The structural organization of this class of deeply conserved vertebrate enhancers is under strong selective constraints, and, even in light of binding site redundancies exhibited by transcription factors, few sequence changes are present. Hence, the ZRS has remained highly invariant against a backdrop of major evolutionary changes to the anatomy of the appendicular skeleton, which includes the transition of fish fins to tetrapod limbs ( Gehrke and Shubin, 2016). This enhancer is ∼770 bp in length and shows a high degree of similarity in vertebrates across a lengthy evolutionary timescale, including sharks and rays ( Dahn et al., 2007), and, in accord, the mouse shows >70% similarity with the coelacanth (lobe-finned fish) sequence ( Figure S1). Here we focus on a highly conserved element called the zone of polarizing activity regulatory sequence (ZRS) that is responsible for the spatiotemporal expression of Shh during limb bud development ( Lettice et al., 2003, Sagai et al., 2005) and is essential for specifying digit identity and number. One class of enhancers that operates during embryogenesis are those that are highly conserved, acting at long distances from their target genes ( Visel et al., 2009). Despite these important roles, major unanswered questions remain about the information encoded by the enhancer sequence and the importance of the overall structural architecture to enhancer activity. These cis-acting elements, or enhancers, are fundamental to evolution and disease. The basis of embryonic development lies in the spatiotemporal control of gene expression, which is mediated by remote cis-regulatory elements. We propose a collective model for enhancer activity in which function is an integration of discrete expression activities and redundant components that drive robust expression.

Substantial portions of conserved sequence are dispensable, indicating the presence of sequence redundancy. The number of HOXD binding sites regulates expression levels incrementally. Spatial restriction of Shh expression is mediated by a discrete repressor module, whereas levels of gene expression are controlled by large overlapping domains containing varying numbers of HOXD binding sites. We show that enhancer activity is a consolidation of distinct activity domains. Here, we examined the endogenous ZRS in mice, using genome editing to establish the relationship between enhancer composition and embryonic phenotype. Expression of sonic hedgehog ( Shh) in the limb bud is regulated by an enhancer called the zone of polarizing activity regulatory sequence (ZRS), which, in evolution, belongs to an ancient group of highly conserved cis regulators found in all classes of vertebrates.

Sonic windows zone adjust controls