↗
PubMed2006

Ectopic pancreas formation in Hes1 -knockout mice reveals plasticity of endodermal progenitors of the gut, bile duct, and pancreas.

Ectopic pancreas is a developmental anomaly occasionally found in humans. Hes1, a main effector of Notch signaling, regulates the fate and differentiation of many cell types during development. To gain insights into the role of the Notch pathway in pancreatic fate determination, we combined the use of Hes1-knockout mice and lineage tracing employing the Cre/loxP system to specifically mark pancreatic precursor cells and their progeny in Ptf1a-cre and Rosa26 reporter mice. We show that inactivation of Hes1 induces misexpression of Ptf1a in discrete regions of the primitive stomach and duodenum and throughout the common bile duct. All ectopic Ptf1a-expressing cells were reprogrammed, or transcommitted, to multipotent pancreatic progenitor status and subsequently differentiated into mature pancreatic exocrine, endocrine, and duct cells. This process recapitulated normal pancreatogenesis in terms of morphological and genetic features. Furthermore, analysis of Hes1/Ptf1a double mutants revealed that ectopic Ptf1a-cre lineage-labeled cells adopted the fate of region-appropriate gut epithelium or endocrine cells similarly to Ptf1a-inactivated cells in the native pancreatic buds. Our data demonstrate that the Hes1-mediated Notch pathway is required for region-appropriate specification of pancreas in the developing foregut endoderm through regulation of Ptf1a expression, providing novel insight into the pathogenesis of ectopic pancreas development in a mouse model.

Animals↗
PubMed2006

Notch signaling via Hes1 transcription factor maintains survival of melanoblasts and melanocyte stem cells.

Melanoblasts (Mbs) are thought to be strictly regulated by cell-cell interactions with epidermal keratinocytes, although the precise molecular mechanism of the regulation has been elusive. Notch signaling, whose activation is mediated by cell-cell interactions, is implicated in a broad range of developmental processes. We demonstrate the vital role of Notch signaling in the maintenance of Mbs, as well as melanocyte stem cells (MSCs). Conditional ablation of Notch signaling in the melanocyte lineage leads to a severe defect in hair pigmentation, followed by intensive hair graying. The defect is caused by a dramatic elimination of Mbs and MSCs. Furthermore, targeted overexpression of Hes1 is sufficient to protect Mbs from the elimination by apoptosis. Thus, these data provide evidence that Notch signaling, acting through Hes1, plays a crucial role in the survival of immature Mbs by preventing initiation of apoptosis.

Animals↗
PubMed2006

Real-time imaging of the somite segmentation clock: revelation of unstable oscillators in the individual presomitic mesoderm cells.

Notch signaling components such as the basic helix-loop-helix gene Hes1 are cyclically expressed by negative feedback in the presomitic mesoderm (PSM) and constitute the somite segmentation clock. Because Hes1 oscillation occurs in many cell types, this clock may regulate the timing in many biological systems. Although the Hes1 oscillator is stable in the PSM, it damps rapidly in other cells, suggesting that the oscillators in the former and the latter could be intrinsically different. Here, we have established the real-time bioluminescence imaging system of Hes1 expression and found that, although Hes1 oscillation is robust and stable in the PSM, it is unstable in the individual dissociated PSM cells, as in fibroblasts. Thus, the Hes1 oscillators in the individual PSM cells and fibroblasts are intrinsically similar, and these results, together with mathematical simulation, suggest that cell-cell communication is essential not only for synchronization but also for stabilization of cellular oscillators.

Animals↗
PubMed2006

Temporal regulation of Cre recombinase activity in neural stem cells.

