Evolution of salivary glue genes in Drosophila species.

BACKGROUND: At the very end of the larval stage Drosophila expectorate a glue secreted by their salivary glands to attach themselves to a substrate while pupariating. The glue is a mixture of apparently unrelated proteins, some of which are highly glycosylated and possess internal repeats. Because species adhere to distinct substrates (i.e. leaves, wood, rotten fruits), glue genes are expected to evolve rapidly. RESULTS: We used available genome sequences and PCR-sequencing of regions of interest to investigate the glue genes in 20 Drosophila species. We discovered a new gene in addition to the seven glue genes annotated in D. melanogaster. We also identified a phase 1 intron at a conserved position present in five of the eight glue genes of D. melanogaster, suggesting a common origin for those glue genes. A slightly significant rate of gene turnover was inferred. Both the number of repeats and the repeat sequence were found to diverge rapidly, even between closely related species. We also detected high repeat number variation at the intrapopulation level in D. melanogaster. CONCLUSION: Most conspicuous signs of accelerated evolution are found in the repeat regions of several glue genes.

Hobbit regulates intracellular trafficking to drive insulin-dependent growth during Drosophila development.

All animals must coordinate growth rate and timing of maturation to reach the appropriate final size. Here, we describe hobbit, a novel and conserved gene identified in a forward genetic screen for Drosophila animals with small body size. hobbit is highly conserved throughout eukaryotes, but its function remains unknown. We demonstrate that hobbit mutant animals have systemic growth defects because they fail to secrete insulin. Other regulated secretion events also fail in hobbit mutant animals, including mucin-like 'glue' protein secretion from the larval salivary glands. hobbit mutant salivary glands produce glue-containing secretory granules that are reduced in size. Importantly, secretory granules in hobbit mutant cells lack essential membrane fusion machinery required for exocytosis, including Synaptotagmin 1 and the SNARE SNAP-24. These membrane fusion proteins instead accumulate inside enlarged late endosomes. Surprisingly, however, the Hobbit protein localizes to the endoplasmic reticulum. Our results suggest that Hobbit regulates a novel step in intracellular trafficking of membrane fusion proteins. Our studies also suggest that genetic control of body size, as a measure of insulin secretion, is a sensitive functional readout of the secretory machinery.

Molecular mechanisms of developmentally programmed crinophagy in Drosophila.

At the onset of metamorphosis, Drosophila salivary gland cells undergo a burst of glue granule secretion to attach the forming pupa to a solid surface. Here, we show that excess granules evading exocytosis are degraded via direct fusion with lysosomes, a secretory granule-specific autophagic process known as crinophagy. We find that the tethering complex HOPS (homotypic fusion and protein sorting); the small GTPases Rab2, Rab7, and its effector, PLEKHM1; and a SNAP receptor complex consisting of Syntaxin 13, Snap29, and Vamp7 are all required for the fusion of secretory granules with lysosomes. Proper glue degradation within lysosomes also requires the Uvrag-containing Vps34 lipid kinase complex and the v-ATPase proton pump, whereas Atg genes involved in macroautophagy are dispensable for crinophagy. Our work establishes the molecular mechanism of developmentally programmed crinophagy in Drosophila and paves the way for analyzing this process in metazoans.

Members of the salivary gland surface protein (SGS) family are major immunogenic components of mosquito saliva.

