title: A series of ENU-induced single-base substitutions in a long-range cis-element altering Sonic hedgehog expression in the developing mouse limb bud.
abstract: Mammal-fish-conserved-sequence 1 (MFCS1) is a highly conserved sequence that acts as a limb-specific cis-acting regulator of Sonic hedgehog (Shh) expression, residing 1 Mb away from the Shh coding sequence in mouse. Using gene-driven screening of an ENU-mutagenized mouse archive, we obtained mice with three new point mutations in MFCS1: M101116, M101117, and M101192. Phenotype analysis revealed that M101116 mice exhibit preaxial polydactyly and ectopic Shh expression at the anterior margin of the limb buds like a previously identified mutant, M100081. In contrast, M101117 and M101192 show no marked abnormalities in limb morphology. Furthermore, transgenic analysis revealed that the M101116 and M100081 sequences drive ectopic reporter gene expression at the anterior margin of the limb bud, in addition to the normal posterior expression. Such ectopic expression was not observed in the embryos carrying a reporter transgene driven by M101117. These results suggest that M101116 and M100081 affect the negative regulatory activity of MFCS1, which suppresses anterior Shh expression in developing limb buds. Thus, this study shows that gene-driven screening for ENU-induced mutations is an effective approach for exploring the function of conserved, noncoding sequences and potential cis-regulatory elements.
authors.affiliation: Mouse Functional Genomics Research Group, RIKEN GSC 3-1-1 Kouyadai, Tsukuba, Ibaraki 305-0074, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Masuya
authors.forename: Hideki
authors.initials: H
authors.lastname: Sezutsu
authors.forename: Yoshiyuki
authors.initials: Y
authors.lastname: Sakuraba
authors.forename: Tomoko
authors.initials: T
authors.lastname: Sagai
authors.forename: Masaki
authors.initials: M
authors.lastname: Hosoya
authors.forename: Hideki
authors.initials: H
authors.lastname: Kaneda
authors.forename: Ikuo
authors.initials: I
authors.lastname: Miura
authors.forename: Kimio
authors.initials: K
authors.lastname: Kobayashi
authors.forename: Kenta
authors.initials: K
authors.lastname: Sumiyama
authors.forename: Aya
authors.initials: A
authors.lastname: Shimizu
authors.forename: Junko
authors.initials: J
authors.lastname: Nagano
authors.forename: Haruka
authors.initials: H
authors.lastname: Yokoyama
authors.forename: Satoko
authors.initials: S
authors.lastname: Kaneko
authors.forename: Noriko
authors.initials: N
authors.lastname: Sakurai
authors.forename: Yuka
authors.initials: Y
authors.lastname: Okagaki
authors.forename: Tetsuo
authors.initials: T
authors.lastname: Noda
authors.forename: Shigeharu
authors.initials: S
authors.lastname: Wakana
authors.forename: Yoichi
authors.initials: Y
authors.lastname: Gondo
authors.forename: Toshihiko
authors.initials: T
authors.lastname: Shiroishi
mesh_terms: D000818:Animals; D001483:Base Sequence; D017124:Conserved Sequence; D017931:DNA Primers / Q000235:genetics; D004742:Enhancer Elements, Genetic; D005038:Ethylnitrosourea; D005121:Extremities / Q000196:embryology*; D005260:Female; D018507:Gene Expression Regulation, Developmental; D005809:Genes, Regulator; D017930:Genes, Reporter; D005816:Genetic Complementation Test; D053823:Hedgehog Proteins / Q000235:genetics*; D017403:In Situ Hybridization; D017880:Limb Deformities, Congenital / Q000196:embryology / Q000235:genetics; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008811:Mice, Inbred DBA; D008817:Mice, Mutant Strains; D008822:Mice, Transgenic; D010641:Phenotype; D017354:Point Mutation; D020641:Polymorphism, Single Nucleotide; D011247:Pregnancy
chemical_list: D017931:DNA Primers; D053823:Hedgehog Proteins; C465570:Shh protein, mouse; D005038:Ethylnitrosourea