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.

Fringe differentially modulates Jagged1 and Delta1 signalling through Notch1 and Notch2.

Proteins encoded by the fringe family of genes are required to modulate Notch signalling in a wide range of developmental contexts. Using a cell co-culture assay, we find that mammalian Lunatic fringe (Lfng) inhibits Jagged1-mediated signalling and potentiates Delta1-mediated signalling through Notch1. Lfng localizes to the Golgi, and Lfng-dependent modulation of Notch signalling requires both expression of Lfng in the Notch-responsive cell and the Notch extracellular domain. Lfng does not prevent binding of soluble Jagged1 or Delta1 to Notch1-expressing cells. Lfng potentiates both Jagged1- and Delta1-mediated signalling via Notch2, in contrast to its actions with Notch1. Our data suggest that Fringe-dependent differential modulation of the interaction of Delta/Serrate/Lag2 (DSL) ligands with their Notch receptors is likely to have a significant role in the combinatorial repertoire of Notch signalling in mammals.

Expression and activity of the POU transcription factor SCIP.

POU proteins have been shown to transcriptionally active cell-specific genes and to participate in the determination of cell fate. It is therefore thought that these proteins function in development through the stable activation of genes that define specific developmental pathways. Evidence is provided here for an alternative mode of action. The primary structure of SCIP, a POU protein expressed by developing Schwann cells of the peripheral nervous system, was deduced and SCIP activity was studied. Both in normal development and in response to nerve transection, SCIP expression was transiently activated only during the period of rapid cell division that separates the premyelinating and myelinating phases of Schwann cell differentiation. In cotransfection assays, SCIP acted as a transcriptional repressor of myelin-specific genes.

SCIP: a glial POU domain gene regulated by cyclic AMP.

We have isolated cDNA clones encoding SCIP, a POU domain gene expressed by myelin-forming glial of the central and peripheral nervous systems. In purified Schwann cells cultured in the absence of neurons, expression of SCIP is suppressed. This suppression is relieved by cAMP, and induction of SCIP mRNA by this second messenger precedes cAMP induction of myelin-specific genes. Similarly, SCIP expression in vivo precedes full expression of myelin-specific genes in developing oligodendrocytes and Schwann cells. The sequence of the SCIP POU domain is identical to that of Tst-1, a recently identified member of a family of POU domain genes expressed by restricted subsets of neurons. Our results demonstrate that SCIP is also expressed by myelin-forming glia and suggest that it plays a central role in the progressive determination of these cells and their commitment to myelination.

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.

Notch signal transduction: a real rip and more.

The Notch signaling pathway functions in a wide variety of processes that regulate tissue patterning and morphogenesis in developing vertebrates and invertebrates. Research on the mechanism of ligand-induced Notch signal transduction has revealed a novel and essential element in the signal cascade. Some recent findings support a model in which sequential proteolytic cleavage serves to regulate Notch signal transduction.

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.

Nuclear Notch1 signaling and the regulation of dendritic development.

To understand the function of Notch in the mammalian brain, we examined Notch1 signaling and its cellular consequences in developing cortical neurons. We found that the cytoplasmic domain of endogenous Notch1 translocated to the nucleus during neuronal differentiation. Notch1 cytoplasmic-domain constructs transfected into cortical neurons were present in multiple phosphorylated forms, localized to the nucleus and could induce CBF1-mediated transactivation. Molecular perturbation experiments suggested that Notch1 signaling in cortical neurons promoted dendritic branching and inhibited dendritic growth. These observations show that Notch1 signaling to the nucleus exerts an important regulatory influence on the specification of dendritic morphology in neurons.

The neural RNA-binding protein Musashi1 translationally regulates mammalian numb gene expression by interacting with its mRNA.

