Nutrient-dependent expression of insulin-like peptides from neuroendocrine cells in the CNS contributes to growth regulation in Drosophila

BACKGROUND: The insulin/IGF-1 signaling pathway controls cellular and organismal growth in many multicellular organisms. In Drosophila, genetic defects in components of the insulin signaling pathway produce small flies that are delayed in development and possess fewer and smaller cells as well as female sterility, reminiscent of the phenotypes of starved flies. RESULTS: Here we establish a causal link between nutrient availability and insulin-dependent growth. We show that in addition to the Drosophila insulin-like peptide 2 (dilp2) gene, overexpression of dilp1 and dilp3-7 is sufficient to promote growth. Three of the dilp genes are expressed in seven median neurosecretory cells (m-NSCs) in the brain. These m-NSCs possess axon terminals in the larval endocrine gland and on the aorta, from which DILPs may be released into the circulatory system. Although expressed in the same cells, the expression of the three genes is controlled by unrelated cis-regulatory elements. The expression of two of the three genes is regulated by nutrient availability. Genetic ablation of these neurosecretory cells mimics the phenotype of starved or insulin signaling mutant flies. CONCLUSIONS: These results point to a conserved role of the neuroendocrine axis in growth control in multicellular organisms.     

Patterns of Nucleotide Diversity at the Regions Encompassing the Drosophila Insulin-Like Peptide (dilp) Genes: Demography vs. Positive Selection in Drosophila melanogaster

In Drosophila, the insulin-signaling pathway controls some life history traits, such as fertility and lifespan, and it is considered to be the main metabolic pathway involved in establishing adult body size. Several observations concerning variation in body size in the Drosophila genus are suggestive of its adaptive character. Genes encoding proteins in this pathway are, therefore, good candidates to have experienced adaptive changes and to reveal the footprint of positive selection. The Drosophila insulin-like peptides (DILPs) are the ligands that trigger the insulin-signaling cascade. In Drosophila melanogaster, there are several peptides that are structurally similar to the single mammalian insulin peptide. The footprint of recent adaptive changes on nucleotide variation can be unveiled through the analysis of polymorphism and divergence. With this aim, we have surveyed nucleotide sequence variation at the dilp1-7 genes in a natural population of D. melanogaster. The comparison of polymorphism in D. melanogaster and divergence from D. simulans at different functional classes of the dilp genes provided no evidence of adaptive protein evolution after the split of the D. melanogaster and D. simulans lineages. However, our survey of polymorphism at the dilp gene regions of D. melanogaster has provided some evidence for the action of positive selection at or near these genes. The regions encompassing the dilp1-4 genes and the dilp6 gene stand out as likely affected by recent adaptive events.     

Evidence for redundancy but not trans factor-cis element coevolution in the regulation of Drosophila Yp genes.

In Drosophila melanogaster and the endemic Hawaiian species D. grimshawi three Yolk protein (Yp) genes are expressed in a similar sex- and tissue-specific pattern. In contrast, DNA sequence comparisons of promoter/enhancer regions show low levels of similarity. We tested the functional significance of these observations by transforming D. melanogaster with the genomic region that includes the divergently transcribed D. grimshawi DgYp1 and DgYp2 genes; we found that the introduced genes were expressed in female fat body and in ovaries but not in males. Moreover, we found D. grimshawi proteins in the hemolymph and accumulating in ovaries. Using reporter constructs we showed that the intergenic region from D. grimshawi was sufficient to drive accurate expression, but some low level of ectopic expression was seen in males. Transforming D. melanogaster with constructs bearing deletions within the D. grimshawi intergenic region revealed only subtle effects in the overall level of expression, suggesting a high level of redundancy. Testing mutants in the sex-specific regulator doublesex revealed that it is capable of repressing the DgYp genes in males. Together, these data show that D. melanogaster trans-acting factors can regulate the in vivo pattern of DgYp expression and support the notion of a redundant and complex system of cis-acting elements.     

