Structure of the Pitx2 gene and its mRNAs and Pitx2 gene expression in normal murine fetal liver. (A) Genomic organization of the Pitx2 gene. Exon sequences are represented by white boxes and are numbered. The position of the homeodomain coding sequences is indicated (hatched bars). Pitx2a and Pitx2b mRNAs are transcribed using promoter 1 and generated by alternative splicing. Pitx2c mRNA is transcribed using internal promoter 2. For RT-PCR, Pitx2a and Pitx2b primers (•) amplify 274-bp and 412-bp products, respectively. Pitx2c primers (gray circles) amplify a 535 bp product and, for real-time RT-PCR. Pitx2 primers (○) amplify a 233-bp product common to all Pitx2 mRNA isoforms. (B) RT-PCR analysis of Pitx2a and Pitx2c mRNA isoforms during normal fetal liver development from 13 days after coitus to birth (NB) and adult life. d indicates days after coitus. (C) Evolution of Pitx2 mRNA accumulation during normal fetal liver development, from 10 days after coitus to adult life, studied by quantitative real-time RT-PCR amplifying all isoforms of Pitx2 mRNA (see panel A). Results are expressed as the percentage of the Pitx2 mRNA level detected in the fetal liver at 10 days after coitus. (D) FACS analysis of normal 13.5 days after coitus fetal liver cells stained by anti-CD45– FITC (panhematopoietic except erythroblasts) and anti-TER119–PE (late erythroid differentiation marker) antibodies. (E) RT-PCR analysis of Pitx2a and Pitx2c isoform expression in the 3 populations seen in panel D after cell sorting.