Characterizing the Protein Isoforms of foraging (for), the PKGI Ortholog in Drosophila melanogaster.

The foraging (for) gene of Drosophila melanogaster encodes a cGMP-dependent protein kinase (PKG), which is a major effector of the cGMP signaling pathway involved in the regulation of behaviour and metabolic traits. Despite being well studied at the transcript level, little is known about the for gene at the protein level. Here, we provide a detailed characterization of the for gene protein (FOR) products and present new tools for their study, including five isoform-specific antibodies and a transgenic strain that carries an HA-labelled for allele (forBAC::HA). Our results showed that multiple FOR isoforms were expressed in the larval and adult stages of D. melanogaster and that the majority of whole-body FOR expression arises from three (P1, P1α, and P3) of eight putative protein isoforms. We found that FOR expression differed between the larval and adult stages and between the dissected larval organs we analyzed, which included the central nervous system (CNS), fat body, carcass, and intestine. Moreover, we showed that the FOR expression differed between two allelic variants of the for gene, namely, fors (sitter) and forR (rover), that are known to differ in many food-related traits. Together, our in vivo identification of FOR isoforms and the existence of temporal, spatial, and genetic differences in their expression lay the groundwork for determining their functional significance.

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.

How Social Experience and Environment Impacts Behavioural Plasticity in Drosophila.

An organism's behaviour is influenced by its social environment. Experiences such as social isolation or crowding may have profound short or long-term effects on an individual's behaviour. The composition of the social environment also depends on the genetics and previous experiences of the individuals present, leading to additional potential outcomes from each social interaction. In this article, we review selected literature related to the social environment of the model organism Drosophila melanogaster, and how Drosophila respond to variation in their social experiences throughout their lifetimes. We focus on the effects of social environment on behavioural phenotypes such as courtship, aggression, and group dynamics, as well as other phenotypes such as development and physiology. The consequences of phenotypic plasticity due to social environment are discussed with respect to the ecology and evolution of Drosophila. We also relate these studies to laboratory research practices involving Drosophila and other animals.

Autosomal sex-associated co-methylated regions predict biological sex from DNA methylation.

Sex is a modulator of health that has been historically overlooked in biomedical research. Recognizing this knowledge gap, funding agencies now mandate the inclusion of sex as a biological variable with the goal of stimulating efforts to illuminate the molecular underpinnings of sex biases in health and disease. DNA methylation (DNAm) is a strong molecular candidate for mediating such sex biases; however, a robust and well characterized annotation of sex differences in DNAm is yet to emerge. Beginning with a large (n = 3795) dataset of DNAm profiles from normative adult whole blood samples, we identified, validated and characterized autosomal sex-associated co-methylated genomic regions (sCMRs). Strikingly, sCMRs showed consistent sex differences in DNAm over the life course and a subset were also consistent across cell, tissue and cancer types. sCMRs included sites with known sex differences in DNAm and links to health conditions with sex biased effects. The robustness of sCMRs enabled the generation of an autosomal DNAm-based predictor of sex with 96% accuracy. Testing this tool on blood DNAm profiles from individuals with sex chromosome aneuploidies (Klinefelter [47,XXY], Turner [45,X] and 47,XXX syndrome) revealed an intimate relationship between sex chromosomes and sex-biased autosomal DNAm.

A cGMP-dependent protein kinase, encoded by the Drosophila foraging gene, regulates neurotransmission through changes in synaptic structure and function.

A cGMP-dependent protein kinase (PKG) encoded by the Drosophila foraging (for) gene regulates both synaptic structure (nerve terminal growth) and function (neurotransmission) through independent mechanisms at the Drosophila larval neuromuscular junction (nmj). Glial for is known to restrict nerve terminal growth, whereas presynaptic for inhibits synaptic vesicle (SV) exocytosis during low frequency stimulation. Presynaptic for also facilitates SV endocytosis during high frequency stimulation. for's effects on neurotransmission can occur independent of any changes in nerve terminal growth. However, it remains unclear if for's effects on neurotransmission affect nerve terminal growth. Furthermore, it's possible that for's effects on synaptic structure contribute to changes in neurotransmission. In the present study, we examined these questions using RNA interference to selectively knockdown for in presynaptic neurons or glia at the Drosophila larval nmj. Consistent with our previous findings, presynaptic knockdown of for impaired SV endocytosis, whereas knockdown of glial for had no effect on SV endocytosis. Surprisingly, we found that knockdown of either presynaptic or glial for increased neurotransmitter release in response to low frequency stimulation. Knockdown of presynaptic for did not affect nerve terminal growth, demonstrating that for's effects on neurotransmission does not alter nerve terminal growth. In contrast, knockdown of glial for enhanced nerve terminal growth. This enhanced nerve terminal growth was likely the cause of the enhanced neurotransmitter release seen following knockdown of glial for. Overall, we show that for can affect neurotransmitter release by regulating both synaptic structure and function.

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.

Genes and environments, development and time.

