Fig. 1. Gene targeting and Pitx2 isoform expression. (A,B) 8.5 dpc expression of Pitx2c (A), Pitx2a and Pitx2b (B). Arrow denotes sinous venosus and arrowhead indicates lateral mesoderm. (C) 9.0 dpc expression of Pitx2c. bw, body wall; sp, splanchnopleure. (D,E) 10.5 dpc expression of Pitx2c in wild-type (D) and 
ab;ab embryos (E). lb, lung bud; s, stomach. (F) X-gal staining in ab; ab guts. c, cecal diverticulum; d, duodenum; mg, midgut; sma, superior mesenteric artery. (G,H) 10.5 dpc expression of Pitx2c (G), Pitx2a and Pitx2b (H). oe, oral ectoderm; pm, periocular mesenchyme. (I,J) Eye phenotypes (arrowhead) of wild-type (I) and ab;ab (J) embryos. (K) Exon usage of Pitx2 isoforms. (L) Pitx2 genomic structure and targeting strategy. The boxes represent exons and straight lines introns. The exons are not drawn to scale. (M) Targeted allele before and after removal of the PGKneomycin cassette. At the bottom is the Pitx2-null allele that was previously generated (Lu et al., 1999). (N) Southern blot with flanking probes: tail DNA probed with the 5' flanking probe (left); tail DNA probed with the 3' flanking probe (center). The right panel shows a Southern blot probed with an internal lacZ probe after crossing the abhypoc +/- mice to the CMV cre recombinase deletor strain to generate ab +/- mice. After recombination, and PGKneomycin removal, the lacZ probe hybridizes to a 2 kb fragment, while in mice that still retain the PGKneomycin, the lacZ probe hybridizes to a 3 kb fragment. In the right-hand panel, + above the lanes denotes mice that have retained PGKneomycin and – denotes a mouse that has deleted PGKneomycin. (O) Diagram of riboprobe that distinguishes between isoforms and the Pitx2c-protected fragment. (P) Ribonuclease protection assay of mRNA from Pitx2 allelic combinations: 1, probe; 2,3, tRNA; 4,5, wild type; 6, abcnull heterozygous; 7,8, abcnull; ab; 9,10, abcnull; abhypoc; 11,12, abcnull; abcnull; 13, markers. (Q) Quantitation of Pitx2c mRNA levels in the different Pitx2 allelic combinations.