Molecular evolution of Drosophila odorant receptor genes

A total of 752 odorant receptor (Or) genes, including pseudogenes, were identified in 11 Drosophila species and named after their orthologs in Drosophila melanogaster. The 813 Or genes, including 61 from D. melanogaster, were classified into 59 orthologous groups that are well supported by gene phylogeny. By reconciling with the gene family phylogeny, we estimated the number of gene duplication/loss events and intron gain/loss events in the species phylogeny. We found that these events are particularly frequent in Drosophila grimshawi, Drosophila willistoni, and obscura group. More than half of the duplicated genes stay as tandem arrays, whose size range from 2 to 8. These genes vary in sequence and some likely underwent positive selection, indicating that the gene duplication was important for flies to acquire new olfactory functions. We hypothesize that Or genes conferred the basic olfactory repertoire to ancestral flies before the speciation of the Drosophila and Sophophora subgenera about 40 Mya. This repertoire has been largely maintained in the current species, whereas lineage-specific gene duplication seems to have led to additional specialization in some species in response to specific ecological conditions.     

Tissue-Specific Expression Phenotypes of Hawaiian Drosophila Adh Genes in Drosophila Melanogaster Transformants

Interspecific differences in the tissue-specific patterns of expression displayed by the alcohol dehydrogenase (Adh) genes within the Hawaiian picture-winged Drosophila represent a rich source of evolutionary variation in gene regulation. Study of the cis-acting elements responsible for regulatory differences between Adh genes from various species is greatly facilitated by analyzing the behavior of the different Adh genes in a homogeneous background. Accordingly, the Adh gene from Drosophila grimshawi was introduced into the germ line of Drosophila melanogaster by means of P element-mediated transformation, and transformants carrying this gene were compared to transformants carrying the Adh genes from Drosophila affinidisjuncta and Drosophila hawaiiensis. The results indicate that the D. affinidisjuncta and D. grimshawi genes have relatively higher levels of expression and broader tissue distribution of expression than the D. hawaiiensis gene in larvae. All three genes are expressed at similar overall levels in adults, with differences in tissue distribution of enzyme activity corresponding to the pattern in the donor species. However, certain systematic differences between Adh gene expression in transformants and in the Hawaiian Drosophila are noted along with tissue-specific position effects in some cases. The implications of these findings for the understanding of evolved regulatory variation are discussed.     

Evolution of the Autosomal Chorion Locus in Drosophila. I. General Organization of the Locus and Sequence Comparisons of Genes S15 and S19 in Evolutionarily Distant Species

We have isolated clones corresponding to the autosomal chorion locus of Drosophila melanogaster, from two distantly (D. virilis and D. grimshawi) and one closely (D. subobscura) related species. In all the species the locus is unique within the genome and encompasses the same four chorion genes and an adjacent nonchorion gene, in the same order. In all species the locus specifically amplifies in the ovary, as in D. melanogaster. We present the nucleotide sequences of DNA segments that total 8.3 kb in length and include gene s15-1 from D. subobscura, D. virilis, and D. grimshawi as well as gene s19-1 from D. subobscura and D. grimshawi. They show clearly nonuniform rates of divergence, both within and outside the limits of the genes. Highlighted by a background of extensive sequence divergence elsewhere in the extragenic region, highly conserved elements are observed in the 5' flanking DNA and might represent regulatory elements.     

Lifespan extension by increased expression of the Drosophila homologue of the IGFBP7 tumour suppressor

Mammals possess multiple insulin-like growth factor (IGF) binding proteins (IGFBPs), and related proteins, that modulate the activity of insulin/IGF signalling (IIS), a conserved neuroendocrine signalling pathway that affects animal lifespan. Here, we examine if increased levels of an IGFBP-like protein can extend lifespan, using Drosophila as the model organism. We demonstrate that Imaginal morphogenesis protein-Late 2 (IMP-L2), a secreted protein and the fly homologue of the human IGFBP7 tumour suppressor, is capable of binding at least two of the seven Drosophila insulin-like peptides (DILPs), namely native DILP2 and DILP5 as present in the adult fly. Increased expression of Imp-L2 results in phenotypic changes in the adult consistent with down-regulation of IIS, including accumulation of eIF-4E binding protein mRNA, increase in storage lipids, reduced fecundity and enhanced oxidative stress resistance. Increased Imp-L2 results in up-regulation of dilp2, dilp3 and dilp5 mRNA, revealing a feedback circuit that is mediated via the fly gut and/or fat body. Importantly, over-expression of Imp-L2, ubiquitous or restricted to DILP-producing cells or gut and fat body, extends lifespan. This enhanced longevity can also be observed upon adult-onset induction of Imp-L2, indicating it is not attributable to developmental changes. Our findings point to the possibility that an IGFBP or a related protein, such as IGFBP7, plays a role in mammalian aging.     

