TCTAGATATGTAAAGTGACG (TE1135); mutation #7, 50- GGGCTCGCTATTAAGCTTTAAAGTGACGTTTGATCG (TE1147). In vitro transcription RNA used for micro-injection was obtained by in vitro transcription using linearized pCS2 vectors. The vectors were pCS2-lacZ, pCS2-xSmad1 (from J. Thomsen), pCS2- zSmad5 (from M. Mullins), pCS2-xSmad6 (from J. Christian), pCS2-xSmad8 (34) and pCS2-BMP4 (from C. Wright) were used as templates for in vitro transcription. One microgram of linearized template was used to generate capped mRNAs with the mMessage mMachine kit (Ambion), followed by precip- itation with LiCl. RNA was quantiﬁed by optical density, and integrity was conﬁrmed by gel electrophoresis. Microinjection and luciferase assays Eggs were obtained from female Xenopus by standard gonadotropin induction protocols. Eggs were fertilized in vitro and dejellied in a solution of 2% cysteine (pH 8.0). Microinjection was performed with embryos in 0.1· MBS and 5% Ficoll. Each injection mix contained 62.5 pg of RNA encoding either BMP-4 or an irrelevant control RNA (such as lacZ), 25 pg of luciferase reporter DNA and 0.10 pg of RNA encoding Renilla luciferase (to normalize reporter activity). In some experiments, 250 pg of RNA encoding either xSmad1, zSmad5 or xSmad8 was included in place of BMP-4 RNA. In a standard assay, mixes were injected into the two dorsal blas- tomeres of a four-cell stage embryo. To generate lysates for gel mobility shift experiments, RNA was injected at the two-cell stage. Embryos were staged according to Nieuwkoop and Faber (35). Following microinjection, embryos were cultured in 0.1· MBS until stage 13, at which point embryos were col- lected and lysed in 1· Passive Lysis Buffer (Promega). Luciferase assays were performed according to the manufac- turer’s instructions. For each assay, four embryos were lysed in 100 ml of buffer, and typically 3–5 sets were used to gen- erate independent data points for each independent experi- ment, using 5 ml of lysate per 50 ml of luciferase substrate, measured in a Turner TD-20e luminometer. The ﬁreﬂy luciferase values were divided by corresponding values for the control Renilla luciferase. Normalized data from embryos injected with BMP-4 was divided by normalized numbers obtained from embryos injected with control RNA to deter- mine ‘BMP-4 fold induction’. All luciferase data represent averages of at least three independent microinjection experi- ments. Error bars in the ﬁgures indicate standard error of the mean. Preparation of Xenopus nuclear and total cell extracts Total cell extracts were prepared by freezing embryos in liquid N2, followed by homogenization in total cell extract buffer (50 mM Tris–HCl, pH 8.0, 400 mM KCl, 1 mM DTT, 25% glycerol, 50 mM NaF and protease inhibitors). Extracts were cleared by centrifugation three times for 10 min at 8160 g 10–20 mg per reaction was used in gel mobility shift reactions. To generate nuclear extracts, a nuclear pellet was obtained from stage 13 embryos as described (36). The pellet was resuspended in Buffer C (20 mM HEPES–KOH, 25% Nucleic Acids Research, 2005, Vol. 33, No. 13 4359 glycerol, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT and protease inhibitors) on ice and centrifuged at 10 000 g in a microfuge for 1 min at 4˚C to clear contami- nating pigment granules: 1–5 mg was used for each gel mobil- ity shift assay. To obtain lysates from embryos expressing BMP-4, embryos were injected at the two-cell stage with 500 pg of RNA and harvested at stage 13. Total protein con- centration for all experiments was obtained using a modiﬁed Bradford assay (BioRad). Gel mobility shift assays Oligomers were gel-puriﬁed prior to use as probes or cold competitors. For probes, the top strand was end-labeled with [g-32P]ATP using T4 polynucleotide kinase, followed by puriﬁcation thorough a G-25 Sepharose column. The radio- labeled top-strand was annealed with the complementary bot- tom strand by heating to 95 C for 5 min in 0.1 M NaCl, and then gradually cooling the reaction to room temperature. Cold competitors were prepared identically, without labeling. For super-shift experiments, reactions included 0.5 mg of anti- Oct-1 antibody (sc–8024, Santa Cruz) or 0.5 mg of control isotype-matched antibody (various, but the one shown is anti-Chicken Gata2, sc-267). Gels were run and analyzed as described (37). The sequences of the top strand of the oligomers are as follows: 763 to 737, 50-AGCTTATTAATATGTAAA- GTGACGTTTGATA (TE1175); 763 to 737 (mutated), 50- AGCTTATTAAGCATTAAAGTGACGTTTGATA (TE1191); Octamer consensus, 50-CGAATATGCAAATAAGGC (TE1189); CCAAT, 50-ACTGGCTGGCGGAGGCTTGTGA- TTGGCTGGCCCGG (TE1207). RESULTS The gata2 promoter is induced by BMP signaling The gata2 gene is activated at the transcriptional level by BMP signaling, but the mechanism for induction is not known. Previous transgenic reporter experiments demonstrated that 7.3 kb of sequences upstream of the zebraﬁsh gata2 initiation ATG are sufﬁcient to direct appropriate expression of a reporter to the early ventral domain (38). We sought to deﬁne the speciﬁc DNA regulatory elements that mediate transduc- tion of the BMP signal to the gata2 gene. The gata2 gene structure has been studied in a variety of organisms, including human (39,40), mouse (41), chicken (42) and Xenopus (39,43). Both the mouse and the chicken genes contain two alternative ﬁrst exons (proximal and distal), and the different transcrip- tional start sites are used depending on the cell type (41,42). Alternative ﬁrst exons are not described for either the Xenopus or human genes, but it remains possible that multiple promot- ers are a common feature of the gata2 locus, and this could complicate any mapping experiments. Therefore, before attempting to map BMP responsive sequences, we ﬁrst iden- tiﬁed the transcriptional start site for the zgata2 gene in order to deﬁne the proximal promoter. Sequence comparison of the zebraﬁsh gata2 putative pro- moter region with DNA sequences from exon 1 of the gata2 gene from a variety of species failed to identify regions of homology (data not shown). Therefore, to identify the