An insulin-like growth factor-like peptide promotes ovarian development in the silkmoth Bombyx mori.

Insulin family peptides are known to be key regulators of growth and metabolism in insects and vertebrates. Insects have two types of insulin family peptides: insulin-like peptides and insulin-like growth factor (IGF)-like peptides (IGFLPs). We recently demonstrated that an IGFLP in the silkmoth, Bombyx mori (BIGFLP) promotes the growth of the genital imaginal disc ex vivo. However, the role of BIGFLP in the regulation of insect growth remains unclear because no in vivo study has been performed. Therefore, we analysed the functions of BIGFLP in vivo by constructing BIGFLP knock-out (KO) B. mori using the clustered regularly interspaced palindromic repeats (CRISPR) and CRISPR-associated protein 9 (CRISPR-Cas9) system. The KO moths exhibited decreased body weights and size of the appendages compared wild-type (wt) moths. Interestingly, KO females also had drastically lower ovary weights and number of eggs than wt females. However, mutant ovaries that were transplanted into wt host pupae reached a similar weight to wt ovaries that were transplanted into the wt hosts, suggesting that IGFLP in the haemolymph promotes ovarian development. These findings show that BIGFLP regulates the growth and development of adult organs, particularly the ovaries, in B. mori.

Identification and Analysis of a Gene with Potential to Repress the IMD Pathway in Drosophila melanogaster.

The IMD pathway is one of the signal transduction pathways that regulates innate immune responses in Drosophila. To understand the regulation mechanism of the IMD pathway, we performed a mis-expression screening and identified a gene, Dyro, which potentially repress the expression of the downstream target genes of the IMD pathway. We confirmed that Dyro was expressed in the fat body where the IMD pathway is functional and that the overexpression of Dyro increases susceptibility. However, we detected neither an increased expression of target genes nor reduced susceptibility in mutants. During the analysis, we observed that the Dyro mutant exhibits a female sterile phenotype, and observed oogenesis defects. The results suggest that Dyro have potential to suppress immune response but its main role is regulation of oogenesis.

Microtubule-dependent balanced cell contraction and luminal-matrix modification accelerate epithelial tube fusion.

Connection of tubules into larger networks is the key process for the development of circulatory systems. In Drosophila development, tip cells of the tracheal system lead the migration of each branch and connect tubules by adhering to each other and simultaneously changing into a torus-shape. We show that as adhesion sites form between fusion cells, myosin and microtubules form polarized bundles that connect the new adhesion site to the cells' microtubule-organizing centres, and that E-cadherin and retrograde recycling endosomes are preferentially deposited at the new adhesion site. We demonstrate that microtubules help balancing tip cell contraction, which is driven by myosin, and is required for adhesion and tube fusion. We also show that retrograde recycling and directed secretion of a specific matrix protein into the fusion-cell interface promote fusion. We propose that microtubule bundles connecting these cell-cell interfaces coordinate cell contractility and apical secretion to facilitate tube fusion.

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 ß-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.

Antiviral autophagy restrictsRift Valley fever virus infection and is conserved from flies to mammals.

Autophagy has been implicated as a component of host defense, but the significance of antimicrobial autophagy in vivo and the mechanism by which it is regulated during infection are poorly defined. Here we found that antiviral autophagy was conserved in flies and mammals during infection with Rift Valley fever virus (RVFV), a mosquito-borne virus that causes disease in humans and livestock. In Drosophila, Toll-7 limited RVFV replication and mortality through activation of autophagy. RVFV infection also elicited autophagy in mouse and human cells, and viral replication was increased in the absence of autophagy genes. The mammalian Toll-like receptor adaptor, MyD88, was required for anti-RVFV autophagy, revealing an evolutionarily conserved requirement for pattern-recognition receptors in antiviral autophagy. Pharmacologic activation of autophagy inhibited RVFV infection in mammalian cells, including primary hepatocytes and neurons. Thus, autophagy modulation might be an effective strategy for treating RVFV infection, which lacks approved vaccines and therapeutics.

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.

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.

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.

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-kappaB (NF-kappaB)-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-kappaB-related transcription factors and the basal transcription machinery.

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.

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-kappaB 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.

