Analysis of the zebrafish spi1 locus and dissection of the promoter region. (A) Intron-exon structure of the spi1 gene. Solid bars represent translated exonic sequence, and the shaded bar shows the 3′ UTR. The 5′ UTR is too small to be visualized at this scale. (B) Linear depiction of the spi1 genomic locus. Letters indicate restriction enzyme sites (A = ApaI, B = BglII, Ba = BamHI, E = EcoRI, H = HindIII, P = PstI, S = SphI). (C) Activity of spi1 promoter fragments in transient expression assays. Embryos injected with the constructs indicated at the left were examined at 12, 24, and 32 hpf for patterns of EGFP expression that largely recapitulated endogenous spi1 expression pattern. This was scored as present (+) or absent (–). Nonspecific (n/s) expression at other sites (typically, the skeletal muscle and the eye) was similarly scored. All constructs were injected on at least 2 independent occasions, with 20% to 40% of injected embryos showing EGFP fluorescence in each case. Promoter-only injection controls gave no fluorescence. (D) Sequence analysis of the “core” spi1 promoter. Bases correspond to region of the spi1 promoter cloned upstream of EGFP in pA304, which produced early myeloid expression. Consensus Spi1 and c/ebpα sites are bolded and underlined, respectively, and the transcription start is indicated by an arrow.