Notch signaling in the development of the inner ear: Lessons from Drosophila

The sensory patches in the ear of a vertebrate can be compared with the mechanosensory bristles of a fly. This comparison has led to the discovery that lateral inhibition mediated by the Notch cell–cell signaling pathway, first characterized in Drosophila and crucial for bristle development, also has a key role in controlling the pattern of sensory hair cells and supporting cells in the ear. We review the arguments for considering the sensory patches of the vertebrate ear and bristles of the insect to be homologous structures, evolved from a common ancestral mechanosensory organ, and we examine more closely the role of Notch signaling in each system. Using viral vectors to misexpress components of the Notch pathway in the chick ear, we show that a simple lateral-inhibition model based on feedback regulation of the Notch ligand Delta is inadequate for the ear just as it is for the fly bristle. The Notch ligand Serrate1, expressed in supporting cells in the ear, is regulated by lateral induction, not lateral inhibition; commitment to become a hair cell is not simply controlled by levels of expression of the Notch ligands Delta1, Serrate1, and Serrate2 in the neighbors of the nascent hair cell; and at least one factor, Numb, capable of blocking reception of lateral inhibition is concentrated in hair cells. These findings reinforce the parallels between the vertebrate ear and the fly bristle and show how study of the insect system can help us understand the vertebrate.

Incipient speciation by sexual isolation in Drosophila: Concurrent evolution at multiple loci

Drosophila melanogaster from Zimbabwe and nearby regions shows strong but asymmetric sexual isolation from its cosmopolitan counterparts. By creating stable chromosome-substitution lines, earlier studies were able to show that the two major autosomes have very large effects on both male mating success and female mating preference. In this study, we genetically dissect this sexual isolation by recombination analysis between a whole-chromosome substitution line (which carries a Zimbabwe-derived third chromosome) and a strain with seven visible markers on that chromosome. Four loci are responsible for male mating success and three others are found to control female mating preference. Because male and female traits are not closely linked, their strong association among isofemale lines is most likely a reflection of sexual selection in nature. The results suggest that a large number of behavioral loci may evolve concurrently in the incipient stage of speciation before other aspects of reproductive isolation (such as hybrid sterility) have become evident. The results shed light on the population genetic processes underlying the formation of nascent species, as well as modes of speciation.

A locus for female discrimination behavior causing sexual isolation in Drosophila

The genetic basis of sexual isolation that contributes to speciation is one of the unsolved questions in evolutionary biology. Drosophila ananassae and Drosophila pallidosa are closely related, and postmating isolation has not developed between them. However, females of both species discriminate their mating partners, and this discrimination contributes to strong sexual isolation between them. By using surgical treatments, we demonstrate that male courtship songs play a dominant role in female mate discrimination. The absence of the song of D. pallidosa dramatically increased interspecies mating with D. ananassae females but reduced intraspecies mating with D. pallidosa females. Furthermore, genetic analysis and chromosomal introgression by repeated backcrosses to D. pallidosa males identified possible loci that control female discrimination in each species. These loci were mapped on distinct positions near the Delta locus on the middle of the left arm of the second chromosome. Because the mate discrimination we studied is well developed and is the only known mechanism that prevents gene flow between them, these loci may have played crucial roles in the evolution of reproductive isolation, and therefore, in the speciation process between these two species.

Dribble, the Drosophila KRR1p Homologue, Is Involved in rRNA Processing

The Drosophila dribble (dbe) gene encodes a KH domain protein, homologous to yeast KRR1p. Expression of dbe transcripts is ubiquitous during embryogenesis. Overexpressed Dribble protein is localized in the nucleus and in some cell types in a subregion of the nucleolus. Homozygous dbe mutants die at first instar larval stage. Clonal analyses suggest that dbe+ is required for survival of dividing cells. In dbe mutants, a novel rRNA-processing defect is found and accumulation of an abnormal rRNA precursor is detected.

