Notch Locus Research Articles

Notch Signaling Modulates Sleep Homeostasis and Learning after Sleep Deprivation in Drosophila

The role of the transmembrane receptor Notch in the adult brain is poorly understood. Here, we provide evidence that bunched, a negative regulator of Notch, is involved in sleep homeostasis. Genetic evidence indicates that interfering with bunched activity in the mushroom bodies (MBs) abolishes sleep homeostasis. Combining bunched and Delta loss-of-function mutations rescues normal homeostasis, suggesting that Notch signaling may be involved in regulating sensitivity to sleep loss. Preventing the downregulation of Delta by overexpressing a wild-type transgene in MBs reduces sleep homeostasis and, importantly, prevents learning impairments induced by sleep deprivation. Similar resistance to sleep loss is observed with Notch(spl-1) gain-of-function mutants. Immunohistochemistry reveals that the Notch receptor is expressed in glia, whereas Delta is localized in neurons. Importantly, the expression in glia of the intracellular domain of Notch, a dominant activated form of the receptor, is sufficient to prevent learning deficits after sleep deprivation. Together, these results identify a novel neuron-glia signaling pathway dependent on Notch and regulated by bunched. These data highlight the emerging role of neuron-glia interactions in regulating both sleep and learning impairments associated with sleep loss.

The antimorphic mode of action of lethal Abruptex alleles of the Notch locus in Drosophila melanogaster

The mode of action of lethal Abruptex alleles (Ax59b8.1, Ax59d5, and Ax75c24) of the Notch locus of Drosophila melanogaster was investigated by comparing the phenotypes of flies with different doses of the mutant alleles. The expression of the Abruptex phenotype was found to increase with increasing dose of the mutant gene, and to decrease with increasing dose of the wild type gene. It was thus concluded that the lethal Abruptex mutations are antimorphic. Different models for the explanation of antimorphic gene action are discussed. Special emphasis is laid on the “antagonistic functions” model of the Notch gene based on the assumption that the Notch locus is a regulative locus having both activator and repressor functions.

Temperature-sensitivity of the pleiotropic effects of three lethal Abruptex mutations at the Notch locus of Drosophila melanogaster

In heterozygous condition, lethal Abruptex alleles of the Notch complex locus of Drosophila melanogaster affect wing venation, and number of bristles on the head and thorax. All of these pleiotropic effects are temperature-sensitive, suggesting that the Abruptex mutations are missense mutations, and that the same gene product is involved in all the pleiotropic effects. There is a tendency of the phenes in the thorax to respond differently to temperature than do phenes in the head. This possibly suggests autonomy of the pleiotropic effects.

Effect of temperature on interaction of Abruptex and recessive alleles of the Notch complex locus of Drosophila melanogaster

Interaction of recessive visible alleles (fag, fano, spl, nd, nd2) and different Abruptex (Ax) alleles of the Notch locus of Drosophila melanogaster was analysed at 18°C. 25°C and 29°C in transheterozygotes. The homozygous viable Abruptex alleles complement most of the recessives at all temperatures. The expression of hypomorphic Abruptex alleles (AxE2, Ax16172, Ax71d) remains constant at all temperatures, whereas the expression of hypermorphic alleles (Ax28, Ax9B2) is enhanced at elevated temperatures. The homozygous lethal Abruptex alleles (Ax59b8.1, Ax59d5) not only complement all of the recessives at 18°C, but their own phenotypic expression is usually very strong. At 25°C they fail to complement spl and fa8, and their own phenotypic expression is reduced. At 29°C the lethal Abruptex alleles do not complement any of the recessives except nd, and their own expression is reduced or almost absent. At low temperatures the lethal Abruptex alleles compete efficiently with the wild-type allele, but at elevated temperatures their competetive ability is decreased. The results strongly support the view that the Notch complex gene is a single cistron, but that the Abruptex site may possibly have three functionally different mutant forms: hypomorphic, hypermorphic, and antimorphic. The morphism may, however, he different in different functions of the gene which may suggest independence of the pleiotropic effects of the gene.

