Blood-feeding insects, including the malaria mosquito Anopheles gambiae, use highly specialized and sensitive olfactory systems to locate their hosts. This is accomplished by detecting and following plumes of volatile host emissions, which include carbon dioxide (CO2). CO2 is sensed by a population of olfactory sensory neurons in the maxillary palps of mosquitoes and in the antennae of the more genetically tractable fruitfly, Drosophila melanogaster. The molecular identity of the chemosensory CO2 receptor, however, remains unknown. Here we report that CO2-responsive neurons in Drosophila co-express a pair of chemosensory receptors, Gr21a and Gr63a, at both larval and adult life stages. We identify mosquito homologues of Gr21a and Gr63a, GPRGR22 and GPRGR24, and show that these are co-expressed in A. gambiae maxillary palps. We show that Gr21a and Gr63a together are sufficient for olfactory CO2-chemosensation in Drosophila. Ectopic expression of Gr21a and Gr63a together confers CO2 sensitivity on CO2-insensitive olfactory neurons, but neither gustatory receptor alone has this function. Mutant flies lacking Gr63a lose both electrophysiological and behavioural responses to CO2. Knowledge of the molecular identity of the insect olfactory CO2 receptors may spur the development of novel mosquito control strategies designed to take advantage of this unique and critical olfactory pathway. This in turn could bolster the worldwide fight against malaria and other insect-borne diseases.

Carbon dioxide (CO 2 ) elicits different olfactory behaviors across species. In Drosophila, neurons that detect CO 2 are located in the antenna, form connections in a ventral glomerulus in the antennal lobe, and mediate avoidance. By contrast, in the mosquito these neurons are in the maxillary palps (MPs), connect to medial sites, and promote attraction. We found in Drosophila that loss of a microRNA, miR-279, leads to formation of CO 2 neurons in the MPs. miR-279 acts through downregulation of the transcription factor Nerfin-1. The ectopic neurons are hybrid cells. They express CO 2 receptors and form connections characteristic of CO 2 neurons, while exhibiting wiring and receptor characteristics of MP olfactory receptor neurons (ORNs). We propose that this hybrid ORN reveals a cellular intermediate in the evolution of species-specific behaviors elicited by CO 2 .In insects, both the position of CO 2 neurons and the behavior elicited by CO 2 differ among species. For example, olfactory detection of CO 2 through neurons positioned in or around the mouthparts of an insect, such as maxillary palps (MPs) and labial palps, correlates with feedingrelated behaviors. Indeed, in some blood-feeding insects such as mosquitoes and tsetse flies, these neurons are harbored in the MPs and are important in locating hosts via plumes of CO 2 that they emit (1-3). The hawkmoth, Manduca sexta, monitors nectar profitability of newly opened Datura wrightii flowers through CO 2 receptor neurons located in their labial palps (4,5). In these examples, CO 2 acts as an attractant. Conversely, in Drosophila CO 2 is a component of a stress-induced odor that triggers avoidance behavior (6). This repellent response is driven by antennal neurons expressing the CO 2 receptor complex 8). How did these diverse behavioral responses to CO 2 arise during insect evolution? We propose that this diversity emerged through multiple steps, including changes in cellular position (arising from elimination of CO 2 neurons in one appendage and generation of these neurons in another) and changes in circuitry.In the course of a genetic screen for mutants disrupting the organization of the olfactory system, we isolated a mutant (S0962−07) that resulted in the formation of ectopic Gr21a-expressing §To whom correspondence should be addressed.

Individual members of the E2F/DP protein family control cell cycle progression by acting predominantly as an activator or repressor of transcription. In Drosophila melanogaster the E2f1, E2f2, Dp, and Rbf1 genes all contribute to replication control in ovarian follicle cells, which become 16C polyploid and subsequently undergo chorion gene amplification late in oogenesis. Mutation of E2f2, Dp, or Rbf1 causes ectopic DNA replication throughout the follicle cell genome during gene amplification cycles. Here we show by both reverse transcription-PCR and DNA microarray analysis that the transcripts of prereplication complex (pre-RC) genes are elevated compared to the wild type in E2f2, Dp, and Rbf1 mutant follicle cells. For some genes the magnitude of this transcriptional derepression is greater in Rbf1 than in E2f2 mutants. These differences correlate with differences in the magnitude of the replication defects in follicle cells, which attain an inappropriate 32C DNA content in both Rbf1 and Dp mutants but not in E2f2 mutants. The ectopic genomic replication of E2f2 mutant follicle cells can be suppressed by reducing the Orc2, Orc5, or Mcm2 gene dose by half, indicating that small changes in pre-RC gene expression can affect DNA synthesis in these cells. We conclude that RBF1 forms complexes with both E2F1/DP and E2F2/DP that cooperate to repress the expression of pre-RC genes, which helps confine DNA synthesis to sites of gene amplification. In contrast, E2F1 and E2F2 repressors function redundantly for some genes in the embryo. Thus, the relative functional contributions of E2F1 and E2F2 to gene expression and cell cycle control depends on the developmental context.