Mosquitoes transmit Plasmodium and certain arboviruses during blood feeding, when they are injected along with saliva. Mosquito saliva interferes with the host's hemostasis and inflammation response and influences the transmission success of some pathogens. One family of mosquito salivary gland proteins, named SGS, is composed of large bacterial-type proteins that in Aedes aegypti were implicated as receptors for Plasmodium on the basal salivary gland surface. Here, we characterize the biology of two SGSs in the malaria mosquito, Anopheles gambiae, and demonstrate their involvement in blood feeding. Western blots and RT-PCR showed that Sgs4 and Sgs5 are produced exclusively in female salivary glands, that expression increases with age and after blood feeding, and that protein levels fluctuate in a circadian manner. Immunohistochemistry showed that SGSs are present in the acinar cells of the distal lateral lobes and in the salivary ducts of the proximal lobes. SDS-PAGE, Western blots, bite blots, and immunization via mosquito bites showed that SGSs are highly immunogenic and form major components of mosquito saliva. Last, Western and bioinformatic analyses suggest that SGSs are secreted via a non-classical pathway that involves cleavage into a 300-kDa soluble fragment and a smaller membrane-bound fragment. Combined, these data strongly suggest that SGSs play an important role in blood feeding. Together with their role in malaria transmission, we propose that SGSs could be used as markers of human exposure to mosquito bites and in the development of disease control strategies.

Drosophila suppressor of sable protein [Su(s)] promotes degradation of aberrant and transposon-derived RNAs.

RNA-binding proteins act at various stages of gene expression to regulate and fine-tune patterns of mRNA accumulation. One protein in this class is Drosophila Su(s), a nuclear protein that has been previously shown to inhibit the accumulation of mutant transcripts by an unknown mechanism. Here, we have identified several additional RNAs that are downregulated by Su(s). These Su(s) targets include cryptic wild-type transcripts from the developmentally regulated Sgs4 and ng1 genes, noncoding RNAs derived from tandemly repeated alphabeta/alphagamma elements within an Hsp70 locus, and aberrant transcripts induced by Hsp70 promoter transgenes inserted at ectopic sites. We used the alphabeta RNAs to investigate the mechanism of Su(s) function and obtained evidence that these transcripts are degraded by the nuclear exosome and that Su(s) promotes this process. Furthermore, we showed that the RNA binding domains of Su(s) are important for this effect and mapped the sequences involved to a 267-nucleotide region of an alphabeta element. Taken together, these results suggest that Su(s) binds to certain nascent transcripts and stimulates their degradation by the nuclear exosome.

A dual role for the adaptor protein DRK in Drosophila olfactory learning and memory.

Participation of RAS, RAF, and mitogen-activated protein kinase (MAPK) in learning and memory has been demonstrated in a number of studies, but the molecular events requisite for cascade activation and regulation have not been explored. We demonstrate that the adapter protein DRK (downstream of receptor kinase) which is essential for signaling to RAS in developmental contexts, is preferentially distributed in the adult mushroom bodies, centers for olfactory learning and memory. We demonstrate that drk mutant heterozygotes exhibit deficits in olfactory learning and memory, apparent under limited training conditions, but are not impaired in sensory responses requisite for the association of the stimuli, or brain neuroanatomy. Furthermore, we demonstrate that the protein is required acutely within mushroom body neurons to mediate efficient learning, a process that requires RAF activation. Importantly, 90 min memory remained impaired, even after differential training yielding equivalent learning in animals with compromised DRK levels and controls and did not require RAF. Sustained MAPK activation is compromised in drk mutants and surprisingly is negatively regulated by constitutive RAF activity. The data establish a role for DRK in Drosophila behavioral neuroplasticity and suggest a dual role for the protein, first in RAF activation-dependent learning and additionally in RAF-inhibition dependent sustained MAPK activation essential for memory formation or stability.

A novel ecdysone receptor mediates steroid-regulated developmental events during the mid-third instar of Drosophila.

The larval salivary gland of Drosophila melanogaster synthesizes and secretes glue glycoproteins that cement developing animals to a solid surface during metamorphosis. The steroid hormone 20-hydroxyecdysone (20E) is an essential signaling molecule that modulates most of the physiological functions of the larval gland. At the end of larval development, it is known that 20E--signaling through a nuclear receptor heterodimer consisting of EcR and USP--induces the early and late puffing cascade of the polytene chromosomes and causes the exocytosis of stored glue granules into the lumen of the gland. It has also been reported that an earlier pulse of hormone induces the temporally and spatially specific transcriptional activation of the glue genes; however, the receptor responsible for triggering this response has not been characterized. Here we show that the coordinated expression of the glue genes midway through the third instar is mediated by 20E acting to induce genes of the Broad Complex (BRC) through a receptor that is not an EcR/USP heterodimer. This result is novel because it demonstrates for the first time that at least some 20E-mediated, mid-larval, developmental responses are controlled by an uncharacterized receptor that does not contain an RXR-like component.

