Transcriptomic effects of the foraging gene shed light on pathways of pleiotropy and plasticity.

Genes are often pleiotropic and plastic in their expression, features which increase and diversify the functionality of the genome. The foraging (for) gene in Drosophila melanogaster is highly pleiotropic and a long-standing model for studying individual differences in behavior and plasticity from ethological, evolutionary, and genetic perspectives. Its pleiotropy is known to be linked to its complex molecular structure; however, the downstream pathways and interactors remain mostly elusive. To uncover these pathways and interactors and gain a better understanding of how pleiotropy and plasticity are achieved at the molecular level, we explore the effects of different for alleles on gene expression at baseline and in response to 4 h of food deprivation, using RNA sequencing analysis in different Drosophila larval tissues. The results show tissue-specific transcriptomic dynamics influenced by for allelic variation and food deprivation, as well as genotype by treatment interactions. Differentially expressed genes yielded pathways linked to previously described for phenotypes and several potentially novel phenotypes. Together, these findings provide putative genes and pathways through which for might regulate its varied phenotypes in a pleiotropic, plastic, and gene-structure-dependent manner.

The cap epitranscriptome: Early directions to a complex life as mRNA.

Animal, protist and viral messenger RNAs (mRNAs) are most prominently modified at the beginning by methylation of cap-adjacent nucleotides at the 2'-O-position of the ribose (cOMe) by dedicated cap methyltransferases (CMTrs). If the first nucleotide of an mRNA is an adenosine, PCIF1 can methylate at the N6 -position (m6 A), while internally the Mettl3/14 writer complex can methylate. These modifications are introduced co-transcriptionally to affect many aspects of gene expression including localisation to synapses and local translation. Of particular interest, transcription start sites of many genes are heterogeneous leading to sequence diversity at the beginning of mRNAs, which together with cOMe and m6 Am could constitute an extensive novel layer of gene expression control. Given the role of cOMe and m6 A in local gene expression at synapses and higher brain functions including learning and memory, such code could be implemented at the transcriptional level for lasting memories through local gene expression at synapses.

Glial control of sphingolipid levels sculpts diurnal remodeling in a circadian circuit.

Structural plasticity in the brain often necessitates dramatic remodeling of neuronal processes, with attendant reorganization of the cytoskeleton and membranes. Although cytoskeletal restructuring has been studied extensively, how lipids might orchestrate structural plasticity remains unclear. We show that specific glial cells in Drosophila produce glucocerebrosidase (GBA) to locally catabolize sphingolipids. Sphingolipid accumulation drives lysosomal dysfunction, causing gba1b mutants to harbor protein aggregates that cycle across circadian time and are regulated by neural activity, the circadian clock, and sleep. Although the vast majority of membrane lipids are stable across the day, a specific subset that is highly enriched in sphingolipids cycles daily in a gba1b-dependent fashion. Remarkably, both sphingolipid biosynthesis and degradation are required for the diurnal remodeling of circadian clock neurites, which grow and shrink across the day. Thus, dynamic sphingolipid regulation by glia enables diurnal circuit remodeling and proper circadian behavior.

The Drosophila foraging gene plays a vital role at the start of metamorphosis for subsequent adult emergence.

The foraging (for) gene has been extensively studied in many species for its functions in development, physiology, and behavior. It is common for genes that influence behavior and development to be essential genes, and for has been found to be an essential gene in both fruit flies and mammals, with for mutants dying before reaching the adult stage. However, the biological process underlying the lethality associated with this gene is not known. Here, we show that in Drosophila melanogaster, some but not all gene products of for are essential for survival. Specifically, we show that promoter 3 of for, but not promoters 1, 2, and 4 are required for survival past pupal stage. We use full and partial genetic deletions of for, and temperature-restricted knock-down of the gene to further investigate the stage of lethality. While deletion analysis shows that flies lacking for die at the end of pupal development, as pharate adults, temperature-restricted knock-down shows that for is only required at the start of pupal development, for normal adult emergence (AE) and viability. We further show that the inability of these mutants to emerge from their pupal cases is linked to deficiencies in emergence behaviors, caused by a possible energy deficiency, and finally, that the lethality of for mutants seems to be linked to protein isoform P3, transcribed from for promoter 3.