Drosophila insulin‐like peptide dilp1 increases lifespan and glucagon‐like Akh expression epistatic to dilp2

Insulin/IGF signaling (IIS) regulates essential processes including development, metabolism, and aging. The Drosophila genome encodes eight insulin/IGF‐like peptide (dilp) paralogs, including tandem‐encoded dilp1 and dilp2. Many reports show that longevity is increased by manipulations that decrease DILP2 levels. It has been shown that dilp1 is expressed primarily in pupal stages, but also during adult reproductive diapause. Here, we find that dilp1 is also highly expressed in adult dilp2 mutants under nondiapause conditions. The inverse expression of dilp1 and dilp2 suggests these genes interact to regulate aging. Here, we study dilp1 and dilp2 single and double mutants to describe epistatic and synergistic interactions affecting longevity, metabolism, and adipokinetic hormone (AKH), the functional homolog of glucagon. Mutants of dilp2 extend lifespan and increase Akh mRNA and protein in a dilp1‐dependent manner. Loss of dilp1 alone has no impact on these traits, whereas transgene expression of dilp1 increases lifespan in dilp1 ? dilp2 double mutants. On the other hand, dilp1 and dilp2 redundantly or synergistically interact to control circulating sugar, starvation resistance, and compensatory dilp5 expression. These interactions do not correlate with patterns for how dilp1 and dilp2 affect longevity and AKH. Thus, repression or loss of dilp2 slows aging because its depletion induces dilp1, which acts as a pro‐longevity factor. Likewise, dilp2 regulates Akh through epistatic interaction with dilp1. Akh and glycogen affect aging in Caenorhabditis elegans and Drosophila. Our data suggest that dilp2 modulates lifespan in part by regulating Akh, and by repressing dilp1, which acts as a pro‐longevity insulin‐like peptide.     

The hemoglobin genes of Drosophila

We recently reported the unprecedented occurrence of a hemoglobin gene (glob1) in the fruitfly Drosophila melanogaster. Here we investigate the structure and evolution of the glob1 gene in other Drosophila species. We cloned and sequenced glob1 genes and cDNA from D. pseudoobscura and D. virilis, and identified the glob1 gene sequences of D. simulans, D. yakuba, D. erecta, D. ananassae, D. mojavensis and D. grimshawi in the databases. Gene structure (introns in helix positions D7.0 and G7.0), gene synteny and sequence of glob1 are highly conserved, with high ds/dn ratios indicating strong purifying selection. The data suggest an important role of the glob1 protein in Drosophila, which may be the control of oxygen flow from the tracheal system. Furthermore, we identified two additional globin genes (glob2 and glob3) in the Drosophilidae. Although the sequences are highly derived, the amino acids required for heme- and oxygen-binding are conserved. In contrast to other known insect globin, the glob2 and glob3 genes harbour both globin-typical introns at positions B12.2 and G7.0. Both genes are conserved in various drosophilid species, but only expression of glob2 could be demonstrated by western blotting and RT-PCR. Phylogenetic analyses show that the clade leading to glob2 and glob3, which are sistergroups, diverged first in the evolution of dipteran globins. glob1 is closely related to the intracellular hemoglobin of the botfly Gasterophilus intestinalis, and the extracellular hemoglobins from the chironomid midges derive from this clade.     

