A major challenge in modern biology is to understand how naturally occurring variation in DNA sequences affects complex organismal traits through networks of intermediate molecular phenotypes. This question is best addressed in a genetic mapping population in which all molecular polymorphisms are known and for which molecular endophenotypes and complex traits are assessed on the same genotypes. Here, we performed deep RNA sequencing of 200 Drosophila Genetic Reference Panel inbred lines with complete genome sequences and for which phenotypes of many quantitative traits have been evaluated. We mapped expression quantitative trait loci for annotated genes, novel transcribed regions, transposable elements, and microbial species. We identified host variants that affect expression of transposable elements, independent of their copy number, as well as microbiome composition. We constructed sex-specific expression quantitative trait locus regulatory networks. These networks are enriched for novel transcribed regions and target genes in heterochromatin and euchromatic regions of reduced recombination, as well as genes regulating transposable element expression. This study provides new insights regarding the role of natural genetic variation in regulating gene expression and generates testable hypotheses for future functional analyses.

Food consumption is an essential component of animal fitness; however, excessive food intake in humans increases risk for many diseases. The roles of neuroendocrine feedback loops, food sensing modalities, and physiological state in regulating food intake are well understood, but not the genetic basis underlying variation in food consumption. Here, we applied ten generations of artificial selection for high and low food consumption in replicate populations of Drosophila melanogaster. The phenotypic response to selection was highly asymmetric, with significant responses only for increased food consumption and minimal correlated responses in body mass and composition. We assessed the molecular correlates of selection responses by DNA and RNA sequencing of the selection lines. The high and low selection lines had variants with significantly divergent allele frequencies within or near 2,081 genes and 3,526 differentially expressed genes in one or both sexes. A total of 519 genes were both genetically divergent and differentially expressed between the divergent selection lines. We performed functional analyses of the effects of RNAi suppression of gene expression and induced mutations for 27 of these candidate genes that have human orthologs and the strongest statistical support, and confirmed that 25 (93%) affected the mean and/or variance of food consumption.

There is a growing need to understand the potential neurotoxicity of organophosphate flame retardants (OPFRs) and plasticizers because use and, consequently, human exposure, is rapidly expanding. We have previously shown in rats that developmental exposure to the commercial flame retardant mixture Firemaster 550 (FM 550), which contains OPFRs, results in sex-specific behavioral effects, and identified the placenta as a potential target of toxicity. The placenta is a critical coordinator of fetal growth and neurodevelopment, and a source of neurotransmitters for the developing brain. We have shown in rats and humans that flame retardants accumulate in placental tissue, and induce functional changes, including altered neurotransmitter production. Here, we sought to establish if OPFRs (triphenyl phosphate and a mixture of isopropylated triarylphosphate isomers) alter placental function and fetal forebrain development, with disruption of tryptophan metabolism as a primary pathway of interest. Wistar rat dams were orally exposed to OPFRs (0, 500, 1000, or 2000 μg/day) or a serotonin (5-HT) agonist 5-methoxytryptamine for 14 days during gestation and placenta and fetal forebrain tissues collected for analysis by transcriptomics and metabolomics. Relative abundance of genes responsible for the transport and synthesis of placental 5-HT were disrupted, and multiple neuroactive metabolites in the 5-HT and kynurenine metabolic pathways were upregulated. In addition, 5-HTergic projections were significantly longer in the fetal forebrains of exposed males. These findings suggest that OPFRs have the potential to impact the 5-HTergic system in the fetal forebrain by disrupting placental tryptophan metabolism.

