Hes1 suppresses acute myeloid leukemia development through FLT3 repression.

In leukemogenesis, Notch signaling can be up and downregulated in a context-dependent manner. The transcription factor hairy and enhancer of split-1 (Hes1) is well-characterized as a downstream target of Notch signaling. Hes1 encodes a basic helix-loop-helix-type protein, and represses target gene expression. Here, we report that deletion of the Hes1 gene in mice promotes acute myeloid leukemia (AML) development induced by the MLL-AF9 fusion protein. We then found that Hes1 directly bound to the promoter region of the FMS-like tyrosine kinase 3 (FLT3) gene and downregulated the promoter activity. FLT3 was consequently upregulated in MLL-AF9-expressing immortalized and leukemia cells with a Hes1- or RBPJ-null background. MLL-AF9-expressing Hes1-null AML cells showed enhanced proliferation and ERK phosphorylation following FLT3 ligand stimulation. FLT3 inhibition efficiently abrogated proliferation of MLL-AF9-induced Hes1-null AML cells. Furthermore, an agonistic anti-Notch2 antibody induced apoptosis of MLL-AF9-induced AML cells in a Hes1-wild type but not a Hes1-null background. We also accessed two independent databases containing messenger RNA (mRNA) expression profiles and found that the expression level of FLT3 mRNA was negatively correlated with those of HES1 in patient AML samples. These observations demonstrate that Hes1 mediates tumor suppressive roles of Notch signaling in AML development, probably by downregulating FLT3 expression.

HES1 and HES5 are dispensable for cartilage and endochondral bone formation.

Notch signalling, via its downstream mediators HES1 and HES5, regulates development of several different tissues. In vitro studies suggest that these genes are also involved in chondrogenesis and endochondral bone formation. In order to investigate the importance of HES1 and HES5 for these developmental processes, mice lacking chondrogenic expression of HES1 and HES5 were constructed by interbreeding HES5(-/-) mice homozygous for the floxed HES1 allele (HES1(flox/flox)) with COL2A1-Cre transgenic mice, creating conditional HES1;HES5 double mutant mice. The formation of cartilage and endochondral bone was studied in these mice using histological and immunohistochemical stainings, including Alcian Blue van Gieson, Safranin-O, modified Mallory Aniline Blue, tartrate-resistant acid phosphatase and collagen type II stainings. The mice were also studied using several different morphometrical analyses and the differentiation potential of the chondrocytes was evaluated in vitro. Unexpectedly, the conditional HES1;HES5 double mutant mice did not display impaired development of cartilage or endochondral bone. Lack of altered phenotype in the conditional HES1;HES5 double mutant mice can be explained either by the HES1 and HES5 genes not being involved in cartilage and endochondral bone development or by functional redundancy between the genes belonging to the family of HES genes: that is, disruption of one gene could be compensated for by the activity of another. Our results further shed light on the compensatory reserves available during the developing cartilage and bone.

Ultradian oscillators in somite segmentation and other biological events.

Somite formation occurs every 2 hours in mouse embryos by periodic segmentation of the anterior ends of the presomitic mesoderm, and this process is controlled by a biological clock called the segmentation clock. During this process, the basic helix-loop-helix gene Hes7 is cyclically expressed, and each cycle leads to generation of a bilateral pair of somites. Both sustained expression and loss of expression of Hes7 result in severe somite fusion, indicating that Hes7 constitutes an essential component of the segmentation clock. Interestingly, expression of the related gene Hes1 also oscillates with a periodicity of about 2 hours in many cell types. Both sustained Hes1 expression and loss of Hes1 activity lead to retardation of the G(1) phase of the cell cycle, suggesting that Hes1 oscillation with an ultradian rhythm is required for efficient cell proliferation. Both Hes1 and Hes7 oscillations are regulated by negative feedback and rapid degradation of their gene products. Strikingly, expression of other factors such as Stat-Socs and Smad signaling molecules also display ultradian rhythms. All of these data suggest that ultradian oscillations are more general responses than were previously thought and that oscillatory and sustained gene expression results in different biological outcomes.

Math6, a bHLH gene expressed in the developing nervous system, regulates neuronal versus glial differentiation.

