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First published online January 27, 2005
doi: 10.1242/10.1242/dev.01613

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Elimination of a long-range cis-regulatory module causes complete loss of limb-specific Shh expression and truncation of the mouse limb

Mammalian Genetics Laboratory, National Institute of Genetics, Yata-1111 Mishima, Shizuoka-ken 411-8540, Japan

View larger version (10K): [in a new window]   Fig. 1. Three blocks of non-coding sequence are conserved among mammals and the teleost fish medaka. Intervening sequences between the Shh coding region and the Lmbr1 gene are compared between human, mouse and medaka genomes using the VISTA program. The genomic sequences used for comparison of the three species are 1,088,638 bp of human sequence (Ensemble, chromosome 7, 155013840-156102478), 992,498 bp of mouse sequence (Ensemble, chromosome 5, 26711297-27703795), and 203,199 bp of medaka fish sequence (scaffold 35). The boxes and circles depict the conserved fragments that show more than 75% identity over 100 bp of sequence among the three species. The green boxes depict the exons of Shh, and blue boxes the exons of Lmbr1. The orange circles depict conserved non-coding sequences. Notably, the Shh coding region and the conserved sequence blocks are physically linked on the same chromosomes in mouse, human, and medaka fish.

View larger version (37K): [in a new window]   Fig. 2. VISTA data of MFCS1. The VISTA search for mouse-human and mouse-medaka homologous sequence revealed the full extent and core sequence of the MFCS1. In the VISTA search, we used 50 bp window length and 75% conservation level. The sites of two mouse mutations (red arrows), Hx and M100081, and four human PPD mutations (black arrows) are superimposed on MFCS1. A DNA segment (1167 bp in length) that includes the entire MFCS1 was eliminated by gene targeting in this study.

View larger version (29K): [in a new window]   Fig. 3. Generation of mutant mice lacking MFCS1. (A) Design of the targeting vector. A neomycin (Neo) cassette used for positive selection was flanked by a 5652 bp long arm (AB114903 98833-104485) on the 5' end, a 2357 bp short arm (AB114903 95309-97666), and a diphteria toxin (DT) cassette used for negative selection on the 3' end. Homologous recombination results in deletion of 1167 bp of MFCS1. (B) PCR primers p1 and p2 were used for screening recombined ES clones. (C) Genotyping of mice was carried out using the p3 and p4 primers for the wild-type allele, and (D) the p5 and p6 primers for the MFCS1 KO allele. Ba, BamH1; N, Not1; H, Hpa1.

View larger version (89K): [in a new window]   Fig. 4. External phenotype of the MFCS1 KO mouse. (A,B) The limbs of the MFCS1 KO embryos at E12.5 are extremely reduced. (C,D) In the KO embryos at E14.5, the distal ends of the limbs becomes very thin. (E) A 10-day post-natal MFCS1 KO homozygote appears quite healthy, except for the limb abnormality. (F) The body size of a 3-week-old MFCS1 KO homozygote (right) is comparable to that of wild-type (left). (G-J) Magnified pictures of the forelimbs (G,I) and hindlimbs (H,J) in the MFCS1 KO homozygote. The autopod is not recognizable in the forelimbs. In the hindlimbs, only one digit with a nail is observed (H). On the surface of the ventral side of the digit, pad-like tissue is observed (J).

View larger version (65K): [in a new window]   Fig. 5. Skeletal phenotypes of MFCS1 and Shh KO mutants. (A-C) Skeletal preparations of E14.5 embryos were stained with alcian blue. (A,B) Lateral view of a wild-type and MFCS1 KO homozygote. (C) Dorsal view of a Shh KO homozygote. (D-I) Skeletal preparations of 3-week-old mice were stained with alizarin red and alcian blue. The forelimb and hindlimb of the wild-type mouse are shown in (F) and (I) respectively. (D,E) Forelimbs of MFCS1 KO homozygotes. (G,H) Hindlimbs of MFCS1 KO homozygotes. cl, clavicle; eb, elbow joint; fb, floating skeletal element; fe, femur; fi, fibula; h, humerus; mc, metacarpals; mt; metatarsal bones; ph, phalanges; r, radius; s, scapla; sp, stylopod; t, tibia; ts, tarsal bones; u, ulna; zg, zeugopod; zv, zeugopodial vestiges.

View larger version (70K): [in a new window]   Fig. 6. Expression patterns of marker genes in the limb buds at E10.5. In all figures except for A, D and G, anterior side is top. In wild-type (A-C) and MFCS1 KO heterozygous (D-F) embryos, Shh expression is observed in the posterior mesenchyme of the limb buds. In the limb buds of MFCS1 KO homozygous embryos (G-I), Shh expression is not detected. Expression of marker genes, dHand (J-M) and Gli3 (N-Q), Fgf8 (R-U) and Fgf4 (V-Y) were examined at a stage prior to Shh expression.