Toshiro Aigaki

Eiger, a TNF superfamily ligand that triggers the Drosophila JNK pathway

Drosophila provides a powerful genetic model for studying the in vivo regulation of cell death. In our large‐scale gain‐of‐function screen, we identified Eiger, the first invertebrate tumor necrosis factor (TNF) superfamily ligand that can induce cell death. Eiger is a type II transmembrane protein with a C‐terminal TNF homology domain. It is predominantly expressed in the nervous system. Genetic evidence shows that Eiger induces cell death by activating the Drosophila JNK pathway. Although this cell death process is blocked by Drosophila inhibitor‐of‐apoptosis protein 1 (DIAP1), it does not require caspase activity. We also show genetically that Eiger is a physiological ligand for the Drosophila JNK pathway. Our findings demonstrate that Eiger can initiate cell death through an IAP‐sensitive cell death pathway via JNK signaling.

Dally regulates Dpp morphogen gradient formation in the Drosophila wing

Decapentaplegic (Dpp), a Drosophila TGFβ/bone morphogenetic protein homolog, functions as a morphogen to specify cell fate along the anteroposterior axis of the wing. Dpp is a heparin-binding protein and Dpp signal transduction is potentiated by Dally, a cell-surface heparan sulfate proteoglycan, during assembly of several adult tissues. However, the molecular mechanism by which the Dpp morphogen gradient is established and maintained is poorly understood. We show evidence that Dally regulates both cellular responses to Dpp and the distribution of Dpp morphogen in tissues. In the developing wing, dally expression in the wing disc is controlled by the same molecular pathways that regulate expression of thickveins, which encodes a Dpp type I receptor. Elevated levels of Dally increase the sensitivity of cells to Dpp in a cell autonomous fashion. In addition, dally affects the shape of the Dpp ligand gradient as well as its activity gradient. We propose that Dally serves as a co-receptor for Dpp and contributes to shaping the Dpp morphogen gradient.

Drosophila Nedd4 Regulates Endocytosis of Notch and Suppresses Its Ligand-Independent Activation

BACKGROUNDnLigand-induced proteolytic cleavage and internalization of the plasma membrane receptor Notch leads to its activation. Ligand-independent, steady-state internalization of Notch, however, does not lead to activation. The mechanism by which downstream effectors discriminate between these bipartite modes of Notch internalization is not understood. Nedd4 is a HECT domain-containing E3 ubiquitin ligase that targets transmembrane receptors containing the PPSY motif for endocytosis. Deltex is a positive Notch signaling regulator that encodes a putative ubiquitin ligase of the ring finger type.nnnRESULTSnWe used the Drosophila system to show that Notch is ubiquitinated and destabilized by Nedd4 in a manner requiring the PPSY motif in the Notch intracellular domain. Loss of Nedd4 function dominantly suppresses the Notch and Deltex mutant phenotypes, and its hyperactivation attenuates Notch activity. In tissue culture cells, the dominant-negative form of Nedd4 blocks steady-state Notch internalization and activates Notch signaling independently of ligand binding. This effect was further potentiated by Deltex. Nedd4 destines Deltex for degradation in a Notch-dependent manner.nnnCONCLUSIONSnNedd4 antagonizes Notch signaling by promoting degradation of Notch and Deltex. This Nedd4 function may be important for protecting unstimulated cells from sporadic activation of Notch signaling.

Alternative trans‐splicing: a novel mode of pre‐mRNA processing

Alternative splicing is an important process contributing to proteome diversity without involving an increase in the number of genes. In some cases, alternative splicing is carried out under ‘trans‐mode’, called alternative trans‐splicing, in which exons located on separate pre‐mRNA molecules are selectively joined to produce mature mRNAs encoding proteins with distinct structures and functions. However, it is not known how widespread or how frequently trans‐splicing occurs in vivo. Recently, trans‐allelic trans‐splicing has been unambiguously demonstrated in Drosophila using a SNP (single nucleotide polymorphism) as a marker. In this review, we provide an overview of alternative trans‐splicing in Drosophila and mammals, and discuss its mechanisms.

Alternative splicing of lola generates 19 transcription factors controlling axon guidance in Drosophila.