Genes and biological processes controlled by the Drosophila FOXA orthologue Fork head.

The larval salivary glands of Drosophila express the FOXA transcription factor Fork head (Fkh) before, but not after, puparium formation. Forced expression of Fkh in late prepupae prevents the programmed destruction of the tissue, which normally occurs in the early pupa. Using Affymetrix GeneChips, we analysed changes in gene expression brought about by Fkh when expressed shortly before the normal time of salivary gland death. Genes identified as responsive to Fkh include not only cell death genes, but also genes involved in autophagy, phospholipid metabolism and hormone-controlled signalling pathways. In addition, Fkh changed the expression of genes involved in glucose and fatty acid metabolism that are known to be target genes of the FOXAs in vertebrates. Premature loss of fkh induced by RNAi and gain of Fkh by ectopic expression at earlier times of development confirmed that genes identified in the microarray study are under normal developmental control by Fkh. These genes include Eip63F-1, which is expressed in both salivary glands and Malpighian tubules, suggesting that Fkh controls common aspects of the secretory function of the two organs. Eip63F-1 is one of many genes controlled by the steroid hormone 20-hydroxyecdysone that appear to be co-regulated by Fkh.

Downregulation of the tissue-specific transcription factor Fork head by Broad-Complex mediates a stage-specific hormone response.

Drosophila development is coordinated by pulses of the steroid hormone 20-hydroxyecdysone (20E). During metamorphosis, the 20E-inducible Broad-Complex (BR-C) gene plays a key role in the genetic hierarchies that transduce the hormone signal, being required for the destruction of larval tissues and numerous aspects of adult development. Most of the known BR-C target genes, including the salivary gland secretion protein (Sgs) genes, are terminal differentiation genes that are thought to be directly regulated by BR-C-encoded transcription factors. Here, we show that repression of Sgs expression is indirectly controlled by the BR-C through transcriptional down-regulation of fork head, a tissue-specific gene that plays a central role in salivary gland development and is required for Sgs expression. Our results demonstrate that integration of a tissue-specific regulatory gene into a 20E-controlled genetic hierarchy provides a mechanism for hormonal repression. Furthermore, they suggest that the BR-C is placed at a different position within the 20E-controlled hierarchies than previously assumed, and that at least part of its pleiotropic functions are mediated by tissue-specific regulators.

Ecdysone-controlled mRNA stability in Drosophila salivary glands: deadenylation-independent degradation of larval glue protein gene message during the larval/prepupal transition.

20-Hydroxyecdysone induces poly(A) shortening and the subsequent degradation of transcripts encoding the larval glue protein LGP-1 in Drosophila virilis late third larval instar salivary glands. Degradation concurs with the transient increase of ribonucleolytic activities in the gland cells. In vitro nuclease assays using crude cytoplasmic extracts of ecdysone-treated salivary glands demonstrate degradation to be deadenylation-independent and that the induced ribonucleolytic activities initiate the degradation of the Lgp-1 transcripts in putative single-stranded loop regions. The independence of degradation from deadenylation is also found in vivo in transformed D. melanogaster carrying a modified Lgp-1 gene.

Spt5 and spt6 are associated with active transcription and have characteristics of general elongation factors in D. melanogaster.

