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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

The early care environment and DNA methylome variation in childhood.

Prenatal adversity shapes child neurodevelopment and risk for later mental health problems. The quality of the early care environment can buffer some of the negative effects of prenatal adversity on child development. Retrospective studies, in adult samples, highlight epigenetic modifications as sentinel markers of the quality of the early care environment; however, comparable data from pediatric cohorts are lacking. Participants were drawn from the Maternal Adversity Vulnerability and Neurodevelopment (MAVAN) study, a longitudinal cohort with measures of infant attachment, infant development, and child mental health. Children provided buccal epithelial samples (mean age = 6.99, SD = 1.33 years, n = 226), which were used for analyses of genome-wide DNA methylation and genetic variation. We used a series of linear models to describe the association between infant attachment and (a) measures of child outcome and (b) DNA methylation across the genome. Paired genetic data was used to determine the genetic contribution to DNA methylation at attachment-associated sites. Infant attachment style was associated with infant cognitive development (Mental Development Index) and behavior (Behavior Rating Scale) assessed with the Bayley Scales of Infant Development at 36 months. Infant attachment style moderated the effects of prenatal adversity on Behavior Rating Scale scores at 36 months. Infant attachment was also significantly associated with a principal component that accounted for 11.9% of the variation in genome-wide DNA methylation. These effects were most apparent when comparing children with a secure versus a disorganized attachment style and most pronounced in females. The availability of paired genetic data revealed that DNA methylation at approximately half of all infant attachment-associated sites was best explained by considering both infant attachment and child genetic variation. This study provides further evidence that infant attachment can buffer some of the negative effects of early adversity on measures of infant behavior. We also highlight the interplay between infant attachment and child genotype in shaping variation in DNA methylation. Such findings provide preliminary evidence for a molecular signature of infant attachment and may help inform attachment-focused early intervention programs.

Fetal growth interacts with multilocus genetic score reflecting dopamine signaling capacity to predict spontaneous sugar intake in children.

BACKGROUND: We have shown that intrauterine growth restriction (IUGR) leads to increased preference for palatable foods at different ages in both humans and rodents. In IUGR rodents, altered striatal dopamine signaling associates with a preference for palatable foods. OBJECTIVES: Our aim was to investigate if a multilocus genetic score reflecting dopamine-signaling capacity is differently associated with spontaneous palatable food intake in children according to the fetal growth status. METHODS: 192 four-year old children from a community sample from Montreal and Hamilton, Canada, were classified according to birth weight and administered a snack test meal containing regular as well as palatable foods. Intrauterine growth restriction was based on the birth weight ratio below 0.85; children were genotyped for polymorphisms associated with dopamine (DA) signaling, with the hypofunctional variants (TaqIA-A1 allele, DRD2-141C Ins/Ins, DRD4 7-repeat, DAT1-10-repeat, Met/Met-COMT) receiving the lowest scores, and a composite score was calculated reflecting the total number of the five genotypes. Macronutrient intake during the Snack Test was the outcome. RESULTS: Adjusting for z-score BMI at 48 months and sex, there was a significant interaction of the genetic profile and fetal growth on sugar intake [߈ = -4.56, p = 0.04], showing a positive association between the genetic score and sugar intake in IUGR children, and no association in non-IUGR children. No significant interactions were seen in other macronutrients. CONCLUSIONS: Variations in a genetic score reflecting DA signaling are associated with differences in sugar intake only in IUGR children, suggesting that DA function is involved in this behavioral feature in these children. This may have important implications for obesity prevention in this population.

Both maternal care received and genotype influence stress-related phenotype in female rats.

Rat dams differ naturally in the level of maternal care they provide to their offspring within the same litter. We explored possible mechanisms of differential maternal care focused on genetic variation. We examined single nucleotide polymorphisms in the glucocorticoid receptor, FK506-binding protein, and serotonin transporter genes in two separate cohorts, and the relationship between differential maternal care received, genotype, and offspring phenotype. Allelic variation in all three genes was significantly associated with levels of maternal care received by offspring and behavioral and endocrine stress responses in adulthood. Differences in pup behavior were also associated with allelic variation in these genes. Together, these results indicate that the dam/pup interaction is dynamic and implicate the genotype of the offspring in influencing the level of maternal care received. They further suggest that some genotypes may have a dampening effect on the impact of maternal care on stress-related phenotypes in adulthood.

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.

The Drosophila foraging gene human orthologue PRKG1 predicts individual differences in the effects of early adversity on maternal sensitivity.

There is variation in the extent to which childhood adverse experience affects adult individual differences in maternal behavior. Genetic variation in the animal foraging gene, which encodes a cGMP-dependent protein kinase, contributes to variation in the responses of adult fruit flies, Drosophila melanogaster, to early life adversity and is also known to play a role in maternal behavior in social insects. Here we investigate genetic variation in the human foraging gene (PRKG1) as a predictor of individual differences in the effects of early adversity on maternal behavior in two cohorts. We show that the PRKG1 genetic polymorphism rs2043556 associates with maternal sensitivity towards their infants. We also show that rs2043556 moderates the association between self-reported childhood adversity of the mother and her later maternal sensitivity. Mothers with the TT allele of rs2043556 appeared buffered from the effects of early adversity, whereas mothers with the presence of a C allele were not. Our study used the Toronto Longitudinal Cohort (N=288 mother-16 month old infant pairs) and the Maternal Adversity and Vulnerability and Neurodevelopment Cohort (N=281 mother-18 month old infant pairs). Our findings expand the literature on the contributions of both genetics and gene-environment interactions to maternal sensitivity, a salient feature of the early environment relevant for child neurodevelopment.