Musashi1 (Msi1) is an RNA-binding protein that is highly expressed in neural progenitor cells, including neural stem cells. In this study, the RNA-binding sequences for Msi1 were determined by in vitro selection using a pool of degenerate 50-mer sequences. All of the selected RNA species contained repeats of (G/A)U(n)AGU (n = 1 to 3) sequences which were essential for Msi1 binding. These consensus elements were identified in some neural mRNAs. One of these, mammalian numb (m-numb), which encodes a membrane-associated antagonist of Notch signaling, is a likely target of Msi1. Msi1 protein binds in vitro-transcribed m-numb RNA in its 3'-untranslated region (UTR) and binds endogenous m-numb mRNA in vivo, as shown by affinity precipitation followed by reverse transcription-PCR. Furthermore, adenovirus-induced Msi1 expression resulted in the down-regulation of endogenous m-Numb protein expression. Reporter assays using a chimeric mRNA that combined luciferase and the 3'-UTR of m-numb demonstrated that Msi1 decreased the reporter activity without altering the reporter mRNA level. Thus, our results suggested that Msi1 could regulate the expression of its target gene at the translational level. Furthermore, we found that Notch signaling activity was increased by Msi1 expression in connection with the posttranscriptional down-regulation of the m-numb gene.

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.

Modifications of tumor histology by point mutations in the v-fps oncogene: possible role of extracellular matrix.

Fujinami sarcoma virus (FSV) encodes a protein-tyrosine kinase, p130gag-fps, whose enzymatic activity and ability to transform cultured cells to a neoplastic phenotype are reduced by substitution of the major autophosphorylation site tyrosine-1073 with other amino acids. We compared the histopathology of tumors formed in syngeneic immunocompetent rats by Rat-2 cells and by Rat-2 cells transformed in culture with (a) wild type (wt) FSV, (b) mutant FSV where the codon for tyrosine-1073 of p130gag-fps had been changed to codons for phenylalanine or serine, and (c) a revertant FSV, genotypically identical to wt FSV, in which the codon for tyrosine-1073 had been restored. Latency periods from cell inoculation to tumor formation were 12-29 weeks with Rat-2 cells, 6-8 weeks with mutant-transformed Rat-2 cells, and 2-4 weeks with wt FSV- and revertant FSV-transformed Rat-2 cells. Untransfected Rat-2 cells formed tumors that histologically resembled low grade fibrosarcomas or fibromas and were characterized by uniform fusiform cells in parallel arrays with a prominent collagenous stroma. The growth pattern of tumors produced by mutant FSV-transformed cells was generally similar, although cellular forms and intercellular organization were less uniform. In contrast, Rat-2 cells transformed with either wt FSV or revertant FSV produced tumors that resembled highly malignant sarcomas and were composed of diffuse sheets of pleomorphic, disorganized cells and stroma rich in hyaluronate but poor in fibrous components. Local invasion occurred in 25% of tumors produced by Rat-2 cells and in 53 and 36% of tumors formed by mutant FSV- and wt FSV-transformed cells, respectively. In culture, Rat-2 cells and mutant FSV-transformed cells produced fibrillar pericellular matrices of collagen I and fibronectin. From 5 to 15% of protein secreted by these cells was collagen. Cultures of wt FSV- and revertant FSV-transformed cells lacked collagen and fibronectin matrices and collagen secretion was reduced to 0-2%. These results show that clinically relevant histological characteristics of malignant tumors can correlate with single amino acid substitutions previously shown to affect the enzymatic activity and transforming ability of an oncogenic protein tyrosine kinase. The mechanisms underlying some of the histological differences in this system may be related to differences in the production of extracellular matrix components among the transformed cells.

Transcription factor expression and Notch-dependent regulation of neural progenitors in the adult rat spinal cord.

Recent studies have demonstrated that neural stem cells and other progenitors are present in the adult CNS. Details of their properties, however, remain poorly understood. Here we examined the properties and control mechanisms of neural progenitors in the adult rat spinal cord at the molecular level. Adult and embryonic progenitors commonly expressed various homeodomain-type (Pax6, Pax7, Nkx2.2, and Prox1) and basic helix-loop-helix (bHLH)-type (Ngn2, Mash1, NeuroD1, and Olig2) transcriptional regulatory factors in vitro. Unlike their embryonic counterparts, however, adult progenitors could not generate specific neurons that expressed markers appropriate for spinal motoneurons or interneurons, including Islet1, Lim1, Lim3, and HB9. Cells expressing the homeodomain factors Pax6, Pax7, and Nkx2.2 also emerged in vivo in response to injury and were distributed in unique patterns in the lesioned spinal cord. However, neither the expression of the neurogenic bHLH factors including Ngn2, Mash1, and NeuroD1 nor subsequent generation of new neurons could be detected in injured tissue. Our results suggest that signaling through the cell-surface receptor Notch is involved in this restriction. The expression of Notch1 in vivo was enhanced in response to injury. Furthermore, activation of Notch signaling in vitro inhibited differentiation of adult progenitors, whereas attenuation of Notch signals and forced expression of Ngn2 significantly enhanced neurogenesis. These results suggest that both the intrinsic properties of adult progenitors and local environmental signals, including Notch signaling, account for the limited regenerative potential of the adult spinal cord.