The Spt4, Spt5, and Spt6 proteins are conserved throughout eukaryotes and are believed to play critical and related roles in transcription. They have a positive role in transcription elongation in Saccharomyces cerevisiae and in the activation of transcription by the HIV Tat protein in human cells. In contrast, a complex of Spt4 and Spt5 is required in vitro for the inhibition of RNA polymerase II (Pol II) elongation by the drug DRB, suggesting also a negative role in vivo. To learn more about the function of the Spt4/Spt5 complex and Spt6 in vivo, we have identified Drosophila homologs of Spt5 and Spt6 and characterized their localization on Drosophila polytene chromosomes. We find that Spt5 and Spt6 localize extensively with the phosphorylated, actively elongating form of Pol II, to transcriptionally active sites during salivary gland development and upon heat shock. Furthermore, Spt5 and Spt6 do not colocalize widely with the unphosphorylated, nonelongating form of Pol II. These results strongly suggest that Spt5 and Spt6 play closely related roles associated with active transcription in vivo.

Structure and regulation of the salivary gland secretion protein gene Sgs-1 of Drosophila melanogaster.

The Drosophila melanogaster gene Sgs-1 belongs to the secretion protein genes, which are coordinately expressed in salivary glands of third instar larvae. Earlier analysis had implied that Sgs-1 is located at the 25B2-3 puff. We cloned Sgs-1 from a YAC covering 25B2-3. Despite using a variety of vectors and Escherichia coli strains, subcloning from the YAC led to deletions within the Sgs-1 coding region. Analysis of clonable and unclonable sequences revealed that Sgs-1 mainly consists of 48-bp tandem repeats encoding a threonine-rich protein. The Sgs-1 inserts from single lambda clones are heterogeneous in length, indicating that repeats are eliminated. By analyzing the expression of Sgs-1/lacZ fusions in transgenic flies, cis-regulatory elements of Sgs-1 were mapped to lie within 1 kb upstream of the transcriptional start site. Band shift assays revealed binding sites for the transcription factor fork head (FKH) and the factor secretion enhancer binding protein 3 (SEBP3) at positions that are functionally relevant. FKH and SEBP3 have been shown previously to be involved in the regulation of Sgs-3 and Sgs-4. Comparison of the levels of steady state RNA and of the transcription rates for Sgs-1 and Sgs-1/lacZ reporter genes indicates that Sgs-1 RNA is 100-fold more stable than Sgs-1/lacZ RNA. This has implications for the model of how Sgs transcripts accumulate in late third instar larvae.

The helix-loop-helix proteins dAP-4 and daughterless bind both in vitro and in vivo to SEBP3 sites required for transcriptional activation of the Drosophila gene Sgs-4.

The expression of Sgs genes in the salivary gland of the third instar larva of Drosophila is a spatially restricted response to signalling by the steroid hormone 20-hydroxyecdysone. For Sgs-4, we have previously demonstrated that its strictly tissue and stage-specific expression is the result of combined action of the ecdysone receptor and secretion enhancer binding proteins (SEBPs). One of these SEBPs, SEBP2, was shown to be the product of the homeotic gene fork head. Together with SEBP3, SEBP2 appears to be responsible for the spatial restriction of the hormone response of Sgs-4. Here, we show that SEBP3 is a heterogeneous binding activity that consists of different helix-loop-helix (HLH) proteins. We cloned the Drosophila homologue of human transcription factor AP-4 (dAP-4) and identified it as one of these HLH proteins. The dAP-4 protein shows great similarity to its human and Caenorhabditis counterparts within the bHLHZip domain, the second leucine zipper dimerization motif, and a third region of unknown function. The expression pattern of dAP-4 indicates that it is a ubiquitously expressed HLH protein in Drosophila. As a second component of SEBP3 we identified the Daughterless (Da) protein, which is also ubiquitously expressed and binds to SEBP3 sites independent of dAP-4. Since both dAP-4 and Da can be detected in situ at transposed Sgs-4 transcriptional control elements in polytene salivary gland chromosomes, we propose that each of the two proteins contributes to the transcriptional control of Sgs-4.

