Developmental Alcohol Exposure in Drosophila: Effects on Adult Phenotypes and Gene Expression in the Brain

Abstract

Fetal alcohol exposure can lead to developmental abnormalities, intellectual disability, and behavioral changes, collectively termed fetal alcohol spectrum disorder (FASD). In 2015, the Centers for Disease Control found that 1 in 10 pregnant women report alcohol use and more than 3 million women in the USA are at risk of exposing their developing fetus to alcohol. Drosophila melanogaster is an excellent genetic model to study developmental effects of alcohol exposure because many individuals of the same genotype can be reared rapidly and economically under controlled environmental conditions. Flies exposed to alcohol undergo physiological and behavioral changes that resemble human alcohol-related phenotypes. Here, we show that adult flies that developed on ethanol-supplemented medium have decreased viability, reduced sensitivity to ethanol, and disrupted sleep and activity patterns. To assess the effects of exposure to alcohol during development on brain gene expression, we performed single cell RNA sequencing and resolved cell clusters with differentially expressed genes which represent distinct neuronal and glial populations. Differential gene expression showed extensive sexual dimorphism with little overlap between males and females. Gene expression differences following developmental alcohol exposure were similar to previously reported differential gene expression following cocaine consumption, suggesting that common neural substrates respond to both drugs. Genes associated with glutathione metabolism, lipid transport, glutamate and GABA metabolism, and vision feature in sexually dimorphic global multi-cluster interaction networks. Our results provide a blueprint for translational studies on alcohol-induced effects on gene expression in the brain that may contribute to or result from FASD in human populations.

Keywords: behavioral genetics; fetal alcohol spectrum disorder; interaction networks; model organism; single cell RNA sequencing; transcriptomics.

Figure 1

Diagram of the experimental design.

Figure 2

Effects of developmental alcohol exposure on viability and behavioral phenotypes in adult flies. (A) Boxplots of viability (n = 12 reps of 50 embryos per treatment), (B) Ethanol sensitivity (n = 43–49, 3–5 day old flies per sex per treatment), (C) Activity, (D) Proportion of daytime sleep, (E) Activity bouts during the day. (F) Proportion of night time sleep, (G) Activity bouts during the night. Day hours are from 7 a.m. to 7 p.m., lights on 7 h after hour zero. Gray boxes indicate flies reared on medium supplemented with 10% (v/v) ethanol and white boxes indicate control flies grown on regular medium. n = 57–64 flies per sex per treatment for all sleep and activity phenotypes. *p < 0.05, **p < 0.01, ***p < 0.001. Actograms are shown in Supplementary Figure 1.

Figure 3

Uniformity across samples of single cell transcriptomes. Gene expression patterns of single cells (n = 108, 571) from all eight samples are represented in low dimensional space using a graph-based, non-linear dimensionality reduction method (UMAP). Individual dots represent the transcriptome of each cell and the colors of the dots represent the samples to which the cells belong.

Figure 4

UMAP visualization and annotation of cell clusters. Cells were clustered based on their expression pattern using the unsupervised shared nearest neighbor (SNN) clustering algorithm. Individual dots represent each cell and the colors of the dots represent the cluster to which the cells belong. Annotation of cell types from clusters was performed by cross-referencing cluster-defining genes across FlyBase (30) and published literature (Supplementary Table 3).

Figure 5

Differentially expressed genes across clusters in males (A) and females (B) after developmental alcohol exposure. Differentially expressed genes are listed on the top (columns) and cell clusters are represented by the rows. Upregulated genes are indicated with orange and downregulated genes are indicated with purple. Differentially expressed genes are filtered at |log_eFC| > 0.25 and a Bonferroni adjusted p < 0.05. Differentially expressed genes that survive a threshold of |log_eFC| > 1.0 with a Bonferroni adjusted p < 0.05 are shown in Supplementary Figure 2.

Figure 6

Global interaction networks of differentially expressed gene products in males (A) and females (B) following developmental alcohol exposure. Colors of the nodes correspond to the clusters in which expression of the gene is altered after growth on alcohol-supplemented medium.

Figure 7

Venn diagrams indicating the proportions of differentially regulated genes after exposure to alcohol during development or acute consumption of cocaine for males (A) and females (B). Data for cocaine exposure are from ref 19. See also Supplementary Table 7.