Underexpression of the plant <Emphasis Type="Italic">NOTCHLESS</Emphasis> gene,encoding a WD-repeat protein,causes pleitropic phenotype during plant development

WD-repeat proteins are involved in a breadth of cellular processes. While the WD-repeat protein encoding gene NOTCHLESS has been involved in the regulation of the Notch signaling pathway in Drosophila, its yeast homolog Rsa4p was shown to participate in 60S ribosomal subunit biogenesis. The plant homolog ScNLE was previously characterized in Solanum chacoense (ScNLE) as being involved in seed development. However, expression data and reduced size of ScNLE underexpressing plants suggested in addition a role during shoot development. We here report the detailed phenotypic characterization of ScNLE underexpressing plants during shoot development. ScNLE was shown to be expressed in actively dividing cells of the shoot apex. Consistent with this, ScNLE underexpression caused pleiotropic defects such as a reduction in aerial organ size, a reduction in some organ numbers, delayed flowering, and an increase in stomatal index. Analysis of adaxial epidermal cells revealed that both cell number and cell size were reduced in mature leaves of ScNLE underexpressing lines. Two-hybrid screens with the Nle domain and the WD-repeat domain of ScNLE allowed the isolation of homologs of yeast MIDASIN and NSA2 genes, the products of which are involved in 60S ribosomal subunit biogenesis in yeast. A ScNLE-GFP chimeric protein was localized in both the cytoplasm and nucleus. These data altogether suggest that ScNLE likely plays a role in 60S ribosomal subunit biogenesis, which is essential for proper cellular growth and proliferation during plant development.

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Notch and Wingless Regulate Expression of Cuticle Patterning Genes

The cell surface receptor Notch is required during Drosophila embryogenesis for production of epidermal precursor cells. The secreted factor Wingless is required for specifying different types of cells during differentiation of tissues from these epidermal precursor cells. The results reported here show that the full-length Notch and a form of Notch truncated in the amino terminus associate with Wingless in S2 cells and in embryos. In S2 cells, Wingless and the two different forms of Notch regulate expression of Dfrizzled 2, a receptor of Wg; hairy, a negative regulator of achaete expression; shaggy, a negative regulator of engrailed expression; and patched, a negative regulator of wingless expression. Analyses of expression of the same genes in mutant N embryos indicate that the pattern of gene regulations observed in vitro reflects regulations in vivo. These results suggest that the strong genetic interactions observed between Notch and wingless genes during development of Drosophila is at least partly due to regulation of expression of cuticle patterning genes by Wingless and the two forms of Notch.     

O-linked-N-acetylglucosamine modification of mammalian Notch receptors by an atypical O-GlcNAc transferase Eogt1

O-linked-β-N-acetylglucosamine (O-GlcNAc) modification is a unique cytoplasmic and nuclear protein modification that is common in nearly all eukaryotes, including filamentous fungi, plants, and animals. We had recently reported that epidermal growth factor (EGF) repeats of Notch and Dumpy are O-GlcNAcylated by an atypical O-GlcNAc transferase, EOGT, in Drosophila. However, no study has yet shown whether O-GlcNAcylation of extracellular proteins is limited to insects such as Drosophila or whether it occurs in other organisms, including mammals. Here, we report the characterization of A130022J15Rik, a mouse gene homolog of Drosophila Eogt (Eogt 1). Enzymatic analysis revealed that Eogt1 has a substrate specificity similar to that of Drosophila EOGT, wherein the Thr residue located between the fifth and sixth conserved cysteines of the folded EGF-like domains is modified. This observation is supported by the fact that the expression of Eogt1 in Drosophila rescued the cell-adhesion defect caused by Eogt downregulation. In HEK293T cells, Eogt1 expression promoted modification of Notch1 EGF repeats by O-GlcNAc, which was further modified, at least in part, by galactose to generate a novel O-linked-N-acetyllactosamine structure. These results suggest that Eogt1 encodes EGF domain O-GlcNAc transferase and that O-GlcNAcylation reaction in the secretory pathway is a fundamental biochemical process conserved through evolution.     

Notch2: a second mammalian Notch gene.

