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At least 19 recordsLinked to original sources
PubMed2000

Expression of notch receptors, notch ligands, and fringe genes in hematopoiesis.

OBJECTIVE: Hematopoiesis is the process by which mature blood cell types are generated from a small population of pluripotent hematopoietic stem cells. How these cells undergo fate selection, however, is not fully understood. The Notch signaling system is known to mediate cell fate decisions of multipotent precursors in a wide range of complex animals throughout development. As Notch signaling involves cell-cell interactions, we sought to determine the expression of Notch receptors, ligands, and regulators in individual cell populations along the hematopoietic differentiation pathway. MATERIALS: Described here is a single cell RT-PCR analysis of Notch1, Notch3, Notch4, Notch ligands (Dll1 and Jagged1), and Fringe gene expression in cells of the blood system. As previously described, single cell globally amplified cDNA was generated by RT-PCR from various hematopoietic precursor cells whose potential was known from sibling analysis. A precursor hierarchy slot blot was created containing these cDNAs as well as samples from maturing blood cell populations and two fibroblast cell lines. The precursor slot blot was screened with probes for each of the candidate genes. RESULTS: Macrophage precursors expressed high levels of Notch1 transcript, while maturing macrophages expressed high levels of both Notch1 and Notch4. The Jagged 1 ligand transcript was highly expressed in terminally maturing cells including mast cells and megakaryocytes. In contrast, the Manic Fringe gene was highly expressed in uncommitted bi- and tri-potential precursors as well as in committed neutrophil and macrophage precursors. CONCLUSIONS: Distinct expression patterns of Jagged1 and Manic Fringe suggest that their corresponding proteins could regulate cell fate choices during hematopoiesis and may be responsible for regulating communication between lineage compartments during hematopoietic development.

Animals↗
PubMed2000

Calcium depletion dissociates and activates heterodimeric notch receptors.

Notch receptors participate in a highly conserved signaling pathway that regulates morphogenesis in multicellular animals. Maturation of Notch receptors requires the proteolytic cleavage of a single precursor polypeptide to produce a heterodimer composed of a ligand-binding extracellular domain (N(EC)) and a single-pass transmembrane signaling domain (N(TM)). Notch signaling has been correlated with additional ligand-induced proteolytic cleavages, as well as with nuclear translocation of the intracellular portion of N(TM) (N(ICD)). In the current work, we show that the N(EC) and N(TM) subunits of Drosophila Notch and human Notch1 (hN1) interact noncovalently. N(EC)-N(TM) interaction was disrupted by 0.1% sodium dodecyl sulfate or divalent cation chelators such as EDTA, and stabilized by millimolar Ca(2+). Deletion of the Ca(2+)-binding Lin12-Notch (LN) repeats from the N(EC) subunit resulted in spontaneous shedding of N(EC) into conditioned medium, implying that the LN repeats are important in maintaining the interaction of N(EC) and N(TM). The functional consequences of EDTA-induced N(EC) dissociation were studied by using hN1-expressing NIH 3T3 cells. Treatment of these cells for 10 to 15 min with 0.5 to 10 mM EDTA resulted in the rapid shedding of N(EC), the transient appearance of a polypeptide of the expected size of N(ICD), increased intranuclear anti-Notch1 staining, and the transient activation of an Notch-sensitive reporter gene. EDTA treatment of HeLa cells expressing endogenous Notch1 also stimulated reporter gene activity to a degree equivalent to that resulting from exposure of the cells to the ligand Delta1. These findings indicate that receptor activation can occur as a consequence of N(EC) dissociation, which relieves inhibition of the intrinsically active N(TM) subunit.

3T3 Cells↗
PubMed2000

MAML1, a human homologue of Drosophila mastermind, is a transcriptional co-activator for NOTCH receptors.

