Transcripts encoding the 2 main isoforms of runx1 are differentially expressed from the 2 alternative promoters, P1 and P2, during development. (A) RT-PCR of runx1 P1 and P2 isoforms of RNA extracted from wild-type zebrafish unfertilized oocytes and embryos at different stages, with ef1α as a control. The runx1P1 isoform is maternally expressed, whereas the P2 isoform is predominantly expressed from 14 hpf. Vertical lines have been inserted to indicate a repositioned gel lane. (B) Genomic organization of the zebrafish runx1 locus and strategy for generating promoter constructs for transgenesis. The diagram shows the structure of the runx1 gene and the regions of the promoters used to generate the runx1::EGFP transgenics. Exons and UTRs are shown as filled and unfilled boxes, respectively. A 12-kb fragment of the P1 promoter was cloned by restriction digest and PCR and then inserted into a Tol2 transposon vector. The frequency of germline integration was 42%. An earlier protocol, with a germline integration frequency here of 5%, was used to generate the P2- transgenic line with EGFP driven by an 8-kb SacI-BamHI fragment.