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G Weinmaster

Publications and source records attributed to G Weinmaster.

At least 37 records · Page 2Linked to original sources
PubMed2000

Rapid Notch1 nuclear translocation after ligand binding depends on presenilin-associated gamma-secretase activity.

Recent data suggest an intimate relationship between the familial Alzheimer disease gene presenilin 1 (PS1) and proteolytic processing of both the amyloid precursor protein (APP) and the important cell signaling molecule, Notch1. We now show, using mammalian cells transfected with full-length Notch1, that the C terminal domain of Notch1 rapidly translocates to the nucleus upon stimulation with the physiologic ligand Delta and initiates a CBF1-dependent signal transduction cascade. Using this assay, we demonstrate that the same aspartate mutations in PS1 that block APP processing also prevent Notch1 cleavage and translocation to the nucleus. Moreover, we show that two APP gamma-secretase inhibitors also diminish Notch1 nuclear translocation in a dose-dependent fashion. However, Notch1 signaling, assessed by measuring the activity of CBF1, a downstream gene, was reduced but not completely abolished in the presence of either aspartate mutations or gamma-secretase inhibitors. Our results support the hypothesis that similar PS1-related enzymatic activity is necessary for both APP and Notch1 processing, yet suggest that Notch signaling may remain relatively preserved with moderate levels of gamma-secretase inhibition.

Alzheimer Disease↗
PubMed2000

SKIP, a CBF1-associated protein, interacts with the ankyrin repeat domain of NotchIC To facilitate NotchIC function.

Notch proteins are transmembrane receptors that mediate intercell communication and direct individual cell fate decisions. The activated intracellular form of Notch, NotchIC, translocates to the nucleus, where it targets the DNA binding protein CBF1. CBF1 mediates transcriptional repression through the recruitment of an SMRT-histone deacetylase-containing corepressor complex. We have examined the mechanism whereby NotchIC overcomes CBF1-mediated transcriptional repression. We identified SKIP (Ski-interacting protein) as a CBF1 binding protein in a yeast two-hybrid screen. Both CBF1 and SKIP are highly conserved evolutionarily, and the SKIP-CBF1 interaction is also conserved in assays using the Caenorhabditis elegans and Drosophila melanogaster SKIP homologs. Protein-protein interaction assays demonstrated interaction between SKIP and the corepressor SMRT. More surprisingly, SKIP also interacted with NotchIC. The SMRT and NotchIC interactions were mutually exclusive. In competition binding experiments SMRT displaced NotchIC from CBF1 and from SKIP. Contact with SKIP is required for biological activity of NotchIC. A mutation in the fourth ankyrin repeat that abolished Notch signal transduction did not affect interaction with CBF1 but abolished interaction with SKIP. Further, NotchIC was unable to block muscle cell differentiation in myoblasts expressing antisense SKIP. The results suggest a model in which NotchIC activates responsive promoters by competing with the SMRT-corepressor complex for contacts on both CBF1 and SKIP.

Amino Acid Sequence↗
PubMed2000

Independent development of pancreatic alpha- and beta-cells from neurogenin3-expressing precursors: a role for the notch pathway in repression of premature differentiation.