SDS-PAGE pattern polymorphism of X-chromosomal glue proteins in natural populations of two Drosophila nasuta subgroup species.

For the first time, the glue protein pattern polymorphism in natural populations of D. n. nasuta and D. s. neonasuta has been analyzed by SDS-PAGE. The study involving 200 and 185 isofemale lines comprising 2028 and 1900 individuals of D. n. nasuta and D. s. neonasuta, respectively, revealed the occurrence of eight variant glue protein phenotypes in D. n. nasuta and seven in D. s. neonasuta. The number and frequency of variant phenotypes in different populations of both species were found to vary. Analysis of glue protein patterns in the F1 progeny of crosses involving parents with variant glue protein phenotypes revealed that the polymorphic fractions are produced by co-dominant genes located on the X chromosome. More than 87% of the naturally inseminated adult females were found to produce polymorphic progeny. The heterozygous female larvae were found to exceed the homozygotes in the isofemale line progeny of most of the populations.

The transcriptional switch between the Drosophila genes Pig-1 and Sgs-4 depends on a SEBP1 binding site within a shared enhancer region.

The Drosophila genes Pig-1 and Sgs-4 are adjacent and oppositely transcribed genes which share a common enhancer region located in the intergenic region between the two transcription start sites. Both genes are transcribed in the same tissue, the larval salivary gland, but they are expressed at different times, the expression switching from Pig-1 to Sgs-4 expression during development of the third-instar larva. Here we describe partial purification and functional analysis of a DNA-binding protein, secretion enhancer binding protein 1 (SEBP1), from nuclear extracts of whole Drosophila larvae. SEBP1 binds to a single site within the Pig-1/Sgs-4 intergenic region. Point mutations that disrupt this site had no significant effect on activation of the Sgs-4 promoter in constructs lacking the Pig-1 promoter. However, if the Pig-1 promoter was present, the same mutations led to a strong attenuation of Sgs-4 promoter activity, suggesting that binding of SEBP1 is necessary for the Pig-1/Sgs-4 transcriptional switch. The data are consistent with a model in which the SEBP1 binding site acts as a transient silencing element that facilitates the reorganization of promoter-enhancer interactions by disrupting pre-existing interactions, thus enabling stage-specific activators of Sgs-4 to bind to their enhancer target sites.

Relationships between protein isoforms and genetic functions demonstrate functional redundancy at the Broad-Complex during Drosophila metamorphosis.

Metamorphosis in holometabolous insects is an ecdysone-dependent process by which the larval form is replaced by a reproductive, adult form. At the onset of metamorphosis ecdysone induces a set of early genes which coordinate tissue-specific responses to hormone. The Broad-Complex (BR-C) early gene, which acts as a global regulator of tissue-specific responses to ecdysone, encodes a family of zinc-finger DNA binding proteins known as Z1, Z2, Z3, and Z4. Genetically the BR-C encodes three complementing functions, br, rbp, and 2Bc, and a class of npr1 alleles that fail to complement any of the other genetic functions. The effects of BR-C mutations on metamorphic development are highly pleiotropic, yet little is known about the roles of individual BR-C proteins in directing the required responses to ecdysone. Because the BR-C is a vital regulator of metamorphosis it is essential to establish the relationships between BR-C genetic functions and protein products. We present here the first general and definitive study of these relationships. Using heat-inducible transgenes we have rescued lethality associated with each of the complementing genetic functions and have restored transcriptional activity of tissue-specific BR-C(+)-dependent target genes. Our data lead us to conclude that br+ function is only provided by the Z2 isoform. We find that Z1 transgenes provide full rbp+ function, while Z4 provides partial function. Likewise, while Z3 provides full 2Bc+ function, Z2 also provides partial function. These results indicate possible functional redundancy or regulatory dependence (via autoregulation) associated with the rbp+ and 2Bc+ functions. The establishment of these relationships between BR-C genetic functions and protein isoforms is an important step toward understanding the roles of BR-C proteins in directing metamorphic responses to ecdysone.