A Tropomyosin-2 Mutation Suppresses a Troponin I Myopathy in Drosophila

A suppressor mutation, D53, of the held-up2 allele of the Drosophila melanogaster Troponin I (wupA) gene is described. D53, a missense mutation, S185F, of the tropomyosin-2, Tm2, gene fully suppresses all the phenotypic effects of held-up2, including the destructive hypercontraction of the indirect flight muscles (IFMs), a lack of jumping, the progressive myopathy of the walking muscles, and reductions in larval crawling and feeding behavior. The suppressor restores normal function of the IFMs, but flight ability decreases with age and correlates with an unusual, progressive structural collapse of the myofibrillar lattice starting at the center. The S185F substitution in Tm2 is close to a troponin T binding site on tropomyosin. Models to explain suppression by D53, derived from current knowledge of the vertebrate troponin-tropomyosin complex structure and functions, are discussed. The effects of S185F are compared with those of two mutations in residues 175 and 180 of human α-tropomyosin 1 which cause familial hypertrophic cardiomyopathy (HCM).

The Friend of GATA proteins U-shaped, FOG-1, and FOG-2 function as negative regulators of blood, heart, and eye development in Drosophila

Friend of GATA (FOG) proteins regulate GATA factor-activated gene transcription. During vertebrate hematopoiesis, FOG and GATA proteins cooperate to promote erythrocyte and megakaryocyte differentiation. The Drosophila FOG homologue U-shaped (Ush) is expressed similarly in the blood cell anlage during embryogenesis. During hematopoiesis, the acute myeloid leukemia 1 homologue Lozenge and Glial cells missing are required for the production of crystal cells and plasmatocytes, respectively. However, additional factors have been predicted to control crystal cell proliferation. In this report, we show that Ush is expressed in hemocyte precursors and plasmatocytes throughout embryogenesis and larval development, and the GATA factor Serpent is essential for Ush embryonic expression. Furthermore, loss of ush function results in an overproduction of crystal cells, whereas forced expression of Ush reduces this cell population. Murine FOG-1 and FOG-2 also can repress crystal cell production, but a mutant version of FOG-2 lacking a conserved motif that binds the corepressor C-terminal binding protein fails to affect the cell lineage. The GATA factor Pannier (Pnr) is required for eye and heart development in Drosophila. When Ush, FOG-1, FOG-2, or mutant FOG-2 is coexpressed with Pnr during these developmental processes, severe eye and heart phenotypes result, consistent with a conserved negative regulation of Pnr function. These results indicate that the fly and mouse FOG proteins function similarly in three distinct cellular contexts in Drosophila, but may use different mechanisms to regulate genetic events in blood vs. cardial or eye cell lineages.

Evolutionary EST analysis identifies rapidly evolving male reproductive proteins in Drosophila

Sequence comparisons of genomes or expressed sequence tags (ESTs) from related organisms provide insight into functional conservation and diversification. We compare the sequences of ESTs from the male accessory gland of Drosophila simulans to their orthologs in its close relative Drosophila melanogaster, and demonstrate rapid divergence of many of these reproductive genes. Nineteen (∼11%) of 176 independent genes identified in the EST screen contain protein-coding regions with an excess of nonsynonymous over synonymous changes, suggesting that their divergence has been accelerated by positive Darwinian selection. Genes that encode putative accessory gland-specific seminal fluid proteins had a significantly elevated level of nonsynonymous substitution relative to nonaccessory gland-specific genes. With the 57 new accessory gland genes reported here, we predict that ∼90% of the male accessory gland genes have been identified. The evolutionary EST approach applied here to identify putative targets of adaptive evolution is readily applicable to other tissues and organisms.

