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First published online November 3, 2003
doi: 10.1242/10.1242/dev.00861

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Replicated anterior zeugopod (raz): a polydactylous mouse mutant with lowered Shh signaling in the limb bud

Ottheinz Krebs1,*, Claire M. Schreiner2, William J. Scott, Jr2, Sheila M. Bell2, David J. Robbins3, John A. Goetz3, Heidi Alt1, Norm Hawes4, Eckhard Wolf1 and Jack Favor5

1 Institute of Molecular Animal Breeding and Biotechnology, Gene Center, Ludwig-Maximilian University, Munich, Germany
2 Division of Developmental Biology, Children's Hospital Research Foundation, Department of Pediatrics, University of Cincinnati, College of Medicine, Cincinnati, OH 45229, USA
3 Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati, College of Medicine, Cincinnati, OH 45267, USA
4 The Jackson Laboratory, Bar Harbor, ME 04609, USA
5 GSF – National Research Center for Environment and Health, Institute of Human Genetics, Neuherberg, Germany



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  		Fig. 1. Chromosomal organization of the raz mutation. (A) Results of DNA typing of backcross progeny. Markers shown from chromosome 5 are those that have been typed on 553 backcross DNAs. Values at the bottom of the figure are the number of progeny inheriting the indicated chromosome haplotype from the F1 parent. White squares represent the C3H/HeJ allele; black squares represent the BALB/c allele. (B) Ideogram of chromosome 5. Representation of the microsatellites used for linkage analysis with their physical and genetic positions. Also shown are genes involved in limb morphogenesis that are located within the inversion.

 


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  		Fig. 2. Skeletal phenotype of the forelimb. Dorsal view of the left forelimb of wild-type, wild-type/raz and raz/raz embryos. Anterior is upwards in A-F and towards the right in G-I. (C) There are two holes in the scapula as well as a notch (arrow) at the vertebral border in raz/raz fetuses. The olecranon process of the ulna (arrow) is present in wild-type/wild-type fetuses (A,D) but not in raz/raz fetuses (C,F). Note the styloid process of radius and ulna arises from different sites, peripherally from the radius and centrally from the ulna in wild-type/wild-type (G') and wild-type/raz (H) fetuses. However, in raz/raz fetuses the styloid process of both bones originates peripherally (I), indicating both bones have characteristics of a radius. Note cartilage bar in carpus of raz/raz (red arrow in I). Nomenclature in G is according to Davis and Capecchi (Davis and Capecchi, 1994Go). Scale bars: in C, 1.0 mm for A-C; in F, 1.0 mm for D-F; in I, 1.0 mm for G-I.

 


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  		Fig. 3. Skeletal phenotype of the hindlimb. Dorsal view of right hindlimbs of wild-type, wild-type/raz and raz/raz embryos. Anterior is upwards in A-I and towards the left in J-L. Note the presence of an enlarged talus, the anterior bone in the proximal tarsus, in raz/raz hindlimbs (F,L), and the absence (F) or remnant (L) of a calcaneus, the posterior bone of the proximal tarsus. The distal end of the zeugopod of wild-type/wild-type hindlimbs has distinctive malleoli characteristic of the tibia and fibula (G), whereas the raz/raz hindlimb has malleoli characteristic of the anterior bone, the tibia (I). The bar of distal cartilage from which the peripheral digits arise in the autopod of the raz/raz limb (red arrow in L). Scale bars: in C, 1.0 mm for A-C; in F, 1.0 mm for D-F; in I, 1.0 mm for G-I; in L, 1.0 mm for K-L.

 


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  		Fig. 4. Forelimb autopod of raz/raz fetus. Dorsal view of right forelimb autopod from raz/raz fetus. Anterior is towards the left. Note the metacarpals and phalanges arising from different dorsal (D) and ventral (V) planes. Scale bar: 0.25 mm.

 


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  		Fig. 5. Shh and Ptch expression. Whole-mount in situ hybridization of sonic hedgehog (Shh) and patched 1 (Ptch) expression in embryos (A-C,J-L) or left forelimbs (D-I,M-R) from wild-type, wild-type/raz and raz/raz embryos at different developmental stages. The left forelimbs (D-I,M-R) are viewed from the dorsal surface so that anterior is towards the top. Note the absence of Shh expression in raz/raz limbs (compare D with F and G with I), but the presence of normal expression throughout the remainder the raz/raz embryo (compare A with C). Ptch expression is also absent in the raz/raz limb bud (compare M with O and P with R), but is present throughout the remainder of the raz/raz embryo (compare J to L).

 


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  		Fig. 6. Shh/Shh in limb buds. (A) PCR analysis of forelimbs, hindlimbs, heads and bodies from E11.5 embryos of wild-type/wild-type, wild-type/raz and raz/raz genotypes. Note the low, but definitive band in lanes with raz/raz limb bud samples (lanes 2, 3 and 10). (B) Band quantitation by image analysis of the gel in A. (C) Western blot of E11.5 forelimbs and hindlimbs from wild-type/wild-type, wild-type/raz and raz/raz embryos. (D) Image analysis of bands in C based on tubulin content of each sample.

 


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  		Fig. 7. Polarizing activity and Shh activity in the limb. Polarizing activity (red) and Shh signaling activity (blue) are plotted with the mean from wild-type/wild-type samples given a value of 100. The number above each column represents the number of assays (n) conducted.

 


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  		Fig. 8. Gli1 and Gli3 expression. (A-C) Whole-mount in situ hybridization to reveal Gli1 expression in the left forelimb of E11.5 wild-type/wild-type, wild-type/raz and raz/raz embryos. Anterior is upwards. Note the posterior expression of Gli1 in wild-type/wild-type limbs (A), and absence of expression in raz/raz limbs (C). (D-I) Gli3 expression in forelimbs (D-F) or left forelimb (G-I) of wild-type/wild-type, wild-type/raz and raz/raz embryos. Scale bar: 0.25 mm. (J) PCR analysis for Gli3 mRNA of E11.5 forelimbs (FL), hindlimbs (HL) and remainder of embryo (body) of wild-type/wild-type, wild-type/raz and raz/raz embryos. Limb buds were split longitudinally to provide anterior (Ant.) and posterior (Post.) samples. (K) Image quantitation of PCR analyses with wild-type/wild-type body given a value of 100 and other tissues normalized to that value. Results are mean±s.d.

 

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© The Company of Biologists Ltd 2003