Single embryo-resolution quantitative analysis of reporters permits multiplex spatial cis-regulatory analysis

•A novel high-throughput method for spatial cis-regulatory analysis is developed.•The method uses quantitative profiles of single-embryo resolution reporter assays.•The method can rapidly predict germ layer-specific CRM activities.•The method can detect spatial responses of CRMs to gene perturbations.•The method can be scaled up to many hundreds of CRMs in a single experiment.

Cis-regulatory modules (CRMs) control spatiotemporal gene expression patterns in embryos. While measurement of quantitative CRM activities has become efficient, measurement of spatial CRM activities still relies on slow, one-by-one methods. To overcome this bottleneck, we have developed a high-throughput method that can simultaneously measure quantitative and spatial CRM activities. The new method builds profiles of quantitative CRM activities measured at single-embryo resolution in many mosaic embryos and uses these profiles as a 'fingerprint' of spatial patterns. We show in sea urchin embryos that the new method, Multiplex and Mosaic Observation of Spatial Information encoded in Cis-regulatory modules (MMOSAIC), can efficiently predict spatial activities of new CRMs and can detect spatial responses of CRMs to gene perturbations. We anticipate that MMOSAIC will facilitate systems-wide functional analyses of CRMs in embryos.