The E-Protein Daughterless Regulates Olfactory Learning of Adult Drosophila melanogaster

Emc overexpression impairs Da transcriptional activity in vivo. a, Light micrographs of adult flies; control, elavC155-Gal4/white*; Da^OE, elavC155-Gal4 > da; Emc^OE, elavC155-Gal4 > emc; Da^OE, Emc^OE, elavC155-Gal4 > Da,Emc. b, Schematic representation of in vivo luciferase reporter assay (created using BioRender.com). Transgenic flies containing minimal promoter (min) and a firefly luciferase reporter gene (Firefly luciferase) for min-luc or 12xCAGCTG E-boxes (12xE-box), minimal promoter, and a firefly luciferase reporter gene for 12xE-box-min-luc were used. Emc was overexpressed (Emc^OE) using elavC155-Gal4 driver. As a control, elavC155-Gal4 and reporter construct containing flies were crossed to white* flies (control; min-luc and control;12xE-box-min-luc). c, Results of the luciferase reporter assay. Luciferase activities were measured and shown as fold change compared with the control, the replicates are shown as individual shapes, and error bars indicate standard error of the mean (SEM); n = 4, two-tailed Student’s paired t test.

Widespread transcriptional changes after pan-neuronal Da inhibition by Emc. a, Differentially expressed genes (DEGs) in the brains of Emc^OE (elavC155-Gal4 > emc) flies compared with the control (elavC155-Gal4xwhite*). On x-axis, fold changes are shown in log₂ scale, and on y-axis adjusted p values (BH normalization, DESeq2) are shown in −log₁₀ scale. Dotted lines represent cutoff values for ±0.2 log₂ fold change and 0.05 adj. p value. Significantly upregulated and downregulated genes are listed in Extended Data Figure 3-1. Levels of emc, da, and Class 2 bHLH protein mRNAs are shown in Extended Data Figure 3-2. b, Summary of DEGs from the RNA-seq experiment, above and below zero, indicates up- and downregulated genes, respectively. c, GO terms and (d) Reactome Pathway defined pathways dysregulated by Emc overexpression; color gradient represents adjusted p values; red and blue indicate up- and downregulated gene cohorts, respectively. Biological processes affected by Da inhibition are shown in Extended Data Figure 3-3 and Reactome Pathways in Extended Data Figure 3-4.

Pathways directly regulated by Da. a, Visualization of anti-FLAG ChIP-seq data from adult Drosophila heads of 3xFLAG-Da and control flies and RNA-seq data from adult brains of Emc^OE (elavC155-Gal4 > emc) flies compared with control (elavC155-Gal4xwhite*) flies. ChIP-Seq data were visualized as fold enrichment over input and RNA-Seq data as counts per million aligned reads. Biological replicates were merged for visualization, with n = 2 for ChIP-Seq and n = 4 for RNA-Seq. As an example, stripe (sr) gene locus is shown, where 3xFLAG-Da binds to the promoter regions, and expression in RNA-Seq is lower in Emc^OE. b, Venn diagram of common statistically significant genes from 3xFLAG-Da ChIP-seq and Emc^OE RNA-seq datasets. For 3xFLAG-Da ChIP-seq, statistically significant genes were defined as log₂ fold change > 1 (compared with white*) and false discovery rate ≤ 0.05. For Emc^OE RNA-seq, statistically significant genes were determined as follows: counts ≥20 at least in control or Emc^OE samples, log₂ fold change >0.2 or <−0.2 and p-adjusted values ≤ 0.05. Genes that were upregulated or downregulated by Emc overexpression and that contained FLAG-Da ChIP peaks are listed in Extended Data Figure 4-1. c, GO terms and (d) Reactome Pathway terms for common genes. Color gradient represents adjusted p values (BH); red and blue indicate up- and downregulated gene cohorts in the RNA-seq data, respectively. Biological processes directly regulated by Da are shown in Extended Data Figure 4-2 and Reactome Pathways in Extended Data Figure 4-3.

Appetitive associative learning is impaired in adult Drosophila with pan-neuronal Da inhibition by Emc. Performance index of 1-d-old Emc^OE (elavC155-Gal4 > emc) flies is significantly reduced compared with control (elavC155-Gal4xwhite*) flies (a), and performance index of da^RNAi (nSyb-Gal4 > da^RNAi) flies is significantly reduced compared with control (nSyb-Gal4xwhite*) flies (e). Preference index toward MCH is shown on b and f, OCT (3-octanol) on c and g, and sucrose on d and h. Performance and preference indexes are visualized using box-whisker plots that show the median, the 25% and 75% quartiles (hinges); the upper whisker extends from the hinge to the largest value no further than 1.5 of the interquartile range from the hinge; the lower whisker extends from the hinge to the smallest value at most 1.5 * interquartile range of the hinge, individual data points are presented as small dots; n = 12 for a and e; n = 8 for b, c, d, f, g, and h. p values were calculated using two-sided Wilcoxon rank-sum test. Expression patterns of pan-neuronal Gal4 drivers used are shown in Extended Data Figure 5-1.

Inhibiting Da in the adult brains after pupariation does not significantly effect memory of the flies. a, Performance index of ts-Gal80;elavC155-Gal4 > emc flies starved in 29°C for 24 h is not significantly changed compared with flies starved in 18°C. Performance indexes are visualized using box-whisker plots that show the median, the 25% and 75% quartiles (hinges); the upper whisker extends from the hinge to the largest value no further than 1.5 of the interquartile range from the hinge; the lower whisker extends from the hinge to the smallest value at most 1.5 * interquartile range of the hinge; individual data points are presented as small dots; n = 12 p value was calculated using two-sided Wilcoxon rank-sum test. b, qPCR results of cDNA from ts-Gal80;elavC155-Gal4 > emc flies kept in 29 or 18°C for 24 h. Relative emc mRNA levels were calculated and shown in fold change compared with flies in 18°C. Replicates are shown as individual shapes, and error bars indicate SEM; n = 3, two-tailed Student’s paired t test.