Organ-specific patterns of gene expression in the reproductive tract of Drosophila are regulated by the sex-determination genes

The sex-determination genes of Drosophila act to repress the developmental pathway for the internal somatic reproductive organs of the opposite sex. By misregulating this pathway during preadult development, the organ-specific expression pattern of the glucose dehydrogenase gene (Gld) in the reproductive tract of adult flies has been changed without a concomitant sexual transformation of the reproductive organs. Misregulation of the tra, tra-2, and dsx genes leads to very similar patterns of ectopic expression of Gld. The induced ectopic patterns of Gld expression at the adult stage occur in a small subset of organs which all normally express the Gld gene during their morphogenesis. These ectopic patterns are irrevocably set during late larval-early pupal development. The normal pattern of Gld expression in several other Drosophila species is quite similar to the ectopic patterns which we have generated in D. melanogaster, suggesting that the interspecific variation in Gld expression may result from variation in the expression of the sex-determination genes.     

Coevolution of the glucose dehydrogenase gene and the ejaculatory duct in the genus Drosophila

The glucose dehydrogenase gene (Gld) in Drosophila melanogaster exhibits a unique spatial and temporal pattern of expression. GLD expression switches from a non-sex-limited state at the pupal stage to a male-limited state at the adult stage. At the adult stage, the enzyme is restricted to the ejaculatory duct. Within the genus Drosophila, the ejaculatory duct has undergone a simple morphological divergence. In order to determine whether correlated changes in GLD expression had occurred, GLD activity during the pupal and adult stages was determined for several Drosophila species. It was found that virtually all of the species exhibit pupal GLD activity, whereas only those species with an expanded ejaculatory duct express male-limited GLD. The results of interspecific genital imaginal disc transplantation experiments indicate that the expanded morphology and GLD expression do not require any species- or sex-specific diffusible factors. An apparent regulatory polymorphism exists within the D. takahashii species with respect to male-limited GLD expression.      

Developmental Expression of the Glucose Dehydrogenase Gene in Drosophila Melanogaster

The Gld gene of Drosophila melanogaster is transiently expressed during every stage of development. The temporal pattern of Gld expression is highly correlated with that of ecdysteroids. Exogeneous treatment of third instar larvae with 20-hydroxyecdysone induces the accumulation of Gld mRNA in the hypoderm and anterior spiracular gland cells. During metamorphosis Gld is expressed in a variety of tissues derived from the ectoderm. In the developing reproductive tract, Gld mRNA accumulates in the female spermathecae and oviduct and in the male ejaculatory duct and ejaculatory bulb. These four organs are derived from closely related cell lineages in the genital imaginal disc. Since the expression of Gld is not required for the development of these reproductive structures, this spatial pattern of expression is most likely a fortuitous consequence of a shared regulatory factor in this cell lineage. At the adult stage a high level of the Gld mRNA is only observed in the male ejaculatory duct.     

Correlated evolution of the cis-acting regulatory elements and developmental expression of the Drosophila Gld gene in seven species from the subgroup melanogaster

The tissue-specific expression patterns of glucose dehydrogenase (GLD) exhibit a high degree of inter specific variation in the adult reproductive tract among the species in the genus Drosophila. We chose to focus on the evolution of GLD expression and the evolution of the Gld promoter in seven closely related species in the mela-nogaster subgroup as a means of elucidating the relationship of changes in cis-acting regulatory elements in the Gld promoter region with changes in tissue-specific expression. Although little variation in tissue-specific patterns of GLD was found in nonreproductive tissues during development, a surprisingly high level of variation was observed in the expression of GLD in both developing and ma-ture reproductive organs. In some cases this variation is correlated with changes in sequence elements in the Gld promoter which were previously shown to direct tissue-specific expression in the reproductive tract. In particular D. teissieri adult males do not express GLD in their ejaculatory ducts, atypical of the melanogaster subgroup species. The Gld promoter region of D. teissieri specifically lacks all three of the TTAGA regulatory elements present in D. melanogaster. The TTAGA elements were previously shown to direct reporter gene expression to the ejaculatory duct. Together these data suggest the absence or presence of the TTAGA elements may be responsible for variation in the absence or presence of GLD in the ejaculatory duct among species. © 1994 Wiley-Liss, Inc.     