A now substantial body of science implicates a dynamic interplay between genetic and environmental variation in the development of individual differences in behavior and health. Such outcomes are affected by molecular, often epigenetic, processes involving gene-environment (G-E) interplay that can influence gene expression. Early environments with exposures to poverty, chronic adversities, and acutely stressful events have been linked to maladaptive development and compromised health and behavior. Genetic differences can impart either enhanced or blunted susceptibility to the effects of such pathogenic environments. However, largely missing from present discourse regarding G-E interplay is the role of time, a "third factor" guiding the emergence of complex developmental endpoints across different scales of time. Trajectories of development increasingly appear best accounted for by a complex, dynamic interchange among the highly linked elements of genes, contexts, and time at multiple scales, including neurobiological (minutes to milliseconds), genomic (hours to minutes), developmental (years and months), and evolutionary (centuries and millennia) time. This special issue of PNAS thus explores time and timing among G-E transactions: The importance of timing and timescales in plasticity and critical periods of brain development; epigenetics and the molecular underpinnings of biologically embedded experience; the encoding of experience across time and biological levels of organization; and gene-regulatory networks in behavior and development and their linkages to neuronal networks. Taken together, the collection of papers offers perspectives on how G-E interplay operates contingently within and against a backdrop of time and timescales.

Behavior-related gene regulatory networks: A new level of organization in the brain.

Neuronal networks are the standard heuristic model today for describing brain activity associated with animal behavior. Recent studies have revealed an extensive role for a completely distinct layer of networked activities in the brain-the gene regulatory network (GRN)-that orchestrates expression levels of hundreds to thousands of genes in a behavior-related manner. We examine emerging insights into the relationships between these two types of networks and discuss their interplay in spatial as well as temporal dimensions, across multiple scales of organization. We discuss properties expected of behavior-related GRNs by drawing inspiration from the rich literature on GRNs related to animal development, comparing and contrasting these two broad classes of GRNs as they relate to their respective phenotypic manifestations. Developmental GRNs also represent a third layer of network biology, playing out over a third timescale, which is believed to play a crucial mediatory role between neuronal networks and behavioral GRNs. We end with a special emphasis on social behavior, discuss whether unique GRN organization and cis-regulatory architecture underlies this special class of behavior, and review literature that suggests an affirmative answer.

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.

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.

Distinct functions of a cGMP-dependent protein kinase in nerve terminal growth and synaptic vesicle cycling.

Sustained neurotransmission requires the tight coupling of synaptic vesicle (SV) exocytosis and endocytosis. The mechanisms underlying this coupling are poorly understood. We tested the hypothesis that a cGMP-dependent protein kinase (PKG), encoded by the foraging (for) gene in Drosophila melanogaster, is critical for this process using a for null mutant, genomic rescues and tissue-specific rescues. We uncoupled the exocytic and endocytic functions of FOR in neurotransmission using a temperature-sensitive shibire mutant in conjunction with fluorescein-assisted light inactivation of FOR. We discovered a dual role for presynaptic FOR, in which FOR inhibits SV exocytosis during low-frequency stimulation by negatively regulating presynaptic Ca2+ levels and maintains neurotransmission during high-frequency stimulation by facilitating SV endocytosis. Additionally, glial FOR negatively regulated nerve terminal growth through TGF-ß signalling, and this developmental effect was independent of the effects of FOR on neurotransmission. Overall, FOR plays a critical role in coupling SV exocytosis and endocytosis, thereby balancing these two components to maintain sustained neurotransmission.

Self-regulation and the foraging gene (PRKG1) in humans.

Foraging is a goal-directed behavior that balances the need to explore the environment for resources with the need to exploit those resources. In Drosophila melanogaster, distinct phenotypes have been observed in relation to the foraging gene (for), labeled the rover and sitter. Adult rovers explore their environs more extensively than do adult sitters. We explored whether this distinction would be conserved in humans. We made use of a distinction from regulatory mode theory between those who "get on with it," so-called locomotors, and those who prefer to ensure they "do the right thing," so-called assessors. In this logic, rovers and locomotors share similarities in goal pursuit, as do sitters and assessors. We showed that genetic variation in PRKG1, the human ortholog of for, is associated with preferential adoption of a specific regulatory mode. Next, participants performed a foraging task to see whether genetic differences associated with distinct regulatory modes would be associated with distinct goal pursuit patterns. Assessors tended to hug the boundary of the foraging environment, much like behaviors seen in Drosophila adult sitters. In a patchy foraging environment, assessors adopted more cautious search strategies maximizing exploitation. These results show that distinct patterns of goal pursuit are associated with particular genotypes of PRKG1, the human ortholog of for.

A Neuroethics Backbone for the Evolving Canadian Brain Research Strategy.

Building on Canada's strong traditions in neuroscience and ethics, neuroethics provides a backbone for the evolving Canadian Brain Research Strategy (CBRS) that, from the outset, incorporates ethically responsible discoveries in brain science into clinical, societal, educational, and commercial innovation.

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.