Neural stem cells are known to give rise to distinct subtypes of neurons and glial cells over time by changing their competency. However, precise characterization of neural stem cells at various developmental stages remains to be performed. For such analysis, a tool to manipulate neural stem cells at different time points is necessary. Here, we generated transgenic mice that express Cre-ER(T2) in the ventricular zone of the developing nervous system under the control of the nestin promoter and enhancer (Nes-CreER(T2)). In mice expressing Cre-ER(T2) at appropriate levels, Cre recombinase activity was mostly inactive but efficiently activated by tamoxifen within 1 day. When such mice were crossed with the ROSA-26 or Z/EG reporter mice, neural stem cells were permanently labeled after administration of tamoxifen. Thus, Nes-CreER(T2) mice offer a powerful tool to manipulate neural stem cells genetically at desired time points.

Animals↗
PubMed2006

Notch1 expression is spatiotemporally correlated with neurogenesis and negatively regulated by Notch1-independent Hes genes in the developing nervous system.

In the developing nervous system, neural stem cells initially proliferate by a symmetric cell division and then undergo an asymmetric cell division, which makes one neuron (or neuronal precursor) and one progenitor. It remains to be determined how the switch from the symmetric to asymmetric cell divisions is regulated. Here, we found that Notch1 is expressed in the regions where neurogenesis occurs actively but not in the regions where neurogenesis does not yet occur. Furthermore, in Hes-mutant mice where neurogenesis is accelerated, Notch1 expression is also accelerated. Thus, Notch1 expression is negatively regulated by Hes genes and is spatiotemporally correlated with neurogenesis, suggesting that the neural stem cells that undergo asymmetric cell divisions express Notch1, whereas those that undergo symmetric cell divisions do not. We propose that initiation of Notch1 expression is one of the key features for switch from the symmetric to asymmetric cell divisions of neural stem cells and that this process is negatively regulated by Notch1-independent Hes genes.

Animals↗
PubMed2006

Hes1 and Hes5 regulate the development of the cranial and spinal nerve systems.

The basic helix-loop-helix genes Hes1 and Hes5, known Notch effectors, regulate the maintenance of neural stem cells and the development of the central nervous system (CNS). In the absence of Hes1 and Hes5, the size, shape and cytoarchitecture of the CNS are severely disorganized, but the development of the peripheral nervous system remains to be analyzed. Here, we found that in Hes1;Hes5 double-mutant mice, the cranial and spinal nerve systems are also severely disorganized. In these mutant mice, axonal projections from the mesencephalic neurons to the trigeminal (V) ganglion become aberrant and the proximal parts of the glossopharyngeal (IX) and vagus (X) nerves are fused. The hypoglossal (XII) nerve is also formed poorly. Furthermore, the dorsal root ganglia are fused with the spinal cord, and the dorsal and ventral roots of the spinal nerves are lacking in many segments. These results indicate that Hes1 and Hes5 play an important role in the formation of the cranial and spinal nerve systems.

Animals↗
PubMed2005

Target selectivity of vertebrate notch proteins. Collaboration between discrete domains and CSL-binding site architecture determines activation probability.

All four mammalian Notch proteins interact with a single DNA-binding protein (RBP-jkappa), yet they are not equivalent in activating target genes. Parallel assays of three Notch-responsive promoters in several cell lines revealed that relative activation strength is dependent on protein module and promoter context more than the cellular context. Each Notch protein reads binding site orientation and distribution on the promoter differently; Notch1 performs extremely well on paired sites, and Notch3 prefers single sites in conjunction with a proximal zinc finger transcription factor. Although head-head sites can elicit a Notch response on their own, use of CBS (CSL binding site) in tail-tail orientation is context-dependent. Bias for specific DNA elements is achieved by interplay between the N-terminal RAM (RBP-jkappa-associated molecule/ankyrin region), which interprets CBS proximity and orientation, and the C-terminal transactivation domain that interacts specifically with the transcription machinery or nearby factors. To confirm the prediction that modular design underscores the evolution of functional divergence between Notch proteins, we generated a synthetic Notch protein (Notch1 ankyrin with Notch3 transactivation domain) that displayed superior signaling strength on the hes5 promoter. Consistent with the prediction that "preferred" targets (Hes1) should respond faster and at lower Notch concentration than other targets, we showed that Hes5-GFP was extinguished fast and recovered slowly, whereas Hes1-GFP was inhibited late and recovered quickly after a pulse of DAPT in metanephroi cultures.

