The Female Post-Mating Response Requires Genes Expressed in the Secondary Cells of the Male Accessory Gland in Drosophila melanogaster

Seminal proteins from the Drosophila male accessory gland induce post-mating responses (PMR) in females. The PMR comprise behavioral and physiological changes that include increased egg laying, decreased receptivity to courting males, and changes in the storage and use of sperm. Many of these changes are induced by a “sex peptide” (SP) and are maintained by SP’s binding to, and slow release from, sperm. The accessory gland contains two secretory cell types with distinct morphological and developmental characteristics. Products of these “main” and “secondary” cells work interdependently to induce and maintain the PMR. To identify individual genes needed for the morphology and function of secondary cells, we studied iab-6^cocu males, whose secondary cells have abnormal morphology and fail to provide products to maintain the PMR. By RNA-seq, we identified 77 genes that are downregulated by a factor of >5× in iab-6^cocu males. By functional assays and microscopy, we tested 20 candidate genes and found that at least 9 are required for normal storage and release of SP in mated females. Knockdown of each of these 9 genes consequently leads to a reduction in egg laying and an increase in receptivity over time, confirming a role for the secondary cells in maintaining the long-term PMR. Interestingly, only 1 of the 9 genes, CG3349, encodes a previously reported seminal fluid protein (Sfp), suggesting that secondary cells may perform essential functions beyond the production and modification of known Sfps. At least 3 of the 9 genes also regulate the size and/or abundance of secondary cell vacuoles, suggesting that the vacuoles’ contents may be important for the machinery used to maintain the PMR.     

Dissection of cis-regulatory elements in the C. elegans Hox gene egl-5 promoter

Hox genes are highly conserved segmental identity genes well known for their complex expression patterns and divergent targets. Here we present an analysis of cis-regulatory elements in the Caenorhabditis elegans Hox gene egl-5, which is expressed in multiple tissues in the posterior region of the nematode. We have utilized phylogenetic footprinting to efficiently identify cis-regulatory elements and have characterized these with gfp reporters and tissue-specific rescue experiments. We have found that the complex expression pattern of egl-5 is the cumulative result of the activities of multiple tissue or local region-specific activator sequences that are conserved both in sequence and near-perfect order in the related nematode Caenorhabditis briggsae. Two conserved regulatory blocks analyzed in detail contain multiple sites for both positively and negatively acting factors. One of these regions may promote activation of egl-5 in certain cells via the Wnt pathway. Positively acting regions are repressed in inappropriate tissues by additional negative pathways acting at other sites within the promoter. Our analysis has allowed us to implicate several new regulatory factors significant to the control of egl-5 expression.     

Conserved regulation of the Caenorhabditis elegans labial/Hox1 gene ceh-13

Caenorhabditis elegans contains a set of six cluster-type homeobox (Hox) genes that are required during larval development. Some of them, but unlike in flies not all of them, are also required during embryogenesis. It has been suggested that the control of the embryonic expression of the worm Hox genes might differ from that of other species by being regulated in a lineal rather than a regional mode. Here, we present a trans-species analysis of the cis-regulatory region of ceh-13, the worm ortholog of the Drosophila labial and the vertebrate Hox1 genes, and find that the molecular mechanisms that regulate its expression may be similar to what has been found in species that follow a regulative, non-cell-autonomous mode of development. We have identified two enhancer fragments that are involved in different aspects of the embryonic ceh-13 expression pattern. We show that important features of comma-stage expression depend on an autoregulatory input that requires ceh-13 and ceh-20 functions. Our data show that the molecular nature of Hox1 class gene autoregulation has been conserved between worms, flies, and vertebrates. The second regulatory sequence is sufficient to drive correct early embryonic expression of ceh-13. Interestingly, this enhancer fragment acts as a response element of the Wnt/WG signaling pathway in Drosophila embryos.     

Regulation of gene expression in the distal region of the Drosophila leg by the Hox11 homolog, C15

The distal region of the Drosophila leg, the tarsus, is divided into five segments (ta I-V) and terminates in the pretarsus, which is characterized by a pair of claws. Several homeobox genes are expressed in distinct regions of the tarsus, including aristaless (al) and lim1 in the pretarsus, Bar (B) in ta IV and V, and apterous (ap) in ta IV. This pattern is governed by regulatory interactions between these genes; for example, Al and B are mutually antagonistic resulting in exclusion of B expression from the pretarsus. Although Al is necessary, it is not sufficient to repress B, indicating another factor is required. Here, this factor is identified as the product of the C15 gene, which is another homeodomain protein, a homolog of the human Hox11 oncogene. C15 is expressed in the same cells as al and, together, C15 and Al appear to directly repress B. C15/Al also act indirectly to repress ap in ta V, i.e., in surrounding cells. To do this, C15/Al autonomously repress expression of the gene encoding the Notch ligand Delta (Dl) in the pretarsus, restricting Dl to ta V and creating a Dl+/Dl- border at the interface between ta V and the pretarsus. This results in upregulation of Notch signaling, which induces expression of the bowl gene, the product of which represses ap.     