New Twists in Detecting mRNA Modification Dynamics.

Modified nucleotides in mRNA are an essential addition to the standard genetic code of four nucleotides in animals, plants, and their viruses. The emerging field of epitranscriptomics examines nucleotide modifications in mRNA and their impact on gene expression. The low abundance of nucleotide modifications and technical limitations, however, have hampered systematic analysis of their occurrence and functions. Selective chemical and immunological identification of modified nucleotides has revealed global candidate topology maps for many modifications in mRNA, but further technical advances to increase confidence will be necessary. Single-molecule sequencing introduced by Oxford Nanopore now promises to overcome such limitations, and we summarize current progress with a particular focus on the bioinformatic challenges of this novel sequencing technology.

Drosophila melanogaster foraging regulates a nociceptive-like escape behavior through a developmentally plastic sensory circuit.

Painful or threatening experiences trigger escape responses that are guided by nociceptive neuronal circuitry. Although some components of this circuitry are known and conserved across animals, how this circuitry is regulated at the genetic and developmental levels is mostly unknown. To escape noxious stimuli, such as parasitoid wasp attacks, Drosophila melanogaster larvae generate a curling and rolling response. Rover and sitter allelic variants of the Drosophila foraging (for) gene differ in parasitoid wasp susceptibility, suggesting a link between for and nociception. By optogenetically activating cells associated with each of for's promoters (pr1-pr4), we show that pr1 cells regulate larval escape behavior. In accordance with rover and sitter differences in parasitoid wasp susceptibility, we found that rovers have higher pr1 expression and increased sensitivity to nociception relative to sitters. The for null mutants display impaired responses to thermal nociception, which are rescued by restoring for expression in pr1 cells. Conversely, knockdown of for in pr1 cells phenocopies the for null mutant. To gain insight into the circuitry underlying this response, we used an intersectional approach and activity-dependent GFP reconstitution across synaptic partners (GRASP) to show that pr1 cells in the ventral nerve cord (VNC) are required for the nociceptive response, and that multidendritic sensory nociceptive neurons synapse onto pr1 neurons in the VNC. Finally, we show that activation of the pr1 circuit during development suppresses the escape response. Our data demonstrate a role of for in larval nociceptive behavior. This function is specific to for pr1 neurons in the VNC, guiding a developmentally plastic escape response circuit.

The role of the genome in experience-dependent plasticity: Extending the analogy of the genomic action potential.

Our past experiences shape our current and future behavior. These experiences must leave some enduring imprint on our brains, altering neural circuits that mediate behavior and contributing to our individual differences. As a framework for understanding how experiences might produce lasting changes in neural circuits, Clayton [D. F. Clayton, Neurobiol. Learn. Mem. 74, 185-216 (2000)] introduced the concept of the genomic action potential (gAP)-a structured genomic response in the brain to acute experience. Similar to the familiar electrophysiological action potential (eAP), the gAP also provides a means for integrating afferent patterns of activity but on a slower timescale and with longer-lasting effects. We revisit this concept in light of contemporary work on experience-dependent modification of neural circuits. We review the "Immediate Early Gene" (IEG) response, the starting point for understanding the gAP. We discuss evidence for its involvement in the encoding of experience to long-term memory across time and biological levels of organization ranging from individual cells to cell ensembles and whole organisms. We explore distinctions between memory encoding and homeostatic functions and consider the potential for perpetuation of the imprint of experience through epigenetic mechanisms. We describe a specific example of a gAP in humans linked to individual differences in the response to stress. Finally, we identify key objectives and new tools for continuing research in this area.

