Yoshimasa Yagi

A Fat Body-Derived IGF-like Peptide Regulates Postfeeding Growth in Drosophila

Members of the insulin family of peptides have conserved roles in the regulation of growth and metabolism in a wide variety of metazoans. Here we show that Drosophila insulin-like peptide 6 (DILP6), which is structurally similar to vertebrate insulin-like growth factor (IGF), is predominantly expressed in the fat body, a functional equivalent of the vertebrate liver and adipocytes. This expression occurs during the postfeeding stage under the direct regulation of ecdysteroid. We further reveal that dilp6 mutants show growth defects during the postfeeding stage, which results in reduced adult body size through a decrease in cell number. This phenotype is rescued by fat body-specific expression of dilp6. These data indicate that DILP6 is a functional, as well as a structural, counterpart of vertebrate IGFs. Our data provide in vivo evidence for a role of ILPs in determining adult body size through the regulation of postfeeding growth.

The Drosophila Toll-9 activates a constitutive antimicrobial defense.

The Toll family of transmembrane proteins participates in signaling infection during the innate immune response. We analyzed the nine Drosophila Toll proteins and found that wild‐type Toll‐9 behaves similar to gain‐of‐function Toll‐1. Toll‐9 activates strongly the expression of drosomycin, and utilizes similar signaling components to Toll‐1 in activating the antifungal gene. The predicted protein sequence of Toll‐9 contains a tyrosine residue in place of a conserved cysteine, and this residue switch is critical for the high activity of Toll‐9. The Toll‐9 gene is expressed in adult and larval stages prior to microbial challenge, and the expression correlates with the high constitutive level of drosomycin mRNA in the animals. The results suggest that Toll‐9 is a constitutively active protein, and implies its novel function in protecting the host by maintaining a substantial level of antimicrobial gene products to ward off the continuous challenge of microorganisms.

A Drosophila p38 orthologue is required for environmental stress responses

The p38 mitogen‐activated protein kinase (MAPK) cascade is an evolutionarily conserved signalling mechanism involved in processes as diverse as apoptosis, cell fate determination, immune function and stress response. Aberrant p38 signalling has been implicated in many human diseases, including heart disease, cancer, arthritis and neurodegenerative diseases. To further understand the role of p38 in these processes, we generated a Drosophila strain that is null for the D‐p38a gene. Mutants are homozygous viable and show no observable developmental defects. However, flies lacking D‐p38a are susceptible to some environmental stresses, including heat shock, oxidative stress and starvation. These phenotypes only partially overlap those caused by mutations in D‐MEKK1 and dTAK1, suggesting that the D‐p38a gene is required to mediate some, but not all, of the functions ascribed to p38 signalling.

Expression and characterization of the Drosophila X11‐like/Mint protein during neural development

The X11‐like (X11L) protein was originally isolated as a protein bound to the cytoplasmic domain of the β‐amyloid precursor protein (APP), which is associated with Alzheimers disease. In mammals, X11L is believed to play an important role in the regulation of APP metabolism. Here we isolated and characterized the Drosophila X11L (dX11L) protein, also may be referred to this protein as Drosophila Mint (dMint), Lin 10 (dLin10) or X11 (dX11), is thought to be expressed in neuronal tissues from late embryonic through to the adult stages of the fly. The phosphotyrosine interaction domain of dX11L interacts with the cytoplasmic domain of the Drosophila amyloid precursor protein‐like (APPL) similar to the way human X11L (hX11L) interacts with APP. Overexpression of dX11L on post‐mitotic neurons had a lethal effect on flies and, when it was localized to the eye imaginal disc, disruption of compound eye morphology due to enhanced apoptosis of neuronal cells was observed. Overexpression of hX11L and the PDZ domain of dX11L resulted in identical eye phenotypes. The PDZ domain is highly conserved between Drosophila and human, and appears to be responsible for this phenotype. Our findings suggest that the X11L family may be involved with the regulation of apoptosis during neural cell development and that aberrant X11L function could be contribute in this way to the neuronal degeneration observed in Alzheimers disease.

Functional analysis of Toll-related genes in Drosophila

The Drosophila genome encodes a total of nine Toll and related proteins. The immune and developmental functions of Toll and 18Wheeler (18W) have been analyzed extensively, while the in vivo functions of the other Toll‐related proteins require further investigation. We performed transgenic experiments and found that overexpression of Toll‐related genes caused different extents of lethality and developmental defects. Moreover, 18w, Toll‐6, Toll‐7 and Toll‐8 often caused related phenotypic changes, consistent with the idea that these four genes have more conserved molecular structure and thus may regulate similar processes in vivo. Deletion alleles of Toll‐6, Toll‐7 and Toll‐8 were generated by targeted homologous recombination or P element excision. These mutant alleles were viable, fertile, and had no detectable defect in the inducible expression of antimicrobial peptide genes except for the Toll‐8 mutant had some defects in leg development. The expression of 18w, Toll‐7 and Toll‐8 mRNA showed wide and overlapping patterns in imaginal discs and the 18w, Toll‐8 double and Toll‐7, Toll‐8 double mutants showed substantially increased lethality. Overall our results suggest that some of the Toll‐related proteins, such as 18W, Toll‐7 and Toll‐8, may have redundant functions in regulating developmental processes.