Increased sexual activity reduces male immune function in Drosophila melanogaster

Despite the benefits of resistance, susceptibility to infectious disease is commonplace. Although specific susceptibility may be considered an inevitable consequence of the co-evolutionary arms race between parasite and host, a more general constraint may arise from the cost of an immune response. This “cost” hypothesis predicts a tradeoff between immune defense and other components of fitness. In particular, a tradeoff between immunity and sexually selected male behavior has been proposed. Here we provide experimental support for the direct phenotypic tradeoff between sexual activity and immunity by studying the antibacterial immune response in Drosophila melanogaster. Males exposed to more females showed a reduced ability to clear a bacterial infection, an effect that we experimentally link to changes in sexual activity. Our results suggest immunosuppression is an important cost of reproduction and that immune function and levels of disease susceptibility will be influenced by sexual selection.

Drosophila Heterochromatin Protein 1 (HP1)/Origin Recognition Complex (ORC) Protein Is Associated with HP1 and ORC and Functions in Heterochromatin-induced Silencing

Heterochromatin protein 1 (HP1) is a conserved component of the highly compact chromatin of higher eukaryotic centromeres and telomeres. Cytogenetic experiments in Drosophila have shown that HP1 localization into this chromatin is perturbed in mutants for the origin recognition complex (ORC) 2 subunit. ORC has a multisubunit DNA-binding activity that binds origins of DNA replication where it is required for origin firing. The DNA-binding activity of ORC is also used in the recruitment of the Sir1 protein to silence nucleation sites flanking silent copies of the mating-type genes in Saccharomyces cerevisiae. A fraction of HP1 in the maternally loaded cytoplasm of the early Drosophila embryo is associated with a multiprotein complex containing Drosophila melanogaster ORC subunits. This complex appears to be poised to function in heterochromatin assembly later in embryonic development. Here we report the identification of a novel component of this complex, the HP1/ORC-associated protein. This protein contains similarity to DNA sequence-specific HMG proteins and is shown to bind specific satellite sequences and the telomere-associated sequence in vitro. The protein is shown to have heterochromatic localization in both diploid interphase and mitotic chromosomes and polytene chromosomes. Moreover, the gene encoding HP1/ORC-associated protein was found to display reciprocal dose-dependent variegation modifier phenotypes, similar to those for mutants in HP1 and the ORC 2 subunit.

Physical and functional interactions between Drosophila TRAF2 and Pelle kinase contribute to Dorsal activation

Signaling through the Toll receptor is required for dorsal/ventral polarity in Drosophila embryos, and also plays an evolutionarily conserved role in the immune response. Upon ligand binding, Toll appears to multimerize and activate the associated kinase, Pelle. However, the immediate downstream targets of Pelle have not been identified. Here we show that Drosophila tumor necrosis factor receptor-associated factor 2 (dTRAF2), a homologue of human TRAF6, physically and functionally interacts with Pelle, and is phosphorylated by Pelle in vitro. Importantly, dTRAF2 and Pelle cooperate to activate Dorsal synergistically in cotransfected Schneider cells. Deletion of the C-terminal TRAF domain of dTRAF2 enhances Dorsal activation, perhaps reflecting the much stronger interaction of the mutant protein with phosphorylated, active Pelle. Taken together, our results indicate that Pelle and dTRAF2 physically and functionally interact, and that the TRAF domain acts as a regulator of this interaction. dTRAF2 thus appears to be a downstream target of Pelle. We discuss these results in the context of Toll signaling in flies and mammals.

Sexually antagonistic coevolution of a postmating-prezygotic reproductive character in desert Drosophila

Rapid divergence in postmating-prezygotic characters suggests that selection may be responsible for generating reproductive barriers between closely related species. Theoretical models indicate that this rapid divergence could be generated by a series of male adaptations and female counteradaptations by means of sexual selection or conflict, but empirical tests of particular mechanisms are generally lacking. Moreover, although a male–female genotypic interaction in mediating sperm competition attests to an active role of females, molecular or morphological evidence of the female's participation in the coevolutionary process is critically needed. Here we show that postmating-prezygotic variation among populations of cactophilic desert Drosophila reflects divergent coevolutionary trajectories between the sexes. We explicitly test the female's role in intersexual interactions by quantifying differences in a specific postmating-prezygotic reproductive character, the insemination reaction mass, in two species, Drosophila mojavensis and Drosophila arizonae. A series of interpopulation crosses confirmed that population divergence was propelled by male–female interactions, a prerequisite if the selective forces derive from sexual conflicts. An association between the reaction mass and remating and oviposition behavior argues that divergence has been propelled by sexually antagonistic coevolution, and potentially has important implications for speciation.

