Characterization of transcriptomics during aging and genes required for lifespan in Drosophila intestine

Abstract

Aging is closely associated with imbalanced transcription. Regulated transcription in different organs is significantly different during aging, indicating that organ-specific transcriptomics is critical for understanding this process. Here we analyze the transcriptomics of the intestines of 3-, 15-, 30-, 40- and 50-days old female flies, which include young, middle-aged, and old flies. We find that the differential expression of protein-coding genes and lncRNAs is significant in aging, and fly age is characterized by well-separated gene expression trajectories. The highly clustered differentially expressed genes are connected to specific biological processes and signalling pathways. In particular, the Imd and Toll pathways are the top two immune signalling pathways that are highly regulated, and members with increased expression in the Imd pathway span all upstream activating events and include many ubiquitylation-associated factors and regulators of NF-κB factor Relish. Increased expression of Toll pathway members includes sensing mediators for all kinds of microorganisms and multiple proteases in the proteolytic processing cascade. Moreover, the expression of molecular markers of intestinal cells is greatly changed. Enterocyte markers are the most significantly influenced, and enteroendocrine markers AstA and NPF, as well as intestinal stem cell (ISC)/enteroblast (EB) markers Esg and Klu are expressed at low levels in young flies and much higher levels in aged flies. Furthermore, lncRNAs show similar expression trends and clustering patterns to those of protein-coding genes. Lastly, we find that ISC/EB-specific knock-down of 13 out of 19 genes that are highly differentially expressed reduces the lifespan of the fly. Together, the characterized transcriptomics and newly identified functional genes in aging will provide potential targets for preventing intestinal aging and associated disorders.

Keywords: Drosophila; Aging; Intestine; Lifespan; Transcriptomics; lncRNA.

Fig. 1

Differential expression of protein-coding genes during aging process in fly gut. (A) Scheme of workflow. (B) Principal component analysis (PCA) plot of protein-coding genes expression. Samples from different aging timepoints were presented with unique colors. (C) Heatmap illustrating correlation of gene expression at various timepoints. (D) Volcano plot showing relative expression of protein-coding genes in comparisons between different timepoints. Number of differentially expressed genes (DEGs) were shown. The y-axis represents statistical significance as −log10 (adjusted p value), and the x-axis represents log2 fold change. Up-regulated genes were marked in red, down-regulated genes were marked in blue.

Fig. 2

Distinct expression trends are connected with specific biological processes and signalling pathways. (A) Mfuzz of differentially expressed genes showing 8 clusters of expression trend. Number of DEGs were shown in bracket. The black line represents an average expression trend of all genes within a cluster, a shift of red region indicates contribution to the expression trend, and colored lines indicate the membership degree of individual gene within a cluster. (B) GO (Gene Ontology) and C. KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analysis of DEGs. The top-seven significantly enriched categories were presented in color charts.

Fig. 3

Gene expression trends of immune-related signalling pathways. (A–F). Distribution maps illustrating the expression patterns of DEGs across immune-related signalling pathways.

Fig. 4

Gene expression trends of molecular markers of intestinal cells and hormones. (A–D). Distribution maps illustrating the expression patterns of molecular markers and hormones in designated cells.

Fig. 5

ISC/EB-specific knockdown of genes expression reduces lifespan. Survival curves of flies with indicated genotypes (n = 150 per group) following ISC/EB-specific knockdown driven by esg ^ts > Gal4. Survival curves of flies were shown as the following: fit (A), CG17321 (B), CG17264 (C), CNMa (D), aurB (E), me31B (F), CG42324 (G), CG42700 (H), CG11555 (I), Idgf5 (J), eIF5 (K), nec (L), CG10581 (M), Hsdl2 (N), Kr (O), CCHa1 (P), sc (Q), Mp20 (R) and Mcm7 (S).

Fig. 6

Dysregulation of aging-related genes breaks intestinal integrity. Proportion of Smurf flies of indicated genotypes on specified days after birth, CNMa (A), fit (B), idgf5 (C), and me31B (D).

Fig. 7

Differential expression of lncRNAs during aging process in fly gut. (A) Principal component analysis (PCA) plot of lncRNAs expression. Samples from different aging timepoints were presented with unique color. (B) Heatmap illustrating correlation of lncRNAs expression at various timepoints. (C) Volcano plot showing relative expression of lncRNAs in comparisons between different timepoints. Number of differentially expressed lncRNAs (DERs) were shown. The y-axis represents statistical significance as −log10 (adjusted p value), and the x-axis represents log2 fold change. Up-regulated lncRNAs were marked in red, down-regulated lncRNAs were marked in blue.

Fig. 8

Expression trends of differentially expressed lncRNAs. Mfuzz of DERs showing 8 clusters of expression trend. The number of DERs were shown in bracket.