Abstract Understanding spatial gene expression and regulation is key to uncovering developmental and physiological processes, during homeostasis and disease. Numerous techniques exist to gain gene expression and regulation information, but very few utilise intuitive true-to-life three-dimensional representations to analyze and visualize results. Here we combined spatial transcriptomics with 3D modelling to represent and interrogate, transcriptome-wide, three-dimensional gene expression and location in the mouse adult heart. Our study has unveiled specific subsets of genes that display complex spatial expression in organ sub-compartments. Also, we created a web-based user interface for spatial transcriptome analysis and visualization. The application may be accessed from http://3d-cardiomics.erc.monash.edu/ .

3D-Cardiomics: A spatial transcriptional atlas of the mammalian heart