Locus-Specific Decoupling of Base Composition Evolution at Synonymous Sites and Introns along the Drosophila melanogaster and Drosophila sechellia Lineages

Selection is thought to be partially responsible for patterns of molecular evolution at synonymous sites within numerous Drosophila species. Recently, “per-site” and likelihood methods have been developed to detect loci for which positive selection is a major component of synonymous site evolution. An underlying assumption of these methods, however, is a homogeneous mutation process. To address this potential shortcoming, we perform a complementary analysis making gene-by-gene comparisons of paired synonymous site and intron substitution rates toward and away from the nucleotides G and C because preferred codons are G or C ending in Drosophila. This comparison may reduce both the false-positive rate (due to broadscale heterogeneity in mutation) and false-negative rate (due to lack of power comparing small numbers of sites) of the per-site and likelihood methods. We detect loci with patterns of evolution suggestive of synonymous site selection pressures predominately favoring unpreferred and preferred codons along the Drosophila melanogaster and Drosophila sechellia lineages, respectively. Intron selection pressures do not appear sufficient to explain all these results as the magnitude of the difference in synonymous and intron evolution is dependent on recombination environment and chromosomal location in a direction supporting the hypothesis of selectively driven synonymous fixations. This comparison identifies 101 loci with an apparent switch in codon preference between D. melanogaster and D. sechellia, a pattern previously only observed at the Notch locus.

A male‐specific effect of dominant‐negative Fos

The transcription factor Fos contains a basic DNA binding domain combined with a leucine zipper (bZip). Expression of a truncated form of Fos in Drosophila that contains only the bZip region (Fos bZip) elicits phenotypes resembling fos mutations. These effects presumably derive from competition between wild-type and truncated forms for dimerization partners, with the truncation acting in a dominant-negative manner. We found that expression of Fos bZip elicits male-specific phenotypes. Moreover, genetic interactions occur between Fos bZip and mutations in loci encoding the X chromosome dosage compensation complex. Fos bZip effects are correlated with aberrant male X chromosome structure and depressed signaling through the X-linked Notch locus. Unexpectedly, the male-specific effects are not reproduced with Fos RNAi, suggesting that Fos bZip can be neomorphic in nature. These results provide insight into how mutations in bZip proteins can exhibit gain of function activity.

Cis-trans tests at the Notch locus of Drosophila melanogaster suggesting multiple cis-acting control sites inside the locus

Tests were made of expression of Abruptex in compounds of recessive mutations and dominant antimorphic Abruptex (Ax) mutations at the Notch locus of Drosophila melanogaster. In combination with split, expression of Abruptex was stronger in cis-heterozygotes than in trans-heterozygotes, whereas the split homozygotes tended to be intermediate. In combination with facet-glossy, the expression of Abruptex was stronger in trans-heterozygotes than in cis-heterozygotes and the facet-glossy homozygotes tended to be intermediate. The Abruptex mutations usually have a stronger expression in all combinations with split than with facet-glossy. Split showed a pseudodominant expression both in cis-and trans-heterozygotes but facet glossy only in trans-heterozygotes. The +Ax++ heterozygotes were most expressed of all genotypes in respect to wing venation, but approximately at the same level as the two trans-heterozygotes in the bristle phenotype. These results tentatively suggest that the Notch locus can be divided into a structural element consisting of a right-most fifth of the locus including the site of Abruptex, and a control element apparently including several cis-acting sites. In cis-compounds split enhances and facet-glossy suppresses the expression of the presumed structural element. The homologous chromosome, however, also seems to be affected by these control sites, which suggests that the effect of the cis-acting control sites would be at the level of transcription.

Gene dosage studies of a temperature sensitive Abruptex mutation of the Notch locus of Drosophila melanogaster

Gene dosage studies of a temperature sensitive Abruptex mutation of the Notch locus of Drosophila melanogaster revealed, firstly, that hemizygous females were lethal at all temperatures studied, while hemizygous males were subvital at 18 degrees C, semilethal at 25 degrees C, and lethal at 29 degrees C. This is due to dosage compensation of the Notch locus. Secondly, it was observed that in general an increasing dose of the wild allele decreased the expression of Abruptex at all temperatures. An exception was the Axtsl/Axtsl/ + genotype, which was more expressed than Axtsl/Axtsl at low temperatures. This suggests antimorphism as the most likely mode of action of the temperature sensitive Abruptex mutation. Thirdly, it was observed that the expression of Abruptex phenotype increased from 18 degrees C to 25 degrees C but decreased from 25 degrees C to 29 degrees C if the wild type allele was involved in the genotype. This suggests that the gene product of the temperature sensitive Abruptex allele loses its function at high temperatures. The putative antimorphic mode of action of the temperature sensitive Abruptex mutation suggests that the Notch gene product interacts with products of other neurogenic loci or with itself.

