Distinct activation patterns of EGF receptor signaling in the homoplastic evolution of eggshell morphology in genus Drosophila

Homoplasy is a phenomenon in which organisms in different phylogenetic groups independently acquire similar traits. However, it is largely unknown how developmental mechanisms are altered to give rise to homoplasy. In the genus Drosophila, all species of the subgenus Sophophora, including Drosophila (D.) melanogaster, have eggshells with two dorsal appendages (DAs); most species in the subgenus Drosophila, including D. virilis, and in the subgenus Dorsilopha, have four-DAs. D. melanica belongs to the Drosophila subgenus, but has two-DAs, and phylogenetic analyses suggest that it acquired this characteristic independently. The patterning of the DAs is tightly regulated by epidermal growth factor receptor (EGFR) signaling in D. melanogaster. Previous studies suggested that a change in the EGFR signal activation pattern could have led to the divergence in DA number between D. melanogaster and D. virilis. Here, we compared the patterns of EGFR signal activation across the Drosophila subgenera by immunostaining for anti-activated MAP kinase (MAPK). Our analysis revealed distinct patterns of EGFR signal activation in each subgenus that was consistent with their phylogenetic relationship. In addition, the number of DAs always corresponded to the number of EGFR signaling activation domains in two, three, and four-DA species. Despite their common two-DA characteristic, the EGFR signaling activation pattern in D. melanica diverged significantly from that of species in the subgenus Sophophora. Our results suggest that acquisition of the homoplastic two-DA characteristic could be explained by modifications of the EGFR signaling system in the genus Drosophila that occurred independently and at least twice during evolution.     

Species-specific activation of EGF receptor signaling underlies evolutionary diversity in the dorsal appendage number of the genus Drosophila eggshells

In Drosophila melanogaster, the patterning of dorsal appendages on the eggshell is strictly controlled by EGFR signaling. However, the number of dorsal appendages is remarkably diverse among Drosophila species. For example, D. melanogaster and D. virilis have two and four dorsal appendages, respectively. Here we show that during oogenesis the expression patterns of rhomboid (rho) and argos (aos), positive and negative regulators of EGFR signaling, respectively, were substantially different between D. melanogaster and D. virilis. Importantly, the number and position of both the rho expression and MAPK activation were consistent with those of the dorsal appendages in each species. Despite the differences in the spatial expression, these results suggest that the function of EGFR signaling in dorsal appendage formation is largely conserved between these two species. Thus, our results link the species-specific activation of EGFR signaling and the evolution of eggshell morphology in Drosophila.     

Satellite Dnas in the Embryos of Various Species of the Genus Drosophila

A tentative evolutionary pattern has been found for two classes of the multiple satellite DNA's found in the genus Drosophila. The satellite DNA's from five Drosophila species (D. melanogaster, D. simulans, D. nasuta, D. virilis and D. hydei) were analyzed and found to fall into three arbitrary CsCl buoyant density classes: Class I, rho = 1.661-1.669 g cm(-3), DNA molecules composed of primarily dA and dT moieties; Class II, rho = 1.685 and rho = 1.692, DNA molecules of low GC content; and Class III, rho = 1.711, a DNA of high GC composition. The dAT satellite DNA's appear in all the species studied except D. hydei, the species of most recent evolutionary divergence, whereas the heavy satellite appears only in the two species of most recent divergence, D. virilis and D. hydei.     

Comparison of the gap segmentation gene hunchback between Drosophila melanogaster and Drosophila virilis reveals novel modes of evolutionary change.

We have cloned and sequenced a large portion of the hunchback (hb) locus from Drosophila virilis. Comparison with the Drosophila melanogaster hb sequence shows multiple strong homologies in the upstream and downstream regions of the gene, including most of the known functional parts. The coding sequence is highly conserved within the presumptive DNA-binding finger regions, but more diverged outside of them. The regions of high divergence are correlated with regions which are rich in short direct repeats (regions of high 'cryptic simplicity'), suggesting a significant influence of slippage-like mechanisms in the evolutionary divergence of the two genes. Staining of early D.virilis embryos with an hb antibody reveals conserved and divergent features of the spatial expression pattern at blastoderm stage. It appears that the basic expression pattern, which serves as the gap gene function of hb, is conserved, while certain secondary expression patterns, which have separate functions for the segmentation process, are partly diverged. Thus, both slippage driven mutations in the coding region, which are likely to occur at higher rates than point mutations and the evolutionary divergence of secondary expression patterns may contribute to the evolution of regulatory genes.     

