Evolution of single and double Wolbachia symbioses during speciation in the Drosophila simulans complex.

Maternally inherited bacteria of the genus Wolbachia are responsible for the early death of embryos in crosses between uninfected females and infected males in several insect species. This phenomenon, known as cytoplasmic incompatibility, also occurs between strains infected by different symbionts in some species, including Drosophila simulans. Wolbachia was found in two species closely related to D. simulans, Drosophila mauritiana, and Drosophila sechellia, and shown to cause incompatibility in the latter species but not in D. mauritiana. Comparison of bacterial and mtDNA history clarifies the origins of bacterial and incompatibility polymorphisms in D. simulans. Infection in D. mauritiana is probably the result of introgression of an infected D. simulans cytoplasm. Some D. simulans and D. sechellia cytoplasmic lineages harbor two bacteria as a consequence of a double infection which probably occurred in a common ancestor. The descendant symbionts in each species are associated with similar incompatibility relationships, which suggests that little variation of incompatibility types has occurred within maternal lineages beyond that related to the density of symbionts in their hosts.

Functional domain analysis of glass, a zinc-finger-containing transcription factor in Drosophila.

The glass gene is required for proper photo-receptor differentiation during development of the Drosophila eye glass codes for a DNA-binding protein containing five zinc fingers that we show is a transcriptional activator. A comparison of the sequences of the glass genes from two species of Drosophila and a detailed functional domain analysis of the Drosophila melanogaster glass gene reveal that both the DNA-binding domain and the transcriptional-activation domain are highly conserved between the two species. Analysis of the DNA-binding domain of glass indicates that the three carboxyl-terminal zinc fingers alone are necessary and sufficient for DNA binding. We also show that a deletion mutant of glass containing only the DNA-binding domain can behave in a dominant-negative manner both in vivo and in a cell culture assay that measures transcriptional activation.

The Ste locus, a component of the parasitic cry-Ste system of Drosophila melanogaster, encodes a protein that forms crystals in primary spermatocytes and mimics properties of the beta subunit of casein kinase 2.

Males of Drosophila melanogaster lacking the Y chromosome-linked crystal locus show multiple meiotic alterations including chromosome disorganization and prominent crystal formation in primary spermatocytes. These alterations are due to the derepression of the X chromosome-linked Stellate sequences. To understand how the derepression of the Stellate elements gives rise to these abnormalities, we have expressed the protein encoded by the Stellate sequences in bacteria and produced an antibody against the fusion protein. Immunostaining of crystal- testes has clearly shown that the Stellate protein is a major component of the crystals. Moreover, in vitro experiments have shown that this protein can interact with the catalytic alpha subunit of casein kinase 2 enzyme, altering its activity.

Two Drosophila nervous system antigens, Nervana 1 and 2, are homologous to the beta subunit of Na+,K(+)-ATPase.

A nervous system-specific glycoprotein antigen from adult Drosophila heads, designated Nervana (Nrv), has been purified on the basis of reactivity of its carbohydrate epitope(s) with anti-horseradish peroxidase (HRP) antibodies that are specific markers for Drosophila neurons. Anti-Nrv monoclonal antibodies (mAbs), specific for the protein moiety of Nrv, were used to screen a Drosophila embryo cDNA expression library. Three cDNA clones (designated Nrv1, Nrv2.1, and Nrv2.2) were isolated that code for proteins recognized by anti-Nrv mAbs on Western blots. DNA sequencing and Southern blot analyses established that the cDNA clones are derived from two different genes. In situ hybridization to Drosophila polytene chromosomes showed that the cDNA clones map to the third chromosome near 92C-D. Nrv1 and Nrv2.1/2.2 have open reading frames of 309 and 322/323 amino acids, respectively, and they are 43.4% identical at the amino acid level. The proteins deduced from these clones exhibit significant homology in both primary sequence and predicted topology to the beta subunit of Na+,K(+)-ATPase. Immunoaffinity-purified Nrv is associated with a protein (M(r) 100,000) recognized on Western blots by anti-ATPase alpha-subunit mAb. Our results suggest that the Drosophila nervous system-specific antigens Nrv1 and -2 are neuronal forms of the beta subunit of Na+,K(+)-ATPase.

Drosophila 5-HT2 serotonin receptor: coexpression with fushi-tarazu during segmentation.