BACKGROUND: Whereas multiple basic helix-loop-helix (bHLH) genes are expressed in the developing nervous system, they account for the differentiation of only subsets of neurones, suggesting that there may be as-yet unidentified bHLH genes. RESULTS: We have isolated a novel bHLH gene, designated Math6, a distant mammalian homologue of the Drosophila proneural gene atonal. Structural analysis of the Math6 gene demonstrated that the coding region is divided into three exons, whereas that of other atonal homologues is present in a single exon, indicating that the genomic structure of Math6 is unique among the atonal homologues. Math6 is initially expressed by neural precursor cells in the ventricular zone, but later by subsets of differentiating and mature neurones such as hippocampal neurones and cerebellar Purkinje cells. Mis-expression of Math6 with retrovirus in the developing retina induced neurogenesis, while inhibiting gliogenesis, without affecting cell proliferation and death. CONCLUSIONS: These results show that cells which would normally differentiate into glia adopted the neuronal fate by mis-expression of Math6, indicating that Math6 promotes neuronal vs. glial fate determination in the nervous system.

Induction of photoreceptor-specific phenotypes in adult mammalian iris tissue.

We show that iris tissue in the adult rat eye, which is embryonically related to the neural retina, can generate cells expressing differentiated neuronal antigens. In addition, the Crx gene transfer induced the specific antigens for rod photoreceptors in the iris-derived cells, which was not seen in the adult hippocampus-derived neural stem cells. Our findings demonstrate a remarkable plasticity of adult iris tissue with potential clinical applications, as autologous iris tissue can be feasibly obtained with peripheral iridectomy.

Dynamic expression and essential functions of Hes7 in somite segmentation.

The basic helix-loop-helix (bHLH) gene Hes7, a putative Notch effector, encodes a transcriptional repressor. Here, we found that Hes7 expression oscillates in 2-h cycles in the presomitic mesoderm (PSM). In Hes7-null mice, somites are not properly segmented and their anterior-posterior polarity is disrupted. As a result, the somite derivatives such as vertebrae and ribs are severely disorganized. Although expression of Notch and its ligands is not affected significantly, the oscillator and Notch modulator lunatic fringe is expressed continuously throughout the mutant PSM. These results indicate that Hes7 controls the cyclic expression of lunatic fringe and is essential for coordinated somite segmentation.

Hes1 and Hes3 regulate maintenance of the isthmic organizer and development of the mid/hindbrain.

The isthmic organizer, which is located at the midbrain-hindbrain boundary, plays an essential role in development of the midbrain and anterior hindbrain. It has been shown that homeobox genes regulate establishment of the isthmic organizer, but the mechanism by which the organizer is maintained is not well understood. Here, we found that, in mice doubly mutant for the basic helix-loop-helix genes Hes1 and Hes3, the midbrain and anterior hindbrain structures are missing without any significant cell death. In these mutants, the isthmic organizer cells prematurely differentiate into neurons and terminate expression of secreting molecules such as Fgf8 and Wnt1 and the paired box genes Pax2/5, all of which are essential for the isthmic organizer function. These results indicate that Hes1 and Hes3 prevent premature differentiation and maintain the organizer activity of the isthmic cells, thereby regulating the development of the midbrain and anterior hindbrain.

Roles of the basic helix-loop-helix genes Hes1 and Hes5 in expansion of neural stem cells of the developing brain.

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- and Hes5- expressing cells are maintained as neural stem cells during embryogenesis. Conversely, in the absence of Hes1 and 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.

Hes1 and Hes5 activities are required for the normal development of the hair cells in the mammalian inner ear.

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.

BMP2-mediated alteration in the developmental pathway of fetal mouse brain cells from neurogenesis to astrocytogenesis.

We show that when telencephalic neural progenitors are briefly exposed to bone morphogenetic protein 2 (BMP2) in culture, their developmental fate is changed from neuronal cells to astrocytic cells. BMP2 significantly reduced the number of cells expressing microtubule-associated protein 2, a neuronal marker, and cells expressing nestin, a marker for undifferentiated neural precursors, but BMP2 increased the number of cells expressing S100-beta, an astrocytic marker. In telencephalic neuroepithelial cells, BMP2 up-regulated the expression of negative helix-loop-helix (HLH) factors Id1, Id3, and Hes-5 (where Hes is homologue of hairy and Enhancer of Split) that inhibited the transcriptional activity of neurogenic HLH transcription factors Mash1 and neurogenin. Ectopic expression of either Id1 or Id3 (where Id is inhibitor of differentiation) inhibited neurogenesis of neuroepithelial cells, suggesting an important role for these HLH proteins in the BMP2-mediated changes in the neurogenic fate of these cells. Because gliogenesis in the brain and spinal cord, derived from implanted neural stem cells or induced by injury, is responsible for much of the failure of neuronal regeneration, this work may lead to a therapeutic strategy to minimize this problem.

