Genome-wide identification of signaling center enhancers in the developing limb

Abstract

The limb iswidely used as amodel developmental systemand changes to gene expression patterns in its signaling centers, notably the zone of polarizing activity (ZPA) and the apical ectodermal ridge (AER), are known to cause limbmalformations and evolutionary differences in limb morphology. Although several genes that define these limb signaling centers have been described, the identification of regulatory elements that are active within these centers has been limited. By dissecting mouseE11.5 limbs that fluorescentlymark the ZPAorAER, followed by fluorescence-activated cell sorting and low-cell H3K27ac ChIP-seq, we identified thousands of specific signaling-center enhancers. Our ChIP-seq datasets show strong correlation with ZPA- and AERexpressed genes, previously characterized functional ZPA and AER enhancers and enrichment for relevant biological terms related to limb development and malformation for the neighboring genes. Using transgenic assays, we show that several of these sequences function as ZPA and AER enhancers. Our results identify novel ZPA and AER enhancers that could be important regulators of genes involved in the establishment of these specialized regions and the patterning of tetrapod limbs.