Notch receptors are involved in cell-fate determination in organisms as diverse as flies, frogs and humans. In Drosophila melanogaster , loss-of-function mutations of Notch produce a 'neurogenic' phenotype in which cells destined to become epidermis switch fate and differentiate to neural cells. Upon ligand activation, the intracellular domain of Notch (ICN) translocates to the nucleus, and interacts directly with the DNA-binding protein Suppressor of hairless (Su(H)) in flies, or recombination signal binding protein Jkappa (RBP-Jkappa) in mammals, to activate gene transcription. But the precise mechanisms of Notch-induced gene expression are not completely understood. The gene mastermind has been identified in multiple genetic screens for modifiers of Notch mutations in Drosophila. Here we clone MAML1, a human homologue of the Drosophila gene Mastermind, and show that it encodes a protein of 130 kD localizing to nuclear bodies. MAML1 binds to the ankyrin repeat domain of all four mammalian NOTCH receptors, forms a DNA-binding complex with ICN and RBP-Jkappa, and amplifies NOTCH-induced transcription of HES1. These studies provide a molecular mechanism to explain the genetic links between mastermind and Notch in Drosophila and indicate that MAML1 functions as a transcriptional co-activator for NOTCH signalling.

3T3 Cells↗
PubMed2001

Role of Deltex-1 as a transcriptional regulator downstream of the Notch receptor.

Intercellular signaling through the cell-surface receptor Notch plays important roles in a variety of developmental processes as well as in pathogenesis of some human cancers and genetic disorders. However, the mechanisms by which Notch signals are transduced into cells still remain elusive. Here we investigated the signaling mechanisms for Notch in the cell fate control of neural progenitor cells. We show that Deltex-1 (DTX1), a mammalian homolog of Drosophila Deltex, mediates a Notch signal to block differentiation of neural progenitor cells. We found that a significant fraction of DTX1 proteins were localized in the nucleus and physically interacted with the transcriptional coactivator p300. Through its binding to p300, DTX1 inhibited transcriptional activation by the neural-specific helix-loop-helix-type transcription factor MASH1, and this mechanism is likely responsible for the differentiation inhibition of neural progenitor cells. Our results further suggest that DTX1 regulates transcription independently of the previously characterized Notch signaling pathway involving RBP-J and HES1/HES5. Thus, DTX1 serves as an important signaling component downstream of Notch that regulates transcription in the nucleus.

Animals↗
PubMed2001

Notch receptors and hematopoiesis.

Notch receptors are involved in a variety of cell-fate decisions that affect the development and function of many organs, including hematopoiesis and the immune system. There are four mammalian Notch receptors that have only partially overlapping functions despite sharing similar structures and ligands. The ligands for Notch are transmembrane proteins expressed on adjacent cells, including Jagged and Delta, and it is quite possible that signaling is bidirectional. A large Notch precursor protein is proteolytically cleaved to form the mature cell-surface receptor. Ligand binding induces additional proteolytic events followed by translocation of the intracellular domain to the nucleus. There, Notch interacts with transcription factors such as RBPJ kappa, activating transcription of basic helix-loop-helix genes such as HES1. These in turn regulate expression of tissue-specific transcription factors that influence lineage commitment and other events. In this review, the details of Notch signaling will be discussed, with a focus on what is known about the role of Notch in hematopoiesis.

Animals↗
PubMed1996

[Role of the Notch receptors in intercellular communication].

The Notch gene was discovered in Drosophila at the beginning of the century and is currently the subject of intensive investigation, not only in invertebrates but also in vertebrates where remarkably well conserved homologues have been recently found. Notch encodes a new kind of cellular receptor whose functioning is still unclear and plays a role in a large number of cell interactions throughout development and in tissue renewal in the adult. Detailed study in invertebrates of some of these interactions has led to the identification of other genes required for transduction of the signal initiated by the receptor. Notch is always involved in processes where cells have the potential to choose between several different programmes of differentiation. Cells adopt a specific developmental pathway as a result of the inhibition of some programmes through Notch signalling. In this review we discuss the contribution of different experimental models to an understanding of the role of Notch in intercellular signalling.

Animals↗
PubMed1999

Mouse jagged1 physically interacts with notch2 and other notch receptors. Assessment by quantitative methods.