Hes7: a bHLH-type repressor gene regulated by Notch and expressed in the presomitic mesoderm.

BACKGROUND: Whereas Notch signalling is essential for somitogenesis, mice deficient for the basic helix-loop-helix (bHLH) genes Hes1 and Hes5, downstream Notch effectors, display normal somite formation, indicating that there may be an as-yet unidentified Hes1-related bHLH gene. RESULTS: We identified a novel bHLH gene, designated Hes7, from mouse embryos. Hes7 has a conserved bHLH domain in the amino-terminal region and the WRPW domain at the carboxy-terminal end, like Hes1. The mouse Hes7 gene is located next to Aloxe3, which is mapped to a position 37.0 cM from the centromere on chromosome 11. In a transfection analysis, Hes7 represses transcription from the N box- and E box-containing promoters. In addition, Hes7 suppresses the E47-induced transcriptional activation. Promoter analysis indicated that Hes7 expression is controlled by Notch signalling. Strikingly, Hes7 is specifically expressed in the presomitic mesoderm in a dynamic manner. We also identified two related bHLH genes from human: one is closely related to mouse Hes7 and therefore designated hHes7 and the other designated hHes4. CONCLUSION: The structure, transcriptional activity and expression pattern in the presomitic mesoderm of Hes7 are very similar to those of Hes1, suggesting that Hes7, together with Hes1, may play a role in somite formation under the control of Notch signalling.

Roles of homeobox and bHLH genes in specification of a retinal cell type.

Previous analysis of mutant mice has revealed that the bHLH genes Mash1 and Math3, and the homeobox gene Chx10 are essential for generation of bipolar cells, the interneurons present in the inner nuclear layer of the retina. Thus, a combination of the bHLH and homeobox genes should be important for bipolar cell genesis, but the exact functions of each gene remain largely unknown. We have found that in Mash1-Math3 double-mutant retina, which exhibits a complete loss of bipolar cells, Chx10 expression did not disappear but remained in Müller glial cells, suggesting that Chx10 expression per se is compatible with gliogenesis. In agreement with this, misexpression of Chx10 alone with retrovirus in the retinal explant cultures induced generation of the inner nuclear layer cells, including Müller glia, but few of them were mature bipolar cells. Misexpression of Mash1 or Math3 alone did not promote bipolar cell genesis either, but inhibited Müller gliogenesis. In contrast, misexpression of Mash1 or Math3 together with Chx10 increased the population of mature bipolar cells and decreased that of Müller glia. Thus, the homeobox gene provides the inner nuclear layer-specific identity while the bHLH genes regulate the neuronal versus glial fate determination, and these two classes of genes together specify the bipolar cell fate. Moreover, Mash1 and Math3 promoted the bipolar cell fate, but not the other inner nuclear layer-specific neuronal subtypes in the presence of Chx10, raising the possibility that the bHLH genes may be involved in neuronal subtype specification, in addition to simply making the neuronal versus glial fate choice.

Significance of proneural basic helix-loop-helix transcription factors in neuroendocrine differentiation of fetal lung epithelial cells and lung carcinoma cells.

In this brief review article, we describe how cell fate determination by which the airway epithelial cells become neuroendocrine or non-neuroendocrine is regulated by a network of basic helix-loop-helix transcription (bHLH) factors in a similar manner to neuronal differentiation, and how this system could work to determine cell differentiation of human lung carcinomas. Immunohistochemical studies reveal that mammalina achaete-scute complex homologue (Mash)1 is expressed in pulmonary neuroendocrine cells (PNEC), while hairy and Enhancer of split (Hes)1 is expressed in pulmonary non-neuroendocrine cells (non-PNEC). Studies using gene-deficient mice for the bHLH factors revealed that in Mash1 homozygous null mice no PNEC are detected, while PNEC increase markedly in Hes1 homozygous null mice. These observations suggest that Mash1 is an essential positive factor for neuroendocrine differentiation of lung epithelium, and that Hes1 is one of the repressive factors for neuroendocrine differentiation. Moreover, immunohistochemical studies revealed that Notch receptors are detected in non-PNEC, and thus the Notch signalling pathway could play a role in the determination of airway epithelial cell differentiation. In human lung carcinomas, a similar bHLH network should operate to determine cell differentiation phenotypes. Generally, expression of the human homologue of Mash1 (HASH1) is detected in small cell carcinoma and carcinoids, while Hes1 seems to be expressed mainly in non-small cell carcinoma. Thus, proneuronal bHLH factors may play roles in cell fate determination of the airway epithelial system, and may regulate human airway epithelial cells in diseased conditions.

