Abstract

Critical developmental control genes sometimes contain “shadow” enhancers that can be located in remote positions, including the introns of neighboring genes [1Hong J.W. Hendrix D.A. Levine M.S. Shadow enhancers as a source of evolutionary novelty.Science. 2008; 321: 1314Crossref PubMed Scopus (321) Google Scholar]. They nonetheless produce patterns of gene expression that are the same as or similar to those produced by more proximal primary enhancers. It was suggested that shadow enhancers help foster robustness in gene expression in response to environmental or genetic perturbations [2Boettiger A.N. Levine M. Synchronous and stochastic patterns of gene activation in the Drosophila embryo.Science. 2009; 325: 471-473Crossref PubMed Scopus (193) Google Scholar, 3Perry M.W. Cande J.D. Boettiger A.N. Levine M. Evolution of insect dorsoventral patterning mechanisms.Cold Spring Harb. Symp. Quant. Biol. 2009; 74: 275-279Crossref PubMed Scopus (16) Google Scholar]. We critically tested this hypothesis by employing a combination of bacterial artificial chromosome (BAC) recombineering and quantitative confocal imaging methods [2Boettiger A.N. Levine M. Synchronous and stochastic patterns of gene activation in the Drosophila embryo.Science. 2009; 325: 471-473Crossref PubMed Scopus (193) Google Scholar, 4Venken K.J. He Y. Hoskins R.A. Bellen H.J. P[acman]: A BAC transgenic platform for targeted insertion of large DNA fragments in D. melanogaster.Science. 2006; 314: 1747-1751Crossref PubMed Scopus (578) Google Scholar]. Evidence is presented that the snail gene is regulated by a distal shadow enhancer located within a neighboring locus. Removal of the proximal primary enhancer does not significantly perturb snail function, including the repression of neurogenic genes and formation of the ventral furrow during gastrulation at normal temperatures. However, at elevated temperatures, there is sporadic loss of snail expression and coincident disruptions in gastrulation. Similar defects are observed at normal temperatures upon reductions in the levels of Dorsal, a key activator of snail expression (reviewed in [5Hong J.W. Hendrix D.A. Papatsenko D. Levine M.S. How the Dorsal gradient works: Insights from postgenome technologies.Proc. Natl. Acad. Sci. USA. 2008; 105: 20072-20076Crossref PubMed Scopus (97) Google Scholar]). These results suggest that shadow enhancers represent a novel mechanism of canalization whereby complex developmental processes “bring about one definite end-result regardless of minor variations in conditions” [6Waddington C.H. Canalization of development and the inheritance of acquired characters.Nature. 1942; 150: 563-565Crossref Scopus (2002) Google Scholar].