Persistent locus-specific instability of yellow 2-717 and Notch Uc-1 in Drosophila melanogaster coincides with hobo multiplication

According to FISH data the presence of multiple hobo element copies in the unstable yellow and Notch loci in y(2-717) and Uc-1 Drosophila melanogaster stocks, respectively, was found. Locus-specific instability in these strains is caused by hobo multiplication in the respective loci and its subsequent recombination with neighboring hobo copies rather than its insertion-excision.

Maximum Likelihood Estimation of Ancestral Codon Usage Bias Parameters in Drosophila

We present a likelihood method for estimating codon usage bias parameters along the lineages of a phylogeny. The method is an extension of the classical codon-based models used for estimating dN/dS ratios along the lineages of a phylogeny. However, we add one extra parameter for each lineage: the selection coefficient for optimal codon usage (S), allowing joint maximum likelihood estimation of S and the dN/dS ratio. We apply the method to previously published data from Drosophila melanogaster, Drosophila simulans, and Drosophila yakuba and show, in accordance with previous results, that the D. melanogaster lineage has experienced a reduction in the selection for optimal codon usage. However, the D. melanogaster lineage has also experienced a change in the biological mutation rates relative to D. simulans, in particular, a relative reduction in the mutation rate from A to G and an increase in the mutation rate from C to T. However, neither a reduction in the strength of selection nor a change in the mutational pattern can alone explain all of the data observed in the D. melanogaster lineage. For example, we also confirm previous results showing that the Notch locus has experienced positive selection for previously classified unpreferred mutations.

Multiple Signatures of Positive Selection Downstream of Notch on the X Chromosome in Drosophila melanogaster

To identify genomic regions affected by the rapid fixation of beneficial mutations (selective sweeps), we performed a scan of microsatellite variability across the Notch locus region of Drosophila melanogaster. Nine microsatellites spanning 60 kb of the X chromosome were surveyed for variation in one African and three non-African populations of this species. The microsatellites identified an approximately 14-kb window for which we observed relatively low levels of variability and/or a skew in the frequency spectrum toward rare alleles, patterns predicted at regions linked to a selective sweep. DNA sequence polymorphism data were subsequently collected within this 14-kb region for three of the D. melanogaster populations. The sequence data strongly support the initial microsatellite findings; in the non-African populations there is evidence of a recent selective sweep downstream of the Notch locus near or within the open reading frames CG18508 and Fcp3C. In addition, we observe a significant McDonald-Kreitman test result suggesting too many amino acid fixations species wide, presumably due to positive selection, at the unannotated open reading frame CG18508. Thus, we observe within this small genomic region evidence for both recent (skew toward rare alleles in non-African populations) and recurring (amino acid evolution at CG18508) episodes of positive selection.

Genetic evidence that Notch affects independently the development of machrochaeta and wing venation in Drosophila melanogaster.

Temperature sensitive experiments on the interaction of Abruptex mutations of the Notch locus and Enhancer of split indicate that the effect of temperature on the development of macrochaeta and wing venation is different. This suggest that Notch affects the development of macrochaeta and wing venation in different and independent ways being in accordance with the proposition that the signal pathway from the Notch receptor at the cell membrane to Enhancer of split complex in the nucleus is different in the case of the development of macrochaeta and wing venation. The Abruptex mutations as gain-of-function mutations of the Notch locus cannot rescue the Enhancer of split phenotype.

DNA variability and divergence at the notch locus in Drosophila melanogaster and D. simulans: a case of accelerated synonymous site divergence.

DNA diversity in two segments of the Notch locus was surveyed in four populations of Drosophila melanogaster and two of D. simulans. In both species we observed evidence of non-steady-state evolution. In D. simulans we observed a significant excess of intermediate frequency variants in a non-African population. In D. melanogaster we observed a disparity between levels of sequence polymorphism and divergence between one of the Notch regions sequenced and other neutral X chromosome loci. The striking feature of the data is the high level of synonymous site divergence at Notch, which is the highest reported to date. To more thoroughly investigate the pattern of synonymous site evolution between these species, we developed a method for calibrating preferred, unpreferred, and equal synonymous substitutions by the effective (potential) number of such changes. In D. simulans, we find that preferred changes per "site" are evolving significantly faster than unpreferred changes at Notch. In contrast we observe a significantly faster per site substitution rate of unpreferred changes in D. melanogaster at this locus. These results suggest that positive selection, and not simply relaxation of constraint on codon bias, has contributed to the higher levels of unpreferred divergence along the D. melanogaster lineage at Notch.