dlk inhibits stem cell factor-induced colony formation of murine hematopoietic progenitors: Hes-1-independent effect.

Delta-like (dlk) is a family of transmembrane proteins containing epidermal growth factor-like repeat motifs homologous to the notch/delta/serrate family. Recent studies suggest that dlk is a negative regulator of adipocyte differentiation, a promoting factor of cobblestone area colony formation, and a molecule which influences stromal cell-pre-B cell interactions and augments cellularity of developing thymocytes. However, the role of dlk in regulating the growth and differentiation of hematopoietic progenitors remains unclear. In the present study, we examined the effect of dlk on the proliferation of murine hematopoietic progenitors by hematopoietic growth factors. Soluble dlk-IgG Fc chimeric protein completely inhibited the colony formation of lineage-marker negative (Lin-) bone marrow cells by GM-CSF, G-CSF, or macrophage-CSF (M-CSF) in the presence of stem cell factor (SCF). However, dlk failed to inhibit the colony formation of Lin- bone marrow cells by CSF, as described above, or M-CSF plus interleukin 3. Furthermore, dlk failed to inhibit the colony formation of Hes-1-null fetal liver cells by M-CSF in the presence of SCF. These findings suggest that dlk is an important regulator of hematopoietic progenitor proliferation. Depending on the presence of SCF, dlk may act as a growth inhibitor, although dlk signaling does not mediate Hes-1 transcription factor.

The basic helix-loop-helix gene hesr2 promotes gliogenesis in mouse retina.

Members of a subclass of hairy/Enhancer of split [E(spl)] homologs, called hesr genes, are structurally related to another subclass of hairy/E(spl) homologs, Hes genes, which play an important role in neural development. To characterize the roles of hesr genes in neural development, we used the retina as a model system. In situ hybridization analysis indicated that all hesr genes are expressed in the developing retina, but only hesr2 expression is associated spatially with gliogenesis. Each member was then misexpressed with retrovirus in the retinal explant cultures prepared from mouse embryos or neonates, which well mimic in vivo retinal development. Interestingly, hesr2 but not hesr1 or hesr3 promoted gliogenesis while inhibiting rod genesis without affecting cell proliferation or death, suggesting that the cells that normally differentiate into rods adopted the glial fate by misexpression of hesr2. The gliogenic activity of hesr2 was more profound when it was misexpressed postnatally than prenatally. In addition, double mutation of the neuronal determination genes Mash1 and Math3, which increases Müller glia at the expense of bipolar cells, upregulated hesr2 expression. These results indicate that, among structurally related hesr genes, only hesr2 promotes glial versus neuronal cell fate specification in the retina and that antagonistic regulation between hesr2 and Mash1-Math3 may determine the ratios of neurons and glia.

A novel cis-acting element regulates HES-1 gene expression in P19 embryonal carcinoma cells treated with retinoic acid.

The regulatory mechanisms of mammalian hairy and Enhancer of split homologue-1 (HES-1) genes were examined in mouse P19 embryonic carcinoma cells (P19 cells). Undifferentiated P19 stem cells expressed a basal level of the HES-1 gene, whereas the expression of this gene was increased upon induction of the cells to a neural cell lineage using retinoic acid (RA). Reporter co-transfection analysis identified an activating region within the upstream promoter region of HES-1 from nucleotides -201 to -172. This activating region, called activating region X (ARX), shows a high GC content and contains both an AP-2 binding motif and a CCAAT box. An electrophoretic mobility shift assay using nuclear proteins extracted from P19 cells showed that ARX forms a specific DNA-protein complex. Importantly, ARX-dependent transcription, as well as ARX-binding activity, was significantly increased in P19 cells treated with RA. These results indicate that ARX transduces signals that up-regulate HES-1 gene expression in response to RA-treatment. Thus, a novel cis-acting element involved in HES-1 gene regulation that plays a role in RA-induced neural differentiation of P19 cells has been identified.

