A parallel reporter assay for zebrafish

Cis-regulatory modules (CRMs) are central to gene regulation, yet in vivo authentication of candidate CRMs remains a major bottleneck. Here, we introduce a parallel reporter assay for zebrafish that enables simultaneous, quantitative analysis of multiple transcriptional reporters using standard Tol2-mediated transgenesis. Activity of individual reporters is established through detection of a molecular barcode, allowing direct comparison of CRM activity within the same biological sample. We find that barcoded reporters function independently and that variability arising from mosaicism and genomic position effects is effectively mitigated by analyzing modest numbers of embryos routinely obtained in transient transgenesis experiments. Using whole embryos, we demonstrate robust detection of reporter activity across CRMs with distinct levels and spatial domains of activity. We further show that pre-enrichment enhances sensitivity. This enables cell type-specific comparisons of CRM activity and reveals context-dependent differences in amplitude. Together, these findings demonstrate that conventional zebrafish transgenesis supports reliable, multiplexed analysis of transcriptional reporters. This approach provides a practical framework for higher-throughput authentication of candidate CRMs in vivo and complements existing strategies for detailed spatial characterization.