The Drosophila melanogaster transcription factor Lola (longitudinals lacking) is a pivotal regulator of neural wiring that sets the precise expression levels of proteins that execute specific axon guidance decisions. Lola has a zinc finger DNA binding domain and a BTB (for Broad-complex, Tramtrack and Bric a brac) dimerization motif. We now show that alternative splicing of the lola gene creates a family of 19 transcription factors. All lola isoforms share a common dimerization domain, but 17 have their own unique DNA-binding domains. Seven of these 17 isoforms are present in the distantly-related Dipteran Anopheles gambiae, suggesting that the properties of specific isoforms are likely to be critical to lola function. Analysis of the expression patterns of individual splice variants and of the phenotypes of mutants lacking single isoforms supports this idea and establishes that the alternative forms of lola are responsible for different functions of this gene. Thus, in this system, the alternative splicing of a key transcription factor helps to explain how a small genome encodes all the information that is necessary to specify the enormous diversity of axonal trajectories.

Disruption of the microsomal glutathione S-transferase-like gene reduces life span of Drosophila melanogaster.

Microsomal glutathione S-transferase-I (MGST-I) has been thought to be important for protecting the cell from oxidative damages and/or xenobiotics. We have previously identified the Microsomal glutathione S-transferase-like (Mgstl) gene, a Drosophila homologue of human MGST-I. To investigate the function of the enzyme using Drosophila as a model system, we examined the expression pattern of Mgstl during development, and generated loss-of-function mutants to assess its in-vivo function. Mgstl was expressed in all developmental stages. It is expressed ubiquitously with the highest expression in the larval fat body, an insect organ thought to be functionally corresponding to mammalian liver, while relatively low in the central nervous system. This tissue distribution is consistent with that of MGST-I in humans or Rats. Mgstl null mutants generated from a P element insertion line showed no obvious defects in morphology, indicating that it is not essential for the development. However, their life span was significantly reduced compared to control flies, suggesting that the MGSTL protein is involved in processes somehow contributing to aging. We found an Mgstl pseudogene, which is apparently derived through the reverse transcription of Mgstl mRNA and subsequent integration into the genome.

Longevity determination genes in Drosophila melanogaster

Identification of longevity mutants is crucial for genetic approach to dissect the molecular mechanism of aging and longevity determination. In Drosophila melanogaster, several mutations have been shown to extend the longevity: methuselah encoding a putative G-protein coupled receptor, Indy encoding a sodium dicarboxylate cotransporter, chico encoding insulin receptor substrate, and InR encoding the insulin-like receptor. Extended longevity phenotypes were also observed in transgenic flies overexpressing antioxidant enzymes, Cu/Zn superoxide dismutase and Catalase, Cu/Zn SOD only, or a molecular chaperone, hsp70. Pleiotropism of mutations is a limitation associated with conventional mutagenesis for efficient detection of longevity determination genes. Using a conditional misexpression system, we identified Drosophila POSH (DPOSH), a scaffold protein containing RING finger and four SH3 domains, whose ubiquitous overexpression in adult stage extends the longevity. Neural-specific overexpression of DPOSH is sufficient to extend the longevity, whereas overexpression in non-neural tissues during development induces apoptosis through activation of JNK/SAPK pathway.

Drosophila lola encodes a family of BTB-transcription regulators with highly variable C-terminal domains containing zinc finger motifs.

Alternative splicing is an important mechanism contributing to the increased proteome diversity in higher eukaryotes. We have explored the alternative splicing events in the Drosophila longitudinals lacking (lola) gene by means of 5 RACE, 3 RACE, genome sequence searches, and EST sequencing. We demonstrated that the lola locus is comprised of 32 exons spanning over 60 kb, and encodes a total of 80 alternatively spliced variants consisting of 5 and 3 variable sequences and constitutive common exons. All the variants shared a common sequence (exons 5-8) encoding the N-terminal region containing the BTB domain, but both the 5 and 3 ends were variable. There were four promoters responsible for the variation in the 5 end (exons 1-4). Alternative splicing was involved in the variation in the 3 end corresponding to the C-terminal variable region, which was encoded by one or two exons that were selected from 20 groups of exons in a mutually exclusive manner (exons 9-32). Seventeen of the 20 isoforms contained C(2)H(2)-like zinc finger motifs in the C-terminal variable region. Analyses of the 3 variant-specific cDNA pools revealed that all combinations of 5 and 3 variable sequences were expressed in both the embryonic and third instar larval stages. Since the BTB domain mediates dimerization, lola encodes a family of transcription regulators with a large variety of DNA- or protein-binding specificities, and could be involved in various developmental processes, including the embryonic neural pathfindings. We also showed that the structures of Lola isoforms were highly conserved in Drosophila pseudoobscura.