Fibroblast growth factors (FGFs) are important regulators of retinal development and survival. We examined the expression and distribution of FGF9 and its preferred receptors FGFR2IIIc and FGFR3IIIc in this tissue. FGF9 transcripts in whole rat retina were detected by RT-PCR but were not present in purified cultured Muller glia. Transcripts appeared as 3.2-kb and 4.0-kb bands on Northern blots, and Western blotting of whole retina revealed FGF9-immunoreactive bands at 30 and 55 kDa. FGF9 mRNA demonstrated a biphasic expression profile, elevated at birth and adulthood, but relatively decreased during terminal retinal differentiation (4-14 days postnatal). Antibody labeling broadly reflected these findings: staining in vivo was observed mainly in the inner retina (and outer plexiform layer in adults) whereas FGF9 was not detectable in cultured Muller glia. In adults, FGF9 in situ hybridization also showed a detectable signal in inner retina. FGFR2IIIc and FGFR3IIIc were detected by RT-PCR, and Western blotting showed both FGFRs existed as multiple forms between approximately 100-200 kDa. FGFR2 and FGFR3 antibodies showed prominent labeling in the inner retina, especially in proliferating cultured Muller glia. Exogenous FGF9 elicited a dose-dependent increase in Muller glial proliferation in vitro. These data suggest a role for FGF9 in retinal differentiation and maturation, possibly representing a neuronally derived factor acting upon glial (and other) cells.

E2F transcription factors are thought to influence the G1-S cell-cycle transition by controlling expression of genes required for growth and DNA synthesis. But emerging evidence suggests E2F complexes can control the cell cycle independently of transcription by directly regulating DNA replication origin usage during S phase.

Human beta-catenin and its fly homolog Armadillo are best known for their roles in cadherin-based cell-cell adhesion and in transduction of Wingless/Wnt signals. It has been hypothesized that beta-catenin may also regulate cell migration and cell shape changes, possibly by regulating the microtubule cytoskeleton via interactions with APC. This hypothesis was based on experiments in which a hyperstable mutant form of beta-catenin was expressed in MDCK cells, where it altered their migratory properties and their ability to send out long cellular processes. We tested the generality of this hypothesis in vivo in Drosophila. We utilized three model systems in which cell migration and/or process extension are known to play key roles during development: the migration of the border cells during oogenesis, the extension of axons in the nervous system, and the migration and cell process extension of tracheal cells. In all cases, cells expressing activated Armadillo were able to migrate and extend cell processes essentially normally. The one alteration from normal involved an apparent cell fate change in certain tracheal cells. These results suggest that only certain cells are affected by activation of Armadillo/beta-catenin, and that Armadillo/beta-catenin does not play a general role in inhibiting cell migration or process extension.

Drosophila contains two members of the E2F transcription factor family (E2f and E2f2), which controls the expression of genes that regulate the G1-S transition of the cell cycle. Previous genetic analyses have indicated that E2f is an essential gene that stimulates DNA replication. We show that loss of E2f2 is viable, but causes partial female sterility associated with changes in the mode of DNA replication in the follicle cells that surround the developing oocyte. Late in wild-type oogenesis, polyploid follicle cells terminate a program of asynchronous endocycles in which the euchromatin is entirely replicated, and then confine DNA synthesis to the synchronous amplification of specific loci, including two clusters of chorion genes that encode eggshell proteins. E2f2 mutant follicle cells terminate endocycles on schedule, but then fail to confine DNA synthesis to sites of gene amplification and inappropriately begin genomic DNA replication. This ectopic DNA synthesis does not represent a continuation of the endocycle program, as the cells do not complete an entire additional S phase. E2f2 mutant females display a 50% reduction in chorion gene amplification, and lay poorly viable eggs with a defective chorion. The replication proteins ORC2, CDC45L and ORC5, which in wild-type follicle cell nuclei localize to sites of gene amplification, are distributed throughout the entire follicle cell nucleus in E2f2 mutants, consistent with their use at many genomic replication origins rather than only at sites of gene amplification. RT-PCR analyses of RNA purified from E2f2 mutant follicle cells indicate an increase in the level of Orc5 mRNA relative to wild type. These data indicate that E2f2 functions to inhibit widespread genomic DNA synthesis in late stage follicle cells, and may do so by repressing the expression of specific components of the replication machinery.

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