Role of the Drosophila EGF receptor in determination of the dorsoventral domains of escargot expression during primary neurogenesis

Background : Primary neurogenesis in the central nervous system of insects and vertebrates occurs in three dorsoventral domains in each side of the neuroectoderm. Among the three dorsoventral domains of the Drosophila neuroectoderm, the medial and lateral columns express the zinc‐finger gene escargot (esg), whereas the intermediate column does not. We studied esg expression as a probe to investigate the mechanism of neuroectoderm patterning.

Toll and Toll-9 in Drosophila innate immune response.

In both insects and mammals, members of the Toll receptor family play important roles in the initial events leading to the activation of immunity genes. The prototypic Toll in Drosophila appears to be activated by a host protein ligand after microbial stimulation. The cellular events and the biological response after Toll activation, however, require further investigation. We used transgenic Drosophila strains expressing NF-κB and Toll proteins to investigate innate immune response in whole larvae and dissected larval fat bodies. Substantial activation of antimicrobial peptide genes was observed after septic injury. To circumvent the contribution of injury-induced response, we used dissected larval fat bodies to show that commercially available microbial compounds were able to alter the cellular distribution of Toll. The results also demonstrate that complex cellular events, including receptor trafficking, likely take place after stimulation of the larval immune tissue. By genome-wide expression analysis, we further show that Toll and Toll-9 may utilize the same signaling pathway in activating many immunity genes. Thus, the innate immune response in Drosophila is regulated by complex mechanisms, which involve Toll and other Toll-related proteins.

Helicase89B is a Mot1p/BTAF1 homologue that mediates an antimicrobial response in Drosophila.

We have identified a novel component, Helicase89B, that is required for the inducible antimicrobial response in Drosophila larvae by means of a P‐element insertional genetic screen. Helicase89B belongs to the Mot1p/BTAF1 subfamily of SNF2‐like ATPases. This subfamily can interact with TATA‐binding proteins, but whether the interaction leads to gene activation or repression is being debated. We found that Helicase89B is required for the inducible expression of antimicrobial peptide genes but not for the inducible expression of heat‐shock genes. The antimicrobial peptide genes are activated by the Toll and immune deficiency (IMD) signalling pathways. Genetic experiments show that Helicase89B acts downstream of DIF and Relish, the two nuclear factor‐κB (NF‐κB)‐related transcription factors that mediate Toll‐ and IMD‐stimulated antimicrobial response. Thus, Helicase89B positively regulates gene expression during innate immune response and may act as a link between NF‐κB‐related transcription factors and the basal transcription machinery.

fat facets induces polyubiquitination of Imd and inhibits the innate immune response in Drosophila

The IMD pathway is one of the major regulators of the innate immune response in Drosophila. Although extensive analysis of the IMD pathway has been carried out, precise mechanisms for how each target gene of the pathway is down‐regulated remain to be clarified. Here, we carried out genetic screening and found that fat facets (faf), which encodes a deubiquitinating enzyme, inhibited the expression of the target genes of the IMD pathway. Overexpression of faf suppressed the infection‐induced expression of Diptericin and increased susceptibility to bacterial infection in flies, whereas faf loss‐of‐function mutants decreased susceptibility. Time course analysis revealed that specific subsets of the target genes of the IMD pathway were affected by faf. Biochemical analysis showed that Faf made a complex with Imd, and both Faf and Imd were polyubiquitinated when they were co‐overexpressed. Given that faf‐dependent Imd polyubiquitination did not seem to cause protein degradation of Imd, Faf might inhibit the IMD pathway by modulating the state of Imd ubiquitination and/or stability.

Cellular Defense and Sensory Cell Survival Require Distinct Functions of ebi in Drosophila

The innate immune response and stress-induced apoptosis are well-established signaling pathways related to cellular defense. NF-κB and AP-1 are redox-sensitive transcription factors that play important roles in those pathways. Here we show that Ebi, a Drosophila homolog of the mammalian co-repressor molecule transducin β-like 1 (TBL1), variously regulates the expression of specific genes that are targets of redox-sensitive transcription factors. In response to different stimuli, Ebi activated gene expression to support the acute immune response in fat bodies, whereas Ebi repressed genes that are involved in apoptosis in photoreceptor cells. Thus, Ebi seems to act as a regulatory switch for genes that are activated or repressed in response to different external stimuli. Our results offer clear in vivo evidence that the Ebi-containing co-repressor complex acts in a distinct manner to regulate transcription that is required for modulating the output of various processes during Drosophila development.