The Delta/Serrate/LAG-2 (DSL) domain containing proteins are considered to be ligands for Notch receptors. However, the physical interaction between DSL proteins and Notch receptors is poorly understood. In this study, we cloned a cDNA for mouse Jagged1 (mJagged1). To identify the receptor interacting with mJagged1 and to gain insight into its binding characteristics, we established two experimental systems using fusion proteins comprising various extracellular parts of mJagged1, a "cell" binding assay and a "solid-phase" binding assay. mJagged1 physically bound to mouse Notch2 (mNotch2) on the cell surface and to a purified extracellular portion of mNotch2, respectively, in a Ca(2+)-dependent manner. Scatchard analysis of mJagged1 binding to BaF3 cells and to the soluble Notch2 protein demonstrated dissociation constants of 0.4 and 0.7 nM, respectively, and that the number of mJagged1-binding sites on BaF3 is 5,548 per cell. Furthermore, deletion mutant analyses showed that the DSL domain of mJagged1 is a minimal binding unit and is indispensable for binding to mNotch2. The epidermal growth factor-like repeats of mJagged1 modulate the affinity of the interaction, with the first and second repeats playing a major role. Finally, solid-phase binding assay showed that Jagged1 binds to Notch1 and Notch3 in addition to Notch2, suggesting that mJagged1 is a ligand for multiple Notch receptors.

Amino Acid Sequence↗
PubMed1997

Diverse roles for the Notch receptor in the development of D. melanogaster.

Notch proteins appear to be involved in cell fate commitments with deep evolutionary roots. Homologues have been shown to play key roles in the development of nematodes, insects, amphibia, and mammals. Activity of the Notch receptor has been observed in the patterning of ectoderm, mesoderm, and endoderm, indicating an origin prior to the functional differentiation of these germ layers. To understand how a single receptor can participate so widely in development, we have been examining the role of specific extracellular segments of Notch. Early studies of mutations affecting widely separated EGF-like elements of Notch first raised the possibility for interaction with multiple ligands. Biochemical approaches, and exhaustive structure function studies in transgenic Drosophila are beginning to reveal how this receptor is activated, and point to a range of physical interactions with other proteins.

Animals↗
PubMed1999

Localization of ADAM10 and Notch receptors in bone.

In Drosophila melanogaster, the role of the metallodisintegrin, Kuzbanian (kuz), is thought to involve activation of the Drosophila Notch receptor that plays a role in cell-fate determination during neurogenesis and myoblast differentiation. To understand the possible function(s) of a-disintegrin and metalloproteinase (ADAM10), the mammalian ortholog of kuz, in the skeleton, we studied its expression as well as the messenger RNA (mRNA) encoding one candidate substrate, the mammalian Notch2 receptor in bone, bone cells, and cartilage. In sections of neonatal rat tibiae, ADAM10 is expressed in specific regions of articular cartilage and metaphyseal bone. Expression of ADAM10 in articular cartilage occurs predominantly in superficial chondrocytes and becomes more sporadic with increasing distance from the articular surface. In bone, ADAM10 is expressed by periosteal cells, osteoblasts, and osteocytes at locations of active bone formation. Osteoclasts did not express ADAM10. Notch2 mRNA expression was not detectable in superficial chondrocytes. However it colocalized at all sites of ADAM10 expression in bone cells. In vitro, both primary human osteoblasts and osteoblast cell lines expressed a single 4.5 kb and 7.5 kb transcript of ADAM10 and the Notch2 receptor homolog, respectively. Subcellular localization of the ADAM10 protein in MG-63 cells was determined using immunofluorescent techniques. These observations showed clearly that the ADAM10 protein was expressed in the trans-Golgi network and on the plasma membrane. Western blot analysis of fractionated cells showed that, in the plasma membrane fraction, the previously characterized 58 kDa and 56 kDa isoforms were present, whereas, in the trans-Golgi network, the ADAM10 protein was present in several additional bands, possibly indicative of further interdomain processing of the ADAM10 protein. The metallodisintegrins (ADAMs) have several putative functions, including modulation of cell adhesion, membrane-associated proteolysis, and cell-cell signaling. These observations suggest that, in bone but not cartilage, ADAM10 has catalytic activity within the transGolgi network and may play a role in the activation of Notch receptor homologs. This implicates ADAM10 in cell-fate determination of osteoblast progenitor cells, possibly during skeletal development and normal bone remodeling. Plasma-membrane-associated ADAM10 may confer alternative functions.

Amino Acid Sequence↗
PubMed1999

Rel/NF-kappaB can trigger the Notch signaling pathway by inducing the expression of Jagged1, a ligand for Notch receptors.