Multimerization and interaction of Toll and Spätzle in Drosophila.

The Toll family of receptors is required for innate immune response to pathogen-associated molecules, but the mechanism of signaling is not entirely clear. In Drosophila the prototypic Toll regulates both embryonic development and adult immune response. We demonstrate here that the host protein Spätzle can function as a ligand for Toll because Spätzle forms a complex with Toll in transgenic fly extracts and stimulates the expression of a Toll-dependent immunity gene, drosomycin, in adult flies. We also show that constitutively active mutants of Toll form multimers that contain intermolecular disulfide linkages. These disulfide linkages are critical for the activity of one of these mutant receptors, indicating that multimerization is essential for the constitutive activity. Furthermore, systematic mutational analysis revealed that a conserved cysteine-containing motif, different from the cysteines used for the intermolecular disulfide linkages, serves as a self-inhibitory module of Toll. Deleting or mutating this cysteine-containing motif leads to constitutive activity. This motif is located just outside the transmembrane domain and may provide a structural hindrance for multimerization and activation of Toll. Together, our results suggest that multimerization may be a regulated, essential step for Toll-receptor activation.

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 beta-amyloid precursor protein (APP), which is associated with Alzheimer's 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 Alzheimer's disease.

Interaction of Alzheimer's beta -amyloid precursor family proteins with scaffold proteins of the JNK signaling cascade.

We have isolated a novel protein based on its association with Drosophila APP-like protein (APPL), a homolog of the beta-amyloid precursor protein (APP) that is implicated in Alzheimer's disease. This novel APPL-interacting protein 1 (APLIP1) contains a Src homology 3 domain and a phosphotyrosine interaction domain and is expressed abundantly in neural tissues. The phosphotyrosine interaction domain of APLIP1 interacts with a sequence containing GYENPTY in the cytoplasmic domain of APPL. APLIP1 is highly homologous to the carboxyl-terminal halves of mammalian c-Jun NH(2)-terminal kinase (JNK)-interacting protein 1b (JIP1b) and 2 (JIP2), which also contain Src homology 3 and phosphotyrosine interaction domains. The similarity of APLIP1 to JIP1b and JIP2 includes interaction with component(s) of the JNK signaling pathway and with the motor protein kinesin and the formation of homo-oligomers. JIP1b interacts strongly with the cytoplasmic domain of APP (APPcyt), as APLIP1 does with APPL, but the interaction of JIP2 with APPcyt is weak. Overexpression of JIP1b slightly enhances the JNK-dependent threonine phosphorylation of APP in cultured cells, but that of JIP2 suppresses it. These observations suggest that the interactions of APP family proteins with APLIP1, JIP1b, and JIP2 are conserved and play important roles in the metabolism and/or the function of APPs including the regulation of APP phosphorylation by JNK. Analysis of APP family proteins and their associated proteins is expected to contribute to understanding the molecular process of neural degeneration in Alzheimer's disease.

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.

The brain neurosecretory cells of the moth Samia cynthia ricini: Immunohistochemical localization and developmental changes of the Samia homologues of the Bombyx prothoracicotropic hormone and bombyxin.

We produced mouse antisera against synthetic peptides corresponding to the sequences of the Samia cynthia ricini homologues of the Bombyx mori PTTH and bombyxin. Immunohistochemical analyses of the Samia cephalic neuroendocrine system using these antisera were performed to identify the neurosecretory cells (NSC) containing the PTTH and bombyxin homologues and to examine the developmental changes in their amounts in the NSC. The results show that the PTTH and bombyxin homologues are produced by two pairs of dorsolateral and 16 pairs of dorsomedial NSC of Samia brain, respectively, and both are transported to, and released from, the corpora allata. No clear-cut correlation was found between the fluctuation in the amount of immunoreactive substances in the brain NSC and the endocrinologically anticipated timings of PTTH secretion. From Samia brain extract, two forms of PTTH activity (∼30 kDa and ∼5 kDa) were resolved through Sephadex gel filtration. The ∼30 kDa and ∼5 kDa PTTH seem to represent the PTTH and bombyxin homologues, respectively. We discuss that the ∼30 kDa PTTH homologue is the true PTTH of Samia.