BACKGROUND: Embryonic development is coordinated by sets of cis-regulatory 
elements that are collectively responsible for the precise spatio-temporal 
organization of regulatory gene networks. There is little information on how 
these elements, which are often associated with highly conserved noncoding 
sequences, are combined to generate precise gene expression patterns in 
vertebrates. To address this issue, we have focused on Six3, an important 
regulator of vertebrate forebrain development.
RESULTS: Using computational analysis and exploiting the diversity of teleost 
genomes, we identified a cluster of highly conserved noncoding sequences 
surrounding the Six3 gene. Transgenesis in medaka fish demonstrates that these 
sequences have enhancer, silencer, and silencer blocker activities that are 
differentially combined to control the entire distribution of Six3.
CONCLUSION: This report provides the first example of the precise regulatory 
code necessary for the expression of a vertebrate gene, and offers a unique 
framework for defining the interplay of trans-acting factors that control the 
evolutionary conserved use of Six3.