Presenilin suppresses the negative complementation at the Notch locus of Drosophila melanogaster, suggesting a mechanism for negative complementation

A loss-of-function mutant form of the Presenilin (Psn) gene from the third chromosome, known to be involved in the proteolytic cleavage of the Notch receptor protein on the cell membrane of Drosophila melanogaster and the access of its intracellular domain to the nucleus, completely rescues the lethality caused by the negative complementation in certain heteroallelic combinations of the Abruptex (Ax) type mutations of the Notch locus in the X chromosome. On the other hand, Presenilin enhances the negative complementation of one particular semilethal Abruptex genotype (Ax28/AxE2). Since the Abruptex mutations are point mutations of the extracellular domain of the Notch receptor located close to the binding site of the Delta ligand, it is suggested that the mechanism of negative complementation at the Notch locus is the impairment of the ligand activation of the Notch receptor, subsequently followed by the failure of the cleavage of the receptor protein and impairment of the access of the intracellular domain into the nucleus.

Numb suppresses the negative complementation at the Notch locus of Drosophila melanogaster, suggesting a putative mechanism for negative complementation.

The mutant form of the intracellular asymmetrically localized Numb membrane-bound protein of Drosophila melanogaster suppresses the negative complementation of certain Abruptex (Ax) mutations of the Notch (N) locus encoding a transmembrane receptor protein in which the Ax mutations are mutations in the epidermal growth factor (EGF)-like repeats of the extracellular domain of the receptor. One model for how Ax mutants affect N function is that they are refractory to an antagonistic signal generated by an excess of N ligands. Genetically numb (nb) is an antagonist of N. In the absence of nb, cells follow the same fate as they would in the presence of a gain-of-function N allele, such as Ax. Numb has been shown to interact with the cytoplasmic domain of Notch. It is therefore suggested that numb counteracts the effect of Abruptex on Notch ligand binding, i.e. that Numb is an antagonist to the activation of the Notch signal generated by Notch ligands. Numb might accomplish this by interfering with the proteolytic cleavage of the Notch intracellular domain at the cell membrane. Thus, it seems possible that the mechanism of negative complementation of certain Ax mutants is the failure of this cleavage. Other possible mechanisms for negative complementation are also discussed.

Development and Assessment of a General Theory of Cervical Carcinogenesis Utilizing a Severe Combined Immunodeficiency Murine–Human Xenograft Model

Objective. Currently, we lack a theoretical explanation for why squamous cell cervical cancer develops predominantly in specific sites (i.e., along the squamocolumnar junction). We therefore implanted human cervical tissues containing the transformation zone in severe combined immunodeficiency (SCID) mice and studied morphology, steroid effects, gene expression, and human papillomavirus (HPV) factors.Methods. Normal and dysplastic human cervical tissues (3 × 2 mm) were placed subcutaneously in SCID-beige mice and later assessed by in situ hybridization for HPV 16/18 DNA and by immunohistochemistry for expression of CD31, keratin, proliferating-cell nuclear antigen, HPV 16 E6, p53, and Notch-1 (a binary cell fate determination protein). Some normal tissues were implanted with either a 90-day release 1.7-mg 17β-estradiol pellet or a 5-mg tamoxifen pellet; others were infected prior to implantation with human recombinant adenovirus 5 vector containing a human cytomegalovirus promoter-driven β-galactosidase gene and later assessed by X-gal staining.Results. Murine and human vessels formed anastomoses by 3 weeks. For at least 11 weeks, normal tissue retained the transformation zone and normal cell-type-specific keratin expression and exhibited normal proliferation; Notch-1 was present only in the basal cell layer. Dysplastic tissues exhibited koilocytosis, increased levels of cellular proliferation, and aberrant keratin, p53, and Notch-1 expression; HPV 16/18 DNA and HPV 16 E6 protein were detected for at least 6 weeks. Squamous metaplasia of normal cervical epithelium resulted from estrogen exposure, and a predominant columnar differentiation pattern was associated with tamoxifen administration. Through stable adenovirus infection, β-galactosidase was expressed for at least 6 weeks.Conclusions. This small manipulatable xenograft model maintains normal and dysplastic human cervical epithelium through neovascularization. Neoplastic tissue retains HPV 16/18 DNA and a premalignant phenotype, including elevated levels of cellular proliferation and aberrant keratin, p53, and Notch-1 expression. These attributes constitute essential features of a biologic model through which one may study HPV-mediated human disease and may be superior to cell culture and transgenic murine systems. Furthermore, this may serve as a model for gene therapy. Finally, we suggest that the normal cervical epithelium is maintained through putative interactions between the Notch locus and cell cycle growth regulators such as p53 and pRb. Neoplastic cervical epithelium may arise through disruption of this pathway. This theory may be testable in our animal model.