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.

Vascular expression of Notch pathway receptors and ligands is restricted to arterial vessels.

Mice with targeted mutations in genes required for Notch signal transduction die during embryogenesis, displaying overt signs of hemorrhage due to defects in their vascular development. Surprisingly, directed expression of a constitutively active form of Notch4 within mouse endothelial cells produces a similar vascular embryonic lethality. Moreover, patients with mutations in Notch3 exhibit the cerebral vascular disorder, cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). These findings underscore the importance of Notch signaling in vascular development; however, they do not identify the specific functional defect. Here, we report that Notch1, Notch3, Notch4, Delta4, Jagged1 and Jagged2 are all expressed in arteries, but are not expressed by veins. These findings identify an aspect of Notch signaling that could contribute to the mechanism by which this pathway modulates vascular morphogenesis.

Expression patterns of Notch1, Notch2, and Notch3 suggest multiple functional roles for the Notch-DSL signaling system during brain development.

The Notch-DSL signaling system consists of multiple receptors and ligands, and plays many roles in development. The function of Notch receptors and ligands in mammalian brain, however, is poorly understood. In the current study, we examined the expression patterns for three receptors of this system, Notch1, 2, and 3, in late embryonic and postnatal rat brain by in situ hybridization. The three receptors have overlapping but different patterns of expression. Messenger RNA for all three proteins is found in postnatal central nervous system (CNS) germinal zones and, in early postnatal life, within numerous cells throughout the CNS. Within zones of cellular proliferation of the postnatal brain, Notch1 mRNA is found in both the subventricular and the ventricular germinal zones, whereas Notch2 and Notch3 mRNAs are more highly localized to the ventricular zones. Both Notch1 and Notch3 mRNAs are expressed along the inner aspect of the dentate gyrus, a site of adult neurogenesis. Notch2 mRNA is expressed in the external granule cell layer of the developing cerebellum. In several brain areas, Notch1 and Notch2 mRNAs are relatively concentrated in white matter, whereas Notch3 mRNA is not. Neurosphere cultures (which contain CNS stem cells), purified astrocyte cultures, and striatal neuron-enriched cultures express Notch1 mRNA. However, in these latter cultures, Notch1 mRNA is produced by nestin-containing cells, rather than by postmitotic neurons. Taken together, these results support multiple roles for Notch1, 2, and 3 receptor activation during CNS development, particularly during gliogenesis.

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.

The many facets of Notch ligands.

The Notch signaling pathway regulates a diverse array of cell types and cellular processes and is tightly regulated by ligand binding. Both canonical and noncanonical Notch ligands have been identified that may account for some of the pleiotropic nature associated with Notch signaling. This review focuses on the molecular mechanisms by which Notch ligands function as signaling agonists and antagonists, and discusses different modes of activating ligands as well as findings that support intrinsic ligand signaling activity independent of Notch. Post-translational modification, proteolytic processing, endocytosis and membrane trafficking, as well as interactions with the actin cytoskeleton may contribute to the recently appreciated multifunctionality of Notch ligands. The regulation of Notch ligand expression by other signaling pathways provides a mechanism to coordinate Notch signaling with multiple cellular and developmental cues. The association of Notch ligands with inherited human disorders and cancer highlights the importance of understanding the molecular nature and activities intrinsic to Notch ligands. Oncogene (2008) 27, 5148-5167; doi:10.1038/onc.2008.229.