Animals↗
PubMed2005

Prethymic T-cell development defined by the expression of paired immunoglobulin-like receptors.

T cells are produced in the thymus from progenitors of extrathymic origin. As no specific markers are available, the developmental pathway of progenitors preceding thymic colonization remains unclear. Here we show that progenitors in murine fetal liver and blood, which are capable of giving rise to T cells, NK cells and dendritic cells, but not B cells, can be isolated by their surface expression of paired immunoglobulin-like receptors (PIR). PIR expression is maintained until the earliest intrathymic stage, then downregulated before the onset of CD25 expression. Unlike intrathymic progenitors, generation of prethymic PIR(+) progenitors does not require Hes1-mediated Notch signaling. These findings disclose a prethymic stage of T-cell development programmed for immigration of the thymus, which is genetically separable from intrathymic stages.

Animals↗
PubMed2005

CCAAT/enhancer-binding protein phosphorylation biases cortical precursors to generate neurons rather than astrocytes in vivo.

The intracellular mechanisms that bias mammalian neural precursors to generate neurons versus glial cells are not well understood. We demonstrated previously that the growth factor-regulated mitogen-activated protein kinase kinase (MEK) and its downstream target, the CCAAT/enhancer-binding protein (C/EBP) family of transcription factors, are essential for neurogenesis in cultured cortical precursor cells (Ménard et al., 2002). Here, we examined a role for this pathway during cortical cell fate determination in vivo using in utero electroporation of the embryonic cortex. These studies demonstrate that inhibition of the activity of either MEK or the C/EBPs inhibits the genesis of neurons in vivo. Moreover, the MEK pathway mediates phosphorylation of C/EBPbeta in cortical precursors, and expression of a C/EBPbeta construct in which the MEK pathway phosphorylation sites are mutated inhibits neurogenesis. Conversely, expression of a C/EBPbeta construct, in which the same sites are mutated to glutamate and therefore are "constitutively" phosphorylated, enhances neurogenesis in the early embryonic cortex. A subpopulation of precursors in which C/EBP activity is inhibited are maintained as cycling precursors in the ventricular/subventricular zone of the cortex until early in postnatal life, when they have an enhanced propensity to generate astrocytes, presumably in response to gliogenic signals in the neonatal environment. Thus, activation of an MEK-C/EBP pathway in cortical precursors in vivo biases them to become neurons and against becoming astrocytes, thereby acting as a growth factor-regulated switch.

Animals↗
PubMed2005

Visualization of embryonic neural stem cells using Hes promoters in transgenic mice.

In the central nervous system, neural stem cells proliferate in the ventricular zone (VZ) and sequentially give rise to both neurons and glial cells in a temporally and spatially regulated manner, suggesting that stem cells may differ from one another in different brain regions and at different developmental stages. For the purpose of marking and purifying neural stem cells to ascertain whether such differences exist, we generated transgenic mice using promoters from Hes genes (pHes1 or pHes5) to drive expression of destabilized enhanced green fluorescent protein. In the developing brains of these transgenic mice, GFP expression was restricted to undifferentiated cells in the VZ, which could asymmetrically produce a Numb-positive neuronal daughter and a GFP-positive progenitor cell in clonal culture, indicating that they retain the capacity to self-renew. Our results suggest that pHes-EGFP transgenic mice can be used to explore similarities and differences among neural stem cells during development.

Animals↗
PubMed2005

Mash1 and Math3 are required for development of branchiomotor neurons and maintenance of neural progenitors.

