Early cAMP signaling orchestrates single-cell synchronicity throughout Dictyostelium development

Synchronicity is a fascinating biological property observed across many organizational levels, from physiological functions such as heartbeat, breathing, and brain activity, to circadian rhythms, and even population dynamics in insects and plankton. It is also a hallmark of developmental biology, evident in the coordinated formation of the vertebrate body axis, the fly eye, and the aggregative development of the social amoeba Dictyostelium discoideum. Despite its prevalence, quantitative and mechanistic analyses of synchronicity at the single-cell level remain rare. Here, we show that developmental synchronicity in D. discoideum is mediated by pulsatile cyclic adenosine monophosphate (cAMP) signaling. Using single-cell RNA sequencing (scRNA-seq) and the Universal Cell Embedding (UCE) model, we quantified transcriptome similarity between individual cells across developmental stages. We found that synchronicity initially declined upon starvation but increased markedly with the onset of cAMP-pulse signaling during aggregation. Synchronicity remained stable throughout development and differed between prespore and prestalk cells, with prespore cells exhibiting greater transcriptome homogeneity. Genetic perturbations in cAMP production and response revealed that cAMP-pulse signaling is essential for establishing and maintaining synchronicity, as its absence led to highly asynchronous development even when morphogenesis was restored. Interestingly, starvation alone induced a modest synchronizing effect in the absence of development in a strain unable to produce cAMP. These findings highlight the central role of early cAMP-pulse signaling in coordinating transcriptomic and morphological synchronicity, and establish scRNA-seq as a powerful tool for quantitative analysis of developmental synchronicity at single-cell resolution. Overall design: A total of 30 samples were prepared for single-cell RNA sequencing using the 10x Genomics Chromium X system. Wild-type AX4, acaA?, and acaA? PkaCoe strains were generated and collected at six time points, spaced four hours apart, up to 20 hours. The AX4 strain expressed three distinct fluorescent proteins, while the acaA? strain expressed four. Each fluorescently tagged strain was cultured and processed independently, then pooled prior to loading onto the Chromium X platform. Fluorescent tag expression was used to distinguish biological replicates. The acaA? PkaCoe strain was analyzed in three biological replicates.