Temporally restricted activities ofEn1regulatory elements underlie distinct limb malformations

Abstract

AbstractThe 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 locusMaenliwhich drivesEn1expression at E9.5, specifically in the limb. Here we addressed howEn1expression is maintained at later developmental stages whenMaenlitranscriptional activity is absent. With a series ofin vivoCRISPR editing, we demonstrate that at later stages E10.5 and E11.5,En1expression is driven by two intergenic enhancer elements, LSEE1 and LSEE2. Upon simultaneous loss of these two enhancers, mice only exhibit a subset of theEn1mutant andMaenli-/-limb malformations. We show that the timing ofEn1misexpression during limb development causes distinct phenotypes. These findings demonstrate that the temporally restricted activities ofcis-regulatory elements, including lncRNA loci and enhancers, may underlie subtle differences in complex disease phenotypes.