The nature and identity of the pancreatic beta-cell precursor has remained elusive for many years. One model envisions an early multihormonal precursor that gives rise to both alpha- and beta-cells and the other endocrine cell types. Alternatively, beta-cells have been suggested to arise late, directly from the GLUT2- and pancreatic duodenal homeobox factor-1 (PDX1)-expressing epithelium, which gives rise also to the acinar cells during this stage. In this study, we have identified a subset of the PDX1+ epithelial cells that are marked by expression of Neurogenin3 (Ngn3). Ngn3, a member of the basic helix-loop-helix (bHLH) family of transcription factors, is suggested to act upstream of NeuroD in a bHLH cascade. Detailed analysis of Ngn3/paired box factor 6 (PAX6) and NeuroD/PAX6 co-expression shows that the two bHLH factors are expressed in a largely nonoverlapping set of cells, but such analysis also suggests that the NeuroD+ cells arise from cells expressing Ngn3 transiently. NeuroD+ cells do not express Ki-67, a marker of proliferating cells, which shows that these cells are postmitotic. In contrast, Ki-67 is readily detected in Ngn3+ cells. Thus, Ngn3+ cells fulfill the criteria for an endocrine precursor cell. These expression patterns support the notion that both alpha- and beta-cells develop independently from PDX1+/Ngn3+ epithelial cells, rather than from GLU+/INS+ intermediate stages. The earliest sign of alpha-cell development appears to be Brain4 expression, which apparently precedes Islet-1 (ISL1) expression. Based on our expression analysis, we propose a temporal sequence of gene activation and inactivation for developing alpha- and beta-cells beginning with activation of NeuroD expression. Endocrine cells leave the cell cycle before NeuroD activation, but re-enter the cell cycle at perinatal stages. Dynamic expression of Notch1 in PDX+ epithelial cells suggests that Notch signaling could inhibit a Ngn-NeuroD cascade as seen in the nervous system and thus prevent premature differentiation of endocrine cells.

Animals↗
PubMed1999

Hepatic jagged1 expression studies.

Mutations in Jagged1, a Notch ligand, have been shown to result in Alagille syndrome (AGS), however, the causal link between haploinsufficiency of Jagged1 and intrahepatic ductal paucity is unknown. This survey was performed to determine the expression pattern of Jagged1 in the fetal and postnatal liver. Reverse transcription polymerase chain reaction (RT-PCR) showed Jagged1 expression in all samples studied including rat liver embryonic days 16 to 21, 1-day-old, 1-week-old, and 2-month-old adult rats. RT-PCR detected Jagged1 in total liver RNA extracted from cadaver organ donor samples from reduced human grafts and explanted native livers from a variety of pediatric disorders including AGS, biliary atresia, congenital hepatic fibrosis, sclerosing cholangitis, cystic fibrosis, fulminant hepatic failure, tyrosinemia, and chronic rejection. Immunohistochemistry showed Jagged1 expression in human fetal samples localized to the ductal plate from 14-week gestation onward. Expression in the postnatal liver was seen in biliary epithelium and zone 3 hepatocytes. In conclusion, these studies show that Jagged1 is expressed in the fetal and postnatal liver in health and disease. We show localization of expression by immunohistochemistry to ductal plate epithelium in human fetal samples and to the biliary epithelium and zone 3 hepatocytes in human postnatal samples. Our results show the localization of Jagged1 in fetal liver and demonstration of Jagged1 expression in postnatal rat and human liver specimens. Further studies of Jagged1 and the Notch signaling pathway are expected to elucidate mechanisms of the regulation of biliary epithelial growth and development.

Aging↗
PubMed1999

The Notch/Jagged pathway inhibits proliferation of human hematopoietic progenitors in vitro.

The cell surface receptor Notch1 is expressed on CD34+ hematopoietic precursors, whereas one of its ligands, Jagged1, is expressed on bone marrow stromal cells. To examine the role of Notch signaling in early hematopoiesis, human CD34+ cells were cultured in the presence or absence of exogenous cytokines on feeder layers that either did or did not express Jagged1. In the absence of recombinant growth factors, Jagged1 decreased myeloid colony formation by CD34+ cells, as well as 3H-thymidine incorporation and entry into S phase. In the presence of a strong cytokine signal to proliferate and mature, (interleukin 3 [IL-3] and IL-6, stem cell factor [SCF], and G-CSF), Jagged1 did not significantly alter either the fold expansion or the types of colonies formed by CD34+ cells. However, in the presence of SCF alone, Jagged1 increased erythroid colony formation twofold. These results demonstrate that Notch can modulate a growth factor signal, and that in the absence of growth factor stimulation, the Jagged1-Notch pathway preserves CD34+ cells in an immature state.

3T3 Cells↗
PubMed1999

Notch signalling pathway mediates hair cell development in mammalian cochlea.