Jagged1 belongs to the DSL family of ligands for Notch receptors that control the proliferation and differentiation of various cell lineages. However, little is known about the transcription factors that regulate its expression. Here, we show that Jagged1 is a Rel/NF-kappaB-responsive gene. Both c-Rel and RelA induced jagged1 gene expression, whereas a mutant defective for transactivation did not. Importantly, jagged1 transcripts were also upregulated by endogenous NF-kappaB activation and this effect was inhibited by a dominant mutant of IkappaBalpha, a physiological inhibitor of NF-kappaB. Cell surface expression of Jagged1 in c-Rel-expressing cell monolayers led to a functional interaction with lymphocytes expressing the Notch1/TAN-1 receptor. This correlated with the initiation of signaling downstream of Notch, as evidenced by increased levels of HES-1 transcripts in co-cultivated T cells and of CD23 transcripts in co-cultivated B cells. Consistent with its Rel/NF-kappaB-dependent induction, Jagged1 was found to be highly expressed in splenic B cells where c-Rel is expressed constitutively. These results demonstrate that c-Rel can trigger the Notch signaling pathway in neighboring cells by inducing jagged1 gene expression, and suggest a role for Jagged1 in B-cell activation, differentiation or function. These findings also highlight the potential for an interplay between the Notch and NF-kappaB signaling pathways in the immune system.

B-Lymphocytes↗
PubMed1996

Functional conservation of mouse Notch receptor family members.

All the known members of the mouse Notch receptor family were examined for their biochemical function by interaction with a DNA binding protein RBP-Jkappa. mNotch2, mNotch3 and int3 (= mNotch4) were shown to interact with RBP-Jkappa by the GST-fusion pull down assay and dominant negative competition with Epstein Barr virus nuclear antigen 2. Furthermore the intracellular region of int3 was shown to transactivate the Epstein Barr virus TP1 promoter. These results indicate that all mouse Notch family members have biochemical functions similar to mNotch1, which transduces proliferative signal by direct interaction with the DNA binding protein RBP-Jkappa.

Amino Acid Sequence↗
PubMed1996

The Drosophila Numb protein inhibits signaling of the Notch receptor during cell-cell interaction in sensory organ lineage.

Specification of unequal daughter cell fates in the Drosophila external sense organ lineage requires asymmetric localization of the intrinsic determinant Numb as well as cell-cell interactions mediated by the Delta ligand and Notch receptor. Previous genetic studies indicated that numb acts upstream of Notch, and biochemical studies revealed that Numb can bind Notch. For a functional assay of the action of Numb on Notch signaling, we expressed these proteins in cultured Drosophila cells and used nuclear translocation of Suppressor of Hairless [Su(H)] as a reporter for Notch activity. We found that Numb interfered with the ability of Notch to cause nuclear translocation of Su(H); both the C-terminal half of the phosphotyrosine binding domain and the C terminus of Numb are required to inhibit Notch. Overexpression of Numb during wing development, which is sensitive to Notch dosage, revealed that Numb is also able to inhibit the Notch receptor in vivo. In the external sense organ lineage, the phosphotyrosine binding domain of Numb was found to be essential for the function but not for asymmetric localization of Numb. Our results suggest that Numb determines daughter cell fates in the external sense organ lineage by inhibiting Notch signaling.

Amino Acid Sequence↗
PubMed1998

Structural conservation of Notch receptors and ligands.

The Notch signalling pathway is an evolutionarily conserved cell-to-cell communication system utilized multiple times and in many tissues during development. The outcome of an interaction between Notch and its ligands is highly influenced by factors both extrinsic and intrinsic to Notch expressing cells, suggesting that Notch functions either directly or in parallel with other signalling systems to regulate cellular differentiation events. Protein domains common to all ligands and receptors of this system suggest conserved functional properties that likely relate to regulatory mechanisms for Notch signalling. Within this review, the known functional properties of these domains are analyzed with respect to their contributions to ligand/receptor interactions and Notch signalling.

Amino Acid Sequence↗
PubMed1999

The Drosophila melanogaster Suppressor of deltex gene, a regulator of the Notch receptor signaling pathway, is an E3 class ubiquitin ligase.

