Phylogenetic conservation of a limb-specific, cis-acting regulator of Sonic hedgehog (Shh) Tomoko Sagai, 1 Hiroshi Masuya, 2 Masaru Tamura, 1 Kunihiko Shimizu, 3 Yukari Yada, 1 Shigeharu Wakana, 2 Yoichi Gondo, 4 Tetsuo Noda, 2 Toshihiko Shiroishi, 1,2 1 Mammalian Genetics Laboratory, National Institute of Genetics, Yata-1111, Mishima, Shizuoka-ken 411-8540, Japan 2 Mouse Functional Genomics Research Group, Riken Genomic Sciences Center, 214 Maeda-cho, Totsuka-ku, Yokohama, Kanagawa 244-0804, Japan 3 Department of Pedodontics, Nihon University Graduate School of Dentistry at Matudo, 2-870-1 Sakaecho-Nishi, Matudo, Chiba, 271, Japan 4 Population and Quantitative Genomics Team, Bioinformatics Group, Riken Genomic Sciences Center, 214 Maeda-cho, Totsuka-ku, Yokohama, Kanagawa 244-0804, Japan Received: 22 July 2003 / Accepted: 2 September 2003 Abstract Polarized expression of the Sonic hedgehog (Shh) gene in the posterior mesenchyme is essential for pattern formation in the appendages of higher ver- tebrates, from teleost fins to tetrapod limb buds. We report on a sequence in intron 5 of the Lmbr1 gene, which resides approximately 1 Mb from the Shh coding region in the mouse genome and is highly conserved among teleost fishes and throughout the tetrapod lineage. Positional cloning revealed that two mouse mutations, Hx and M100081, character- ized by mirror-image digit duplication and ectopic anterior Shh expression, have base substitutions in this sequence. Absence of the conserved sequence in limbless reptiles and amphibians and a cis-trans test using the Hx and Shh KO alleles suggest that the sequence is a cis-acting regulator that controls the polarized expression of Shh. Acquisition of tetrapod limbs is among the most striking events in vertebrate evolution. It is cur- rently thought that paired fins of teleost fishes and tetrapod limbs evolved from a common ancestral appendage and that the emergence of polarized Sonic hedgehog (Shh) expression in the posterior margin of fin buds played a crucial role in freeing the fins from the body axis (Tanaka et al. 2002). This polarized Shh expression in the posterior mesenchyme is con- served evolutionarily throughout teleost fins and tetrapod limbs (Echelard et al. 1993; Endo et al. 1997; Riddle et al. 1993; Torok et al. 1999), but the mechanism of its regulation is poorly understood. Shh encodes a signaling protein expressed in the zone of polarizing activity (ZPA), which is located in the posterior mesenchyme of the limb buds and is known to control the anteroposterior (A/P) patterning of limbs (Johnson and Tabin 1997; Riddle et al. 1993; Saunders and Gasseling 1968). Its function is exerted either directly, by dose-dependent activation of target gene expression (Drossopoulou et al. 2000; Yang et al. 1997) or indirectly, via the regulation of the relative balance of Gli3 transcriptional activation, and re- pression of the Shh downstream genes (Litingtung et al. 2002; te Welsher et al. 2002b; Wang et al. 2000). Preaxial polydactyly with mirror-image digit duplication is a major heritable abnormality of limb development, both in humans and in the mouse. A locus for the major form of human preaxial poly- dactyly (PPD) has been mapped to Chromosome (Chr) 7q36 (Heutink et al. 1994; Tsukurov et al. 1994). In the mouse, two preaxial polydactyly mu- tations, hemimelic extratoes (Hx) and Sasquatch (Ssq), have been mapped to the syntenic region that is closely linked to Shh in Chr 5 (Martin et al. 1990; Sharpe et al. 1999). Recently, a similar mutation, M100081, generated by large-scale ENU mutagenesis in RIKEN GSC, was mapped to the same region (data not shown). The phenotypes of these mutants are confined to the distal limb field of the zeugopods and autopods (Knudsen and Kochhar 1981; Sharpe et al. DOI: 10.1007/s00335-033-2317-5 • Volume 15, 23–34 (2004) •Ó Springer-Verlag New York, Inc. 2004 23 The nucleotide sequence data reported in this paper have been submitted to GenBank and have been assigned the accession number: AB092986 to AB093004, AB093207, and AB114903. Correspondence to: T. Shiroishi; E-mail: tshirois@lab.nig.ac.jp