Notch is a cell surface receptor that mediates a wide variety of cellular interactions that specify cell fate during Drosophila development. Recently, homologs of Drosophila Notch have been isolated from Xenopus, human and rat, and the expression patterns of these vertebrate proteins suggest that they may be functionally analogous to their Drosophila counterpart. We have now identified a second rat gene that exhibits substantial nucleic and amino acid sequence identity to Drosophila Notch. This gene, designated Notch2, encodes a protein that contains all the structural motifs characteristic of a Notch protein. Thus, mammals differ from Drosophila in having more than one Notch gene. Northern and in situ hybridisation analyses in the developing and adult rat identify distinct spatial and temporal patterns of expression for Notch1 and Notch2, indicating that these genes are not redundant. These results suggest that the great diversity of cell-fate decisions regulated by Notch in Drosophila may be further expanded in vertebrates by the activation of distinct Notch proteins.     

Son of Notch, a winged-helix gene involved in boundary formation in the Drosophila wing

A P element enhancer trap screen was conducted to identify genes involved in dorsal-ventral boundary formation in Drosophila. The son of Notch (son) gene was identified by the son(2205) enhancer trap insertion, which is a partial loss-of-function mutation. Based on son(2205) mutant phenotypes and genetic interactions with Notch and wingless mutations, we conclude that son participates in wing development, and functions in the Notch signaling pathway at the dorsal-ventral boundary in the wing. Notch signaling pathway components activate son enhancer trap expression in wing cells. son enhancer trap expression is regulated positively by wingless, and negatively by cut in boundary cells. Ectopic Son protein induces wingless and cut expression in wing discs. We hypothesize that there is positive feedback regulation of son by wingless, and negative regulation by cut at the dorsal-ventral boundary during wing development.     

昆虫Notch信号通路研究进展

Notch 信号通路由 Notch 受体、Notch 配体(DSL 蛋白)、CSL[C promoter binding factor-1(CBF1),Suppressor of hairless(Su(H)),Lag-1]转录因子、其他效应子和Notch调节分子构成,在动物组织的发育和器官的细胞命运决定中起着基础性的...     

Drosophila Ndfip is a novel regulator of Notch signaling

In the Drosophila wing, the Nedd4 ubiquitin ligases (E3s), dNedd4 and Su(dx), are important negative regulators of Notch signaling; they ubiquitinate Notch, promoting its endocytosis and turnover. Here, we show that Drosophila Nedd4 family interacting protein (dNdfip) interacts with the Drosophila Nedd4-like E3s. dNdfip expression dramatically enhances dNedd4 and Su(dx)-mediated wing phenotypes and further disrupts Notch signaling. dNdfip colocalizes with Notch in wing imaginal discs and with the late endosomal marker Rab7 in cultured cells. In addition, dNdfip expression in the wing leads to ectopic Notch signaling. Supporting this, expression of dNdfip suppressed Notch(+/-) wing phenotype and knockdown of dNdfip enhanced the Notch(+/-) wing phenotype. The increase in Notch activity by dNdfip is ligand independent as dNdfip expression also suppressed deltex RNAi and Serrate(+/-) wing phenotypes. The opposing effects of dNdfip expression on Notch signaling and its late endosomal localization support a model whereby dNdfip promotes localization of Notch to the limiting membrane of late endosomes allowing for activation, similar to the model previously shown with ectopic Deltex expression. When dNedd4 or Su(dx) are also present, dNdfip promotes their activity in Notch ubiquitination and internalization to the lysosomal lumen for degradation.     

