Single-nucleus multiome ATAC and RNA sequencing reveals the molecular basis of thermal acclimation in Drosophila melanogaster embryos

Embryogenesis is remarkably robust to temperature variability, yet the homeostatic mechanisms that offset thermal effects during early development remain poorly understood. We measured how acclimation shifts the upper thermal limit and profiled chromatin accessibility and gene expression in D. melanogaster embryos using single-nucleus multiome sequencing. Acclimation preserved shared primordial cell types yet rapidly shifted heat tolerance. Cool-acclimated embryos showed a homeostatic response characterized by increased accessibility at binding motifs for the transcriptional activator Zelda , along with enhanced activity of gene regulatory networks in a subset of primordial cell types including the gut, muscle, and nervous system. Cool-acclimated embryos also had higher expression of ribosomal and oxidative phosphorylation genes, reflecting a coordinated increase in translation and energy production to buffer slower biochemical kinetics in the cold. Our results indicate that maintaining developmental robustness across temperature requires homeostatic gene expression responses that may carry metabolic costs constraining upper thermal limits.