Fig. 1. Disruption of Tbx6 binding sites eliminates Mesp2 expression. (A) Targeting strategy to generate the Mesp2 enhancer knockout mouse (P2EmB1D). A DNA fragment containing mutated Tbx6 binding sites (black ovals with X) was substituted for the wild-type sequence (white ovals) by homologous recombination. The PGK-neoR selection marker was removed by the Cre-loxP system to obtain a neo allele. (B) PCR detection of homozygotes in the P2EmB1D intercross. (C) Impaired skeletal segmentation in the Mesp2 enhancer knockout mouse. The P2EmB1D/P2EmB1D mouse exhibits severe skeletal malformation at E17.5 (centre) identical to that of the Mesp2-null mouse (P2MCM/P2MCM, right). Note the shortened spine with incompletely segmented vertebrae (upper panels) and fused ribs (bracket in lower panels). (D) Expression of Mesp2 and the somite-specific genes Mesp1, Epha4 and Tbx18 in P2EmB1D/+ (left column) and P2EmB1D/P2EmB1D (right column) embryos. Mesp2 mRNA expression is eliminated in the P2EmB1D/P2EmB1D homozygotes. Wild-type (+/+) and heterozygote (P2EmB1D/+) embryos showed varying Mesp2 expression patterns owing to its cyclic expression. Mesp1 is upregulated and Epha4 is not affected, whereas Tbx18 is completely abolished in P2EmB1D/P2EmB1D.