Developmental genes can have complex cis-regulatory regions with multiple enhancers. Early work revealed remarkable modularity of enhancers, whereby distinct DNA regions drive gene expression in defined spatiotemporal domains. Nevertheless, a few reports have shown that enhancers function in multiple developmental stages, implying that enhancers can be pleiotropic. Here, we have studied the activity of the enhancers of the shavenbaby gene throughout D. melanogaster development. We found that all seven shavenbaby enhancers drive expression in multiple tissues and developmental stages. We explored how enhancer pleiotropy is encoded in two of these enhancers. In one enhancer, the same transcription factor binding sites contribute to embryonic and pupal expression, revealing site pleiotropy, whereas for a second enhancer, these roles are encoded by distinct sites. Enhancer pleiotropy may be a common feature of cis-regulatory regions of developmental genes, and site pleiotropy may constrain enhancer evolution in some cases.

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Biological systems display extraordinary robustness. Robustness of transcriptional enhancers results mainly from clusters of binding sites for the same transcription factor, and it is not clear how robust enhancers can evolve loss of expression through point mutations. Here, we report the high-resolution functional dissection of a robust enhancer of the shavenbaby gene that has contributed to morphological evolution. We found that robustness is encoded by many binding sites for the transcriptional activator Arrowhead and that, during evolution, some of these activator sites were lost, weakening enhancer activity. Complete silencing of enhancer function, however, required evolution of a binding site for the spatially restricted potent repressor Abrupt. These findings illustrate that recruitment of repressor binding sites can overcome enhancer robustness and may minimize pleiotropic consequences of enhancer evolution. Recruitment of repression may be a general mode of evolution to break robust regulatory linkages.