DEV026476 Supplementary Material

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• Supplemental Figure S1 -

  Fig. S1. Expression of Ldb1 in the early limb bud is mostly eliminated by the T-Cre activity. (A) Immunohistochemistry with an antibody against Ldb1 in the wild-type and Ldb1^c/−;T-Cre hindlimb buds at 11.5 dpc. Ldb1 expression was significantly reduced in Ldb1^c/−;T-Cre mutant. (B) Sections of the hindlimb bud at 12.5 dpc were stained with Ldb1 antibodies. There were distally located patches of Ldb1⁺ cells in the Ldb1^c/−;T-Cre embryo (arrow). No Ldb1⁺ cells were detected in the mesoderm of the Ldb1^c/−;Ap2-Cre;T-Cre limb. (C) Cells in the Z/AP mice that have escaped Cre-mediated recombination expresses lacZ. The Z/AP mice were mated with the T-Cre mice and the embryos (Z/AP; T-Cre) were stained by X-gal at 11.5 dpc to visualize the lacZ expression (blue). The stained limbs were sectioned and counterstained with neutral red. The boxed region was shown in higher magnification below. Ap2-Cre mice (Li et al., 2005) were crossed with the T-Cre mice to generate the Ap2-Cre; T-Cre mice. These mice were crossed with Ldb1^+/− to obtain the Ldb1^+/−; Ap2-Cre; T-Cre mice. These mice were crossed with the Ldb1^c/c mice to produce the Ldb1^c/−; Ap2-Cre; T-Cre embryos. Ap2-Cre mice were genotyped using these oligos: Cre forward, 5′-GCGCTAACCCAGAGAGTAGCTCC-3′; and Cre reverse, 5′-CGCGAACATCTTCAGGTTCTGCGGG-3′.

  Reference

  Li, C., Xu, X., Nelson, D. K., Williams, T., Kuehn, M. R. and Deng, C. X. (2005). FGFR1 function at the earliest stages of mouse limb development plays an indispensable role in subsequent autopod morphogenesis. Development 132, 4755-4764.

• Supplemental Figure S2 -

  Fig. S2. Partial ventralization in the Ldb1-deficient mutant limbs. Whole-mount in situ hybridization of wild-type and indicated mutant hindlimbs with the Lmx1b probe. (A) Expression of Lmx1b was reduced in patches of the dorsal hindlimb at 12.5 dpc (arrow) but not at 10.5 dpc. (B) Lmx1b expression was more severely reduced in the Ldb1^c/−;Ap2-Cre;T-Cre hindlimb bud than in the Ldb1^c/−; T-Cre one. Limbs shown are at 11.5 dpc. Sections of the limb buds (limb buds were sectioned along the PD axis) are shown in the lower panel. Reduced Lmx1b expression is indicated by arrows.

• Supplemental Figure S3 -

  Fig. S3. Skeletal preparations of the Ldb1^c/−; T-Cre and Ldb1^c/−; Ap2-Cre;T-Cre limbs. (A) At 18.5 dpc, Ldb1^c/−; T-Cre mutant hindlimb showed severely shortened zeugopod and autopod (arrow) and fewer digits. The phenotype in the hindlimb was more severe. (C) At 18.5 dpc, Ldb1^c/−; Ap2-Cre;T-Cre limbs showed more severely shortened zeugopod and autopod (arrow) in both the forelimb (FL) and the hindlimb (HL) compared with Ldb1^c/−; T-Cre mutant limbs. The phenotype in the hindlimb was more severe. The digits were very small and fused with truncated phalanges (arrow). FL, forelimb; HL, hindlimb; H, humerus; R, radius; U, ulna; Fe, femur; T, tibia; Fi, fibula; S, stylopod; Z, zeugopod; A, autopod.

• Supplemental Figure S4 -

  Fig. S4. Cell death in the cultured limb bud. Whole-mount TUNEL procedure was performed to examine cell death in the 10.5 hindlimb that has been cultured in vitro for 24 hours. There is no difference in death between the culture wild type and Ldb1^c/−;T-Cre mutant limb bud.

• Supplemental Figure S5 -

  Fig. S5. Expression patterns of Lhx2 and Lhx9 in Fgf8^c/−;Fgf4^c/−;Msx2-Cre mutant limbs. Whole-mount in situ hybridization of Lhx2 and Lhx9 in wild-type and mutant hindlimbs at several stages. The Lhx9 expression pattern is normal in the mutant limb at 10.5 dpc. Later, both Lhx2 and Lhx9 expression is weaker. This may be caused by the reduction of distal limb mesenchymal cells in the Fgf8^c/−;Fgf4^c/−;Msx2-Cre mutant limb bud at later stages. Fgf8^c/−;Fgf4^c/−;Msx2-Cre mutant mice were generated and genotyped as previously described (Sun et al., 2002).

  Reference

  Sun, X., Mariani, F. V. and Martin, G. R. (2002). Functions of FGF signalling from the apical ectodermal ridge in limb development. Nature 418, 501-508.

• Supplemental Table S1 -

  Table S1. Lhx2, Lhx9 and Lmx1b are the predominant LIM-HD genes expressed during mouse limb development. Expression levels of all mouse LIM-HD genes and Ldb1, Ldb2 in the forelimb and hindlimb of the wild-type embryos at the indicated developmental stages were assessed by quantitative RT-PCR. -, no expression detected; +/−, very weak expression; +, expression; ++, strong expression.