Temporal loss of En1 during limb development causes distinct phenotypes

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 9.5 (E9.5) to E11.5. Previously, we identified the long noncoding RNA locus Maenli, which drives limb-specific En1 expression at E9.5. Here, we uncover two intergenic enhancer elements, LSEE1&2, that are essential for En1 expression at E10.5 and E11.5. We demonstrate that Maenli and LSEE1&2 coordinate two transcriptional waves of En1 expression that are essential for establishing dorsal-ventral limb identity. The early wave, controlled by Maenli, is required for ventral ectoderm and apical ectodermal ridge specification, while the late wave, associated with LSEE1&2, is critical for dorsal-ventral ectoderm specification in mouse limbs. Loss of Maenli versus LSEE1&2 results in distinct developmental phenotypes by altering the spatiotemporal expression of key genes governing axial patterning pathways. Our study underscores how temporal cis-regulatory elements modulate lineage specification and developmental patterning through shaping spatiotemporal gene function during development, representing potential candidates underpinning isolated subphenotypes.