Temporally restricted activities of En1 regulatory elements underlie distinct limb malformations

The precise spatiotemporal expression of developmental genes is required for proper embryonic development. EN1 plays a key role in dorsal-ventral patterning in mouse limb development from embryonic day (E) 9.5 to E11.5. Previously, we identified the lncRNA locus Maenli which drives En1 expression at E9.5, specifically in the limb. Here we addressed how En1 expression is maintained at later developmental stages when Maenli transcriptional activity is absent. With a series of in vivo CRISPR editing, we demonstrate that at later stages E10.5 and E11.5, En1 expression is driven by two intergenic enhancer elements, LSEE1 and LSEE2. Upon simultaneous loss of these two enhancers, mice only exhibit a subset of the En1 mutant and Maenli^-/- limb malformations. We show that the timing of En1 misexpression during limb development causes distinct phenotypes. These findings demonstrate that the temporally restricted activities of cis-regulatory elements, including lncRNA loci and enhancers, may underlie subtle differences in complex disease phenotypes.