Elimination of a long-range cis-regulatory module causes complete loss of limb-specific Shh expression and truncation of the mouse limb.
Mutations in a conserved non-coding region in intron 5 of the Lmbr1 locus, which is 1 Mb away from the sonic hedgehog (Shh) coding sequence, are responsible for mouse and human preaxial polydactyly with mirror-image digit duplications. In the mouse mutants, ectopic Shh expression is observed in the anterior mesenchyme of limb buds. Furthermore, a transgenic reporter gene flanked with this conserved non-coding region shows normal polarized expression in mouse limb buds. This conserved sequence has therefore been proposed to act as a long-range, cis-acting regulator of limb-specific Shh expression. Previous phylogenetic studies have also shown that this sequence is highly conserved among tetrapods, and even in teleost fishes. Paired fins of teleost fishes and tetrapod limbs have evolved from common ancestral appendages, and polarized Shh expression is commonly observed in fins. In this study, we first show that this conserved sequence motif is also physically linked to the Shh coding sequence in a teleost fish, the medaka, by homology search of a newly available genomic sequence database. Next, we show that deletion of this conserved intronic sequence by targeted mutation in the mouse results in a complete loss of Shh expression in the limb bud and degeneration of skeletal elements distal to the stylopod/zygopod junction. This sequence contains a major limb-specific Shh enhancer that is necessary for distal limb development. These results suggest that the conserved intronic sequence evolved in a common ancestor of fishes and tetrapods to control fin and limb development.
Sagai
Tomoko
T
Mammalian Genetics Laboratory, National Institute of Genetics, Yata-1111 Mishima, Shizuoka-ken 411-8540, Japan.
Hosoya
Masaki
M
Mizushina
Youichi
Y
Tamura
Masaru
M
Shiroishi
Toshihiko
T
D000818:Animals; D017124:Conserved Sequence; D005121:Extremities / Q000196:embryology / Q000254:growth & development*; D018507:Gene Expression Regulation, Developmental / Q000235:genetics / Q000502:physiology*; D053823:Hedgehog Proteins; D007438:Introns / Q000235:genetics; D018878:Limb Buds / Q000196:embryology / Q000254:growth & development; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008648:Mesoderm / Q000166:cytology / Q000378:metabolism; D051379:Mice; D009024:Morphogenesis / Q000235:genetics / Q000502:physiology*; D009154:Mutation / Q000235:genetics; D009990:Oryzias / Q000196:embryology / Q000235:genetics / Q000254:growth & development; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*
D053823:Hedgehog Proteins; C417920:Lmbr1 protein, mouse; D008565:Membrane Proteins; C465570:Shh protein, mouse; D015534:Trans-Activators