Paramutation in Drosophila Requires Both Nuclear and Cytoplasmic Actors of the piRNA Pathway and Induces Cis-spreading of piRNA Production

Transposable element activity is repressed in the germline in animals by PIWI-interacting RNAs (piRNAs), a class of small RNAs produced by genomic loci mostly composed of TE sequences. The mechanism of induction of piRNA production by these loci is still enigmatic. We have shown that, in Drosophila melanogaster, a cluster of tandemly repeated P-lacZ-white transgenes can be activated for piRNA production by maternal inheritance of a cytoplasm containing homologous piRNAs. This activated state is stably transmitted over generations and allows trans-silencing of a homologous transgenic target in the female germline. Such an epigenetic conversion displays the functional characteristics of a paramutation, i.e., a heritable epigenetic modification of one allele by the other. We report here that piRNA production and trans-silencing capacities of the paramutated cluster depend on the function of the rhino, cutoff, and zucchini genes involved in primary piRNA biogenesis in the germline, as well as on that of the aubergine gene implicated in the ping-pong piRNA amplification step. The 21-nt RNAs, which are produced by the paramutated cluster, in addition to 23- to 28-nt piRNAs are not necessary for paramutation to occur. Production of these 21-nt RNAs requires Dicer-2 but also all the piRNA genes tested. Moreover, cytoplasmic transmission of piRNAs homologous to only a subregion of the transgenic locus can generate a strong paramutated locus that produces piRNAs along the whole length of the transgenes. Finally, we observed that maternally inherited transgenic small RNAs can also impact transgene expression in the soma. In conclusion, paramutation involves both nuclear (Rhino, Cutoff) and cytoplasmic (Aubergine, Zucchini) actors of the piRNA pathway. In addition, since it is observed between nonfully homologous loci located on different chromosomes, paramutation may play a crucial role in epigenome shaping in Drosophila natural populations.     

Interactions between enhancer of rudimentary and Notch and deltex reveal a regulatory function of enhancer of rudimentary in the Notch signaling pathway in Drosophila melanogaster

Enhancer of rudimentary, e(r), encodes a small nuclear protein, ER, that has been implicated in the regulation of pyrimidine metabolism, DNA replication and cell proliferation. In Drosophila melanogaster, a new recessive Notch allele, N^nd-p, was isolated as a lethal in combination with an e(r) allele, e(r)^p2. Both mutants are viable as single mutants. N^nd-p is caused by a P-element insertion in the 5′ UTR, 378-bp upstream of the start of translation. Together the molecular and genetic data argue that N^nd-p is a hypomorphic allele of N. The three viable notchoid alleles, N^nd-p, N^nd-1 and N^nd-3, are lethal in combination with e(r)⁻ alleles. Our present hypothesis is that e(r) is a positive regulator of the Notch signaling pathway and that the lethality of the N e(r) double mutants is caused by a reduction in the expression of the pathway. This is supported by the rescue of the lethality by a mutation in Hairless, a negative regulator of N, and by the synthetic lethality of dx e(r) double mutants. Further support for the hypothesis is a reduction in E(spl) expression in an e(r)⁻ mutant. Immunostaining localizes ER to the nucleus, suggesting a nuclear function for ER. A role in the Notch signaling pathway, suggests that e(r) may be expressed in the nervous system. This turns out to be the case, as immunostaining of ER shows that ER is localized to the developing CNS.     

The Hox gene Ultrabithorax modulates the shape and size of the third leg of Drosophila by influencing diverse mechanisms

The developmental mechanisms that regulate the relative size and shape of organs have remained obscure despite almost a century of interest in the problem and the fact that changes in relative size represent the dominant mode of evolutionary change. Here, I investigate how the Hox gene Ultrabithorax (Ubx) instructs the legs on the third thoracic segment of Drosophila melanogaster to develop with a different size and shape from the legs on the second thoracic segment. Through loss-of-function and gain-of-function experiments, I demonstrate that different segments of the leg, the femur and the first tarsal segment, and even different regions of the femur, regulate their size in response to Ubx expression through qualitatively different mechanisms. In some regions, Ubx acts autonomously to specify shape and size, whereas in other regions, Ubx influences size through nonautonomous mechanisms. Loss of Ubx autonomously reduces cell size in the T3 femur, but this reduction seems to be partially compensated by an increase in cell numbers, so that it is unclear what effect cell size and number directly have on femur size. Loss of Ubx has both autonomous and nonautonomous effects on cell number in different regions of the basitarsus, but again there is not a strong correlation between cell size or number and organ size. Total organ size appears to be regulated through mechanisms that operate at the level of the entire leg segment (femur or basitarsus) relatively independently of the behavior of individual subpopulations of cells within the segment.     

