Hox genes, arranged in clusters, encode a family of conserved transcription factors that play important roles in specifying regions of the body plan along the anterior-posterior axis. Signalling pathways, including retinoic acid signalling, regulate Hox gene expression patterns along the body axis. How enhancers integrate graded signalling inputs to coordinate the transcription of Hox genes in vertebrates is still unclear. Here, Robb Krumlauf and colleagues use single-molecule approaches to study transcriptional regulation of the Hoxb cluster in individual cells of mouse embryos. They focus on investigating retinoic acid response element (RARE)-dependent enhancers, which are shared by several genes within the Hoxb cluster. First, the authors couple single-molecule fluorescence in situ hybridisation with deep learning to measure transcription of the Hoxb genes. They detect newly synthesized transcripts of only a single Hoxb gene in a cell at a given time, rather than observing simultaneous transcription of multiple Hoxb genes that share RARE enhancers. Then, the authors mutate RARE enhancers, individually or in combination, in mice and detect differential transcriptional changes, suggesting that interactions between these enhancers ensure proper Hoxb transcription. Overall, this work introduces new approaches and provides further insights into the transcriptional mechanisms controlling Hox gene expression during vertebrate development.