Complex organization of promoter and enhancer elements regulate the tissue- and developmental stage-specific expression of the Drosophila melanogaster Gld gene

The Drosophila melanogaster Gld gene has multiple and diverse developmental and physiological functions. We report herein that interactions among proximal promoter elements and a cluster of intronically located enhancers and silencers specify the complex regulation of Gld that underlies its diverse functions. Gld expression in nonreproductive tissues is largely determined by proximal promoter elements with the exception of the embryonic labium where Gld is activated by an enhancer within the first intron. A nuclear protein, GPAL, has been identified that binds the Gpal elements in the proximal promoter region. Regulation of Gld in the reproductive organs is particularly complex, involving interactions among the Gpal proximal promoter elements, a unique TATA box, three distinct enhancer types, and one or more silencer elements. The three somatic reproductive organ enhancers each activate expression in male and female pairs of reproductive organs. One of these pairs, the male ejaculatory duct and female oviduct, are known to be developmentally homologous. We report evidence that the other two pairs of organs are developmentally homologous as well. A comprehensive model to explain the full developmental regulation of Gld and its evolution is presented.     

Evolution of the glucose dehydrogenase gene in Drosophila

The glucose dehydrogenase genes (Gld) of Drosophila melanogaster, of D. pseudoobscura, and of D. virilis have been isolated and compared with each other in order to identify conserved and divergent aspects of their structure and expression. The exon/intron structure of Gld is conserved. The Gld mRNAs are similar, with a range of 2.6-2.8 kb among the three species. All three species exhibit peaks of Gld expression during every major developmental stage, although considerable variation in the precise timing of these peaks exists between species. Interspecific gene transfer experiments demonstrate that the regulation and function of the D. pseudoobscura Gld is similar enough to the homologous gene in D. melanogaster to substitute for its essential role in the eclosion process. Comparison of the putative promoter sequences has identified both shared and divergent sequence elements which are likely responsible, respectively, for the conserved and divergent patterns of expression observed. The entire coding sequences of the pseudoobscura and melanogaster Gld genes are presented and shown to encode a 612-amino-acid pre-protein. The inferred amino acid sequences are 92% conserved between the two species. In general the intronic regions of Gld are unusually well conserved.     

High level of divergence of male-reproductive-tract proteins, between Drosophila melanogaster and its sibling species, D. simulans

We compared male-reproductive-tract polypeptides of Drosophila melanogaster and D. simulans by using two-dimensional gel electrophoresis. Approximately 64% of male-reproductive-tract polypeptides were identical between two randomly chosen isofemale lines from these two species, compared with 83% identity for third-instar imaginal wing-disc polypeptides. Qualitatively similar differences were found between reproductive tracts and imaginal discs when D. sechellia was compared with D. melanogaster and with D. simulans. When genic polymorphism was taken into account, approximately 10% of male- reproductive-tract polypeptides were apparently fixed for different alleles between D. melanogaster and D. simulans; this proportion is the same as that found for soluble enzymes by one-dimensional gel electrophoresis. Strikingly, approximately 20% of male-reproductive- tract polypeptides of either D. melanogaster or D. simulans had no detectable homologue in the other species. We propose that proteins of the Drosophila male reproductive tract may have diverged more extensively between species than have other types of proteins and that much of this divergence may involve large changes in levels of polypeptide expression.      

The Drosophila melanogaster seminal fluid protein Acp62F is a protease inhibitor that is toxic upon ectopic expression.

