Introduction

The aging process affects multiple organs, and the intestine is particularly sensitive due to its barrier function and stem cell dynamics. In Drosophila melanogaster, the gut epithelium renews continuously, making it an excellent model to study organ-specific aging. This article summarizes a Scientific Reports study that mapped gene expression changes in the fly intestine over five adult ages, identifying key pathways and lifespan regulators.

Methods Overview

Researchers dissected female fly guts at days 3, 15, 30, 40, and 50 post-eclosion. Poly-A RNA libraries were sequenced on Illumina HiSeq, generating 40–50 million high-quality reads per sample. Reads were aligned to the Drosophila reference genome and counted. Protein-coding genes with ≥10 reads and lncRNAs detected in all replicates were analyzed with DESeq2 (|log2 fold change| ≥1, adj. p<0.05). Time-series clustering used MFuzz, and functional enrichment applied Gene Ontology and KEGG databases.

Results

Global Gene Expression Shifts

Out of 10,074 protein-coding genes, 4,832 were differentially expressed across age comparisons. Principal component analysis separated samples by age: young (3,15 days), mid-age (30 days), and old (40,50 days).

Eight Temporal Expression Clusters

• Cluster 1: Early peak at day 15 in carbohydrate metabolism genes.
• Cluster 2: Gradual upregulation, enriched in DNA repair and immunity.
• Cluster 4: Midlife peak at day 30 in ribosome biogenesis and oxidative phosphorylation.
• Clusters 5–7: Late-phase shifts including chronic immune activation (Imd/Toll) and muscle decline.

Immune Pathway Activation

The Imd and Toll pathways were the top two upregulated immune cascades. Genes encoding upstream activators, ubiquitylation factors, and Relish regulators showed age-dependent increases. Toll pathway mediators including peptidoglycan- and glucan-binding proteins and proteases also rose in old guts.

Cell-Type Marker Dynamics

Enterocyte markers displayed a steep decline from young to old, while enteroendocrine hormones (AstA, NPF) and ISC/EB markers (esg, klu) increased, reflecting dysregulation of stem cell proliferation and differentiation in aging.

Functional Validation

Using esg-Gal4;UAS-GFP;Gal80ts to drive ISC/EB-specific RNAi, 13 out of 19 candidate genes reduced lifespan when knocked down in adults, and four compromised gut barrier integrity in the Smurf assay.

Implications

This comprehensive map of gut aging highlights phased shifts from metabolic priming to inflammaging. Identification of 13 lifespan regulators offers targets for interventions aimed at preserving intestinal function. The study underscores the value of tissue-specific transcriptomics for aging and longevity research.

Conclusion

By integrating RNA-seq, clustering, and functional assays, this work sets a framework to dissect organ-specific aging mechanisms. Future studies may explore modulators of these pathways to delay age-related gut decline and extend healthspan.

Key points

• Distinct transcriptomic profiles define young, mid-aged, and old fly intestines with 4,832 DEGs across eight expression clusters.
• Imd and Toll immune pathways show progressive activation during gut aging, alongside metabolic and stem cell transcriptome shifts.
• RNAi in intestinal stem cells validated 13 genes as crucial lifespan regulators and four for gut barrier maintenance.