Tissue and stage-specific expression of the Tolls in Drosophila embryos

The Drosophila transmembrane receptor Toll plays a key role in specifying the dorsoventral axis of the embryo. At later stages of development, it controls the immune response of the fly to fungal and Gram-positive bacterial infections. The Drosophila genome has a total of nine Toll-like genes, including the previously characterized Toll (Toll-1) and 18-wheeler (Toll-2). Here we describe the embryonic expression patterns of the seven Toll-like genes Toll-3 through Toll-9. We find that these genes have distinct expression domains and that their expression is dynamically changing throughout embryonic development. This complex and tissue-specific regulation of Toll-like gene expression strongly suggests a role in embryonic development for most Drosophila Tolls. The evolving picture on the Toll family members in Drosophila contrasts with that of mammalian Toll-like receptors, which are predominantly expressed in immune responsive cells where their activation occurs via microbial structural determinants.     

Annotation and expression profiling of apoptosis-related genes in the yellow fever mosquito, Aedes aegypti

Apoptosis has been extensively studied in Drosophila by both biochemical and genetic approaches, but there is a lack of knowledge about the mechanisms of apoptosis regulation in other insects. In mosquitoes, apoptosis occurs during Plasmodium and arbovirus infection in the midgut, suggesting that apoptosis plays a role in mosquito innate immunity. We searched the Aedes aegypti genome for apoptosis-related genes using Drosophila and Anopheles gambiae protein sequences as queries. In this study we have identified eleven caspases, three inhibitor of apoptosis (IAP) proteins, a previously unreported IAP antagonist, and orthologs of Drosophila Ark, Dnr1, and BG4 (also called dFadd). While most of these genes have been previously annotated, we have improved the annotation of several of them, and we also report the discovery of four previously unannotated apoptosis-related genes. We examined the developmental expression profile of these genes in Ae. aegypti larvae, pupae and adults, and we also studied the function of a novel IAP antagonist, IMP. Expression of IMP in mosquito cells caused apoptosis, indicating that it is a functional pro-death protein. Further characterization of these genes will help elucidate the molecular mechanisms of apoptosis regulation in Ae. aegypti.     

Heat shock and ecdysterone activation of the Drosophila melanogaster hsp23 gene; a sequence element implied in developmental regulation.

The regulation of the Drosophila melanogaster hsp23 gene by heat shock and ecdysterone has been analysed by measuring activities of hsp--Escherichia coli beta-galactosidase hybrid genes in transfected hormone-sensitive D. melanogaster cells. Mutation analysis identified multiple, distinct promoter elements. A sequence element, which also occurs in the promoters of several other developmentally regulated Drosophila genes, is present in regions of the hsp23 promoter that are essential for its ecdysterone, but not its heat-regulated activity; this element may represent a binding site for an ecdysterone--receptor complex. Mutant promoters that can be activated only by heat shock or by hormone have been constructed. Thus the two types of regulation of the hsp23 gene can function independently of each other.     

Transfer RNA genes in Drosophila mitochondrial DNA: related 5'' flanking sequences and comparisons to mammalian mitochondrial tRNA genes.

Genes for tRNAgly and tRNAserUCN have been identified within sequences of mtDNA of Drosophila yakuba. The tRNAgly gene lies between the genes for cytochrome c oxidase subunit III and URF3, and all three of these genes are contained in the same strand of the mtDNA molecule. The tRNAserUCN gene is adjacent to the URF1 gene. These genes are contained in opposite strands of the mtDNA molecule and their 3' ends overlap. The structures of the tRNAgly and tRNAserUCN genes, and of the four tRNA genes of D. yakuba mtDNA reported earlier (tRNAile, tRNAgln, tRNAf-met and tRNAval) are compared to each other, to non-organelle tRNAs, and to corresponding mammalian mitochondrial tRNA genes. Within 19 nucleotides upstream from the 5' terminal nucleotide of each of the Drosophila mitochondrial tRNAgly, tRNAserUCN, tRNAile, tRNAgln and tRNAf-met genes occurs the sequence 5'TTTATTAT, or a sequence differing from it by one nucleotide substitution. Upstream from this octanucleotide sequence, and separated from it by 3, 4 and 11 nucleotides, respectively, in the 5' flanking regions of the tRNAile, tRNAserUCN and tRNAgly genes occurs the sequence 5'GATGAG.     