Serotonin, first described as a neurotransmitter in invertebrates, has been investigated mostly for its functions in the mature central nervous system of higher vertebrates. Serotonin receptor diversity has been described in the mammalian brain and in insects. We report the isolation of a cDNA coding for a Drosophila melanogaster serotonin receptor that displays a sequence, a gene organization, and pharmacological properties typical of the mammalian 5-HT2 serotonin receptor subtype. Its mRNA can be detected in the adult fly; moreover, a high level of expression occurs at 3 hr of Drosophila embryogenesis. This early embryonic expression is surprisingly organized in a seven-stripe pattern that appears at the cellular blastoderm stage. In addition, this pattern is in phase with that of the even-parasegment-expressed pair-rule gene fushi-tarazu and is similarly modified by mutations affecting segmentation genes. Simultaneously with this pair-rule expression, the complete machinery of serotonin synthesis is present and leads to a peak of ligand concomitant with a peak of 5-HT2-specific receptor sites in blastoderm embryos.

Misexpression of the white (w) gene triggers male-male courtship in Drosophila.

We report here that the general ectopic expression of a tryptophan/guanine transmembrane transporter gene, white (w), induces male-male courtship in Drosophila. Activation of a hsp-70/miniwhite (mini-w) transgene in mature males results in a marked change in their sexual behavior such that they begin to vigorously court other mature males. In transformant populations containing equal numbers of both sexes, most males participate, thus forming male-male courtship chains, circles, and lariats. Mutations that ablate the w transgene function also abolish this inducible behavior. Female sexual behavior does not appear to be altered by ectopic w expression. By contrast, when exposed to an active homosexual courtship environment, non-transformant males alter their behavior and actively participate in the male-male chaining. These findings demonstrate that, in Drosophila, both genetic and environmental factors play a role in male sexual behavior.

Redistribution of synaptic vesicles and their proteins in temperature-sensitive shibire(ts1) mutant Drosophila.

From an extract of Drosophila melanogaster head homogenates, a membrane fraction can be isolated that has the same sedimentation properties as vertebrate synaptic vesicles and contains Drosophila synaptotagmin. The fraction disappears from homogenates of temperature-sensitive (ts) mutant shibire(ts1) (shi(ts1)) flies paralyzed by exposure to non-permissive temperatures, and reappears on return to permissive temperatures. Since reversible, temperature-dependent depletion of synaptic vesicles is known to occur in shibire(ts1) flies, we conclude that the fraction we have identified contains synaptic vesicles. We have examined the fate of synaptic vesicle membrane proteins in shibire flies at nonpermissive temperatures and found that all of these vesicle antigens are transferred to rapidly sedimenting membranes and codistribute with a plasma membrane marker by both glycerol velocity and metrizamide density sedimentation and by confocal microscopy. Three criteria were used to establish that other neuron-specific antigens--neuronal synaptobrevin and cysteine-string proteins--are legitimate components of synaptic vesicles: cosedimentation with Drosophila synaptotagmin, immunoadsorption, and disappearance of these antigens from the vesicle fractions in paralyzed shibire flies.

Transposable elements are stable structural components of Drosophila melanogaster heterochromatin.

We determined the distribution of 11 different transposable elements on Drosophila melanogaster mitotic chromosomes by using high-resolution fluorescent in situ hybridization (FISH) coupled with charge-coupled device camera analysis. Nine of these transposable elements (copia, gypsy, mdg-1, blood, Doc, I, F, G, and Bari-1) are preferentially clustered into one or more discrete heterochromatic regions in chromosomes of the Oregon-R laboratory stock. Moreover, FISH analysis of geographically distant strains revealed that the locations of these heterochromatic transposable element clusters are highly conserved. The P and hobo elements, which are likely to have invaded the D. melanogaster genome at the beginning of this century, are absent from Oregon-R heterochromatin but clearly exhibit heterochromatic clusters in certain natural populations. Together these data indicate that transposable elements are major structural components of Drosophila heterochromatin, and they change the current views on the role of transposable elements in host genome evolution.

Expression cloning of dSR-CI, a class C macrophage-specific scavenger receptor from Drosophila melanogaster.

Mammalian class A macrophage-specific scavenger receptors (SR-A) exhibit unusually broad binding specificity for a wide variety of polyanionic ligands. The properties of these receptors suggest that they may be involved in atherosclerosis and host defense. We have previously observed a similar receptor activity in Drosophila melanogaster embryonic macrophages and in the Drosophila macrophage-like Schneider L2 cell line. Expression cloning was used to isolate from L2 cells a cDNA that encodes a third class (class C) of scavenger receptor, Drosophila SR-CI (dSR-CI). dSR-CI expression was restricted to macrophages/hemocytes during embryonic development. When expressed in mammalian cells, dSR-CI exhibited high affinity and saturable binding of 125I-labeled acetylated low density lipoprotein and mediated its chloroquine-dependent, presumably lysosomal, degradation. Although the broad polyanionic ligand-binding specificity of dSR-CI was similar to that of SR-A, their predicted protein sequences are not similar. dSR-CI is a 609-residue type I integral membrane protein containing several well-known sequence motifs, including two complement control protein (CCP) domains and somatomedin B, MAM, and mucin-like domains. Macrophage scavenger receptors apparently mediate important, well-conserved functions and may be pattern-recognition receptors that arose early in the evolution of host-defense mechanisms. Genetic and physiologic analysis of dSR-CI function in Drosophila should provide further insights into the roles played by scavenger receptors in host defense and development.