Basic helix-loop-helix (bHLH) transcription factors are known to play important roles in neuronal determination and differentiation. However, their exact roles in neural development still remain to be determined because of the functional redundancy. Here, we examined the roles of neural bHLH genes Mash1 and Math3 in the development of trigeminal and facial branchiomotor neurons, which derive from rhombomeres 2-4. In Math3-null mutant mice, facial branchiomotor neurons are misspecified, and both trigeminal and facial branchiomotor neurons adopt abnormal migratory pathways. In Mash1;Math3 double-mutant mice, trigeminal and facial branchiomotor neurons are severely reduced in number partly because of increased apoptosis. In addition, neurons with migratory defects are intermingled over the midline from either side of the neural tube. Furthermore, oligodendrocyte progenitors of rhombomere 4 are reduced in number. In the absence of Mash1 and Math3, expression of Notch signaling components is severely downregulated in rhombomere 4 and neural progenitors are not properly maintained, which may lead to intermingling of neurons and a decrease in oligodendrocyte progenitors. These results indicate that Mash1 and Math3 not only promote branchiomotor neuron development but also regulate the subsequent oligodendrocyte development and the cytoarchitecture by maintaining neural progenitors through Notch signaling.

Animals↗
PubMed2005

Otx2 regulates subtype specification and neurogenesis in the midbrain.

The transcription factor Otx2 is required to determine mesencephalic versus metencephalic (cerebellum/pons) territory during embryogenesis. This function of Otx2 primarily involves positioning and maintaining the mid-hindbrain organizer at the border between midbrain and anterior hindbrain. Otx2 expression is maintained long after this organizer is established. We therefore generated conditional mutants of Otx2 using the Cre/loxP system to study later roles during rostral brain development. For inactivation of Otx2 in neuronal progenitor cells, we crossed Otx2(flox/flox) animals with Nestin-Cre transgenic animals. In Nestin-Cre/+; Otx2(flox/flox) embryos, Otx2 activity was lost from the ventral midbrain starting at embryonic day 10.5 (E10.5). In these mutant embryos, the mid-hindbrain organizer was properly positioned at E12.5, although Otx2 is absent from the midbrain. Hence, the Nestin-Cre/+; Otx2(flox/flox) animals represent a novel mouse model for studying the role of Otx2 in the midbrain, independently of abnormal development of the mid-hindbrain organizer. Our data demonstrate that Otx2 controls the development of several neuronal populations in the midbrain by regulating progenitor identity and neurogenesis. Dorsal midbrain progenitors ectopically expressed Math1 and generate an ectopic cerebellar-like structure. Similarly, Nkx2.2 ectopic expression ventrally into tegmentum progenitors is responsible for the formation of serotonergic neurons and hypoplasia of the red nucleus in the midbrain. In addition, we discovered a novel role for Otx2 in regulating neurogenesis of dopaminergic neurons. Altogether, these results demonstrate that Otx2 is required from E10.5 onward to regulate neuronal subtype identity and neurogenesis in the midbrain.

Age Factors↗
PubMed2005

Roles of bHLH genes in neural stem cell differentiation.

Neural stem cells change their characteristics over time during development: they initially proliferate only and then give rise to neurons first and glial cells later. In the absence of the repressor-type basic helix-loop-helix (bHLH) genes Hes1, Hes3 and Hes5, neural stem cells do not proliferate sufficiently but prematurely differentiate into neurons and become depleted without making the later born cell types such as astrocytes and ependymal cells. Thus, Hes genes are essential for maintenance of neural stem cells to make cells not only in correct numbers but also in full diversity. Hes genes antagonize the activator-type bHLH genes, which include Mash1, Math and Neurogenin. The activator-type bHLH genes promote the neuronal fate determination and induce expression of Notch ligands such as Delta. These ligands activate Notch signaling and upregulate Hes1 and Hes5 expression in neighboring cells, thereby maintaining these cells undifferentiated. Thus, the activator-type and repressor-type bHLH genes regulate each other, allowing only subsets of cells to undergo differentiation while keeping others to stay neural stem cells. This regulation is essential for generation of complex brain structures of appropriate size, shape and cell arrangement.

Animals↗