Drosophila melanogaster seminal fluid proteins stimulate sperm storage and egg laying in the mated female but also cause a reduction in her life span. We report here that of eight Drosophila seminal fluid proteins (Acps) and one non-Acp tested, only Acp62F is toxic when ectopically expressed. Toxicity to preadult male or female Drosophila occurs upon one exposure, whereas multiple exposures are needed for toxicity to adult female flies. Of the Acp62F received by females during mating, approximately 10% enters the circulatory system while approximately 90% remains in the reproductive tract. We show that in the reproductive tract, Acp62F localizes to the lumen of the uterus and the female's sperm storage organs. Analysis of Acp62F's sequence, and biochemical assays, reveals that it encodes a trypsin inhibitor with sequence and structural similarities to extracellular serine protease inhibitors from the nematode Ascaris. In light of previous results demonstrating entry of Acp62F into the mated female's hemolymph, we propose that Acp62F is a candidate for a molecule to contribute to the Acp-dependent decrease in female life span. We propose that Acp62F's protease inhibitor activity exerts positive protective functions in the mated female's reproductive tract but that entry of a small amount of this protein into the female's hemolymph could contribute to the cost of mating.     

A Rehabilitation of the Genetic Map of the 84b-D Region in DROSOPHILA MELANOGASTER

A reanalysis of the 84B3 to 84D3,5 region of the polytene chromosomes of Drosophila melanogaster has led to the identification and localization of 16 genes. These genes include 11 vital loci, four genes exhibiting nonlethal visible mutant phenotypes and one gene encoding a nonessential enzyme. The identity of the gene products of two of the vital genes has been determined to be alpha-tubulin and glucose dehydrogenase (Gld). Three newly identified genes, sticking (stk), half out (hat) and trapped (ted), as well as Gld are required for eclosion. Among the nonessential genes are roughened eye (roe) and ruffed eye (rue), which affect eye texture. The roe phenotype is greatly enhanced by deletions that simultaneously remove roe and an unidentified locus in 84E. Mutations in another nonessential gene, rotund (rn), are characterized by pattern deletions of most adult appendages.     

Regulatory elements involved in Drosophila Adh gene expression are conserved in divergent species and separate elements mediate expression in different tissues.

Alcohol dehydrogenase (ADH) is expressed in a complex temporal and spatial pattern from tandem promoters (proximal and distal) in Drosophila melanogaster, and from two closely linked genes (Adh-1 and Adh-2) in D. mulleri. The expression patterns of Adh-1 and the proximal promoter, and Adh-2 and the distal promoter are similar, but not identical. We show that the mulleri Adh genes are appropriately expressed when introduced into the melanogaster genome, indicating that the cis- and trans-acting elements which regulate the corresponding promoters are functionally equivalent in the two species. By analyzing the expression of in vitro generated mutants of the mulleri Adh locus, we define at least three regulatory regions of the mulleri Adh genes and show that different control elements mediate the expression of Adh in different tissues.     

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.     

Characterization of defects in adult germline development and oogenesis of sterile and rescued female hybrids in crosses between Drosophila simulans and Drosophila melanogaster

Crosses between Drosophila melanogaster and D. simulans normally result in progeny that are either inviable or sterile. Recent discovery of strains that rescue these inviability and sterility phenotypes has made it possible to study the developmental basis of reproductive isolation between these two species in greater detail. By producing both rescued and unrescued hybrids and examining the protein product staining patterns of genes known to be involved in early germline development and gametogenesis, we have found that in crosses between D. simulans and D. melanogaster, hybrid female sterility results from the improper control of primordial germline proliferation, germline stem cell maintenance, and cystoblast formation and differentiation during early oogenesis. Rescued hybrid females are fertile, yet they generally have lower amounts of adult germline from the outset and show a premature degeneration of adult germline cells with age. In addition, older rescued hybrid females also exhibit mutant egg phenotypes associated with defects in dorso-ventral patterning which may result from the improper partitioning of cytoplasmic factors during early oogenesis that could stem from the early defect. Although a variety of germline and oogenic defects are described for the hybrid females, all of them can potentially result from the same underlying primary defect. Hybrid males from these same crosses, on the other hand, have no detectable germline in adult reproductive tissues, even when hybrid sterility rescue strains are used, indicating that male sterility and female sterility stem from distinctly different developmental defects.     