An evolutionarily conserved function of the Drosophila insulin receptor and insulin-like peptides in growth control

BACKGROUND: Size regulation is fundamental in developing multicellular organisms and occurs through the control of cell number and cell size. Studies in Drosophila have identified an evolutionarily conserved signaling pathway that regulates organismal size and that includes the Drosophila insulin receptor substrate homolog Chico, the lipid kinase PI(3)K (Dp110), DAkt1/dPKB, and dS6K. RESULTS: We demonstrate that varying the activity of the Drosophila insulin receptor homolog (DInr) during development regulates organ size by changing cell size and cell number in a cell-autonomous manner. An amino acid substitution at the corresponding position in the kinase domain of the human and Drosophila insulin receptors causes severe growth retardation. Furthermore, we show that the Drosophila genome contains seven insulin-like genes that are expressed in a highly tissue- and stage-specific pattern. Overexpression of one of these insulin-like genes alters growth control in a DInr-dependent manner. CONCLUSIONS: This study shows that the Drosophila insulin receptor autonomously controls cell and organ size, and that overexpression of a gene encoding an insulin-like peptide is sufficient to increase body size.     

Molecular biology and anatomy of Drosophila olfactory associative learning.

Most of our current knowledge of olfactory associative learning in Drosophila comes from the behavioral and molecular analysis of mutants that fail to learn. The identities of the genes affected in these mutants implicate new signaling pathways as mediators of associative learning. The expression patterns of these genes provide insight into the neuroanatomical areas that underlie learning. In recent years, there have been great strides in understanding the molecular and neuroanatomical basis for olfaction in insects. It is now clear that much of the association between the conditioned stimuli and the unconditioned stimuli in olfactory learning occurs within mushroom bodies - third order olfactory neurons within the central brain. In this review, we discuss the nature of the behavioral tasks, the molecules, and the neuronal circuits involved in olfactory learning in Drosophila.     

Exon-specific RNAi: a tool for dissecting the functional relevance of alternative splicing

The goal of functional genomics is to determine the function of each protein encoded by an organism. Typically, this is done by inactivating individual genes and, subsequently, analyzing the phenotype of the modified organisms. In higher eukaryotes, where a tremendous amount of alternative splicing occurs, such approaches are not feasible because they have the potential to simultaneously affect multiple proteins that could have quite distinct and important functions. Thus, it is necessary to develop techniques that inactivate only a subset of proteins synthesized from genes encoding alternatively spliced mRNAs. Here we demonstrate that RNA interference (RNAi) can be used to selectively degrade specific alternatively spliced mRNA isoforms in cultured Drosophila cells. This is achieved by treating the cells with double-stranded RNA corresponding to an alternatively spliced exon. This technique may prove to be a powerful tool to assess the function of proteins synthesized from alternatively spliced mRNAs. In addition, these results have implications regarding the mechanism of RNAi in Drosophila.     

Germ line imprinting in Drosophila: Epigenetics in search of function

Germ line imprinting produces parent-specific differences in the behavior of chromosomes or expression of genes. Epigenetic marks, placed on chromosomes in the parental germ line, govern classical imprinted effects such as chromosomal inactivation, chromosome elimination and mono-allelic expression. Germ line imprinting occurs in insects, plants and mammals. Several Drosophila systems display imprinted effects. In spite of this, many aspects of imprinting in flies, including the normal function of this process, remain mysterious. Transgenerational inheritance of epigenetic marks is a powerful force in genome regulation. Elucidation of the mechanism of imprint establishment and maintenance in a model organism, such as Drosophila, is thus of great interest. In this review we summarize the primary systems that have been used to study imprinting in flies and speculate on the origin and biological function of imprinting in Drosophila.     

The establishment of segmentation in the Drosophila leg.

Segmentation is a developmental mechanism that subdivides a tissue into repeating functional units, which can then be further elaborated upon during development. In contrast to embryonic segmentation, Drosophila leg segmentation occurs in a tissue that is rapidly growing in size and thus segmentation must be coordinated with tissue growth. I demonstrate that segmentation of the Drosophila leg, as assayed by expression of the key regulators of segmentation, the Notch ligands and fringe, occurs progressively and I define the sequence in which the initial segmental subdivisions arise. I further demonstrate that the proximal-distal patterning genes homothorax and dachshund are positively required, while Distal-less is unexpectedly negatively required, to establish the segmental pattern of Notch ligand and fringe expression. Two Serrate enhancers that respond to regulation by dachshund are also identified. Together, these studies provide evidence that distinct combinations of the proximal-distal patterning genes independently regulate each segmental ring of Notch ligand and fringe expression and that this regulation occurs through distinct enhancers. These studies thus provide a molecular framework for understanding how segmentation during tissue growth is accomplished.     