Enhancer of split [E(spl)(D)] is a gro-independent, hypermorphic mutation in Drosophila.

Enhancer of split [E(spl)] refers to a gene complex in Drosophila melanogaster, which contains a number of target genes of the Notch signaling pathway. The complex was originally identified by a dominant mutation E(spl)(D) that displays allele-specific interactions with a recessive mutation in the Notch locus called split (N(spl)). The spl phenotype is characterized by smaller eyes with irregularly spaced ommatidia, and it is strongly enhanced by E(spl)(D). This enhancement is correlated with a truncation of one of the E(spl) bHLH genes, m8, causing an increased stability of the mutant transcripts and an altered C-terminus in the mutant M8* protein. Concurrently, an insertion of a middle repetitive element in the adjacent groucho (gro) gene was observed. In this work, three different E(spl)(D) revertants (BE22, BE25, BX37), which have lost the ability to enhance N(spl) completely, were analyzed at the molecular level. In each case, the structure of the mutant M8* protein was affected, suggesting a specific involvement of the aberrant protein in the enhancement of the spl phenotype. This hypothesis is supported by the finding that a perfect phenocopy of spl enhancement can be achieved with hybrid constructs, where the altered C-terminus of M8* was fused to other E(spl) bHLH proteins. Thus, the ability to interact with N(spl) is not restricted to M8* but instead can be induced by an appropriate mutation in other E(spl) bHLH genes within the context of N(spl). In a N(spl) background, E(spl)(D) behaves like a hyperactive M8 mutation. However, the mutant M8* protein has lost the ability of binding to the corepressor Gro, which is an essential feature for normal E(spl) activity. Yet, other protein interactions, notably those with other bHLH proteins of either E(spl) or proneural family, are still observed. These findings suggest that the structural changes associated with the E(spl)(D) mutant protein are the primary cause for the phenotypic interactions with the recessive Notch mutation N(spl).

Molecular characterization of hobo-mediated inversions in Drosophila melanogaster.

The structure of chromosomal inversions mediated by hobo transposable elements in the Uc-1 X chromosome was investigated using cytogenetic and molecular methods. Uc-1 contains a phenotypically silent hobo element inserted in an intron of the Notch locus. Cytological screening identified six independent Notch mutations resulting from chromosomal inversions with one breakpoint at cytological position 3C7, the location of Notch. In situ hybridization to salivary gland polytene chromosomes determined that both ends of each inversion contained hobo and Notch sequences. Southern blot analyses showed that both breakpoints in each inversion had hobo-Notch junction fragments indistinguishable in structure from those present in the Uc-1 X chromosome prior to the rearrangements. Polymerase chain reaction amplification of the 12 hobo-Notch junction fragments in the six inversions, followed by DNA sequence analysis, determined that each was identical to one of the two hobo-Notch junctions present in Uc-1. These results are consistent with a model in which hobo-mediated inversions result from homologous pairing and recombination between a pair of hobo elements in reverse orientation.

Paralogy mapping: identification of a region in the human MHC triplicated onto human chromosomes 1 and 9 allows the prediction and isolation of novel PBX and NOTCH loci

Paralogy mapping: identification of a region in the human MHC triplicated onto human chromosomes 1 and 9 allows the prediction and isolation of novel PBX and NOTCH loci

Genetic interactions among vestigial, hairy, and Notch suggest a role of vestigial in the differentiation of epidermal and neural cells of the wing and halter of Drosophila melanogaster.

In this paper we describe the results of genetic analysis of the vestigial locus by studying its interactions with hairy and Notch loci in Drosophila melanogaster. Different vestigial alleles in homo- and heterozygous combination with different hairy alleles show synergism in increasing both cell death and formation of ectopic bristles and produce ectopic veins. Interactions between N and vg also show synergism in increasing cell death and formation of ectopic bristles. Only synergism in cell death is seen between h and N. The interactions indicate that vg product plays a role in the differentiation of epidermal and neural cells of the wing disc by interacting with N and h products either directly or indirectly. Mechanisms of molecular interactions among the three loci are discussed.