4360 Nucleic Acids Research, 2005, Vol. 33, No. 13 Figure 1. Structure of the zebrafish gata2 gene. (A) The diagram illustrates the exon/intron structure at the 50 end of the zebrafish gata2 gene, determined by comparing the 50-RACE product with the genomic sequence. Exons are shown as boxes and +1 indicates the transcription start site. (B) The position of the first intron relative to the genomic sequence is shown, with intronic sequences in lower case, and conserved intronic splice junction sequences in bold. (C) To generate a reporter gene, the sequences from the initiation ATG at +595 (relative to the transcription start site) to 6520 were placed upstream of the coding sequences for the firefly luciferase gene. This construct is analogous to a transgenic reporter shown to recapitulate the early gata2 expression pattern in zebrafish embryos (38). zebraﬁsh gata2 transcriptional start site, 50 rapid ampliﬁcation of cDNA ends (RACE) was performed on RNA isolated from embryos at 75% epiboly, the ﬁrst stage at which gata2 tran- scripts are readily detected (44). A single PCR product was obtained from this analysis, and subsequent sequencing and comparison with the genomic sequence determined that the transcription start site is located 595 bp upstream of the ini- tiation methionine (Figure 1A). The analysis identiﬁes a short non-coding ﬁrst exon and places the ATG initiation codon within the second exon (Figure 1A and B). The cDNA isolated by RACE includes sequences just upstream of an indepen- dently isolated gata2 cDNA clone (45). No alternative 50 sequences that would suggest an alternative exon were identiﬁed in the expressed sequence tag database. The exon 1 sequence does not show homology with gata2 genomic sequences from other species. The result does not rule out that additional promoters might be used in speciﬁc cell types or later stages of development, but indicates that a proxi- mal promoter functions during early embryogenesis, initiating transcription at 595 bp upstream of the initiation ATG. Therefore, we considered the 6.5 kb of sequences upstream of this region as putative regulatory sequences and developed an assay to determine if they are able to mediate a BMP response. Although it should be possible to do this experiment in the homologous zebraﬁsh system, Xenopus embryos provide a unique advantage in that the dorsal/ventral axis is discernable as early as the four-cell stage. Furthermore, the primary components of the early embryonic BMP signal- ing pathway are thought to be highly conserved. Therefore, the genomic sequence including the ATG was cloned upstream of a ﬁreﬂy luciferase reporter gene (Figure 1C) and this plasmid was injected into developing Xenopus embryos. This approach allows activity of the reporter to be analyzed in vivo during the early stages of embryogenesis when gata2 expression is known to be dependent on BMP signaling. In the Xenopus system, blastomeres committed to dorsal-anterior or ventral- posterior fates can be distinguished already at the four-cell stage, by virtue of pigment differences that arise following cortical rotation. A reporter sensitive to the endogenous BMP program is expected to be relatively more active when injected at the four-cell stage into presumptive ventral-posterior blas- tomeres compared to dorsal-anterior blastomeres. The reporter was co-injected with control RNA expressing Renilla luciferase. Preliminary experiments demonstrated that the control luciferase is equally active regardless of the injected blastomere (data not shown). The approach of using Renilla luciferase RNA (rather than a Renilla luciferase expression plasmid) avoids any potential concern that BMP-4 might also inﬂuence a control promoter. Embryos were harvested at stage 12–13 (during early neurulation) and protein lysates were tested for the relative amount of ﬁreﬂy luciferase, normalized to Renilla luciferase. We found that the gata2:luciferase reporter is more active when injected into the two ventral-posterior blastomeres of a four-cell embryo compared to activity when injected into the two dorsal-anterior blastomeres (Figure 2A). The difference is 2-fold, consistent with the gradient of BMP signaling across the dorsal/ventral axis. Importantly, co-injection of RNA encoding Xenopus Smad6 (xSmad6), which block BMP sig- naling (46–48), reduces the activity of the reporter when injected into ventral blastomeres to a level below what is found when the reporter is injected into dorsal-anterior blastomeres (Figure 2A). Thus, the reporter responds to the embryonic BMP program in a manner consistent with the endogenous pathway. To demonstrate directly that the reporter is activated by the BMP pathway, the gata2 and control reporter constructs were co-injected with RNA encoding BMP-4 into the two dorsal blastomeres. In this assay, the activity of the injected BMP-4 is conﬁrmed by the subsequent ventralized phenotype of injected embryos (data not shown). As shown in Figure 2B, BMP-4 expression results in an average 5-fold induction of reporter activity compared to activity of the reporter when coinjected with control RNA. Co-injection of Smad6 is sufﬁcient to block this activation to the basal level activity of the promoter. We conclude that 6.5 kb of upstream gata2 genomic sequence is sufﬁcient to respond to BMP signaling, and that the developing Xenopus embryo can be used as a model system to deﬁne the relevant cis elements. Deletion analysis defines an upstream region required for activation by BMP4 To deﬁne the BREs, a series of progressive 50 deletion con- structs were each injected into developing Xenopus embryos with either control RNA or RNA encoding BMP-4. In each