Novel Gq alpha isoform is a candidate transducer of rhodopsin signaling in a Drosophila testes-autonomous pacemaker.

DGq is the alpha subunit of the heterotrimeric GTPase (G alpha), which couples rhodopsin to phospholipase C in Drosophila vision. We have uncovered three duplicated exons in dgq by scanning the GenBank data base for unrecognized coding sequences. These alternative exons encode sites involved in GTPase activity and G beta-binding, NorpA (phospholipase C)-binding, and rhodopsin-binding. We examined the in vivo splicing of dgq in adult flies and find that, in all but the male gonads, only two isoforms are expressed. One, dgqA, is the original visual isoform and is expressed in eyes, ocelli, brain, and male gonads. The other, dgqB, has the three novel exons and is widely expressed. Remarkably, all three nonvisual B exons are highly similar (82% identity at the amino acid level) to the Gq alpha family consensus, from Caenorhabditis elegans to human, but all three visual A exons are divergent (61% identity). Intriguingly, we have found a third isoform, dgqC, which is specifically and abundantly expressed in male gonads, and shares the divergent rhodopsin-binding exon of dgqA. We suggest that DGqC is a candidate for the light-signal transducer of a testes-autonomous photosensory clock. This proposal is supported by the finding that rhodopsin 2 and arrestin 1, two photoreceptor-cell-specific genes, are also expressed in male gonads.

Cloning of an arylalkylamine N-acetyltransferase (aaNAT1) from Drosophila melanogaster expressed in the nervous system and the gut.

In insects, neurotransmitter catabolism, melatonin precursor formation, and sclerotization involve arylalkylamine N-acetyltransferase (aaNAT, EC 2.3.1.87) activity. It is not known if one or multiple aaNAT enzymes are responsible for these activities. We recently have purified an aaNAT from Drosophila melanogaster. Here, we report the cloning of the corresponding aaNAT cDNA (aaNAT1) that upon COS cell expression acetylates dopamine, tryptamine, and the immediate melatonin precursor serotonin. aaNAT1 represents a novel gene family unrelated to known acetyl-transferases, except in two weakly conserved amino acid motifs. In situ hybridization studies of aaNAT1 mRNA in embryos reveal hybridization signals in the brain, the ventral cord, the gut, and probably in oenocytes, indicating a broad tissue distribution of aaNAT1 transcripts. Moreover, in day/ night studies we demonstrate a diurnal rhythm of melatonin concentration without a clear-cut change in aaNAT1 mRNA levels. The data suggest that tissue-specific regulation of aaNAT1 may be associated with different enzymatic functions and do not exclude the possibility of additional aaNAT genes.

A modular misexpression screen in Drosophila detecting tissue-specific phenotypes.

Genetic screens in Drosophila have lead to the discovery of many genes important for patterning and signal transduction in diverse organisms. Traditionally, the phenotypic effects of loss-of-function mutations are analyzed. As an alternative way to link genes and function, I have developed a versatile misexpression screen in Drosophila, the first such screen in higher eukaryotes. The screen identifies genes that, when over- or misexpressed in a pattern of interest, give a specific phenotype or modulate an existing mutant phenotype. It is based on Gal4 transactivation of a mobile enhancer and promoter that "targets" random endogenous genes for expression. The modular design of the screen allows directed expression in any temporal or spatial pattern. When activated in the developing eye, 4% of target inserts gave dominant phenotypes. One insertion was in the gene encoding Ras GTPase-activating protein; its overexpression phenotype was strongly enhanced by a mutation in Ras1. Thus, biologically relevant phenotypes and genetic interactions are identified using this method. The screen is a powerful new tool for developmental genetics; similar approaches can also be developed for other organisms.