Development of five digits is controlled by a bipartite long-range cis-regulator.
Conservation within intergenic DNA often highlights regulatory elements that control gene expression from a long range. How conservation within a single element relates to regulatory information and how internal composition relates to function is unknown. Here, we examine the structural features of the highly conserved ZRS (also called MFCS1) cis-regulator 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, whereas a second domain is required to promote long-range activity. 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 interpreted differently by the fore limbs and the hind limbs; in the absence of the second domain there is no Shh activity in the fore limb, and in the hind limb low levels of Shh lead to a variant 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.
MRC-Human Genetics Unit, MRC Institute of Genetics and Molecular Medicine, University of Edinburgh, Western General Hospital, Crewe Rd, Edinburgh EH4 2XU, UK.
Laura A
LA
Lettice
Iain
I
Williamson
Paul S
PS
Devenney
Fiona
F
Kilanowski
Julia
J
Dorin
Robert E
RE
Hill
D000818:Animals; D001483:Base Sequence; D033481:Chromosomes, Mammalian / Q000737:chemistry; D004252:DNA Mutational Analysis; D004622:Embryo, Mammalian / Q000378:metabolism; D018507:Gene Expression Regulation, Developmental; D006614:Hindlimb / Q000196:embryology / Q000378:metabolism; D017404:In Situ Hybridization, Fluorescence; D018878:Limb Buds / Q000196:embryology* / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D009690:Nucleic Acid Conformation; D010641:Phenotype; D017354:Point Mutation / Q000235:genetics; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics*; D017384:Sequence Deletion / Q000235:genetics
C417920:Lmbr1 protein, mouse; D008565:Membrane Proteins
Limb development; Long-range regulation; Mouse; Sonic hedgehog; ZRS