Isolation and characterization of recombinant DNA plasmids carrying Drosophila tRNA genes.

Recombinant plasmids carrying Drosophila melanogaster tRNA genes were constructed by ligation of HindIII-cleaved Drosophila DNA to HindIII cut pBR322 DNA. 90 clones were isolated that contained genes for one or more of eleven tRNAs. 43 of the plasmids were characterized by a number of methods: restriction nuclease digestion; agarose gel electrophoresis; hybridization with individual, purified, 125I-labelled Drosophila tRNA molecules and in situ hybridization to Drosophila chromosomes. The results show that several different tRNA genes have been isolated which code for single, specific isoacceptors. The DNAs from 8 plasmids each hybridize to single sites on Drosophila polytene chromosomes. In addition, the data show examples of two different plasmids hybridizing to different loci coding for the same tRNA; this means that we have isolated representatives of tRNA genes which map at widely separated points on the Drosophila genome.     

Drosophila homologs of two mammalian intracellular Ca(2+)-release channels: identification and expression patterns of the inositol 1,4,5-triphosphate and the ryanodine receptor genes.

We have identified and cloned portions of two Drosophila genes homologous to two classes of mammalian intracellular Ca(2+)-release channels, the ryanodine receptor and the inositol 1,4,5-triphosphate (IP3) receptor. The Drosophila ryanodine receptor gene (dry) encodes an approx. 15 kb mRNA. It is expressed in the mesoderm of early stage-9 embryos and subsequently in somatic muscles and their precursor cells. In adults, dry mRNA was detected in tubular muscles and at a lower level in neuronal tissues. Embryonic expression of the Drosophila IP3 receptor gene (dip) appears more dynamic and is associated with developing anterior sense organs. In adults, dip expression occurs in several tissues, and relatively high levels of dip mRNA in adult antennae suggest a role for this gene product during olfactory transduction.     

Loss and gain of domains during evolution of cut superclass homeobox genes

The cut superclass of homeobox genes has been divided into three classes: CUX, ONECUT and SATB. Given the various completed genomes, we have now made a comprehensive survey. We find that there are only two cut domain containing genes in Drosophila, one CUX and one ONECUT type. Caenorhabditis elegans has undergone an expansion of the ONECUT subclass genes and has a gene cluster with three ONECUT class genes, one of which has lost the cut domain. Two of these genes contain a conserved sequence motif, termed OCAM, which also occurs in another gene in C. elegans this motif seems to be nematode specific. A recently uncovered C. elegans CUX gene has sequence conservation in its amino-terminus with vertebrate CUX proteins. Further, the 5' end of this gene containing the conserved region can undergo alternative splicing to give rise to a protein with a different carboxy-terminus lacking the cut- and homeodomain. This protein is conserved in its entirety with vertebrate genes termed CASP--which are also alternative splice products of the CUX genes--and with plant and fungal genes. The highly divergent SATB genes share a conserved amino terminal domain, COMPASS, with the Drosophila defective proventriculus gene and a C. elegans ORF. These two "COMPASS" family genes encode two highly divergent homeodomains, may be homologues of the SATB genes and thus probably belong to the cut superclass, too.     

Autophagy as a trigger for cell death: autophagic degradation of inhibitor of apoptosis dBruce controls DNA fragmentation during late oogenesis in Drosophila

Autophagy has been reported to contribute to cell death, but the underlying mechanisms remain largely unknown and controversial. We have: been studying oogenesis in Drosophila melanogaster as a model system to understand the interplay between autophagy and cell death. Using a novel autophagy reporter we found that autophagy occurs during developmental cell death of nurse cells in late oogenesis. Genetic inhibition: of autophagy-related genes atg1, atg13 and vps34 results in late-stage egg chambers containing persisting nurse cell nuclei without fragmented DNA and attenuation of caspase-3 cleavage. We found that Drosophila inhibitor of apoptosis dBruce is degraded by autophagy and this degradation promotes DNA fragmentation and subsequent nurse cell death. These studies demonstrate that autophagic degradation of an inhibitor: of apoptosis is a novel mechanism of triggering cell death.