During development, the Notch receptor regulates many cell fate decisions by a signaling pathway that has been conserved during evolution. One positive regulator of Notch is Deltex, a cytoplasmic, zinc finger domain protein, which binds to the intracellular domain of Notch. Phenotypes resulting from mutations in deltex resemble loss-of-function Notch phenotypes and are suppressed by the mutation Suppressor of deltex [Su(dx)]. Homozygous Su(dx) mutations result in wing-vein phenotypes and interact genetically with Notch pathway genes. We have previously defined Su(dx) genetically as a negative regulator of Notch signaling. Here we present the molecular identification of the Su(dx) gene product. Su(dx) belongs to a family of E3 ubiquitin ligase proteins containing membrane-targeting C2 domains and WW domains that mediate protein-protein interactions through recognition of proline-rich peptide sequences. We have identified a seven-codon deletion in a Su(dx) mutant allele and we show that expression of Su(dx) cDNA rescues Su(dx) mutant phenotypes. Overexpression of Su(dx) also results in ectopic vein differentiation, wing margin loss, and wing growth phenotypes and enhances the phenotypes of loss-of-function mutations in Notch, evidence that supports the conclusion that Su(dx) has a role in the downregulation of Notch signaling.

Amino Acid Sequence↗
PubMed1993

Analysis of phenotypic abnormalities and cell fate changes caused by dominant activated and dominant negative forms of the Notch receptor in Drosophila development.

The Notch gene of Drosophila plays an important role in cell fate specification throughout development. The Notch protein contains a large extracellular domain of 36 EGF-like repeats as well as 3 Notch/lin-12 repeats and an intracellular domain with 6 cdc10/ankyrin repeats, motifs which are highly conserved in several vertebrate Notch homologues [1-7]. In this review we summarize the results of two recent studies which attempt to establish structure-function relationships of the various domains of the Notch gene product [8, 9]. The functions of various structural domains of the Notch protein in vivo were investigated using a series of deletion mutants which have been ectopically expressed either under the hsp70 heat-shock promoter or under the sevenless eye-specific promoter. Truncation of the extracellular domain of Drosophila Notch produces an activated receptor as judged by its ability to cause phenotypes matching those of gain-of-function alleles or duplications of the Notch locus [8]. Equivalent truncations of vertebrate Notch-related proteins have been associated with malignant neoplasms and other developmental abnormalities [3, 6, 10, 11]. In contrast, dominant negative phenotypes result from overexpression of a protein lacking most intracellular sequences. These results were extended by an analysis of activated Notch function at single-cell resolution in the Drosophila compound eye [9]. It was shown that while overexpression of full-length Notch in defined cell types has no apparent effects, overexpression of activated Notch in the same cells transiently blocks their proper cell-fate commitment, causing them to either adopt incorrect cell fates or to differentiate incompletely. Moreover, an activated Notch protein lacking the transmembrane domain is translocated to the nucleus, raising the possibility that Notch may participate directly in nuclear events.

Animals↗
PubMed1997

Identification and localization of a sea urchin Notch homologue: insights into vegetal plate regionalization and Notch receptor regulation.

The specifications of cell types and germ-layers that arise from the vegetal plate of the sea urchin embryo are thought to be regulated by cell-cell interactions, the molecular basis of which are unknown. The Notch intercellular signaling pathway mediates the specification of numerous cell fates in both invertebrate and vertebrate development. To gain insights into mechanisms underlying the diversification of vegetal plate cell types, we have identified and made antibodies to a sea urchin homolog of Notch (LvNotch). We show that in the early blastula embryo, LvNotch is absent from the vegetal pole and concentrated in basolateral membranes of cells in the animal half of the embryo. However, in the mesenchyme blastula embryo LvNotch shifts strikingly in subcellular localization into a ring of cells which surround the central vegetal plate. This ring of LvNotch delineates a boundary between the presumptive secondary mesoderm and presumptive endoderm, and has an asymmetric bias towards the dorsal side of the vegetal plate. Experimental perturbations and quantitative analysis of LvNotch expression demonstrate that the mesenchyme blastula vegetal plate contains both animal/vegetal and dorsoventral molecular organization even before this territory invaginates to form the archenteron. Furthermore, these experiments suggest roles for the Notch pathway in secondary mesoderm and endoderm lineage segregation, and in the establishment of dorsoventral polarity in the endoderm. Finally, the specific and differential subcellular expression of LvNotch in apical and basolateral membrane domains provides compelling evidence that changes in membrane domain localization of LvNotch are an important aspect of Notch receptor function.

Amino Acid Sequence↗