Biological embedding of experience: A primer on epigenetics.

Biological embedding occurs when life experience alters biological processes to affect later life health and well-being. Although extensive correlative data exist supporting the notion that epigenetic mechanisms such as DNA methylation underlie biological embedding, causal data are lacking. We describe specific epigenetic mechanisms and their potential roles in the biological embedding of experience. We also consider the nuanced relationships between the genome, the epigenome, and gene expression. Our ability to connect biological embedding to the epigenetic landscape in its complexity is challenging and complicated by the influence of multiple factors. These include cell type, age, the timing of experience, sex, and DNA sequence. Recent advances in molecular profiling and epigenome editing, combined with the use of comparative animal and human longitudinal studies, should enable this field to transition from correlative to causal analyses.

The foraging Gene and Its Behavioral Effects: Pleiotropy and Plasticity.

The Drosophila melanogaster foraging (for) gene is a well-established example of a gene with major effects on behavior and natural variation. This gene is best known for underlying the behavioral strategies of rover and sitter foraging larvae, having been mapped and named for this phenotype. Nevertheless, in the last three decades an extensive array of studies describing for's role as a modifier of behavior in a wide range of phenotypes, in both Drosophila and other organisms, has emerged. Furthermore, recent work reveals new insights into the genetic and molecular underpinnings of how for affects these phenotypes. In this article, we discuss the history of the for gene and its role in natural variation in behavior, plasticity, and behavioral pleiotropy, with special attention to recent findings on the molecular structure and transcriptional regulation of this gene.

Deciphering pleiotropy: How complex genes regulate behavior.

The genetic underpinnings of animal behavior are exceedingly complex. Behavioral phenotypes are commonly regulated by many genes, and the behavioral effects of a gene often dependent on environmental conditions and genetic background. To complicate the study of behavioral genetics further, many genes that regulate behavioral phenotypes are themselves very complex genes, with several gene products and functions. One example of such a complex gene is the foraging gene in D. melanogaster. foraging influences many behaviors in the fruit fly, and the key to its effects likely lies in its complex molecular structure. We've recently found that expression levels of a small subset of transcripts of the foraging gene underlie the behavioral differences seen in adult foraging patterns of the rover and sitter D. melanogaster strains. Here we comment on the larger implications of this and other findings on gene regulation and pleiotropy in behavior.

The adult foraging assay (AFA) detects strain and food-deprivation effects in feeding-related traits of Drosophila melanogaster.

We introduce a high-resolution adult foraging assay (AFA) that relates pre- and post-ingestive walking behavior to individual instances of food consumption. We explore the utility of the AFA by taking advantage of established rover and sitter strains known to differ in a number of feeding-related traits. The AFA allows us to effectively distinguish locomotor behavior in Fed and Food-Deprived (FD) rover and sitter foragers. We found that rovers exhibit more exploratory behavior into the center of an arena containing sucrose drops compared to sitters who hug the edges of the arena and exhibit thigmotaxic behavior. Rovers also discover and ingest more sucrose drops than sitters. Sitters become more exploratory with increasing durations of food deprivation and the number of ingestion events also increases progressively with prolonged fasting for both strains. AFA results are matched by strain differences in sucrose responsiveness, starvation resistance, and lipid levels, suggesting that under the same feeding condition, rovers are more motivated to forage than sitters. These findings demonstrate the AFA's ability to effectively discriminate movement and food ingestion patterns of different strains and feeding treatments.

Epigenetic mechanisms modulate differences in Drosophila foraging behavior.