Interaction between Sleep and Metabolism in Drosophila with Altered Octopamine Signaling

Sleep length and metabolic dysfunction are correlated, but the causal relationship between these processes is unclear. Octopamine promotes wakefulness in the fly by acting through the insulin-producing cells (IPCs) in the fly brain. To determine if insulin signaling mediates the effects of octopamine on sleep:wake behavior, we assayed flies in which insulin signaling activity was genetically altered. We found that increasing insulin signaling does not promote wake, nor does insulin appear to mediate the wake-promoting effects of octopamine. Octopamine also affects metabolism in invertebrate species, including, as we show here, Drosophila melanogaster. Triglycerides are decreased in mutants with compromised octopamine signaling and elevated in flies with increased activity of octopaminergic neurons. Interestingly, this effect is mediated at least partially by insulin, suggesting that effects of octopamine on metabolism are independent of its effects on sleep. We further investigated the relative contribution of metabolic and sleep phenotypes to the starvation response of flies with altered octopamine signaling. Hyperactivity (indicative of foraging) induced by starvation was elevated in octopamine receptor mutants, despite their high propensity for sleep, indicating that their metabolic state dictates their behavioral response under these conditions. Moreover, flies with increased octopamine signaling do not suppress sleep in response to starvation, even though they are normally hyper-aroused, most likely because of their high triglyceride levels. Together, these data suggest that observed correlations between sleep and metabolic phenotypes can result from shared molecular pathways rather than causality, and environmental conditions can lead to the dominance of one phenotype over the other.     

Non-neutral processes drive the nucleotide composition of non-coding sequences in Drosophila

The nature of the forces affecting base composition is a key question in genome evolution. There is uncertainty as to whether differences in the GC contents of non-coding sequences reflect differences in mutational bias, or in the intensity of selection or biased gene conversion. We have used a polymorphism dataset for non-coding sequences on the X chromosome of Drosophila simulans to examine this question. The proportion of GC-->AT versus AT-->GC polymorphic mutations in a locus is correlated with its GC content. This implies the action of forces that favour GC over AT base pairs, which are apparently strongest in GC-rich sequences.     

Effectors of alcohol-induced cell killing in Drosophila

Heavy alcohol consumption provokes an array of degenerative pathologies but the signals that couple alcohol exposure to regulated forms of cell death are poorly understood. Using Drosophila as a model, we genetically establish that the severity of ethanol challenge dictates the type of death that occurs. In contrast to responses seen under acute exposure, cytotoxic responses to milder challenges required gene encoding components of the apoptosome, Dronc and Dark. We conducted a genome-wide RNAi screen to capture targets that specifically mediate ethanol-induced cell death. One effector, Drat, encodes a novel protein that contains an ADH domain but lacks essential residues in the catalytic site. In cultured cells and neurons in vivo, depletion of Drat conferred protection from alcohol-induced apoptosis. Adults mutated for Drat showed both improved survival and enhanced propensities toward sedation after alcohol challenge. Together, these findings highlight novel effectors that support regulated cell death incited by alcohol stress in vitro and in vivo.     

Testing limits to adaptation along altitudinal gradients in rainforest Drosophila

Given that evolution can generate rapid and dramatic shifts in the ecological tolerance of a species, what prevents populations adapting to expand into new habitat at the edge of their distributions? Recent population genetic models have focused on the relative costs and benefits of migration between populations. On the one hand, migration may limit adaptive divergence by preventing local populations from matching their local selective optima. On the other hand, migration may also contribute to the genetic variance necessary to allow populations to track these changing optima. Empirical evidence for these contrasting effects of gene flow in natural situations are lacking, largely because it remains difficult to acquire. Here, we develop a way to explore theoretical models by estimating genetic divergence in traits that confer stress resistance along similar ecological gradients in rainforest Drosophila. This approach allows testing for the coupling of clinal divergence with local density, and the effects of genetic variance and the rate of change of the optimum on the response to selection. In support of a swamping effect of migration on phenotypic divergence, our data show no evidence for a cline in stress-related traits where the altitudinal gradient is steep, but significant clinal divergence where it is shallow. However, where clinal divergence is detected, sites showing trait means closer to the presumed local optimum have more genetic variation than sites with trait means distant from their local optimum. This pattern suggests that gene flow also aids a sustained response to selection.