Temporal comparison of Broad-Complex expression during eggshell-appendage patterning and morphogenesis in two Drosophila species with different eggshell-appendage numbers

A central question in biology is how developmental mechanisms are altered to bring about morphological evolution. Drosophilids boast a remarkable diversity in eggshell-appendage number-from as few as one to as many as nine, depending on the species. Appendage patterning in Drosophila melanogaster is well characterized, inviting candidate-gene-based approaches that identify the developmental mechanisms underlying Drosophilid eggshell diversity. Previous studies show that a combination of Epidermal growth factor receptor (EGFR) and TGFbeta/BMP2,4 Decapentaplegic (DPP) signaling determines appendage fate in D. melanogaster. Broad-Complex expression integrates EGFR and DPP signaling and predicts future appendage position. Here we present our confocal analyses of BR-C immunofluorescence and appendage morphogenesis in Drosophila melanogaster (two appendages) and Drosophila virilis (four appendages). Our comparison suggests that differences in BR-C patterns among Drosophilids may be strongly influenced by anterior-posterior information.     

Effects of octopamine on reproduction, juvenile hormone metabolism, dopamine, and 20-hydroxyecdysone contents in Drosophila

The effect of an experimentally increased octopamine content (feeding flies with OA) on the levels of juvenile hormone (JH) degradation, dopamine (DA), and 20-hydroxyecdysone (20E) contents, oogenesis, and fecundity of wild type Drosophila flies has been studied. OA feeding of the flies was found to (1) cause a considerable decrease in JH degradation in females, but not males, of D. melanogaster and D. virilis; (2) have no effect on DA content in D. melanogaster and D. virilis; (3) increase 20E contents in D. virilis females; (4) decrease to a large extent the number of vitellogenic (stages 8-10) and mature (stage 14) oocytes in D. virilis; and (5) decrease the fecundity of D. melanogaster and D. virilis. A possible mechanism of action of OA as a neurohormone on the reproductive function of Drosophila is discussed.     

Laminin A is required for follicle cell-oocyte signaling that leads to establishment of the anterior-posterior axis in Drosophila

The establishment of the anterior-posterior (AP) axis in Drosophila melanogaster requires signaling between the oocyte and surrounding somatic follicle cells during oogenesis [1] [2]. First, a signal from the oocyte (Gurken (Grk), a transforming growth factor-alpha (TGFalpha) homolog) is received by predetermined terminal follicle cells in which the epidermal growth factor receptor (EGFR) pathway is activated and a posterior fate is induced [2] [3] [4]. Later, the posterior follicle cells send an unidentified signal back to the oocyte, which leads to the reorganization of its cytoskeletal polarity. This reorganization is required for proper localization of maternal determinants, such as oskar (osk) and bicoid (bcd) mRNAs, that determine the AP polarity of the oocyte and the subsequent embryo [2]. We show here that when the gene lanA, which encodes the extracellular matrix component laminin A, is mutated in posterior follicle cells, localization of AP determinants is disrupted in the underlying oocyte. Posterior follicle-cell differentiation and follicle cell apical-basal polarity are unaffected in the lanA mutant cells, suggesting that laminin A is required for correct signaling from the posterior follicle cells that polarizes the oocyte. This is the first evidence that the extracellular matrix is involved in the establishment of a major body axis.     

Drosophila virilis has long and highly polymorphic microsatellites

Comparative genomics is a powerful approach to inference of the dynamics of genome evolution. Most information about the evolution of microsatellites in the genus Drosophila has been obtained from Drosophila melanogaster. For comparison, we collected microsatellite data for the distantly related species Drosophila virilis. Screening about 0.5 Mb of nonredundant genomic sequence from GenBank, we identified 239 dinucleotide microsatellites. On average, D. virilis dinucleotides were significantly longer than D. melanogaster microsatellites (7.69 repeats vs. 6.75 repeats). Similarly, direct cloning of microsatellites resulted in a higher mean repeat number in D. virilis than in D. melanogaster (12.7 repeats vs. 12.2 repeats). Characterization of 11 microsatellite loci mapping to division 40-49 on the fourth chromosome of D. virilis indicated that D. virilis microsatellites are more variable than those of D. melanogaster.     