Twin intromittent organs of Drosophila for traumatic insemination

In several animals, male genitalia create insemination wounds in areas outside the genital orifice of females. I report that such traumatic insemination (TI) occurs in the Drosophila bipectinata complex (Diptera: Drosophilidae) and illustrate a previously unknown evolutionary pathway for this behaviour. Flash fixation of mating pairs revealed the dual function of the paired claw-like basal processes, previously misidentified as a bifid aedeagus: (i) penetration of the female body wall near the genital orifice and (ii) sperm transfer into the genital tract through the wounds. Basal processes in closely related species (Drosophila ananassae and Drosophila pallidosa) also wounded females but did not transfer sperm; this represents a transitional state to TI as observed in the bipectinata complex. Copulatory wounding is suggested to occur in other allied species of the Drosophila melanogaster species group, including D. melanogaster. Ubiquitous sexual conflicts over mating may have led to the evolution of novel intromittent organs for insemination.     

The YYRR box: a conserved dipyrimidine-dipurine sequence element in Drosophila and other eukaryotes.

We have discovered a novel DNA sequence element in Drosophila which is based upon a CTGA tandem repeat. This element has been named the YYRR box to emphasize its dipyrimidine-dipurine nature which is predicted to have unusual structural features. Southern hybridization analysis of genomic DNA indicates the presence of 25-30 copies of the YYRR box in each of three Drosophila species (melanogaster, pseudoobscura, and virilis) and conservation of genomic location within species. Similar analysis of human and rat DNA indicates the presence of YYRR related sequences in mammals as well. YYRR boxes have been localized to two genetic loci in Drosophila: Gld and a gene tentative identified as ted. These two genes exhibit correlated patterns of developmental expression and an identical mutant phenotype. Sequence analysis of the Gld YYRR box in three Drosophila species revealed a high degree of conservation despite its intronic location.     

Light wavelength dependency of mating activity in the Drosophila melanogaster species subgroup

The action spectra of mating activity among the six species of the Drosophila melanogaster species subgroup were compared to understand how light wavelength affects mating activity. The species fell into three groups with respect to the action spectrum of mating activity. We chose one representative species from each of the three types for detailed study: D. melanogaster, D. sechellia and D. yakuba. The mating activities were investigated under three different light intensities of three monochromatic lights stimulus. Each species showed a unique spectral and intensity response. To know the evolutionary meaning of the light wavelength dependency of mating activity, we superimposed the type of action spectrum of mating activity in these six species on a cladogram. Mating inhibition under UV was conserved in evolution among these species. Furthermore we clarified that D. melanogaster showed low mating activity under UV because males courted less under UV.     

The Use of Yolk Protein Variations in Drosophila Species to Analyse the Control of Vitellogenesis

The yolk proteins of many insects, including Drosophila , are synthesised in the fat body of adult females and are transported through the haemolymph to be accumulated in the oocytes. We have used differences in the size and number of yolk polypeptides in different species of Drosophila to investigate the role of the ovary and of juvenile hormone in vitellogenesis.
The yolk proteins of eight species of Drosophila were compared with those of Drosophila melanogaster . Only Drosophila simulans had three yolk polypeptides of similar molecular weight to the three polypeptides in D. melanogaster and gave a high degree of cross reactivity with antibody raised against the yolk proteins of D. melanogaster . All other species had one to three bands on a sodium dodecyl sulphate gel representing the yolk polypeptides; they are between 44,000 and 49,500 daltons in molecular weight, showing weak cross reactivity with anti- D. melanogaster yolk antibody. Interspecies ovary transplants established that males of D. arizonensis and D.pseudoobscura which supported vitellogenesis of D. melanogaster ovaries, did so by permitting the implanted ovaries to synthesise their own yolk proteins. The synthetic juvenile hormone, ZR515, was unable to induce ovaries, which failed to develop in other species of males, to undergo vitellogenesis. In females, however, ZR515 was able to induce uptake of the yolk proteins of some of the species into the D. melanogaster donor ovaries, which had failed to develop in the absence of hormone. These interspecies differences in the yolk proteins have therefore been used to investigate the control of vitellogenesis and the role of juvenile hormone in this process in Drosophila .