The mammalian cochlea contains an invariant mosaic of sensory hair cells and non-sensory supporting cells reminiscent of invertebrate structures such as the compound eye in Drosophila melanogaster. The sensory epithelium in the mammalian cochlea (the organ of Corti) contains four rows of mechanosensory hair cells: a single row of inner hair cells and three rows of outer hair cells. Each hair cell is separated from the next by an interceding supporting cell, forming an invariant and alternating mosaic that extends the length of the cochlear duct. Previous results suggest that determination of cell fates in the cochlear mosaic occurs via inhibitory interactions between adjacent progenitor cells (lateral inhibition). Cells populating the cochlear epithelium appear to constitute a developmental equivalence group in which developing hair cells suppress differentiation in their immediate neighbours through lateral inhibition. These interactions may be mediated through the Notch signalling pathway, a molecular mechanism that is involved in the determination of a variety of cell fates. Here we show that genes encoding the receptor protein Notch1 and its ligand, Jagged 2, are expressed in alternating cell types in the developing sensory epithelium. In addition, genetic deletion of Jag2 results in a significant increase in sensory hair cells, presumably as a result of a decrease in Notch activation. These results provide direct evidence for Notch-mediated lateral inhibition in a mammalian system and support a role for Notch in the development of the cochlear mosaic.

Animals↗
PubMed1999

Embryonic lethality and vascular defects in mice lacking the Notch ligand Jagged1.

The Notch signaling pathway is an evolutionarily conserved intercellular signaling mechanism essential for embryonic development in mammals. Mutations in the human JAGGED1 ( JAG1 ) gene, which encodes a ligand for the Notch family of transmembrane receptors, cause the autosomal dominant disorder Alagille syndrome. We have examined the in vivo role of the mouse Jag1 gene by creating a null allele through gene targeting. Mice homozygous for the Jag1 mutation die from hemorrhage early during embryogenesis, exhibiting defects in remodeling of the embryonic and yolk sac vasculature. We mapped the Jag1 gene to mouse chromosome 2, in the vicinity of the Coloboma ( Cm ) deletion. Molecular and complementation analyses revealed that the Jag1 gene is functionally deleted in the Cm mutant allele. Mice heterozygous for the Jag1 null allele exhibit an eye dysmorphology similar to that of Cm /+ heterozygotes, but do not exhibit other phenotypes characteristic of Cm /+ mice or of humans with Alagille syndrome. These results establish the phenotype of Cm /+ mice as a contiguous gene deletion syndrome and demonstrate that Jag1 is essential for remodeling of the embryonic vasculature.

Animals↗
PubMed1999

Notch signaling imposes two distinct blocks in the differentiation of C2C12 myoblasts.

Notch signal transduction regulates expression of downstream genes through the activation of the DNA-binding protein Su(H)/CBF1. In Drosophila most of Notch signaling requires Su(H); however, some Notch-dependent processes occur in the absence of Su(H) suggesting that Notch signaling does not always involve activation of this factor. Using constitutively active forms of Notch lacking CBF1-interacting sequences we identified a Notch signaling pathway that inhibits myogenic differentiation of C2C12 myoblasts in the absence of CBF1 activation. Here we show that ligand-induced Notch signaling suppresses myogenesis in C2C12 myoblasts that express a dominant negative form of CBF1, providing additional evidence for CBF1-independent Notch signal transduction. Surprisingly mutant forms of Notch deficient in CBF1 activation are unable to antagonize MyoD activity, despite the fact that they inhibit myogenesis. Moreover, Notch-induced antagonism of MyoD requires CBF1 suggesting that the CBF1-dependent pathway mediates a cell-type-specific block in the myogenic program. However, Notch signaling in the absence of CBF1 activation blocks both myogenesis and osteogenesis, indicative of a general block in cellular differentiation. Taken together our data provide evidence for two distinct Notch signaling pathways that function to block differentiation at separate steps during the process of myogenesis in C2C12 myoblasts.

3T3 Cells↗
PubMed1998

Defects in limb, craniofacial, and thymic development in Jagged2 mutant mice.