Little is known about how genetic variation and epigenetic marks interact to shape differences in behavior. The foraging (for) gene regulates behavioral differences between the rover and sitter Drosophila melanogaster strains, but the molecular mechanisms through which it does so have remained elusive. We show that the epigenetic regulator G9a interacts with for to regulate strain-specific adult foraging behavior through allele-specific histone methylation of a for promoter (pr4). Rovers have higher pr4 H3K9me dimethylation, lower pr4 RNA expression, and higher foraging scores than sitters. The rover-sitter differences disappear in the presence of G9a null mutant alleles, showing that G9a is necessary for these differences. Furthermore, rover foraging scores can be phenocopied by transgenically reducing pr4 expression in sitters. This compelling evidence shows that genetic variation can interact with an epigenetic modifier to produce differences in gene expression, establishing a behavioral polymorphism in Drosophila.

An ant-plant mutualism through the lens of cGMP-dependent kinase genes.

In plant-animal mutualisms, how an animal forages often determines how much benefit its plant partner receives. In many animals, foraging behaviour changes in response to foraging gene expression or activation of the cGMP-dependent protein kinase (PKG) that foraging encodes. Here, we show that this highly conserved molecular mechanism affects the outcome of a plant-animal mutualism. We studied the two PKG genes of Allomerus octoarticulatus, an Amazonian ant that defends the ant-plant Cordia nodosa against herbivores. Some ant colonies are better 'bodyguards' than others. Working in the field in Peru, we found that colonies fed with a PKG activator recruited more workers to attack herbivores than control colonies. This resulted in less herbivore damage. PKG gene expression in ant workers correlated with whether an ant colony discovered an herbivore and how much damage herbivores inflicted on leaves in a complex way; natural variation in expression levels of the two genes had significant interaction effects on ant behaviour and herbivory. Our results suggest a molecular basis for ant protection of plants in this mutualism.

Feeding-Related Traits Are Affected by Dosage of the foraging Gene in Drosophila melanogaster.

Nutrient acquisition and energy storage are critical parts of achieving metabolic homeostasis. The foraging gene in Drosophila melanogaster has previously been implicated in multiple feeding-related and metabolic traits. Before foraging's functions can be further dissected, we need a precise genetic null mutant to definitively map its amorphic phenotypes. We used homologous recombination to precisely delete foraging, generating the for0 null allele, and used recombineering to reintegrate a full copy of the gene, generating the {forBAC} rescue allele. We show that a total loss of foraging expression in larvae results in reduced larval path length and food intake behavior, while conversely showing an increase in triglyceride levels. Furthermore, varying foraging gene dosage demonstrates a linear dose-response on these phenotypes in relation to foraging gene expression levels. These experiments have unequivocally proven a causal, dose-dependent relationship between the foraging gene and its pleiotropic influence on these feeding-related traits. Our analysis of foraging's transcription start sites, termination sites, and splicing patterns using rapid amplification of cDNA ends (RACE) and full-length cDNA sequencing, revealed four independent promoters, pr1-4, that produce 21 transcripts with nine distinct open reading frames (ORFs). The use of alternative promoters and alternative splicing at the foraging locus creates diversity and flexibility in the regulation of gene expression, and ultimately function. Future studies will exploit these genetic tools to precisely dissect the isoform- and tissue-specific requirements of foraging's functions and shed light on the genetic control of feeding-related traits involved in energy homeostasis.

Foraging Path-length Protocol for Drosophila melanogaster Larvae.

The Drosophila melanogaster larval path-length phenotype is an established measure used to study the genetic and environmental contributions to behavioral variation. The larval path-length assay was developed to measure individual differences in foraging behavior that were later linked to the foraging gene. Larval path-length is an easily scored trait that facilitates the collection of large sample sizes, at minimal cost, for genetic screens. Here we provide a detailed description of the current protocol for the larval path-length assay first used by Sokolowski. The protocol details how to reproducibly handle test animals, perform the behavioral assay and analyze the data. An example of how the assay can be used to measure behavioral plasticity in response to environmental change, by manipulating feeding environment prior to performing the assay, is also provided. Finally, appropriate test design as well as environmental factors that can modify larval path-length such as food quality, developmental age and day effects are discussed.