Nucleotide variation at the no-on-transient A gene in Drosophila littoralis

The no-on-transient A (nonA) gene encodes a putative RNA-binding protein, and mutations in this gene are known to affect vision, male courtship song and viability in Drosophila melanogaster. Here we have sequenced the coding region of the nonA gene of Drosophila littoralis and compared it with those of Drosophila virilis and D. melanogaster. All portions of nonA appeared to be conserved between D. littoralis and D. virilis, while the 5' region of the gene of these two species showed high divergence from that of a more distantly-related species, D. melanogaster. The same was true for the glycine repeat regions. No significant deviation from neutrality was observed in the analysis of intraspecific nucleotide variation in 5' or 3' region of the nonA gene in D. littoralis population. Also, comparison of D. littoralis sequences with homologous sequence of D. virilis suggests that the gene is evolving neutrally in D. virilis group. Divergence of the 5' regions between D. virilis group species and D. melanogaster could be a result of positive selection, but this finding is obscured by the long divergence time of the species groups.     

Evolution of yellow gene regulation and pigmentation in Drosophila

BACKGROUND: Changes in developmental gene expression are central to phenotypic evolution, but the genetic mechanisms underlying these changes are not well understood. Interspecific differences in gene expression can arise from evolutionary changes in cis-regulatory DNA and/or in the expression of trans-acting regulatory proteins, but few case studies have distinguished between these mechanisms. Here, we compare the regulation of the yellow gene, which is required for melanization, among distantly related Drosophila species with different pigment patterns and determine the phenotypic effects of divergent Yellow expression. RESULTS: Yellow expression has diverged among D. melanogaster, D. subobscura, and D. virilis and, in all cases, correlates with the distribution of black melanin. Species-specific Yellow expression patterns were retained in D. melanogaster transformants carrying the D. subobscura and D. virilis yellow genes, indicating that sequence evolution within the yellow gene underlies the divergence of Yellow expression. Evolutionary changes in the activity of orthologous cis-regulatory elements are responsible for differences in abdominal Yellow expression; however, cis-regulatory element evolution is not the sole cause of divergent Yellow expression patterns. Transformation of the D. melanogaster yellow gene into D. virilis altered its expression pattern, indicating that trans-acting factors that regulate the D. melanogaster yellow gene have also diverged between these two species. Finally, we found that the phenotypic effects of evolutionary changes in Yellow expression depend on epistatic interactions with other genes. CONCLUSIONS: Evolutionary changes in Yellow expression correlate with divergent melanin patterns and are a result of evolution in both cis- and trans-regulation. These changes were likely necessary for the divergence of pigmentation, but evolutionary changes in other genes were also required.     

Analysis of a fushi tarazu autoregulatory element: multiple sequence elements contribute to enhancer activity.

Regulatory sequences or factors involved in the regulation of target genes of Drosophila homeodomain proteins are largely unknown. Here, we identify sequence elements that are involved in the function of the fushi tarazu (ftz) autoregulatory element AE, a direct in vivo target of the homeodomain protein ftz. A systematic deletion analysis of AE in transgenic embryos defines multiple elements that are redundantly involved in enhancer activity. Sequences juxtaposed to ftz binding sites are not strictly required for enhancer function. Several sequence motifs are conserved in other developmentally regulated genes of Drosophila melanogaster and in the AE homologue of Drosophila virilis. The D. virilis AE is functional in D. melanogaster. The sequence motifs identified here are candidate elements contributing to the target specificity of the homeodomain protein ftz.     

Interspecific Comparison of the Transformer Gene of Drosophila Reveals an Unusually High Degree of Evolutionary Divergence

The transformer (tra) gene of Drosophila melanogaster occupies an intermediate position in the regulatory pathway controlling all aspects of somatic sexual differentiation. The female-specific expression of this gene's function is regulated by the Sex lethal (Sxl) gene, through a mechanism involving sex-specific alternative splicing of tra pre-mRNA. The tra gene encodes a protein that is thought to act in conjunction with the transformer-2 (tra-2) gene product to control the sex-specific processing of doublesex (dsx) pre-mRNA. The bifunctional dsx gene carries out opposite functions in the two sexes, repressing female differentiation in males and repressing male differentiation in females. Here we report the results from an evolutionary approach to investigate tra regulation and function, by isolating the tra-homologous genes from selected Drosophila species, and then using the interspecific DNA sequence comparisons to help identify regions of functional significance. The tra-homologous genes from two Sophophoran subgenus species, Drosophila simulans and Drosophila erecta, and two Drosophila subgenus species, Drosophila hydei and Drosophila virilis, were cloned, sequenced and compared to the D. melanogaster tra gene. This comparison reveals an unusually high degree of evolutionary divergence among the tra coding sequences. These studies also highlight a highly conserved sequence within intron one that probably defines a cis-acting regulator of the sex-specific alternative splicing event.     

Mechanism of activation of the Drosophila EGF Receptor by the TGFalpha ligand Gurken during oogenesis.