Notch and PKC Are Involved in Formation of the Lateral Region of the Dorso-Ventral Axis in Drosophila Embryos

The Notch gene encodes an evolutionarily conserved cell surface receptor that generates regulatory signals based on interactions between neighboring cells. In Drosophila embryos it is normally expressed at a low level due to strong negative regulation. When this negative regulation is abrogated neurogenesis in the ventral region is suppressed, the development of lateral epidermis is severely disrupted, and the dorsal aminoserosa is expanded. Of these phenotypes only the anti-neurogenic phenotype could be linked to excess canonical Notch signaling. The other phenotypes were linked to high levels of Notch protein expression at the surface of cells in the lateral regions indicating that a non-canonical Notch signaling activity normally functions in these regions. Results of our studies reported here provide evidence. They show that Notch activities are inextricably linked to that of Pkc98E, the homolog of mammalian PKCδ. Notch and Pkc98E up-regulate the levels of the phosphorylated form of IκBCactus, a negative regulator of Toll signaling, and Mothers against dpp (MAD), an effector of Dpp signaling. Our data suggest that in the lateral regions of the Drosophila embryos Notch activity, in conjunction with Pkc98E activity, is used to form the slopes of the opposing gradients of Toll and Dpp signaling that specify cell fates along the dorso-ventral axis.     

Analysis of Dominant Enhancers and Suppressors of Activated Notch in Drosophila

The Notch receptor controls cell fate decisions throughout Drosophila development. Truncated, ligand-independent forms of this protein delay or block differentiation. We have previously shown that expression of the intracellular domain of the receptor under the control of the sevenless enhancer/promoter induces a rough eye phenotype in the adult fly. Analysis of the resultant cellular transformations suggested that this form of Notch acts as a constitutively activated receptor. To identify gene products that interact with Notch, a second-site mutagenesis screen was performed to isolate enhancers and suppressors of the eye phenotype caused by expression of these activated Notch molecules. We screened 137,000 mutagenized flies and recovered 290 dominant modifiers. Many new alleles of previously identified genes were isolated, as were mutations defining novel loci that may function in the Notch signaling pathway. We discuss the data with respect to known features of Notch receptor signaling and Drosophila eye development.     

Transgenic tobacco plants expressing the Drosophila Polycomb (Pc) chromodomain show developmental alterations: possible role of Pc chromodomain proteins in chromatin-mediated gene regulation in plants.

The chromodomain of the Drosophila Polycomb (Pc) protein has been introduced into tobacco nuclei to determine its location in the nucleus and its effect on plant development. Pc is a repressor of homeotic Drosophila genes that shares a well-conserved, although not identical, chromodomain with a structural heterochromatin component, Heterochromatin Protein 1. The chromodomains might therefore play a common role in chromatin repression. An analysis of transgenic plants expressing the Pc chromodomain, which was linked to the green fluorescent protein, suggested that the Pc chromodomain has distinct target regions in the plant genome. Transgenic plants expressing the Pc chromodomain had phenotypic abnormalities in their leaves and flowers, indicating a disruption in development. In axillary shoot buds of plants displaying altered leaf phenotypes, enhanced expression of a homeodomain gene, which is downregulated in wild-type leaves, was found. In Drosophila, Pc has been shown to possess distinct chromosome binding activity and to be involved in the regulation of development-specific genes. Our results support the assumptions that the heterologous chromodomain affects related functions in Drosophila and in plants, and that chromatin modification mechanisms are involved in the regulation of certain plant genes, in a manner similar to chromatin-mediated gene regulation in Drosophila.     

Identification of Genetic Modifiers of TDP-43 Neurotoxicity in Drosophila

Cytosolic aggregation of the nuclear RNA-binding protein TDP-43 is a histopathologic signature of degenerating neurons in amyotrophic lateral sclerosis (ALS), and mutations in the TARDBP gene encoding TDP-43 cause dominantly inherited forms of this condition. To understand the relationship between TDP-43 misregulation and neurotoxicity, we and others have used Drosophila as a model system, in which overexpression of either wild-type TDP-43 or its ALS-associated mutants in neurons is sufficient to induce neurotoxicity, paralysis, and early death. Using microarrays, we have examined gene expression patterns that accompany TDP-43-induced neurotoxicity in the fly system. Constitutive expression of TDP-43 in the Drosophila compound eye elicited widespread gene expression changes, with strong upregulation of cell cycle regulatory genes and genes functioning in the Notch intercellular communication pathway. Inducible expression of TDP-43 specifically in neurons elicited significant expression differences in a more restricted set of genes. Genes that were upregulated in both paradigms included SpindleB and the Notch target Hey, which appeared to be a direct TDP-43 target. Mutations that diminished activity of Notch or disrupted the function of downstream Notch target genes extended the lifespan of TDP-43 transgenic flies, suggesting that Notch activation was deleterious in this model. Finally, we showed that mutation of the nucleoporin Nup50 increased the lifespan of TDP-43 transgenic flies, suggesting that nuclear events contribute to TDP-43-dependent neurotoxicity. The combined findings identified pathways whose deregulation might contribute to TDP-43-induced neurotoxicity in Drosophila.     

