Dev095430 1715..1725

Conservationwithin intergenicDNAoftenhighlights regulatoryelements that control geneexpression froma long range.Howconservationwithin a single element relates to regulatory information and how internal composition relates to function is unknown. Here, we examine the structural featuresof thehighly conservedZRS (also calledMFCS1) cisregulator responsible for the spatiotemporal control of Shh in the limb bud. By systematically dissecting the ZRS, both in transgenic assays and within in the endogenous locus, we show that the ZRS is, in effect, composed of two distinct domains of activity: one domain directs spatiotemporal activity but functions predominantly from a short range, whereasaseconddomain is required topromote long-rangeactivity.We show further that these two domains encode activities that are highly integrated and that the second domain is crucial in promoting the chromosomal conformational changes correlated with gene activity. During limb bud development, these activities encoded by the ZRS are interpreteddifferentlyby the fore limbsand thehind limbs; in theabsence of the second domain there is no Shh activity in the fore limb, and in the hind limb low levels ofShh lead to avariant digit pattern ranging from two to four digits. Hence, in the embryo, the second domain stabilises the developmental programme providing a buffer for SHH morphogen activity and this ensures that five digits form in both sets of limbs.