We have analyzed the mechanism of activation of the Epidermal growth factor receptor (Egfr) by the transforming growth factor (TGF) alpha-like molecule, Gurken (Grk). Grk is expressed in the oocyte and activates the Egfr in the surrounding follicle cells during oogenesis. We show that expression of either a membrane bound form of Grk (mbGrk), or a secreted form of Grk (secGrk), in either the follicle cells or in the germline, activates the Egfr. In tissue culture cells, both forms can bind to the Egfr; however, only the soluble form can trigger Egfr signaling, which is consistent with the observed cleavage of Grk in vivo. We find that the two transmembrane proteins Star and Brho potentiate the activity of mbGrk. These two proteins collaborate to promote an activating proteolytic cleavage and release of Grk. After cleavage, the extracellular domain of Grk is secreted from the oocyte to activate the Egfr in the follicular epithelium.     

Convergent intron loss of MRP1 in Drosophila and mosquito species

Previous study revealed that the MRP1 gene ortholog DMRP1/CG6214 of Drosophila melanogaster contains 12 exons in the coding region. In the current study, the genes of DMRP1/CG6214 from D. melanogaster and Drosophila virilis were compared, and the result indicated that D. virilis had an extra intron located in exon 2, implying that intron loss or gain might have occurred at this locus. To track the evolution of the extra intron (Intron Z), orthologous nucleotide sequences of 37 arthropod species were cloned or annotated. Based on phylogenetic analysis, we found that Intron Z should present in the common ancestor of arthropod species, more than 420 Ma. In addition, we found that Sophophora subgenus species and mosquito (Culex pipiens) lost Intron Z independently, showing evolutionary convergence.     

Structural and functional conservation of the Drosophila doublesex splicing enhancer repeat elements.

We have compared the RNA sequences and secondary structures of the Drosophila melanogaster and Drosophila virilis doublesex (dsx) splicing enhancers. The sequences of the two splicing enhancers are highly divergent except for the presence of nearly identical 13-nt repeat elements (six in D. melanogaster and four in D. virilis) and a stretch of nucleotides at the 5' and 3' ends of the enhancers. In vitro RNA structure probing of the two enhancers revealed that the 13-nt repeats are predominantly single-stranded. Thus, both the primary sequences and single-stranded nature of the repeats are conserved between the two species. The significance of the primary sequence conservation was demonstrated by showing that the two enhancers are functionally interchangeable in Tra-/Tra2-dependent in vitro splicing. In addition, inhibition of splicing enhancer activity by antisense oligonucleotides complementary to the repeats demonstrated the importance of the conserved single-stranded structure of the repeats. In vitro binding studies revealed that Tra2 interacts with each of the D. melanogaster repeat elements, except for repeat 2, with affinities that are indistinguishable, whereas Tra binds nonspecifically to the enhancer. Taken together, these observations indicate that the organization of sequences within the dsx splicing enhancers of D. melanogaster and D. virilis results in a structure in which each of the repeat elements is single-stranded and therefore accessible for specific recognition by the RNA-binding domain of Tra2.     

A 9.6 kb intervening sequence in D. virilis rDNA, and sequence homology in rDNA interruptions of diverse species of Drosophila and other diptera.

A large proportion of the 28S ribosomal RNA genes in Drosophila virilis are interrupted by a DNA sequence 9.6 kilobase pairs long. As regards both its presence and its position in the 28S gene (about two thirds of the way in), the D. virilis rDNA intervening sequence is similar to that found in D. melanogaster rDNA, but lengths differ markedly between the two species. Degrees of nucleotide sequence homology have been detected bewteen rDNA interruptions of the two species. This homology extends to putative rDNA intervening sequences in diverse higher diptera (other Drosophila species, the house fly and the flesh fly), but hybridization of cloned D. melanogaster and D. virilis rDNA interruption segments to DNA of several lower diptera has been negative. As is the case with melanogaster rDNA interruptions, segments of the virilis rDNA intervening sequence hybridize with non-rDNA components of the virilis genome, and interspecific homology may involve these non-rDNA sequences as well as rDNA interruptions. There is, however, evidence from buoyant density fractionation of DNA that the distributions of interruption-related sequences are distinct in D. melanogaster and D. virilis genomes. Moreover, thermal denaturation studies have indicated differing extents of homology between hybridizable sequences in D. virilis DNA and different segments of the D. melanogaster rDNA intervening sequence. We infer from our studies that rDNA intervening sequences are prevalent among higher diptera; that in the course of the evolution of these organisms, elements of the intervening sequences have been moderately to highly conserved; and that this conservation extends in at least two distantly related species of Drosophila to similar sequences found elsewhere in the genomes.