Temporally restricted activities of En1 regulatory elements underlie distinct limb malformations

The precise spatiotemporal regulation of developmental genes is required for proper organogenesis. Engrailed-1 (En1) is essential for dorsal-ventral patterning during mouse limb development from embryonic day E9.5 to E11.5. Previously, we identified the long non-coding RNA locus Maenli, which drives limb-specific En1 expression at E9.5. In this study, we investigated the regulatory mechanisms sustaining En1 expression at later developmental stages when Maenli transcriptional activity is drastically reduced. Using in vivo CRISPR editing, we identified two intergenic enhancer elements, LSEE1 and LSEE2, that maintain En1 expression at E10.5 and E11.5. Mice lacking these enhancers exhibit only a subset of the limb malformations observed in En1 and Maenli mutants, indicating that the timing of En1 misexpression causes distinct phenotypes. These findings underscore the role of temporally restricted activities of cis-regulatory elements, including lncRNA loci and enhancers, in modulating gene expression and explaining subtle differences in complex disease phenotypes.