Spatial-scERA: A method for reconstructing spatial single-cell enhancer activity in multicellular organisms

Enhancers play an essential role in developmental processes by orchestrating the spatial and temporal regulation of gene expression. However, mapping the location of these regulatory elements in the genome and precisely characterizing their spatial and temporal activity remain important challenges. Here we introduce a novel in vivo and in silico method for spatial single-cell enhancer-reporter assays (spatial-scERA) designed to reconstruct the spatial activity of multiple candidate enhancer regions in parallel in a multicellular organism. Spatial-scERA integrates massively parallel reporter assays coupled with single-cell RNA sequencing (scRNA-seq) and spatial reconstruction using optimal transport, to map cell-type-specific enhancer activity at the single-cell level on a 3D virtual representation of the sample. We evaluated spatial-scERA in stage 6 Drosophila embryos using 25 candidate enhancers (including 19 uncharacterized regions), and validated the robustness of our predicted reconstructions by comparing them to microscopy images generated by in situ hybridization. Remarkably, spatial-scERA faithfully reconstructed the spatial activity of these enhancers, even when the enhancer-reporter construct was expressed in as few as 10 cells. Our results demonstrate the importance of integrating transcriptomic and spatial data for the accurate prediction of enhancer activity patterns in complex multicellular samples. Indeed, we found that chromatin modifications and open chromatin regions are often poor predictors of enhancer activity. Moreover, spatial data can often be essential for the accurate annotation of scRNA-seq clusters. Finally, we showed that spatial-scERA could be a powerful tool to link enhancers with their potential target genes. Overall, spatial-scERA provides a scalable approach to map spatio-temporal enhancer activity at single-cell resolution without the need for imaging or a priori knowledge of embryology and can be applied to any multicellular organism amenable to transgenesis.