Molecular phylogeny and divergence times of drosophilid species

The phylogenetic relationships and divergence times of 39 drosophilid species were studied by using the coding region of the Adh gene. Four genera--Scaptodrosophila, Zaprionus, Drosophila, and Scaptomyza (from Hawaii)--and three Drosophila subgenera--Drosophila, Engiscaptomyza, and Sophophora--were included. After conducting statistical analyses of the nucleotide sequences of the Adh, Adhr (Adh-related gene), and nuclear rRNA genes and a 905-bp segment of mitochondrial DNA, we used Scaptodrosophila as the outgroup. The phylogenetic tree obtained showed that the first major division of drosophilid species occurs between subgenus Sophophora (genus Drosophila) and the group including subgenera Drosophila and Engiscaptomyza plus the genera Zaprionus and Scaptomyza. Subgenus Sophophora is then divided into D. willistoni and the clade of D. obscura and D. melanogaster species groups. In the other major drosophilid group, Zaprionus first separates from the other species, and then D. immigrans leaves the remaining group of species. This remaining group then splits into the D. repleta group and the Hawaiian drosophilid cluster (Hawaiian Drosophila, Engiscaptomyza, and Scaptomyza). Engiscaptomyza and Scaptomyza are tightly clustered. Each of the D. repleta, D. obscura, and D. melanogaster groups is monophyletic. The splitting of subgenera Drosophila and Sophophora apparently occurred about 40 Mya, whereas the D. repleta group and the Hawaiian drosophilid cluster separated about 32 Mya. By contrast, the splitting of Engiscaptomyza and Scaptomyza occurred only about 11 Mya, suggesting that Scaptomyza experienced a rapid morphological evolution. The D. obscura and D. melanogaster groups apparently diverged about 25 Mya. Many of the D. repleta group species studied here have two functional Adh genes (Adh-1 and Adh-2), and these duplicated genes can be explained by two duplication events.      

Two independent duplications forming the Cyp307a genes in Drosophila

The conserved relationship between orthologs of many cytochrome P450 genes involved in ecdysone synthesis is not reflected in the evolution of the Drosophila Cyp307a genes. In Drosophila melanogaster Cyp307a1 (spook) and Cyp307a2 (spookier) both play essential roles in ecdysone synthesis and may possess biochemically redundant functions. Using phylogenetic analyses we show that the Drosophila Cyp307a genes were formed from two independent duplication events depicting a complicated evolutionary scenario. An initial duplication, from a Cyp307a2 ancestral gene produced the Cyp307a1 gene that has been maintained only in the Sophophoran subgenus. A second duplication in the Drosophila subgenus formed an additional paralog, Cyp307a3. Microsynteny is conserved for Cyp307a2 throughout the Drosophila species, but is not conserved between Cyp307a1 and Cyp307a3. These are located in different genomic positions in the Sophophora and Drosophila subgenera, respectively. Cyp307a3 appears to encode a functional gene product and is expressed in a different spatial and temporal manner to Cyp307a1. This suggests some level of functional divergence between the Cyp307a paralogs in different Drosophila species.     

Positive and negative DNA elements of the Drosophila grimshawi s18 chorion gene assayed in Drosophila melanogaster.

Germ line transformation has been used to map the cis regulatory DNA elements responsible for the precise and evolutionarily stable developmental expression of the s18 chorion gene. Constructs containing chimeric combinations of Drosophila melanogaster and D. grimshawi DNA regions, as well as D. grimshawi sequences alone, can direct expression in the follicular epithelium, in an s18-specific temporal and spatial pattern. The results indicate that both positive and negative regulatory elements can function when transferred from D. grimshawi to D. melanogaster. The first ca. 100 bp of the 5'-flanking DNA region constitute a minimal, developmentally regulated promoter, expression of which is inhibited by the next 100-bp DNA segment and activated by positive elements located further upstream. Expression of the minimal promoter can also be enhanced by more distant chorion regulatory elements, provided the inhibitory DNA segment is absent.     

Noncoordinate synthesis of the vitellogenin proteins in tissues of Drosophila grimshawi

In analyzing the in vitro pattern of protein synthesis by the fat body and ovaries of the Hawaiian species Drosophila grimshawi, we have found that the ovaries synthesize much more protein than the female fat body and that the majority of the synthesized proteins are retained by the ovarian tissues. In contrast, the fat body secrets most of the proteins into the culture medium. Vitellogenins are the major class of proteins synthesized and released into the medium by both tissues. The synthesis of the three vitellogenin proteins (V1, V2, V3) is noncoordinate in the two tissues. Ovaries synthesize much more of the V2 protein, less V1 and very little V3, whereas fat body synthesizes more V1 protein with lesser quantities of the other two. The follicle cells were identified as the site of ovarian vitellogenin synthesis in D. grimshawi, confirming the findings in D. melanogaster. In D. grimshawi, the three vitellogenins are synthesized by the follicle cells in a noncoordinate and developmentally regulated manner. V2 and V1 are the predominant proteins at the onset of vitellogenesis (S8-9); their production peaks together with that of V3 a few hours later (S10) and then decreases to quantities equal to that of V3 during early choriogenesis (S11). During active choriogenesis (S12), V2 and V1 cease to be synthesized, but V3 synthesis continues. The vitellogenins synthesized by the follicles in vitro are released into the medium and not incorporated into the oocyte.     