1 2 A major challenge in modern biology is to understand how naturally occurring variation in DNA 3 sequences affects complex organismal traits through networks of intermediate molecular 4 phenotypes. Here, we performed deep RNA sequencing of 200 Drosophila Genetic Reference 5 Panel inbred lines with complete genome sequences, and mapped expression quantitative trait 6 loci for annotated genes, novel transcribed regions (most of which are long noncoding RNAs), 7transposable elements and microbial species. We identified host variants that affect expression 8 of transposable elements, independent of their copy number, as well as microbiome 9 composition. We constructed sex-specific expression quantitative trait locus regulatory 10 networks. These networks are enriched for novel transcribed regions and target genes in 11 heterochromatin and euchromatic regions of reduced recombination, and genes regulating 12 transposable element expression. This study provides new insights regarding the role of natural 13 genetic variation in regulating gene expression and generates testable hypotheses for future 14 functional analyses.15 Introduction 1 2 Understanding how naturally occurring genetic variation affects variation in organismal 3 quantitative traits by modifying underlying molecular networks is a key challenge in modern 4 biology. Most traits are highly polygenic 1-3 and associated molecular variants have small 5 additive effects on trait variation 4 . Most of these variants are in intergenic regions, up-or down-6 stream of coding regions, or in introns, and presumably play a regulatory role in modulating 7 gene expression.8 Systems genetics analysis seeks to determine how naturally occurring molecular 9 variation gives rise to genetic variation in organismal phenotypes by examining genetic variation 10 in gene expression (expression quantitative trait loci, or eQTLs) and other intermediate 11 molecular phenotypes 2, 5-13 . Polymorphic variants associated with variation in gene expression 12 are classified as cis-or trans-eQTLs depending on whether they are proximal or distal to the 13 gene encoding the transcript, respectively. Genetic variation in gene expression is pervasive; 14 cis-eQTLs can have large effects on gene expression that are detectable in small samples; and 15variants associated with human diseases and quantitative traits tend to be enriched for cis-16 eQTLs 2, 5-15 . eQTLs with both cis-and trans-effects can be assembled into directed 17 transcriptional networks of regulator and target genes [16][17][18] . Elucidating such regulatory 18 transcriptional networks will facilitate understanding how the effects of individual variants 19 propagate through the network, and how multiple variants together regulate gene expression 20 and affect complex traits 15-18 . 21 Here, we performed deep RNA sequencing of the Drosophila melanogaster Genetic 22 Reference Panel (DGRP) of inbred lines with complete DNA sequences 19,20 . We mapped eQTLs 23 for annotated genes, novel transcribed region (NTRs, which are largel...

Introduction: Flame retardants (FRs) are common bodily and environmental pollutants, creating concern about their potential toxicity. We and others have found that the commercial mixture, FireMaster® 550 (FM 550) or its individual brominated (BFR) and organophosphate ester (OPFR) components are potential developmental neurotoxicants. Using Wistar rats, we previously reported developmental exposure to FM 550 or its component classes produced sex- and compound-specific effects on adult socioemotional behaviors. The underlying mechanisms driving the behavioral phenotypes are unknown. Methods: To further mechanistic understanding, here we conducted transcriptomics in parallel with a novel lipidomics approach using cortical tissues from newborn siblings of the rats in the published behavioral study. Inclusion of lipid composition is significant because it is rarely examined in developmental neurotoxicity studies. Pups were gestationally exposed via oral dosing to the dam to FM 550 or the BFR or OPFR components at environmentally relevant doses. Results: The neonatal cortex was highly sexually dimorphic in lipid and transcriptome composition, and males were more significantly impacted by FR exposure. Multiple adverse modes of action for the BFRs and OPFRs on neurodevelopment were identified, with the OPFRs more disruptive than the BFRs via multiple mechanisms including dysregulation of mitochondrial function and disruption of cholinergic and glutamatergic systems. Disrupted mitochondrial function by environmental factors has been linked to higher risk of autism spectrum disorders. Impacted lipid classes included the ceramides, sphingomyelins, and triacylglycerides. Robust ceramide upregulation in the OPFR females could suggest heightened risk of brain metabolic disease. Conclusions: This study reveals multiple mechanisms by which the components of a common FR mixture are developmentally neurotoxic and that the OPFRs may be the compounds of greatest concern.