The Notch signaling pathway is a conserved intercellular signaling mechanism that is essential for proper embryonic development in numerous metazoan organisms. We have examined the in vivo role of the Jagged2 (Jag2) gene, which encodes a ligand for the Notch family of transmembrane receptors, by making a targeted mutation that removes a domain of the Jagged2 protein required for receptor interaction. Mice homozygous for this deletion die perinatally because of defects in craniofacial morphogenesis. The mutant homozygotes exhibit cleft palate and fusion of the tongue with the palatal shelves. The mutant mice also exhibit syndactyly (digit fusions) of the fore- and hindlimbs. The apical ectodermal ridge (AER) of the limb buds of the mutant homozygotes is hyperplastic, and we observe an expanded domain of Fgf8 expression in the AER. In the foot plates of the mutant homozygotes, both Bmp2 and Bmp7 expression and apoptotic interdigital cell death are reduced. Mutant homozygotes also display defects in thymic development, exhibiting altered thymic morphology and impaired differentiation of gamma delta lineage T cells. These results demonstrate that Notch signaling mediated by Jag2 plays an essential role during limb, craniofacial, and thymic development in mice.

Animals↗
PubMed1998

Notch receptor activation inhibits oligodendrocyte differentiation.

In this study, we show that oligodendrocyte differentiation is powerfully inhibited by activation of the Notch pathway. Oligodendrocytes and their precursors in the developing rat optic nerve express Notch1 receptors and, at the same time, retinal ganglion cells express Jagged1, a ligand of the Notch1 receptor, along their axons. Jagged1 expression is developmentally regulated, decreasing with a time course that parallels myelination in the optic nerve. These results suggest that the timing of oligodendrocyte differentiation and myelination is controlled by the Notch pathway and raise the question of whether localization of myelination is controlled by this pathway.

Animals↗
PubMed1998

Notch signaling: direct or what?

Notch signaling has been implicated in a wide variety of processes from cell-fate decisions, tissue patterning and morphogenesis to human diseases and cancer. A model for Notch directly regulating gene expression has been proposed and at least two signaling pathways have been identified; however, the molecular mechanism(s) by which Notch signaling produces so many outcomes remains unclear.

Animals↗
PubMed1997

The ins and outs of notch signaling.

The Notch gene encodes a cell surface protein that regulates cell fate choices in vertebrates and invertebrates. Given the wide variety of cell types influenced by Notch, it would seem that the signal relayed through Notch activation is not an instructive one per se. Rather, Notch signaling is thought to influence the cell's ability to respond to instructive signals responsible for specific cell fates. Expression and functional studies of Notch support this idea; however, the possibility of additional functions for Notch cannot be excluded. Much of what we know about the Notch signaling pathway comes from studies with Drosophila Notch and the Caenorhabditis elegans Notch-related genes lin-12 and glp-1. With the isolation of multiple vertebrate Notch genes we are beginning to understand and define Notch signaling in vertebrates as well. A number of excellent reviews have been published summarizing the current status of Notch/LIN-12/GLP-1 signaling in Drosophila and C. elegans, as well as recent findings with the vertebrate counterparts. Here I review the structure of the various Notch proteins and their putative ligands, and discuss possible interactions between Notch, its ligands, and other cellular components that affect Notch signal transduction. A role for Notch signaling during normal development and in disease processes is discussed in an accompanying review by T. Gridley (1997, Mol. Cell. Neurosci. 9: 103-108).

Animals↗
PubMed1997

Epstein-Barr virus immortalization: Notch2 interacts with CBF1 and blocks differentiation.

EBNA2 is essential for immortalization of B cells by Epstein-Barr virus. EBNA2 is tethered to responsive promoters through a cellular factor, CBF1. CBF1 also binds to the activated form of mammalian Notch1, providing a linkage between EBNA2 function and Notch signalling. However, Notch2 is the predominant form expressed in spleen. The degree to which these Notch homologs are functionally convergent is not known. We present evidence that Notch2 also signals through CBF1. As is the case for Notch1, Notch2 interacted with the minimal repression domain of CBF1 and was targeted to CBF1 through the intracellular, subtransmembrane domain. Additional characterization suggested that the interaction domain of Notch may be bipartite. The intracellular domain of Notch2 (Notch2IC) located to the nucleus. This activated form of Notch2 transactivated expression of a target gene containing upstream CBF1 binding sites. The use of CBF1 mutants carrying amino acid substitutions in the transcriptional repression domain revealed that activation of gene expression by Notch2 is also based on masking of CBF1-mediated repression. Targeting of Notch1 and targeting of Notch2 were found to be identical and distinguishable from targeting by EBNA2. Mutation of CBF1 at codons 249 to 251 abolished interaction with both Notch proteins but not with EBNA2. In a biological examination of Notch2 function in muscle cells, Notch2IC activated endogenous HES-1 gene expression and blocked muscle cell differentiation. Overall, the data imply that at least a subset of the intracellular events following signalling in cells expressing Notch2 are common to those in Notch1-expressing cells. The concept that EBNA2 functions by mimicking Notch signalling is therefore viable whether cells are expressing Notch1 or Notch2.