Sexual isolation in Drosophila melanogaster: a possible case of incipient speciation.

It is generally believed that Drosophila melanogaster has no closely related species with which it can produce the viable and fertile hybrids that are essential for the genetic analysis of speciation. Following the recent report of molecular differentiation between a Zimbabwe, Africa, population and two United States populations, we provide evidence that strong sexual isolation exists between the D. melanogaster population in Zimbabwe and populations of other continents. In the presence of males of their own kind, females from most isofemale lines of Zimbabwe would not mate with males from elsewhere; the reciprocal mating is also significantly reduced, but to a lesser degree. The genes for sexual behaviors are apparently polymorphic in Zimbabwe and postmating reproductive isolation between this and other populations has not yet evolved. Whole chromosome substitutions indicate significant genetic contributions to male mating success by both major autosomes, whereas the X chromosome effect is too weak to measure. In addition, the relative mating success between hybrid and pure line males supports the interpretation of strong female choice. These observations suggest that we are seeing the early stages of speciation in this group and that it is driven by sexual selection. The genetic and molecular tractability of D. melanogaster offers great promise for the detailed analysis of this apparent case of incipient speciation.

Acute Synaptic Activity Causes Differential miRNA Expression in The Drosophila melanogaster Larval Central Nervous System

The primary goal of this thesis was to determine if spaced synaptic stimulation induced the differential expression of microRNAs (miRNAs) in the Drosophila melanogaster central nervous system (CNS). Prior to attaining this goal, we needed to identify and validate a spaced stimulation paradigm that could induce the formation of new synaptic growth at a model synapse, the larval neuromuscular junction (NMJ). Both Channelrhodopsin- and high potassium-based stimulation paradigms adapted from (Ataman, et al. 2008) were tested. Once validation of these paradigms was complete, we sought to characterize the miRNA expression profile of the larval CNS by miRNA array. Following attainment of these data, we used quantitative real-time PCR (RT-qPCR) to determine if acute synaptic stimulation caused the differential expression of neuronal miRNAs. We found that upon high potassium spaced training in a wild type (Canton S) genotype, 5 miRNAs showed significant differential expression when normalized to a validated reference gene, the U1 snRNA. Moreover, absolute quantification of our RT-qPCR study implicated one miRNA: miR-958 as being significantly regulated by activity. Investigation into potential targets for miR-958 revealed it to be a potential regular of Dlar, a protein tyrosine phosphatase implicated in synapse development. This investigation provides the foundation to directly test our underlying hypothesis that, following spaced training, differential expression of miRNAs alters the translation of proteins required to induce and maintain these structural changes at the synapse.

Plasma membrane dynamics in the Drosophila embryo

ABSTRACT Convergent extension is a highly conserved process among mammals, in which the tissue narrows in one axis, and extends across another. Tissue elongation is directed by the regulation of cell interface behaviors, which guides cell intercalation and rosette formation. Rosette formation occurs through the contraction of vertically oriented cell interfaces, and the subsequent elongation of new horizontal interfaces. It has been shown that actomyosin-generated tension functions to direct rosette formation. In this thesis, I have tested the function of regulators of F-actin networks, as well as endocytic and exocytic mechanisms, to identify new components that control interface behaviors and cell shape. I have performed a screen of F-actin regulators and nucleators, and pinpointed the specific actin nucleator dPod-1 as a candidate protein that is localized to vertical interfaces during tissue elongation. Furthermore, I have probed the function of endocytosis using the Shibire mutation, and demonstrated that endocytosis is required for vertical interface shrinking. Finally, I have used mutations in components of the Exocyst Complex and the associated protein RalA to inhibit exocytic mechanisms, in order to address their function in directing cell and tissue morphologies.