Genetic characterization of the Drosophila melanogaster Suppressor of deltex gene: A regulator of notch signaling.

The Notch receptor signaling pathway regulates cell differentiation during the development of multicellular organisms. A number of genes are known to be components of the pathway or regulators of the Notch signal. One candidate for a modifier of Notch function is the Drosophila Suppressor of deltex gene [Su(dx)]. We have isolated four new alleles of Su(dx) and mapped the gene between 22B4 and 22C2. Loss-of-function Su(dx) mutations were found to suppress phenotypes resulting from loss-of-function of Notch signaling and to enhance gain-of-function Notch mutations. Hairless, a mutation in a known negative regulator of the Notch pathway, was also enhanced by Su(dx). Phenotypes were identified for Su(dx) in wing vein development, and a role was demonstrated for the gene between 20 and 30 hr after puparium formation. This corresponds to the period when the Notch protein is involved in refining the vein competent territories. Taken together, our data indicate a role for Su(dx) as a negative regulator of Notch function.     

Mosaic patterns of transgene expression in plants

The review describes the phenomenon of mosaic transgene (gene) expression in plants. Parallels with the mosaic transgene (gene) expression in other organisms are presented. Parallels with the mosaic patterns of gene (transgene) expression in other organisms (Drosophila, transgenic animals, and others) are made.     

ATX-1, an Arabidopsis homolog of trithorax,activates flower homeotic genes

BACKGROUND: The genes of the trithorax (trxG) and Polycomb groups (PcG) are best known for their regulatory functions in Drosophila, where they control homeotic gene expression. Plants and animals are thought to have evolved multicellularity independently. Although homeotic genes control organ identity in both animals and plants, they are unrelated. Despite this fact, several plant homeotic genes are negatively regulated by plant genes similar to the repressors from the animal PcG. However, plant-activating regulators of the trxG have not been characterized. RESULTS: We provide genetic, molecular, functional, and biochemical evidence that an Arabidopsis gene, ATX1, which is similar to the Drosophila trx, regulates floral organ development. The effects are specific: structurally and functionally related flower homeotic genes are under different control. We show that ATX1 is an epigenetic regulator with histone H3K4 methyltransferase activity. This is the first example of this kind of enzyme activity reported in plants, and, in contrast to the Drosophila and the yeast trithorax homologs, ATX1 can methylate in the absence of additional proteins. In its ability to methylate H3K4 as a recombinant protein, ATX1 is similar to the human homolog. CONCLUSIONS: ATX1 functions as an activator of homeotic genes, like Trithorax in animal systems. The histone methylating activity of the ATX1-SET domain argues that the molecular basis of these effects is the ability of ATX1 to modify chromatin structure. Our results suggest a conservation of trxG function between the animal and plant kingdoms despite the different structural nature of their targets.     

Diverse functions of Polycomb group proteins during plant development

Polycomb group (PcG) proteins play essential roles in animal and plant life cycles by controlling the expression of important developmental regulators. These structurally heterogeneous proteins form multimeric protein complexes that control higher order chromatin structure and, thereby, the expression state of their target genes. Once established, PcG proteins maintain silent gene expression states over many cell divisions providing a molecular basis for a cellular 'memory.' PcG proteins are best known for their role in the control of homeotic genes in Drosophila and mammals. In addition, they play important roles in the control of cell proliferation in vertebrate and invertebrate systems. Recent studies in plants have shown that PcG proteins regulate diverse developmental processes and, as in animals, they affect both homeotic gene expression and cell proliferation. Thus, the function of PcG proteins has been widely conserved between the plant and animal kingdoms.