Animals↗
PubMed1997

Differential expression of mammalian Numb, Numblike and Notch1 suggests distinct roles during mouse cortical neurogenesis.

During Drosophila neurogenesis, asymmetric cell divisions are achieved by differential segregation of Numb (d-Numb) into one of the daughter cells to cause a bias in the Notch mediated cell-cell interaction. We have isolated a second mammalian gene with significant sequence similarity to d-numb, mouse numblike. When expressed in dividing neural precursors in Drosophila, Numblike is symmetrically distributed in the cytoplasm, unlike endogenous d-Numb or expressed mouse Numb (m-Numb), both of which are asymmetrically localized to one half of the cell membrane. In d-numb loss-of-function mutant embryos, expression of Numblike allows both daughter cells of a neural precursor to adopt the fate of the cell that normally inherits d-Numb. In mice, numblike mRNA is preferentially expressed in adult and embryonic nervous system. In the developing neocortex, Numblike is expressed in postmitotic neurons in the cortical plate, but not in progenitors within the ventricular zone where m-Numb and Notch1 are expressed. We have also found that, in dividing cortical progenitors, Notch1 is distributed around the entire membrane, unlike m-Numb which is asymmetrically localized to the apical membrane. We propose that an interplay between cell-intrinsic mechanisms (executed by m-numb and numblike) and cell-extrinsic mechanisms (mediated by Notch1) may be involved in both progenitor cell proliferation and neuronal differentiation during mammalian cortical neurogenesis.

Amino Acid Sequence↗
PubMed1996

Jagged2: a serrate-like gene expressed during rat embryogenesis.

DSL (Delta, Serrate, Lag-2) ligands activate Notch signaling and thereby regulate the differentiation of many different cell types during development. We have isolated a novel Serrate-like gene, Jagged2, whose amino acid sequence and expression pattern during rat embryogenesis suggest that it functions as a ligand for Notch. In contrast to previously described DSL ligands for Notch, Jagged2 is not widely expressed in the developing central nervous system. However, Jagged2 and Notch1 are coexpressed in the apical ectodermal ridge (AER), suggesting a role for this ligand-receptor pair in limb development. Furthermore, unlike Jagged1, Jagged2 is coexpressed with Notch in the developing thymus, where it may induce Notch signaling to direct T-cell fate. Our data are consistent with the idea that the diversity of cell types regulated by Notch signaling is a consequence of activation of unique Notch isoforms by different DSL ligands.

Amino Acid Sequence↗
PubMed1996

An activated form of Notch influences the choice between CD4 and CD8 T cell lineages.

Notch is a transmembrane receptor that controls cell fate decisions in Drosophila and whose role in mammalian cell fate decisions is beginning to be explored. We are investigating the role of Notch in a well-studied mammalian cell fate decision: the choice between the CD8 and CD4 T cell lineages. Here we report that expression of an activated form of Notch1 in developing T cells of the mouse leads to both an increase in CD8 lineage T cells and a decrease in CD4 lineage T cells. Expression of activated Notch permits the development of mature CD8 lineage thymocytes even in the absence of class I major histocompatability complex (MHC) proteins, ligands that are normally required for the development of these cells. However, activated Notch is not sufficient to promote CD8 cell development when both class I and class II MHC are absent. These results implicate Notch as a participant in the CD4 versus CD8 lineage decision.

Animals↗