A role for Dynamin-dependent endocytosis during Drosophila gastrulation

Gastrulation, a process conserved among many higher organisms, is the directed migration of cells into layers that will establish various tissues targeted to become anatomical structures. This process is accomplished through another conserved morphogenetic event, known as cell intercalation. Early in development, this movement of cells within an organized tissue leads to unique cellular arrangements where neighboring cells contract their shared interfaces in order to meet at a shared vertex. In this thesis, I present work that demonstrates a requirement for Dynamin-dependent endocytosis during these contraction events. Using quantitative analysis, I have identified varied cell behaviors during experiments which knockdown the function of dynamin. In addition, I demonstrate the existence of an antagonistic relationship between Dynamin and the Myosin II motor protein. Lastly, localization and functional studies I performed for this work suggest a role for Sorting Nexin proteins during plasma membrane reorganization required for Dynamin-dependent endocytosis.

Activity-Regulated microRNAs: Modulators of Synaptic Growth at the Drosophila Neuromuscular Junction

It is well established that long-term changes in synaptic structure and function are mediated by rapid activity-dependent gene transcription and new protein synthesis. A growing body of evidence supports the involvement of the microRNA (miRNA) pathway in these processes. We have used the Drosophila neuromuscular junction (NMJ) as a model synapse to characterize activity-regulated miRNAs and their important mRNA targets. Here, we have identified five neuronal miRNAs (miRs-1, -8, -289, -314, and -958) that are significantly downregulated in response to neuronal activity. Furthermore we have discovered that neuronal misexpression of three of these miRNAs (miR-8, -289, and -958) is capable of suppressing new synaptic growth in response to activity suggesting that these miRNAs control the translation of biologically relevant target mRNAs. Putative targets of the activity-regulated miRNAs-8 and -289 are significantly enriched in clusters mapping to functional processes including axon development, pathfinding, and axon growth. We demonstrate that activity-regulated miR-8 regulates the 3'UTR of wingless, a presynaptic regulatory protein involved in the process of activity-dependent axon terminal growth. Additionally, we show that the 3'UTR of the protein tyrosine phosophatase leukocyte antengen related (lar), a protein required for axon guidance and synaptic growth, is regulated by activity-regulated miRNAs-8, -289, and -958 in vitro. Both wg and lar were identified as relevant putative targets for co-regulation based through our functional cluster analysis. One putative target of miR-289 is the Ca2+/calmodulin-dependent protein kinase II (CamKII). While CamKII is not predicted as a target for co-regulation by multiple activity-regulated miRNAs we identified it as an especially pertinent target for analysis in our system for two reasons. First, CamKII has an extremely well characterized role in postsynaptic plasticity, but its presynaptic role is less well characterized and bears further analysis. Second, local translation of CamKII mRNA is regulated in part by the miRNA pathway in an activity-dependent manner in dendrites. We find that the CamKII 3'UTR is regulated by miR-289 in-vitro and this regulation is alleviated by mutating the `seed region' of the miR-289 binding site within the CamKII 3'UTR. Furthermore, we demonstrate a requirement for local translation of CamKII in motoneurons in the process of activity-regulated axon terminal growth.

The white gene and locomotor recovery from anoxia in Drosophila melanogaster

Locomotor recovery from anoxia is complicated and little is known about the molecular and cellular mechanisms regulating anoxic recovery in Drosophila. For this thesis I established a protocol for large-scale analysis of locomotor activity in adult flies with exposure to a transient anoxia. Using this protocol I observed that wild-type Canton-S flies recovered faster and more consistently from anoxia than the white-eyed mutant w1118, which carries a null allele of w1118 in an isogenic genetic background. Both Canton-S and w1118 are commonly used controls in the Drosophila community. Genetic analysis including serial backcrossing, RNAi knockdown, w+ duplication to Y chromosome as well as gene mutation revealed a strong association between the white gene and the timing of locomotor recovery. I also found that the locomotor recovery phenotype is independent of white-associated eye pigmentation, that heterozygous w+ allele was haplo-insufficient to induce fast and consistent locomotor recovery from anoxia in female flies, and that mini-white is insufficient to promote fast and consistent locomotor recovery. Moreover, locomotor recovery was delayed in flies with RNAi knockdown of white in subsets of serotonin neurons in the central nervous system. I further demonstrated that mutations of phosphodiesterase genes (PDE) displayed wild-type-like fast and consistent locomotor recovery, and that locomotor recovery was light-sensitive in the night in w1118. The delayed locomotor recovery and the light sensitivity were eliminated in PDE mutants that were dual-specific or cyclic guanosine monophosphate (cGMP)-specific. Up-regulation of cGMP using multiple approaches including PDE mutation, sildenafil feeding or specific expression of an atypical soluble guanylyl cyclase (Gyc88E) was sufficient to suppress w-RNAi induced delay of locomotor recovery. Taken together, these data strongly support the hypothesis that White transports cGMP and promotes fast and consistent locomotor recovery from anoxia.