Mammalian achaete-scute and atonal homologs regulate neuronal versus glial fate determination in the central nervous system.

Whereas vertebrate achaete-scute complex (as-c) and atonal (ato) homologs are required for neurogenesis, their neuronal determination activities in the central nervous system (CNS) are not yet supported by loss-of-function studies, probably because of genetic redundancy. Here, to address this problem, we generated mice double mutant for the as-c homolog Mash1 and the ato homolog Math3. Whereas in Mash1 or Math3 single mutants neurogenesis is only weakly affected, in the double mutants tectal neurons, two longitudinal columns of hindbrain neurons and retinal bipolar cells were missing and, instead, those cells that normally differentiate into neurons adopted the glial fate. These results indicated that Mash1 and Math3 direct neuronal versus glial fate determination in the CNS and raised the possibility that downregulation of these bHLH genes is one of the mechanisms to initiate gliogenesis.

Hes1 is a negative regulator of inner ear hair cell differentiation.

Hair cell fate determination in the inner ear has been shown to be controlled by specific genes. Recent loss-of-function and gain-of-function experiments have demonstrated that Math1, a mouse homolog of the Drosophila gene atonal, is essential for the production of hair cells. To identify genes that may interact with Math1 and inhibit hair cell differentiation, we have focused on Hes1, a mammalian hairy and enhancer of split homolog, which is a negative regulator of neurogenesis. We report here that targeted deletion of Hes1 leads to formation of supernumerary hair cells in the cochlea and utricle of the inner ear. RT-PCR analysis shows that Hes1 is expressed in inner ear during hair cell differentiation and its expression is maintained in adulthood. In situ hybridization with late embryonic inner ear tissue reveals that Hes1 is expressed in supporting cells, but not hair cells, of the vestibular sensory epithelium. In the cochlea, Hes1 is selectively expressed in the greater epithelial ridge and lesser epithelial ridge regions which are adjacent to inner and outer hair cells. Co-transfection experiments in postnatal rat explant cultures show that overexpression of Hes1 prevents hair cell differentiation induced by Math1. Therefore Hes1 can negatively regulate hair cell differentiation by antagonizing Math1. These results suggest that a balance between Math1 and negative regulators such as Hes1 is crucial for the production of an appropriate number of inner ear hair cells.

Basic helix-loop-helix transcription factors regulate the neuroendocrine differentiation of fetal mouse pulmonary epithelium.

To clarify the mechanisms that regulate neuroendocrine differentiation of fetal lung epithelia, we have studied the expression of the mammalian homologs of achaete-scute complex (Mash1) (Ascl1 - Mouse Genome Informatics); hairy and enhancer of split1 (Hes1); and the expression of Notch/Notch-ligand system in the fetal and adult mouse lungs, and in the lungs of Mash1- or Hes1-deficient mice. Immunohistochemical studies revealed that Mash1-positive cells seemed to belong to pulmonary neuroendocrine cells (PNEC) and their precursors. In mice deficient for Mash1, no PNEC were detected. Hes1-positive cells belong to non-neuroendocrine cells. In the mice deficient in Hes1, in which Mash1 mRNA was upregulated, PNEC appeared precociously, and the number of PNEC was markedly increased. NeuroD (Neurod1 - Mouse Genome Informatics) expression in the lung was detected in the adult, and was enhanced in the fetal lungs of Hes1-null mice. Expression of Notch1, Notch2, Notch3 and Notch4 mRNAs in the mouse lung increased with age, and Notch1 mRNA was expressed in a Hes1-dependent manner. Notch1, Notch2 and Notch3 were immunohistochemically detected in non-neuroendocrine cells. Moreover, analyses of the lungs from the gene-targeted mice suggested that expression of Delta-like 1 (Dll1 - Mouse Genome Informatics) mRNA depends on Mash1. Thus, the neuroendocrine differentiation depends on basic helix-loop-helix factors, and Notch/Notch-ligand pathways may be involved in determining the cell differentiation fate in fetal airway epithelium.