A single lineage of r2 retrotransposable elements is an active, evolutionarily stable component of the Drosophila rDNA locus

R2 elements are non-long-terminal-repeat (non-LTR) retrotransposons that insert specifically in the 28S rRNA genes of many insects. Previous reports concerning this element in the genus Drosophila have suggested that R2 elements are absent from many species of this genus, particularly those species from the subgenus Drosophila. In this report, we present an extensive study of the distribution and evolution of R2 elements in Drosophila. A PCR survey of 59 species from 23 species groups of the two major Drosophila subgenera found that R2 elements are present in all but two species of the melanogaster species subgroup. Phylogenetic analysis based on partial nucleotide sequences of R2 elements from 23 species demonstrates that the relationships of R2 elements are congruent with those of the Drosophila species phylogeny, suggesting that these elements have been vertically inherited since the divergence of this genus some 60 MYA. Sequence variation between different copies of R2 elements within each species was less than 0.16%, indicating that these elements are undergoing concerted evolution similar to that of the 28S genes. Several properties of the R2 sequences suggest that these elements depend on retrotransposition in addition to simple recombination to remain within the rDNA locus: the rates of synonymous substitutions averaged 4.8 times the rate of replacement substitutions, 82 of 83 R2 copies partially sequenced contained intact open reading frames, and, finally, length variation associated with the poly(A) 3' tails indicated that many R2 copies are the direct result of retrotransposition.      

Comparative genomic analysis of allatostatin-encoding (Ast) genes in Drosophila species and prediction of regulatory elements by phylogenetic footprinting

The role of the YXFGLa family of allatostatin (AST) peptides in dipterans is not well-established. The recent completion of sequencing of genomes for multiple Drosophila species provides an opportunity to study the evolutionary variation of the allatostatins and to examine regulatory elements that control gene expression. We performed comparative analyses of Ast genes from seven Drosophila species (Drosophila melanogaster, Drosophila simulans, Drosophila ananassae, Drosophila yakuba, Drosophila pseudoobscura, Drosophila mojavensis, and Drosophila grimshawi) and used phylogenetic footprinting methods to identify conserved noncoding motifs, which are candidates for regulatory regions. The peptides encoded by the Ast precursor are nearly identical across species with the exception of AST-1, in which the leading residue may be either methionine or valine. Phylogenetic footprinting predicts as few as 3, to as many as 17 potential regulatory sites depending on the parameters used during analysis. These include a Hunchback motif approximately 1.2 kb upstream of the open reading frame (ORF), overlapping motifs for two Broad-complex isoforms in the first intron, and a CF2-II motif located in the 3'-UTR. Understanding the regulatory elements involved in Ast expression may provide insight into the function of this neuropeptide family.     

Evolutionary profiling of the U49 snoRNA gene

Small nucleolar RNAs (snoRNAs) are involved in precursor ribosomal RNA (pre-rRNA) processing and rRNA base modifications (2'-O-ribose methylation and pseudouridylation). Their genomic organization show great flexibility: some are individually or polycistronically transcribed, while others are encoded within introns of other genes. Here, we present an evolutionary analysis of the U49 gene in seven species. In all species analyzed, U49 contains the typical hallmarks of C and D box motifs, and a conserved 12-15 nt sequence complementary to rRNA that define them as homologs. In mouse, human, and Drosophila U49 is found encoded within introns of different genes, and in plants it is transcribed polycistronically from four different locations. In addition, U49 has two copies in two different introns of the RpL14 gene in Drosophila. The results indicate a substantial degree of duplication and translocation of the U49 gene in evolution. In light of its variable organization we discuss which of the two proposed mechanisms of rearrangement has acted upon the U49 snoRNA gene: chromosomal duplication or transposition through an RNA intermediate.