Two new regulatory elements controlling the Drosophila Sgs-3 gene are potential ecdysone receptor and fork head binding sites.

We identified two regulatory elements in the upstream region of the Drosophila Sgs-3 gene which are both able to bind the ecdysone receptor (EcR/USP) and the product of the fork head gene. Interestingly, only one of the EcR/USP binding sites is able to recognize in vitro-translated EcR/USP, which provides evidence for the existence of different receptor forms having different DNA binding specificities. Deletions of the elements lead to a reduced accumulation of Sgs-3 mRNA without altering the temporal expression profile of the gene. The data are consistent with the hypothesis that the ecdysone receptor directly contributes to the transcriptional activation of Sgs-3 by binding to at least one of the two elements. Since also the Sgs-4 gene is controlled by a functional EcR/USP binding site, a direct participation of EcR/USP in the formation of regulatory complexes may be of general importance for the hormonal control of Sgs genes.

The fork head product directly specifies the tissue-specific hormone responsiveness of the Drosophila Sgs-4 gene.

Here we describe the identification of four binding sites of secretion enhancer binding protein 2 (SEBP2) in the regulatory region of the Drosophila salivary gland secretion protein gene 4 (Sgs-4) and show that despite these sites' correspondence with previously described Broad-Complex protein binding sites, SEBP2 is a Broad-Complex-independent factor encoded by the region-specific homeotic gene fork head (fkh). Two of the Fork head/SEBP2 binding sites are located within an ecdysone response unit which controls the tissue- and stage-specific responses of Sgs-4 to the steroid hormone 20-hydroxyecdysone. We demonstrate that these binding sites are relevant to the transcriptional activation of Sgs-4 and show that Fork head also binds to the Sgs-4 ecdysone response unit in vivo. Aside from being involved in the control of decisions during embryonic development, fkh thus participates directly in the control of specialized functions of differentiated cells at later stages of development.

The Drosophila fork head factor directly controls larval salivary gland-specific expression of the glue protein gene Sgs3.

The Drosophila Fork head protein participates in salivary gland formation, since salivary glands are missing in fork head embryos. Here we show that the fork head encoded protein binds to an upstream regulatory region of the larval salivary gland glue protein gene Sgs3. Mobility shift assay in the presence of an anti-Fork head antibody demonstrated that the Fork head factor interacts with the TGTTTGC box shown to be involved in tissue-specific Sgs3 expression. Experiments employing a set of oligonucleotide competitors revealed that Fork head binding was prevented by the same single base substitutions that were previously shown to interfere with the TGTTTGC element function in vivo. Furthermore, the anti-Fork head antibody bound to >60 sites of polytene chromosomes, including the puffs of all Sgs genes and Fork head protein was detected in the nuclei of salivary glands of larvae of all examined stages. These data provide experimental evidence for the hypothesis that the protein encoded by the fork head gene is required initially for salivary gland formation and is utilized subsequently in the control of larval genes specifically expressed in this organ.

Drosophila Sgs genes: stage and tissue specificity of hormone responsiveness.

The up- and down-regulation of the salivary gland secretion protein (Sgs) genes during the third larval instar of Drosophila melanogaster are controlled by fluctuations of the titre of the steroid hormone 20-hydroxyecdysone (20E). Induction of these genes by a low hormone titre is a secondary response to 20E mediated by products of 20E-induced 'early' genes. Surprisingly, in the case of the Sgs-4 gene this response also requires a direct contribution of the 20E-receptor complex. A model is presented which proposes that the Sgs genes, and other 20E-regulated genes with similar temporal expression profiles, are regulated by complex hormone response units. The hormonal signal is effectively transmitted by these response units only after binding of additional factors, e.g. secretion enhancer binding proteins, which act together in a synergistic manner with the 20E receptor and early gene products to establish a stage- and tissue-specific expression pattern.